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He Z, Xu Y, Zhang Y, Jin M, Sun Y, Tang F, Qiu C, Junior AMA, Cai Y, Xu X, Chen X, Chen K, Xiang G, Xiao J, Wang J, Wang J, Chen B. Betulinic acid enhances autopahgy to promote microglial M2 polarization and alleviate inflammation via AMPK-HDAC5-KLF2 signaling pathways in spinal cord injury. Int Immunopharmacol 2025; 158:114889. [PMID: 40388862 DOI: 10.1016/j.intimp.2025.114889] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/14/2025] [Revised: 05/13/2025] [Accepted: 05/14/2025] [Indexed: 05/21/2025]
Abstract
Spinal cord injury (SCI) leads to neuroinflammation and activates microglia, which are crucial contributors to neurological deficits. Betulinic acid (BA), a naturally occurring pentacyclic triterpenoid, has demonstrated effectiveness in treating inflammatory and neurological disorders. This study aims to explore the potential role and underlying mechanism of BA in modulating microglial activation and inflammation in the context of SCI. Using a mouse SCI model, we assessed motor recovery via Basso Mouse Scale (BMS) and neuronal survival via H&E/Nissl staining. Western blotting, qPCR, immunofluorescence, and flow cytometry were employed to analyze microglial polarization, autophagy, and AMPK-HDAC5-KLF2 signaling in vivo and in LPS-stimulated BV2 cells. Our findings reveal that BA significantly enhances functional recovery and reduces neuronal apoptosis following SCI. Furthermore, BA facilitates the phenotypic transition of microglia from the M1 to M2 phenotype, thereby decreasing inflammatory factors in both the SCI model and LPS-stimulated BV2 cells. BA treatment restores the disrupted autophagy flux in microglia induced by SCI or LPS, which in turn mitigates M1 polarization and inflammation. Mechanistically, BA restores autophagy flux by activating the AMPK-HDAC5-KLF2 axis, thereby shifting microglia from pro-inflammatory M1 to anti-inflammatory M2 phenotype.
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Affiliation(s)
- Zili He
- Department of Orthopaedics Surgery, The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University, Wenzhou 325035, China; Oujiang Laboratory (Zhejiang Lab for Regenerative Medicine, Vision and Brain Health), School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou 325000, China
| | - Yitie Xu
- Oujiang Laboratory (Zhejiang Lab for Regenerative Medicine, Vision and Brain Health), School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou 325000, China
| | - Yu Zhang
- Oujiang Laboratory (Zhejiang Lab for Regenerative Medicine, Vision and Brain Health), School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou 325000, China
| | - Mengqi Jin
- Oujiang Laboratory (Zhejiang Lab for Regenerative Medicine, Vision and Brain Health), School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou 325000, China
| | - Yinuo Sun
- Department of Wound Healing, Department of Spine Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325035, China
| | - Fangying Tang
- Department of Wound Healing, Department of Spine Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325035, China
| | - Chuangqi Qiu
- Department of Wound Healing, Department of Spine Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325035, China
| | - Abass Mashud Akinfemi Junior
- Department of Wound Healing, Department of Spine Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325035, China
| | - Yunhao Cai
- Department of Wound Healing, Department of Spine Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325035, China
| | - Xiaodan Xu
- Department of Wound Healing, Department of Spine Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325035, China
| | - Xianghang Chen
- Oujiang Laboratory (Zhejiang Lab for Regenerative Medicine, Vision and Brain Health), School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou 325000, China
| | - Kongbin Chen
- Department of Orthopaedics Surgery, The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University, Wenzhou 325035, China
| | - Guangheng Xiang
- Department of Orthopaedics Surgery, The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University, Wenzhou 325035, China
| | - Jian Xiao
- Department of Orthopaedics Surgery, The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University, Wenzhou 325035, China; Oujiang Laboratory (Zhejiang Lab for Regenerative Medicine, Vision and Brain Health), School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou 325000, China; Department of Wound Healing, Department of Spine Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325035, China
| | - Jian Wang
- Department of Wound Healing, Department of Spine Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325035, China.
| | - Jing Wang
- Department of Wound Healing, Department of Spine Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325035, China.
| | - Baoyi Chen
- Department of Orthopaedics Surgery, The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University, Wenzhou 325035, China; Oujiang Laboratory (Zhejiang Lab for Regenerative Medicine, Vision and Brain Health), School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou 325000, China.
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Huang L, Pei Z, Zhang T, Zhang Z, Sun F, Wen L. Targeting TYROBP to influence the immune microenvironment and osteogenic differentiation of mesenchymal stem cells. J Orthop Surg Res 2025; 20:535. [PMID: 40437576 PMCID: PMC12117746 DOI: 10.1186/s13018-025-05925-7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/16/2025] [Accepted: 05/13/2025] [Indexed: 06/01/2025] Open
Abstract
Background Lactate, as an end product of glycolysis, plays an important role in cellular metabolism and signal transduction, and recent studies have shown that it is closely related to cellular differentiation, but its potential role in osteogenic differentiation has not yet been fully investigated. Methods We obtained two datasets containing human mesenchymal stem cells and human osteoblasts, GSE12266 and GSE18043 , from the GEO database, which contained a total of 14 samples with sequencing data, and searched for lactate metabolism-related genes from the Genecards database. Ten differentially expressed core genes related to lactate metabolism were identified by differential expression analysis, protein interaction network analysis, and correlation expression analysis, and determined to play a key role in osteogenic differentiation. The effects of hub genes on the immune microenvironment of osteogenic differentiation were explored by enrichment analysis and immune infiltration analysis, and the significant effects of the key gene TYRO Protein Tyrosine Kinase-Binding Protein(TYROBP) on the characterization of bone marrow mesenchymal stem cells (BMSCs) were experimentally verified, and it was determined by drug sensitivity analysis that TYROBP may be a regulatory target of certain drugs affecting osteogenic differentiation. Result We successfully screened 10 differentially expressed hub genes related to lactate metabolism, and their area under the curve AUC values for predicting osteogenic differentiation were all highly favorable. Enrichment analysis showed that lactate metabolism may affect osteoblast differentiation through immune infiltration, and the immune infiltration results confirmed the strong association between hub genes and osteoblast immune infiltration status. It was verified that decreasing TYROBP expression promoted cell viability, proliferation and migration ability of BMSCs. Drug sensitivity analysis showed that TYROBP may be a major regulator of drug-induced MSC differentiation. Conclusion Our study reveals the critical role of lactate metabolism in osteoblast differentiation, identifies the role of the key gene TYROBP in the regulation of BMSCs, and provides new insights for studies related to the regulation of osteoblast differentiation.
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Affiliation(s)
- Liangkun Huang
- Beijing Hospital, National Center of Gerontology, Institute of Geriatric Medicine, Chinese Academy of Medical Science & Peking Union Medical College, Beijing, China
| | - Zijie Pei
- Beijing Hospital, National Center of Gerontology, Institute of Geriatric Medicine, Chinese Academy of Medical Science & Peking Union Medical College, Beijing, China
| | - Tongyi Zhang
- Beijing Hospital, National Center of Gerontology, Institute of Geriatric Medicine, Chinese Academy of Medical Science & Peking Union Medical College, Beijing, China
| | - Ze Zhang
- Beijing Hospital, National Center of Gerontology, Institute of Geriatric Medicine, Chinese Academy of Medical Science & Peking Union Medical College, Beijing, China.
| | - Fengpo Sun
- Beijing Hospital, National Center of Gerontology, Institute of Geriatric Medicine, Chinese Academy of Medical Science & Peking Union Medical College, Beijing, China.
| | - Liangyuan Wen
- Beijing Hospital, National Center of Gerontology, Institute of Geriatric Medicine, Chinese Academy of Medical Science & Peking Union Medical College, Beijing, China.
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Jiang YS, Wei WS, Xie DT, Guo G. Circular RNAs inducing the osteogenic differentiation of dental mesenchymal stem cells via microRNA sponging. World J Stem Cells 2025; 17:101638. [DOI: 10.4252/wjsc.v17.i5.101638] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/21/2024] [Revised: 11/24/2024] [Accepted: 04/14/2025] [Indexed: 05/26/2025] Open
Abstract
Circular RNAs (circRNAs) are a distinct type of nonlinear and noncoding RNAs endogenously expressed by pre-mRNA back-splicing and crucial in transcriptional and posttranscriptional regulation. CircRNAs can regulate cellular and molecular pathways through various mechanisms, such as microRNA sponging. Numerous studies have indicated the regulatory roles of circRNAs in the osteogenic differentiation of stem cells (SCs) isolated from different sources. Dental tissue-derived mesenchymal SCs (MSCs) have received considerable attention in artificial bone engineering, in which SCs are used to manufacture functional bone tissues to repair bone defects. Recently, studies have reported the regulatory roles of circRNAs in the osteogenic differentiation of dental-derived MSCs, such as apical papillae, dental pulp, and dental follicle SCs. This review aimed to discuss the findings of studies evaluating the contribution of circRNAs to the osteogenic differentiation of dental-derived MSCs.
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Affiliation(s)
- Yong-Song Jiang
- Department of Orthopedic, The Central Hospital of Yongzhou, Yongzhou 425000, Hunan Province, China
- Department of Orthopedic, Yongzhou Hospital Affiliated to University of South China, Yongzhou 425000, Hunan Province, China
| | - Wei-Sheng Wei
- Department of Orthopedic, The Central Hospital of Yongzhou, Yongzhou 425000, Hunan Province, China
- Department of Orthopedic, Yongzhou Hospital Affiliated to University of South China, Yongzhou 425000, Hunan Province, China
| | - Dao-Tao Xie
- Norxin International Technology Innovation Cooperation Platform, Xi’an 710032, Shaanxi Province, China
| | - Gang Guo
- Norxin International Technology Innovation Cooperation Platform, Xi’an 710032, Shaanxi Province, China
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Naidu P, Das M, Hansda S, Prateeksha P, Howlader MSI, Siraj MA, Das H. Mechanisms of Ellagic Acid (EA)-Mediated Osteogenic Differentiation of Human Dental Pulp-Derived Stem Cells. ACS OMEGA 2025; 10:15229-15242. [PMID: 40290905 PMCID: PMC12019503 DOI: 10.1021/acsomega.4c10642] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 12/04/2024] [Revised: 03/05/2025] [Accepted: 03/11/2025] [Indexed: 04/30/2025]
Abstract
Ellagic acid (EA) is a potent antioxidant that reduces oxidative stress and promotes differentiation. By lowering the harmful levels of reactive oxygen species (ROS), EA fosters an environment conducive to the osteoblastic differentiation (OB) of stem cells. In addition, it promotes autophagy and mitophagy, which are vital for promoting differentiation. Effective autophagic activity recycles damaged organelles and proteins, meeting the energy required during differentiation and shielding from apoptosis. However, molecular mechanisms underlying the osteogenic differentiation of mesenchymal stem cells remain inadequately explored. Therefore, the current study aims to define the regulatory role of EA during the OB of dental pulp-derived stem cells (DPSC) and to study how autophagy and mitophagy are being modulated during this differentiation process. Herein, we showed that the expression level of osteoblast-specific markers, autophagy, and mitophagy-associated markers was significantly elevated during EA-mediated OB differentiation of DPSC. Moreover, we found that the EA induced the osteoblastic-specific markers through canonical BMP2 pathway molecules, reduced ROS in both basal and activated states, and induced autophagy and mitophagy molecules along with enhanced mitochondrial functions. Cell cycle analysis revealed that the G1 phase was arrested via phosphorylation of γ-H2AX, ATM, and CHK2 proteins. Furthermore, in silico analysis revealed that EA strongly binds with osteonectin, a crucial noncollagen protein involved in bone remodeling, and confirmed by Western blot analysis. These results support that EA could be a promising natural compound for bone repair and regeneration applications.
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Affiliation(s)
- Prathyusha Naidu
- Department
of Pharmaceutical Sciences, Jerry H. Hodge School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, Texas 79106, United States
| | - Manjusri Das
- Department
of Pharmaceutical Sciences, Jerry H. Hodge School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, Texas 79106, United States
| | - Surajit Hansda
- Department
of Pharmaceutical Sciences, Jerry H. Hodge School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, Texas 79106, United States
| | - Prateeksha Prateeksha
- Department
of Pharmaceutical Sciences, Jerry H. Hodge School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, Texas 79106, United States
| | - Md Sariful Islam Howlader
- Department
of Pharmaceutical Sciences, Jerry H. Hodge School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, Texas 79106, United States
| | - Md Afjalus Siraj
- Department
of Therapeutic Radiology, Yale School of Medicine, Yale University, New Haven, Connecticut 06520, United States
| | - Hiranmoy Das
- Department
of Pharmaceutical Sciences, Jerry H. Hodge School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, Texas 79106, United States
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Ding Y, Ran Y. OGA promotes human dental pulp stem cell senescence and inhibits mitophagy by inhibition of O-GlcNAcylation of KLF2. BMC Oral Health 2025; 25:595. [PMID: 40251583 PMCID: PMC12008942 DOI: 10.1186/s12903-025-05927-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/25/2024] [Accepted: 04/01/2025] [Indexed: 04/20/2025] Open
Abstract
BACKGROUND Dental pulp stem cells (DPSCs) aging impedes its application in tooth regeneration techniques, involving abnormal mitophagy. O-GlcNAcylation is a post-translational modification that regulates various cellular processes. Here, we aimed to investigate the role of O-GlcNAcylation in mitophagy and senescence. METHODS DPSCs were cultured and passaged in vitro, and the 7th (p7) and 15th (p15) generation cells were collected. OGA and KLF2 were knocked down in p15 cells. Cell senescence was evaluated using senescence associated β-galactosidase staining, enzyme-linked immunosorbent assay, and western blotting; mitophagy was evaluated using western blotting. The regulation of OGA on the O-GlcNAcylation of KLF2 was analyzed using immunoprecipitation and western blotting. RESULTS The results showed that p15 cells were more senescent than p7 cells and had poor mitophagy, with the higher expression of OGA. Knockdown of OGA inhibited senescence and promoted mitophagy in DPSCs. Moreover, silencing of KLF2 reversed the effects on senescence and mitophagy mediated by OGA knockdown. Additionally, OGA suppressed the O-GlcNAcylation of KLF2 at S177 site and thus reduced its stability. CONCLUSION Silencing of OGA promotes mitophagy and inhibits DPSC senescence by promoting the O-GlcNAcylation of KLF2, suggesting a novel mechanism underlying DPSC senescence.
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Affiliation(s)
- Yinhao Ding
- Engineering Laboratory for Biomaterials and Tissue Regeneration, Ningbo Stomatology Hospital, No.435, Xinxing Road, Wangchun Street, Haishu District, Ningbo, 315000, Zhejiang, China
| | - Yan Ran
- Engineering Laboratory for Biomaterials and Tissue Regeneration, Ningbo Stomatology Hospital, No.435, Xinxing Road, Wangchun Street, Haishu District, Ningbo, 315000, Zhejiang, China.
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Cheng D, Bao Y, Wang X, Xiang H, Guo T, Du Y, Zhang Z, Guo H. WNT3A promotes the cementogenic differentiation of dental pulp stem cells through the FOXO1 signaling pathway. Eur J Med Res 2025; 30:68. [PMID: 39905528 PMCID: PMC11792666 DOI: 10.1186/s40001-024-02259-8] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/13/2024] [Accepted: 12/23/2024] [Indexed: 02/06/2025] Open
Abstract
BACKGROUND Dental pulp stem cells (DPSCs) possess capability of multidirectional differentiation, and their cementogenic differentiation potential enables them to participate in cementum repair and regeneration. The molecular mechanisms underlying cementogenic differentiation of DPSCs remain unclear. METHODS DPSC data set GSE138179 was retrieved from gene expression omnibus (GEO) database. Weighted gene co-expression network analysis (WGCNA) was employed to identify significant modules. Pathway enrichment exploration was conducted utilizing gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Set Enrichment Analysis (GSEA), and Metascape tools. CIBERSORT was utilized to analyze immune cell infiltration analysis. The comparative toxicogenomics database (CTD) was utilized for the validation of core targets. Subsequently, cell experiments were conducted to validate the core targets. Changes in protein expression related to the FOXO1 signaling pathway, cell cycle, and apoptosis were evaluated using western blotting (WB). RESULTS Differentially expressed genes (DEGs) associated with DPSC cementogenic differentiation were predominantly enriched in crucial pathways such as the signaling pathway, cell apoptosis, and Wnt signaling pathway. Bioinformatics analysis confirmed WNT3A as a pivotal biomarker for DPSC cementogenic differentiation, and WNT3A was highly expressed in the cementogenic differentiation group. Western blotting results demonstrated that compared to the DPSC group, molecules such as Caspase-3, Caspase-9, FAS, P53, and BAX were downregulated in the CDDPSC group, suggesting reduced apoptosis. Furthermore, upregulation of WNT3A expression in CDDPSC-OE further suppressed the expression of these apoptotic molecules, suggesting a mitigated apoptotic response. Downregulation of WNT3A expression in CDDPSC-KO resulted in increased expression of apoptosis-related molecules, thereby enhancing apoptosis. CONCLUSIONS WNT3A is highly expressed in the cementogenic differentiation of DPSC, and WNT3A mediates FOXO1 pathway to promote differentiation of dental pulp stem cells into cementogenic differentiation, thus realizing the formation and maintenance of cementum tissue.
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Affiliation(s)
- Dongmei Cheng
- Department of Oral Medicine, The Second Hospital of Hebei Medical University, Shijiazhuang, 050000, Hebei, China
| | - Yang Bao
- Department of Oral Surgery, The Fourth Hospital of Hebei Medical University, Shijiazhuang, 050000, Hebei, China
| | - Xue Wang
- Department of Oral Medicine, The Second Hospital of Hebei Medical University, Shijiazhuang, 050000, Hebei, China
| | - Haidong Xiang
- Department of Oral Medicine, The Second Hospital of Hebei Medical University, Shijiazhuang, 050000, Hebei, China
| | - Tianyuan Guo
- Department of Plastic Surgery, The Second Hospital of Hebei Medical University, Shijiazhuang, 050000, Hebei, China
| | - Yong Du
- Department of Oral Medicine, The Second Hospital of Hebei Medical University, Shijiazhuang, 050000, Hebei, China
| | - Zhiyong Zhang
- Department of Oral Medicine, The Second Hospital of Hebei Medical University, Shijiazhuang, 050000, Hebei, China
| | - Han Guo
- Department of Oral Medicine, The Second Hospital of Hebei Medical University, Shijiazhuang, 050000, Hebei, China.
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Deng M, Tang R, Xu Y, Xu Y, Chen L. GDF11 promotes osteogenic/odontogenic differentiation of dental pulp stem cells to accelerate dentin restoration via modulating SIRT3/FOXO3-mediated mitophagy. Int Immunopharmacol 2024; 142:113092. [PMID: 39317051 DOI: 10.1016/j.intimp.2024.113092] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/16/2024] [Revised: 08/23/2024] [Accepted: 09/02/2024] [Indexed: 09/26/2024]
Abstract
BACKGROUND Growth differentiation factor 11 (GDF11) is considered to be a potential molecular target for treating pulpitis. However, whether GDF11 regulates osteogenic/odontogenic differentiation of dental pulp stem cells (DPSCs) to mediate pulpitis process remains unclear. METHODS Lipopolysaccharide (LPS) was used to induce inflammation conditions in DPSCs. The levels of GDF11, sirtuin 3 (SIRT3), forkhead box O-3 (FOXO3), osteogenic/odontogenic differentiation-related markers were measured by quantitative real-time PCR (qRT-PCR) and western blot (WB). Immunofluorescence staining was used to measure mitophagy. Mitophagy-related proteins were analyzed by WB, and the levels of inflammation factors were examined using qRT-PCR, ELISA and immunohistochemistry. Alkaline phosphatase activity and alizarin red S intensity were evaluated to assess osteogenic differentiation. Acute pulp (AP) injury rat model was constructed to study the role of oe-GDF11 in vivo. RESULTS GDF11 was downregulated in LPS-induced DPSCs, and LPS suppressed osteogenic/odontogenic differentiation and mitophagy. GDF11 overexpression promoted osteogenic/odontogenic differentiation in DPSCs through the activation of mitophagy. Furthermore, GDF11 upregulated SIRT3 to enhance FOXO3 expression by inhibiting its acetylation. GDF11 ameliorated LPS-induced inflammation and promoted osteogenic/odontogenic differentiation in DPSCs via enhancing SIRT3/FOXO3-mediated mitophagy. Besides, GDF11 overexpression suppressed inflammation and promoted dentin repair in AP rat models. CONCLUSION GDF11 promoted SIRT3/FOXO3-mediated mitophagy to accelerate osteogenic/odontogenic differentiation in DPSCs, providing a novel target for pulpitis treatment.
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Affiliation(s)
- Mingsi Deng
- Department of Stomatology, The Third Xiangya Hospital, Central South University, No. 138 Tongzipo Road, Yuelu District, Changsha City, Hunan Province, PR China; Department of Orthodontics, Changsha Stomatological Hospital, Changsha City, Hunan Province, PR China
| | - Ruimin Tang
- Department of Stomatology, The Third Xiangya Hospital, Central South University, No. 138 Tongzipo Road, Yuelu District, Changsha City, Hunan Province, PR China
| | - Yani Xu
- Department of Orthodontics, Changsha Stomatological Hospital, Changsha City, Hunan Province, PR China
| | - Yafen Xu
- Department of Orthodontics, Changsha Stomatological Hospital, Changsha City, Hunan Province, PR China
| | - Liangjian Chen
- Department of Stomatology, The Third Xiangya Hospital, Central South University, No. 138 Tongzipo Road, Yuelu District, Changsha City, Hunan Province, PR China.
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Shi J, Chen L, Wang X, Ma X. KLF2 Inhibits Ferroptosis and Improves Mitochondrial Dysfunction in Chondrocyte Through SIRT1/GPX4 Signaling to Improve Osteoarthritis. Drug Dev Res 2024; 85:e70015. [PMID: 39527654 DOI: 10.1002/ddr.70015] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/03/2024] [Revised: 10/11/2024] [Accepted: 10/21/2024] [Indexed: 11/16/2024]
Abstract
Osteoarthritis (OA), a disease of articular joints, is the leading cause of disability in the elderly. Repressing ferroptosis and improving mitochondrial function can delay the progression of OA. Kruppel-like factor 2 (KLF2) exerts a protective effect on OA. However, whether KLF2 affects ferroptosis and mitochondrial function during OA remains unknown. The OA in vivo and in vitro models were constructed in this work. The structural damage of knee joint in OA mice was evaluated through Micro-CT scanning. H&E, SOFG, TB, and TUNEL staining were applied for pathological examination of cartilage tissues. ELISA was employed to examine the contents of inflammatory factors. Additionally, iron deposition in cartilage tissues was measured by Prussian blue staining, and the levels of proteins related to ferroptosis were assessed by immunoblotting. Besides, mitochondrial morphology and function were estimated using a transmission electron microscope and JC-1 staining. In interleukin (IL)-1β-treated C28/I2 cells, the levels of inflammatory factors, intracellular ROS, mitochondrial ROS, lipid ROS, and Fe2+ were measured. Mitochondrial function was evaluated by detecting the levels of mitochondrial membrane potential (MMP), ATP, mPTP, and OCR. KLF2 overexpression ameliorated the structural damage of knee cartilage in OA mice. KLF2 upregulation inhibited ferroptosis and alleviated mitochondrial damage in knee cartilage of OA mice and IL-1β-treated C28/I2 cells. Moreover, KLF2 overexpression activated SIRT1/GPX4 signaling in vivo and in vitro. EX527 addition blocked the influences of KLF2 upregulation on ferroptosis and mitochondrial dysfunction in IL-1β-treated C28/I2 cells. Altogether KLF2 inhibits ferroptosis and improves mitochondrial dysfunction in chondrocytes through SIRT1/GPX4 signaling to improve OA.
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Affiliation(s)
- Jiaqi Shi
- Orthopedic Department, Huashan Hospital Affiliated to Fudan University, Shanghai, China
| | - Li Chen
- Orthopedic Department, Huashan Hospital Affiliated to Fudan University, Shanghai, China
| | - Xu Wang
- Orthopedic Department, Huashan Hospital Affiliated to Fudan University, Shanghai, China
| | - Xin Ma
- Orthopedic Department, Huashan Hospital Affiliated to Fudan University, Shanghai, China
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Yan X, An N, Zhang Z, Qiu Q, Yang D, Wei P, Zhang X, Qiu L, Guo J. Graphene Oxide Quantum Dots-Preactivated Dental Pulp Stem Cells/GelMA Facilitates Mitophagy-Regulated Bone Regeneration. Int J Nanomedicine 2024; 19:10107-10128. [PMID: 39381026 PMCID: PMC11460356 DOI: 10.2147/ijn.s480979] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2024] [Accepted: 10/02/2024] [Indexed: 10/10/2024] Open
Abstract
Background In bone tissue engineering (BTE), cell-laden scaffolds offer a promising strategy for repairing bone defects, particularly when host cell regeneration is insufficient due to age or disease. Exogenous stem cell-based BTE requires bioactive factors to activate these cells. Graphene oxide quantum dots (GOQDs), zero-dimensional derivatives of graphene oxide, have emerged as potential osteogenic nanomedicines. However, constructing biological scaffolds with GOQDs and elucidating their biological mechanisms remain critical challenges. Methods We utilized GOQDs with a particle size of 10 nm, characterized by a surface rich in C-O-H and C-O-C functional groups. We developed a gelatin methacryloyl (GelMA) hydrogel incorporated with GOQDs-treated dental pulp stem cells (DPSCs). These constructs were transplanted into rat calvarial bone defects to estimate the effectiveness of GOQDs-induced DPSCs in repairing bone defects while also investigating the molecular mechanism underlying GOQDs-induced osteogenesis in DPSCs. Results GOQDs at 5 μg/mL significantly enhanced the osteogenic differentiation of DPSCs without toxicity. The GOQDs-induced DPSCs showed active osteogenic potential in three-dimensional cell culture system. In vivo, transplantation of GOQDs-preactivated DPSCs/GelMA composite effectively facilitated calvarial bone regeneration. Mechanistically, GOQDs stimulated mitophagy flux through the phosphatase-and-tensin homolog-induced putative kinase 1 (PINK1)/Parkin E3 ubiquitin ligase (PRKN) pathway. Notably, inhibiting mitophagy with cyclosporin A prevented the osteogenic activity of GOQDs. Conclusion This research presents a well-designed bionic GOQDs/DPSCs/GelMA composite scaffold and demonstrated its ability to promote bone regeneration by enhancing mitophagy. These findings highlight the significant potential of this composite for application in BTE and underscore the crucial role of mitophagy in promoting the osteogenic differentiation of GOQDs-induced stem cells.
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Affiliation(s)
- Xiaoyuan Yan
- Department of Endodontics, School and Hospital of Stomatology, China Medical University, Liaoning Provincial Key Laboratory of Oral Diseases, Shenyang, People’s Republic of China
| | - Na An
- Department of Orthodontics, School and Hospital of Stomatology, China Medical University, Liaoning Provincial Key Laboratory of Oral Diseases, Shenyang, People’s Republic of China
| | - Zeying Zhang
- Department of Endodontics, School and Hospital of Stomatology, China Medical University, Liaoning Provincial Key Laboratory of Oral Diseases, Shenyang, People’s Republic of China
| | - Qiujing Qiu
- Department of Endodontics, School and Hospital of Stomatology, China Medical University, Liaoning Provincial Key Laboratory of Oral Diseases, Shenyang, People’s Republic of China
| | - Di Yang
- Department of Endodontics, School and Hospital of Stomatology, China Medical University, Liaoning Provincial Key Laboratory of Oral Diseases, Shenyang, People’s Republic of China
| | - Penggong Wei
- Department of Endodontics, School and Hospital of Stomatology, China Medical University, Liaoning Provincial Key Laboratory of Oral Diseases, Shenyang, People’s Republic of China
| | - Xiyue Zhang
- Department of Endodontics, School and Hospital of Stomatology, China Medical University, Liaoning Provincial Key Laboratory of Oral Diseases, Shenyang, People’s Republic of China
| | - Lihong Qiu
- Department of Endodontics, School and Hospital of Stomatology, China Medical University, Liaoning Provincial Key Laboratory of Oral Diseases, Shenyang, People’s Republic of China
| | - Jiajie Guo
- Department of Endodontics, School and Hospital of Stomatology, China Medical University, Liaoning Provincial Key Laboratory of Oral Diseases, Shenyang, People’s Republic of China
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Xiang T, Yang C, Deng Z, Sun D, Luo F, Chen Y. Krüppel-like factors family in health and disease. MedComm (Beijing) 2024; 5:e723. [PMID: 39263604 PMCID: PMC11387732 DOI: 10.1002/mco2.723] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/19/2024] [Revised: 08/14/2024] [Accepted: 08/14/2024] [Indexed: 09/13/2024] Open
Abstract
Krüppel-like factors (KLFs) are a family of basic transcription factors with three conserved Cys2/His2 zinc finger domains located in their C-terminal regions. It is acknowledged that KLFs exert complicated effects on cell proliferation, differentiation, survival, and responses to stimuli. Dysregulation of KLFs is associated with a range of diseases including cardiovascular disorders, metabolic diseases, autoimmune conditions, cancer, and neurodegenerative diseases. Their multidimensional roles in modulating critical pathways underscore the significance in both physiological and pathological contexts. Recent research also emphasizes their crucial involvement and complex interplay in the skeletal system. Despite the substantial progress in understanding KLFs and their roles in various cellular processes, several research gaps remain. Here, we elucidated the multifaceted capabilities of KLFs on body health and diseases via various compliable signaling pathways. The associations between KLFs and cellular energy metabolism and epigenetic modification during bone reconstruction have also been summarized. This review helps us better understand the coupling effects and their pivotal functions in multiple systems and detailed mechanisms of bone remodeling and develop potential therapeutic strategies for the clinical treatment of pathological diseases by targeting the KLF family.
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Affiliation(s)
- Tingwen Xiang
- Department of Orthopedics Southwest Hospital Third Military Medical University (Army Medical University) Chongqing China
| | - Chuan Yang
- Department of Biomedical Materials Science Third Military Medical University (Army Medical University) Chongqing China
| | - Zihan Deng
- Department of Orthopedics Southwest Hospital Third Military Medical University (Army Medical University) Chongqing China
| | - Dong Sun
- Department of Orthopedics Southwest Hospital Third Military Medical University (Army Medical University) Chongqing China
| | - Fei Luo
- Department of Orthopedics Southwest Hospital Third Military Medical University (Army Medical University) Chongqing China
| | - Yueqi Chen
- Department of Orthopedics Southwest Hospital Third Military Medical University (Army Medical University) Chongqing China
- Department of Orthopedics Chinese PLA 76th Army Corps Hospital Xining China
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11
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Wang Z, Liu Z, Zhou P, Niu X, Sun Z, He H, Zhu Z. The involvement of krüppel-like transcription factor 2 in megakaryocytic differentiation induction by phorbol 12-myrestrat 13-acetate. Biomark Res 2024; 12:65. [PMID: 39014479 PMCID: PMC11253501 DOI: 10.1186/s40364-024-00614-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/23/2024] [Accepted: 07/05/2024] [Indexed: 07/18/2024] Open
Abstract
BACKGROUND Megakaryocytic differentiation is a complicated process regulated by a series of transcription factors in a context- and stage-dependent manner. Recent studies have suggested that krüppel-like transcription factor 2 (KLF2) is involved in the control of embryonic erythroid precursor cell differentiation and maturation. However, the function and mechanism of KLF2 in regulating megakaryocytic differentiation remain unclear. METHODS The expression patterns of krüppel-like transcription factors (KLFs) during megakaryocytic differentiation were identified from public databases. Phorbol 12-myristate 13-acetate (PMA) treatment of the myeloid-erythroid-leukemic cell lines K562 and HEL were used as cellular megakaryocytic differentiation models. A lentiviral transduction system was utilized to achieve the goal of amplifying or reducing KLF2. The expression of KLF2 was examined using real-time PCR and western blot. The impact of KLF2 on the megakaryocytic differentiation of K562 cells was examined by flow cytometry, Giemsa staining, Phalloidin staining and western blot. RNA-sequencing (RNA-seq) and chromatin immunoprecipitation-sequencing (ChIP-seq) technologies were used to identify the KLF2-regulated targets. RESULTS KLF2 is increased in the maturation process of megakaryocytes. KLF2 overexpression accelerated the PMA-induced megakaryocytic differentiation, as reflected by an increased percentage of CD41/CD61 cells, an increased number of polyploid cells, and an elevated expression of P21 and P27. KLF2 knockdown exhibited the opposite results, indicating that KLF2 knockdown suppressed the megakaryocytic differentiation. Further, combination of the RNA-seq and ChIP-seq results suggested that chimerin 1 (CHN1) and potassium voltage-gated channel subfamily Q member 5 (KCNQ5) may be target genes regulated of KLF2. Both CHN1 and KCNQ5 knockdown could block the megakaryocytic differentiation to some content. CONCLUSION This study implicated a regulatory role of KLF2 in megakaryocytic differentiation, which may suggest KLF2 as a target for illness with abnormal megakaryocytic differentiation.
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Affiliation(s)
- Zhen Wang
- Department of Hematology, Zhengzhou University People's Hospital, Henan Provincial People's Hospital, Zhengzhou, Henan, China.
- Henan University, Kaifeng, Henan, China.
- Zhengzhou University, Zhengzhou, Henan, China.
| | - Zhongwen Liu
- Department of Hematology, Zhengzhou University People's Hospital, Henan Provincial People's Hospital, Zhengzhou, Henan, China
- Henan University, Kaifeng, Henan, China
- Zhengzhou University, Zhengzhou, Henan, China
| | - Pan Zhou
- Department of Hematology, Zhengzhou University People's Hospital, Henan Provincial People's Hospital, Zhengzhou, Henan, China
| | - Xiaona Niu
- Department of Hematology, Zhengzhou University People's Hospital, Henan Provincial People's Hospital, Zhengzhou, Henan, China
- Henan University, Kaifeng, Henan, China
- Zhengzhou University, Zhengzhou, Henan, China
| | | | - Huan He
- Zhengzhou University, Zhengzhou, Henan, China
| | - Zunmin Zhu
- Department of Hematology, Zhengzhou University People's Hospital, Henan Provincial People's Hospital, Zhengzhou, Henan, China.
- Henan University, Kaifeng, Henan, China.
- Zhengzhou University, Zhengzhou, Henan, China.
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12
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Du H, Li B, Yu R, Lu X, Li C, Zhang H, Yang F, Zhao R, Bao W, Yin X, Wang Y, Zhou J, Xu J. ETV2 regulating PHD2-HIF-1α axis controls metabolism reprogramming promotes vascularized bone regeneration. Bioact Mater 2024; 37:222-238. [PMID: 38549772 PMCID: PMC10973785 DOI: 10.1016/j.bioactmat.2024.02.014] [Citation(s) in RCA: 2] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/25/2023] [Revised: 01/23/2024] [Accepted: 02/11/2024] [Indexed: 01/05/2025] Open
Abstract
The synchronized development of mineralized bone and blood vessels is a fundamental requirement for successful bone tissue regeneration. Adequate energy production forms the cornerstone supporting new bone formation. ETS variant 2 (ETV2) has been identified as a transcription factor that promotes energy metabolism reprogramming and facilitates the coordination between osteogenesis and angiogenesis. In vitro molecular experiments have demonstrated that ETV2 enhances osteogenic differentiation of dental pulp stem cells (DPSCs) by regulating the ETV2- prolyl hydroxylase 2 (PHD2)- hypoxia-inducible factor-1α (HIF-1α)- vascular endothelial growth factor A (VEGFA) axis. Notably, ETV2 achieves the rapid reprogramming of energy metabolism by simultaneously accelerating mitochondrial aerobic respiration and glycolysis, thus fulfilling the energy requirements essential to expedite osteogenic differentiation. Furthermore, decreased α-ketoglutarate release from ETV2-modified DPSCs contributes to microcirculation reconstruction. Additionally, we engineered hydroxyapatite/chitosan microspheres (HA/CS MS) with biomimetic nanostructures to facilitate multiple ETV2-DPSC functions and further enhanced the osteogenic differentiation. Animal experiments have validated the synergistic effect of ETV2-modified DPSCs and HA/CS MS in promoting the critical-size bone defect regeneration. In summary, this study offers a novel treatment approach for vascularized bone tissue regeneration that relies on energy metabolism activation and the maintenance of a stable local hypoxia signaling state.
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Affiliation(s)
- HaoRan Du
- College & Hospital of Stomatology, Anhui Medical University, Key Lab. of Oral Diseases Research of Anhui Province, Hefei, 230032, China
- Salivary Gland Disease Center and Beijing Key Laboratory of Tooth Regeneration and Function Reconstruction, Beijing Laboratory of Oral Health and Beijing Stomatological Hospital, Capital Medical University, Beijing, 100050, China
| | - Bang Li
- College & Hospital of Stomatology, Anhui Medical University, Key Lab. of Oral Diseases Research of Anhui Province, Hefei, 230032, China
| | - Rui Yu
- College & Hospital of Stomatology, Anhui Medical University, Key Lab. of Oral Diseases Research of Anhui Province, Hefei, 230032, China
| | - Xiaoxuan Lu
- College & Hospital of Stomatology, Anhui Medical University, Key Lab. of Oral Diseases Research of Anhui Province, Hefei, 230032, China
| | - ChengLin Li
- College & Hospital of Stomatology, Anhui Medical University, Key Lab. of Oral Diseases Research of Anhui Province, Hefei, 230032, China
| | - HuiHui Zhang
- College & Hospital of Stomatology, Anhui Medical University, Key Lab. of Oral Diseases Research of Anhui Province, Hefei, 230032, China
| | - Fan Yang
- College & Hospital of Stomatology, Anhui Medical University, Key Lab. of Oral Diseases Research of Anhui Province, Hefei, 230032, China
| | - RongQuan Zhao
- College & Hospital of Stomatology, Anhui Medical University, Key Lab. of Oral Diseases Research of Anhui Province, Hefei, 230032, China
| | - WeiMin Bao
- College & Hospital of Stomatology, Anhui Medical University, Key Lab. of Oral Diseases Research of Anhui Province, Hefei, 230032, China
| | - Xuan Yin
- College & Hospital of Stomatology, Anhui Medical University, Key Lab. of Oral Diseases Research of Anhui Province, Hefei, 230032, China
| | - YuanYin Wang
- College & Hospital of Stomatology, Anhui Medical University, Key Lab. of Oral Diseases Research of Anhui Province, Hefei, 230032, China
| | - Jian Zhou
- Salivary Gland Disease Center and Beijing Key Laboratory of Tooth Regeneration and Function Reconstruction, Beijing Laboratory of Oral Health and Beijing Stomatological Hospital, Capital Medical University, Beijing, 100050, China
- Department of VIP Dental Service, School of Stomatology, Capital Medical University, Beijing, 100050, China
- Laboratory for Oral and General Health Integration and Translation, Beijing Tiantan Hospital, Capital Medical University, Beijing, China
| | - Jianguang Xu
- College & Hospital of Stomatology, Anhui Medical University, Key Lab. of Oral Diseases Research of Anhui Province, Hefei, 230032, China
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13
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Li Q, Chen Y, Chen Y, Hua Z, Gong B, Liu Z, Thiele CJ, Li Z. Novel small molecule DMAMCL induces differentiation in rhabdomyosarcoma by downregulating of DLL1. Biomed Pharmacother 2024; 174:116562. [PMID: 38626518 DOI: 10.1016/j.biopha.2024.116562] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/27/2023] [Revised: 03/27/2024] [Accepted: 04/04/2024] [Indexed: 04/18/2024] Open
Abstract
Rhabdomyosarcoma (RMS), a mesenchymal tumor occurring in the soft tissue of children, is associated with a defect in differentiation. This study unveils a novel anti-tumor mechanism of dimethylaminomicheliolide (DMAMCL), which is a water-soluble derivative of Micheliolide. First, we demonstrate that DMAMCL inhibits RMS cell growth without obvious cell death, leading to morphological alterations, enhanced expression of muscle differentiation markers, and a shift from a malignant to a more benign metabolic phenotype. Second, we detected decreased expression of DLL1 in RMS cells after DMAMCL treatment, known as a pivotal ligand in the Notch signaling pathway. Downregulation of DLL1 inhibits RMS cell growth and induces morphological changes similar to the effects of DMAMCL. Furthermore, DMAMCL treatment or loss of DLL1 expression also inhibits RMS xenograft tumor growth and augmented the expression of differentiation markers. Surprisingly, in C2C12 cells DMAMCL treatment or DLL1 downregulation also induces cell growth inhibition and an elevation in muscle differentiation marker expression. These data indicated that DMAMCL induced RMS differentiation and DLL1 is an important factor for RMS differentiation, opening a new window for the clinical use of DMAMCL as an agent for differentiation-inducing therapy for RMS treatment.
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Affiliation(s)
- Qi Li
- Department of Pediatrics, Shengjing Hospital of China Medical University, Shenyang 110001, China; Medical Research Center, Liaoning Key Laboratory of Research and Application of Animal Models for Environment and Metabolic Diseases, Shengjing Hospital of China Medical University, Shenyang 110004, China
| | - Yexi Chen
- Department of Pediatrics, Shengjing Hospital of China Medical University, Shenyang 110001, China; Medical Research Center, Liaoning Key Laboratory of Research and Application of Animal Models for Environment and Metabolic Diseases, Shengjing Hospital of China Medical University, Shenyang 110004, China
| | - Yang Chen
- Department of Pediatrics, Shengjing Hospital of China Medical University, Shenyang 110001, China; Medical Research Center, Liaoning Key Laboratory of Research and Application of Animal Models for Environment and Metabolic Diseases, Shengjing Hospital of China Medical University, Shenyang 110004, China
| | - Zhongyan Hua
- Department of Pediatrics, Shengjing Hospital of China Medical University, Shenyang 110001, China; Medical Research Center, Liaoning Key Laboratory of Research and Application of Animal Models for Environment and Metabolic Diseases, Shengjing Hospital of China Medical University, Shenyang 110004, China
| | - Baocheng Gong
- Department of Pediatrics, Shengjing Hospital of China Medical University, Shenyang 110001, China; Medical Research Center, Liaoning Key Laboratory of Research and Application of Animal Models for Environment and Metabolic Diseases, Shengjing Hospital of China Medical University, Shenyang 110004, China
| | - Zhihui Liu
- Cell and Molecular Biology Section, Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institute of Health, Bethesda, MD 20892, USA
| | - Carol J Thiele
- Cell and Molecular Biology Section, Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institute of Health, Bethesda, MD 20892, USA
| | - Zhijie Li
- Department of Pediatrics, Shengjing Hospital of China Medical University, Shenyang 110001, China; Medical Research Center, Liaoning Key Laboratory of Research and Application of Animal Models for Environment and Metabolic Diseases, Shengjing Hospital of China Medical University, Shenyang 110004, China.
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14
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Wang H, Han J, Dmitrii G, Ning K, Zhang X. KLF transcription factors in bone diseases. J Cell Mol Med 2024; 28:e18278. [PMID: 38546623 PMCID: PMC10977429 DOI: 10.1111/jcmm.18278] [Citation(s) in RCA: 6] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/11/2023] [Revised: 03/06/2024] [Accepted: 03/15/2024] [Indexed: 04/11/2025] Open
Abstract
Krüppel-like factors (KLFs) are crucial in the development of bone disease. They are a family of zinc finger transcription factors that are unusual in containing three highly conserved zinc finger structural domains interacting with DNA. It has been discovered that it engages in various cell functions, including proliferation, apoptosis, autophagy, stemness, invasion and migration, and is crucial for the development of human tissues. In recent years, the role of KLFs in bone physiology and pathology has received adequate attention. In addition to regulating the normal growth and development of the musculoskeletal system, KLFs participate in the pathological process of the bones and joints and are intimately linked to several skeletal illnesses, such as osteoarthritis (OA), rheumatoid arthritis (RA), osteoporosis (OP) and osteosarcoma (OS). Consequently, targeting KLFs has emerged as a promising therapeutic approach for an array of bone disorders. In this review, we summarize the current literature on the importance of KLFs in the emergence and regulation of bone illnesses, with a particular emphasis on the pertinent mechanisms by which KLFs regulate skeletal diseases. We also discuss the need for KLFs-based medication-targeted treatment. These endeavours offer new perspectives on the use of KLFs in bone disorders and provide prognostic biomarkers, therapeutic targets and possible drug candidates for bone diseases.
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Affiliation(s)
- Haixia Wang
- College of Exercise and HealthShenyang Sport UniversityShenyangLiaoningChina
| | - Juanjuan Han
- College of Exercise and HealthShenyang Sport UniversityShenyangLiaoningChina
- Department of Sport RehabilitationShanghai University of SportShanghaiChina
| | - Gorbachev Dmitrii
- Head of General Hygiene DepartmentSamara State Medical UniversitySamaraRussia
| | - Ke Ning
- College of Exercise and HealthShenyang Sport UniversityShenyangLiaoningChina
| | - Xin‐an Zhang
- College of Exercise and HealthShenyang Sport UniversityShenyangLiaoningChina
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15
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Xiao Y, Chen L, Xu Y, Yu R, Lu J, Ke Y, Guo R, Gu T, Yu H, Fang Y, Li Z, Yu J. Circ-ZNF236 mediates stem cells from apical papilla differentiation by regulating LGR4-induced autophagy. Int Endod J 2024; 57:431-450. [PMID: 38240345 DOI: 10.1111/iej.14021] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/17/2023] [Revised: 11/06/2023] [Accepted: 01/03/2024] [Indexed: 03/07/2024]
Abstract
AIM Human stem cells from the apical papilla (SCAPs) are an appealing stem cell source for tissue regeneration engineering. Circular RNAs (circRNAs) are known to exert pivotal regulatory functions in various cell differentiation processes, including osteogenesis of mesenchymal stem cells. However, few studies have shown the potential mechanism of circRNAs in the odonto/osteogenic differentiation of SCAPs. Herein, we identified a novel circRNA, circ-ZNF236 (hsa_circ_0000857) and found that it was remarkably upregulated during the SCAPs committed differentiation. Thus, in this study, we showed the significance of circ-ZNF236 in the odonto/osteogenic differentiation of SCAPs and its underlying regulatory mechanisms. METHODOLOGY The circular structure of circ-ZNF236 was identified via Sanger sequencing, amplification of convergent and divergent primers. The proliferation of SCAPs was detected by CCK-8, flow cytometry analysis and EdU incorporation assay. Western blotting, qRT-PCR, Alkaline phosphatase (ALP) and Alizarin red staining (ARS) were performed to explore the regulatory effect of circ-ZNF236/miR-218-5p/LGR4 axis in the odonto/osteogenic differentiation of SCAPs in vitro. Fluorescence in situ hybridization, as well as dual-luciferase reporting assays, revealed that circ-ZNF236 binds to miR-218-5p. Transmission electron microscopy (TEM) and mRFP-GFP-LC3 lentivirus were performed to detect the activation of autophagy. RESULTS Circ-ZNF236 was identified as a highly stable circRNA with a covalent closed loop structure. Circ-ZNF236 had no detectable influence on cell proliferation but positively regulated SCAPs odonto/osteogenic differentiation. Furthermore, circ-ZNF236 was confirmed as a sponge of miR-218-5p in SCAPs, while miR-218-5p targets LGR4 mRNA at its 3'-UTR. Subsequent rescue experiments revealed that circ-ZNF236 regulates odonto/osteogenic differentiation by miR-218-5p/LGR4 in SCAPs. Importantly, circ-ZNF236 activated autophagy, and the activation of autophagy strengthened the committed differentiation capability of SCAPs. Subsequently, in vivo experiments showed that SCAPs overexpressing circ-ZNF236 promoted bone formation in a rat skull defect model. CONCLUSIONS Circ-ZNF236 could activate autophagy through increasing LGR4 expression, thus positively regulating SCAPs odonto/osteogenic differentiation. Our findings suggested that circ-ZNF236 might represent a novel therapeutic target to prompt the odonto/osteogenic differentiation of SCAPs.
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Affiliation(s)
- Ya Xiao
- Key Laboratory of Oral Diseases of Jiangsu Province and Stomatological Institute of Nanjing Medical University, Nanjing, China
- Endodontic Department, School of Stomatology, Nanjing Medical University, Nanjing, China
| | - Luyao Chen
- Key Laboratory of Oral Diseases of Jiangsu Province and Stomatological Institute of Nanjing Medical University, Nanjing, China
- Endodontic Department, School of Stomatology, Nanjing Medical University, Nanjing, China
| | - Yunlong Xu
- Endodontic Department, Changzhou Stomatological Hospital, Changzhou, Jiangsu, China
| | - Ruiyang Yu
- School of Pediatrics, Nanjing Medical University, Nanjing, China
| | - Jiamin Lu
- Endodontic Department, Changzhou Stomatological Hospital, Changzhou, Jiangsu, China
| | - Yue Ke
- Key Laboratory of Oral Diseases of Jiangsu Province and Stomatological Institute of Nanjing Medical University, Nanjing, China
- Endodontic Department, School of Stomatology, Nanjing Medical University, Nanjing, China
| | - Rong Guo
- Key Laboratory of Oral Diseases of Jiangsu Province and Stomatological Institute of Nanjing Medical University, Nanjing, China
- Endodontic Department, School of Stomatology, Nanjing Medical University, Nanjing, China
| | - Tingjie Gu
- Key Laboratory of Oral Diseases of Jiangsu Province and Stomatological Institute of Nanjing Medical University, Nanjing, China
- Endodontic Department, School of Stomatology, Nanjing Medical University, Nanjing, China
| | - Haowen Yu
- Key Laboratory of Oral Diseases of Jiangsu Province and Stomatological Institute of Nanjing Medical University, Nanjing, China
- Endodontic Department, School of Stomatology, Nanjing Medical University, Nanjing, China
| | - Yuxin Fang
- Key Laboratory of Oral Diseases of Jiangsu Province and Stomatological Institute of Nanjing Medical University, Nanjing, China
- Endodontic Department, School of Stomatology, Nanjing Medical University, Nanjing, China
| | - Zehan Li
- Key Laboratory of Oral Diseases of Jiangsu Province and Stomatological Institute of Nanjing Medical University, Nanjing, China
- Endodontic Department, School of Stomatology, Nanjing Medical University, Nanjing, China
| | - Jinhua Yu
- Endodontic Department, School of Stomatology, Nanjing Medical University, Nanjing, China
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16
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Li Y, Yang E, Geng Y, Li M, Wang X, Zhang D. TFEB regulates the odontoblastic differentiation of dental pulp stem cells by promoting a positive feedback loop between mitophagy and glycolysis. Arch Oral Biol 2024; 160:105909. [PMID: 38309196 DOI: 10.1016/j.archoralbio.2024.105909] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2023] [Revised: 01/21/2024] [Accepted: 01/28/2024] [Indexed: 02/05/2024]
Abstract
OBJECTIVE To evaluate the regulatory effect of transcription factor EB (TFEB) on the odontoblastic differentiation of dental pulp stem cells(DPSCs) in vivo and in vitro. DESIGNS RNA-seq was used to detect differentially expressed genes in differentiated DPSCs. Lysosomes and the expression of the related gene TFEB were examined in DPSCs. DPSCs were then transfected with lentivirus for TFEB-overexpression. Cell proliferation was detected using CCK-8 and EdU assays, while cell differentiation was detected using ALP and ARS detection kits. Subsequently, mitophagy and cell metabolism were examined using TEM and Seahorse. An odontoblastic differentiation model was constructed subcutaneously in nude mice. Finally, the effects of glycolysis and mitophagy inhibitors were evaluated on odontoblastic differentiation and the associated mechanisms were explored. RESULTS TFEB overexpression promoted a significant increase in ALP activity and the expression of differentiation-related genes in DPSCs, while it inhibited cell proliferation. In vivo, TFEB overexpression caused higher bone volume/trabecular volume(BV/TV), and an increase in collagen formation and heightened DMP-1 expression. Furthermore, Seahorse flux analysis demonstrated that TFEB promoted metabolic reprogramming. Transmission electron microscope(TEM) results indicated an increase in mitochondrial autophagosomes after TFEB overexpression, and the expression of mitophagy-related genes was also elevated. The odontoblastic differentiation of DPSCs promoted by TFEB overexpression was suppressed after the addition of 2-DG and Midiv-1. Addition of Midiv-1 reduced the glycolytic rate of DPSCs, while addition of 2-DG also decreased the mitophagy level of the cells. CONCLUSIONS Our results showed that TFEB promoted the odontoblastic differentiation of DPSCs and identified mitophagy and metabolic reprogramming as a positive feedback loop.
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Affiliation(s)
- Yiming Li
- School and hospital of Stomatology, Cheeloo College of Medicine, Shandong University, China; Department of Oral and Maxillofacial Surgery, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, China
| | - Enli Yang
- School and hospital of Stomatology, Cheeloo College of Medicine, Shandong University, China; Department of Oral and Maxillofacial Surgery, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, China
| | - Yiming Geng
- School and hospital of Stomatology, Cheeloo College of Medicine, Shandong University, China; Department of Oral and Maxillofacial Surgery, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, China
| | - Mingyang Li
- School and hospital of Stomatology, Cheeloo College of Medicine, Shandong University, China; Department of Oral and Maxillofacial Surgery, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, China
| | - Xuan Wang
- Department of Oral and Maxillofacial Surgery, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, China.
| | - Dongsheng Zhang
- School and hospital of Stomatology, Cheeloo College of Medicine, Shandong University, China; Department of Oral and Maxillofacial Surgery, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, China.
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Chen W, Cui Y, Li C, He C, Du L, Liu W, He Z. KLF2 controls proliferation and apoptosis of human spermatogonial stem cells via targeting GJA1. iScience 2024; 27:109024. [PMID: 38352225 PMCID: PMC10863320 DOI: 10.1016/j.isci.2024.109024] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/25/2023] [Revised: 11/14/2023] [Accepted: 01/22/2024] [Indexed: 02/16/2024] Open
Abstract
Human spermatogonial stem cells (SSCs) are essential for spermatogenesis and male fertility. However, molecular mechanisms regulating fate determinations of human SSCs remain elusive. In this study, we revealed that KLF2 decreased the proliferation, DNA synthesis, and colonization of human SSCs as well as increased apoptosis of these cells. We identified and demonstrated that GJA1 was a target gene for KLF2 in human SSCs. Notably, KLF2 overexpression rescued the reduction of proliferation of human SSCs caused by GJA1 silencing as well as the enhancement of apoptosis of human SSCs. Abnormalities in the higher level of KLF2 and/or KIF2 mutations might lead to male infertility. Collectively, these results implicate that KLF2 inhibits proliferation of human SSCs and enhances their apoptosis by targeting GJA1. This study thus provides novel genetic mechanisms underlying human spermatogenesis and azoospermia, and it offers new endogenous targets for treating male infertility.
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Affiliation(s)
- Wei Chen
- Key Laboratory of Model Animals and Stem Cell Biology in Hunan Province, Hunan Normal University School of Medicine, Engineering Research Center of Reproduction and Translational Medicine of Hunan Province, Manufacture-Based Learning and Research Demonstration Center for Human Reproductive Health New Technology of Hunan Normal University, Changsha, China
| | - Yinghong Cui
- Key Laboratory of Model Animals and Stem Cell Biology in Hunan Province, Hunan Normal University School of Medicine, Engineering Research Center of Reproduction and Translational Medicine of Hunan Province, Manufacture-Based Learning and Research Demonstration Center for Human Reproductive Health New Technology of Hunan Normal University, Changsha, China
| | - Chunyun Li
- Key Laboratory of Model Animals and Stem Cell Biology in Hunan Province, Hunan Normal University School of Medicine, Engineering Research Center of Reproduction and Translational Medicine of Hunan Province, Manufacture-Based Learning and Research Demonstration Center for Human Reproductive Health New Technology of Hunan Normal University, Changsha, China
| | - Caimei He
- Key Laboratory of Model Animals and Stem Cell Biology in Hunan Province, Hunan Normal University School of Medicine, Engineering Research Center of Reproduction and Translational Medicine of Hunan Province, Manufacture-Based Learning and Research Demonstration Center for Human Reproductive Health New Technology of Hunan Normal University, Changsha, China
| | - Li Du
- Key Laboratory of Model Animals and Stem Cell Biology in Hunan Province, Hunan Normal University School of Medicine, Engineering Research Center of Reproduction and Translational Medicine of Hunan Province, Manufacture-Based Learning and Research Demonstration Center for Human Reproductive Health New Technology of Hunan Normal University, Changsha, China
| | - Wei Liu
- Key Laboratory of Model Animals and Stem Cell Biology in Hunan Province, Hunan Normal University School of Medicine, Engineering Research Center of Reproduction and Translational Medicine of Hunan Province, Manufacture-Based Learning and Research Demonstration Center for Human Reproductive Health New Technology of Hunan Normal University, Changsha, China
| | - Zuping He
- Key Laboratory of Model Animals and Stem Cell Biology in Hunan Province, Hunan Normal University School of Medicine, Engineering Research Center of Reproduction and Translational Medicine of Hunan Province, Manufacture-Based Learning and Research Demonstration Center for Human Reproductive Health New Technology of Hunan Normal University, Changsha, China
- Shanghai Key Laboratory of Reproductive Medicine, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
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Yuce K, Ozkan AI. The kruppel-like factor (KLF) family, diseases, and physiological events. Gene 2024; 895:148027. [PMID: 38000704 DOI: 10.1016/j.gene.2023.148027] [Citation(s) in RCA: 15] [Impact Index Per Article: 15.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/14/2023] [Revised: 11/06/2023] [Accepted: 11/21/2023] [Indexed: 11/26/2023]
Abstract
The Kruppel-Like Factor family of regulatory proteins, which has 18 members, is transcription factors. This family contains zinc finger proteins, regulates the activation and suppression of transcription, and binds to DNA, RNA, and proteins. Klfs related to the immune system are Klf1, Klf2, Klf3, Klf4, Klf6, and Klf14. Klfs related to adipose tissue development and/or glucose metabolism are Klf3, Klf7, Klf9, Klf10, Klf11, Klf14, Klf15, and Klf16. Klfs related to cancer are Klf3, Klf4, Klf5, Klf6, Klf7, Klf8, Klf9, Klf10, Klf11, Klf12, Klf13, Klf14, Klf16, and Klf17. Klfs related to the cardiovascular system are Klf4, Klf5, Klf10, Klf13, Klf14, and Klf15. Klfs related to the nervous system are Klf4, Klf7, Klf8, and Klf9. Klfs are associated with diseases such as carcinogenesis, oxidative stress, diabetes, liver fibrosis, thalassemia, and the metabolic syndrome. The aim of this review is to provide information about the relationship of Klfs with some diseases and physiological events and to guide future studies.
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Affiliation(s)
- Kemal Yuce
- Selcuk University, Medicine Faculty, Department of Basic Medical Sciences, Physiology, Konya, Turkiye.
| | - Ahmet Ismail Ozkan
- Artvin Coruh University, Medicinal-Aromatic Plants Application and Research Center, Artvin, Turkiye.
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Zhang X, Wang C, Zhou Z, Zhang Q. The mitochondrial-endoplasmic reticulum co-transfer in dental pulp stromal cell promotes pulp injury repair. Cell Prolif 2024; 57:e13530. [PMID: 37493094 PMCID: PMC10771100 DOI: 10.1111/cpr.13530] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/01/2023] [Revised: 07/12/2023] [Accepted: 07/13/2023] [Indexed: 07/27/2023] Open
Abstract
Dental pulp injury remains a clinical challenge with limited therapeutic approaches. In the present study, we sought to prove that dental pulp stromal cells (DPSCs) mitochondrial transfer could promote dental pulp injury repair and endoplasmic reticulum (ER)-mitochondrial contacts have a significant regulatory effect on mitochondrial transfer. Healthy DPSCs were co-cultured directly or indirectly with injured DPSCs in the first molar of 1-2 month SD rats or in vitro. Mitochondrial transfer was observed after 24 h of co-culture using fluorescence microscopy and live cell workstation. After co-culture for 1W, 8-OhdG immunofluorescence, mitochondrial membrane potential and total oxidant status/total antioxidant status were used to detect the mitochondrial function of injured DPSCs before and after mitochondrial transfer. Subsequently, mitochondria-ER co-transfer was regulated by modulating mitochondria-ER binding in healthy DPSCs, and the results of GRP78 and CHOP in DPSCs, and PDI immunofluorescence and haematoxylin and eosin staining of pulp tissue were analysed to clarify the effects of modulating mitochondria-ER co-transfer on endoplasmic reticulum stress (ERS), and on pulp injury repair. Fluorescence microscopy and live cell workstation results showed significant mitochondrial transfer between DPSCs. Meanwhile, mitochondrial transfer significantly restored mitochondrial function in injured DPSCs. By modulating mitochondrial-ER binding, the efficiency of mitochondrial transfer between DPSCs was significantly affected and had an impact on ERS in injured cells. Mitochondrial transfer of DPSCs significantly promotes pulpal injury repair and functional recovery of damaged DPSCs, and mitochondrial transfer of DPSCs is regulated by mitochondria-ER binding.
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Affiliation(s)
- Xiaoyi Zhang
- Department of EndodonticsStomatological Hospital and Dental School of Tongji University, Shanghai Engineering Research Center of Tooth Restoration and RegenerationShanghaiChina
| | - Chunmeng Wang
- Department of EndodonticsStomatological Hospital and Dental School of Tongji University, Shanghai Engineering Research Center of Tooth Restoration and RegenerationShanghaiChina
| | - Zihao Zhou
- Department of EndodonticsStomatological Hospital and Dental School of Tongji University, Shanghai Engineering Research Center of Tooth Restoration and RegenerationShanghaiChina
| | - Qi Zhang
- Department of EndodonticsStomatological Hospital and Dental School of Tongji University, Shanghai Engineering Research Center of Tooth Restoration and RegenerationShanghaiChina
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Howlader MSI, Prateeksha P, Hansda S, Naidu P, Das M, Barthels D, Das H. Secretory products of DPSC mitigate inflammatory effects in microglial cells by targeting MAPK pathway. Biomed Pharmacother 2024; 170:115971. [PMID: 38039760 DOI: 10.1016/j.biopha.2023.115971] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/16/2023] [Revised: 11/21/2023] [Accepted: 11/27/2023] [Indexed: 12/03/2023] Open
Abstract
Activated microglial cells in the central nervous system (CNS) are the main contributors to neurodegenerative disorders such as Alzheimer's disease and Parkinson's disease. Inhibiting their activation will help in reducing inflammation and oxidative stress during pathogenesis, potentially limiting the progression of the diseases. The immunomodulation properties of dental pulp-derived stem cells (DPSC) make it a promising therapy for neurodegenerative disorders. This study aims to determine whether secretory factors of DPSC (DPSC℗) inhibit inflammation and proliferation of microglial cells and define the molecular mechanisms. Our quantitative RT-PCR analysis showed that the DPSC℗ reduced the markers of the inflammation and induced anti-inflammatory molecules in microglial cells. DPSC ℗ reduced the intracellular and mitochondrial reactive oxygen species (ROS) production and mitochondrial membrane potential in microglial cells. In addition, DPSC ℗ decreased the cellular bioenergetics parameters related to oxygen consumption rate (OCAR) and extracellular acidification rate (ECAR). We found that DPSC℗ inhibited microglial cell proliferation by activating a checkpoint molecule, Chk1 leading an arrest at the G1 phase of the cell cycle. To define the mechanism, we performed the western blot analysis and observed that the MAPK P38 pathway was inhibited by DPSC℗. Furthermore, a System biology analysis revealed that the BDNF and GDNF, secretory factors of DPSC, blocked at the phosphorylation site (Tyr 182) of the P38 molecule resulting in the inhibition of downstream signaling of inflammation. These data suggest that the DPSC℗ may be a potential therapeutic agent for neurodegenerative diseases.
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Affiliation(s)
- Md Sariful Islam Howlader
- Department of Pharmaceutical Sciences, Jerry H. Hodge School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX, USA
| | - Prateeksha Prateeksha
- Department of Pharmaceutical Sciences, Jerry H. Hodge School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX, USA
| | - Surajit Hansda
- Department of Pharmaceutical Sciences, Jerry H. Hodge School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX, USA
| | - Prathyusha Naidu
- Department of Pharmaceutical Sciences, Jerry H. Hodge School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX, USA
| | - Manjusri Das
- Department of Pharmaceutical Sciences, Jerry H. Hodge School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX, USA
| | - Derek Barthels
- Department of Pharmaceutical Sciences, Jerry H. Hodge School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX, USA
| | - Hiranmoy Das
- Department of Pharmaceutical Sciences, Jerry H. Hodge School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX, USA.
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21
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Liu H, Xu K, He Y, Huang F. Mitochondria in Multi-Directional Differentiation of Dental-Derived Mesenchymal Stem Cells. Biomolecules 2023; 14:12. [PMID: 38275753 PMCID: PMC10813276 DOI: 10.3390/biom14010012] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/18/2023] [Revised: 12/03/2023] [Accepted: 12/18/2023] [Indexed: 01/27/2024] Open
Abstract
The pursuit of tissue regeneration has fueled decades of research in regenerative medicine. Among the numerous types of mesenchymal stem cells (MSCs), dental-derived mesenchymal stem cells (DMSCs) have recently emerged as a particularly promising candidate for tissue repair and regeneration. In recent years, evidence has highlighted the pivotal role of mitochondria in directing and orchestrating the differentiation processes of DMSCs. Beyond mitochondrial energy metabolism, the multifaceted functions of mitochondria are governed by the mitochondrial quality control (MQC) system, encompassing biogenesis, autophagy, and dynamics. Notably, mitochondrial energy metabolism not only governs the decision to differentiate but also exerts a substantial influence on the determination of differentiation directions. Furthermore, the MQC system exerts a nuanced impact on the differentiation of DMSCs by finely regulating the quality and mass of mitochondria. The review aims to provide a comprehensive overview of the regulatory mechanisms governing the multi-directional differentiation of DMSCs, mediated by both mitochondrial energy metabolism and the MQC system. We also focus on a new idea based on the analysis of data from many research groups never considered before, namely, DMSC-based regenerative medicine applications.
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Affiliation(s)
| | | | - Yifan He
- Hospital of Stomatology, Guanghua School of Stomatology, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Stomatology, Guangzhou 510000, China; (H.L.); (K.X.)
| | - Fang Huang
- Hospital of Stomatology, Guanghua School of Stomatology, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Stomatology, Guangzhou 510000, China; (H.L.); (K.X.)
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Prateeksha P, Howlader MSI, Hansda S, Naidu P, Das M, Abo-Aziza F, Das H. Secretome of Dental Pulp-Derived Stem Cells Reduces Inflammation and Proliferation of Glioblastoma Cells by Deactivating Mapk-Akt Pathway. DISEASES & RESEARCH 2023; 3:74-86. [PMID: 38213319 PMCID: PMC10783424 DOI: 10.54457/dr.202302006] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Indexed: 01/13/2024]
Abstract
Background Dental pulp-derived stem cells (DPSC) is a promising therapy as they modulate the immune response, so we evaluated the inhibitory effect of DPSC secretome (DPSC℗) on the proliferation and inflammation in human glioblastoma (GBM) cells (U-87 MG) and elucidated the concomitant mechanisms involved. Methods The U87-MG cells were cultured with DPSC℗ for 24 h and assessed the expression of inflammatory molecules using quantitative reverse transcription-polymerase chain reaction (qRT-PCR), generation of reactive oxygen species (ROS), and mitochondrial functionality using a seahorse flux analyzer. MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay and cell cycle analysis were performed to evaluate the proliferation and cell cycle. Finally, the protein levels were determined by western blot. Results DPSC℗ reduced the inflammation and proliferation of U-87 MG cells by down-regulating the pro-inflammatory markers and up-regulating anti-inflammatory markers expressions through ROS-mediated signaling. Moreover, DPSC℗ significantly reduced the mitochondrial membrane potential (MMP) in the cells. The cellular bioenergetics revealed that all the parameters of oxygen consumption rate (OCAR) and the extracellular acidification rate (ECAR) were significantly decreased in the GBM cells after the addition of DPSC℗. Additionally, DPSC℗ decreased the GBM cell proliferation by arresting the cell cycle at the G1 phase through activation (phosphorylation) of checkpoint molecule CHK1. Furthermore, mechanistically, we found that the DPSC℗ impedes the phosphorylation of the mitogen-activated protein kinases (P38 MAPK) and protein kinase B (AKT) pathway. Conclusion Our findings lend the first evidence of the inhibitory effects of DPSC℗ on proliferation and inflammation in GBM cells by altering the P38 MAPK-AKT pathway.
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Affiliation(s)
- Prateeksha Prateeksha
- Department of Pharmaceutical Sciences, Jerry H. Hodge School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, Texas 79106, USA
| | - Md Sariful Islam Howlader
- Department of Pharmaceutical Sciences, Jerry H. Hodge School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, Texas 79106, USA
| | - Surajit Hansda
- Department of Pharmaceutical Sciences, Jerry H. Hodge School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, Texas 79106, USA
| | - Prathyusha Naidu
- Department of Pharmaceutical Sciences, Jerry H. Hodge School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, Texas 79106, USA
| | - Manjusri Das
- Department of Pharmaceutical Sciences, Jerry H. Hodge School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, Texas 79106, USA
| | - Faten Abo-Aziza
- Department of Pharmaceutical Sciences, Jerry H. Hodge School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, Texas 79106, USA
| | - Hiranmoy Das
- Department of Pharmaceutical Sciences, Jerry H. Hodge School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, Texas 79106, USA
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Prateeksha P, Naidu P, Das M, Barthels D, Das H. KLF2 Regulates Neural Differentiation of Dental Pulp-derived Stem Cells by Modulating Autophagy and Mitophagy. Stem Cell Rev Rep 2023; 19:2886-2900. [PMID: 37642902 DOI: 10.1007/s12015-023-10607-0] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 08/09/2023] [Indexed: 08/31/2023]
Abstract
BACKGROUND Transplantation of stem cells for treating neurodegenerative disorders is a promising future therapeutic approach. However, the molecular mechanism underlying the neuronal differentiation of dental pulp-derived stem cells (DPSC) remains inadequately explored. The current study aims to define the regulatory role of KLF2 (Kruppel-like factor 2) during the neural differentiation (ND) of DPSC. METHODS We first investigated the transcriptional and translational expression of KLF2, autophagy, and mitophagy-associated markers during the ND of DPSC by using quantitative RT-PCR and western blot methods. After that, we applied the chemical-mediated loss- and gain-of-function approaches using KLF2 inhibitor, GGPP (geranylgeranyl pyrophosphate), and KLF2 activator, GGTI-298 (geranylgeranyl transferase inhibitor-298) to delineate the role of KLF2 during ND of DPSC. The western blot, qRT-PCR, and immunocytochemistry were performed to determine the molecular changes during ND after KLF2 deficiency and KLF2 sufficiency. We also analyzed the oxygen consumption rate (OCR) and the extracellular acidification rate (ECAR) using the Seahorse XFe24 analyzer. RESULTS Our study demonstrated that the expression level of KLF2, autophagy, and mitophagy-associated markers were significantly elevated during the ND of DPSC. Next, we found that the KLF2 inhibitor, GGPP significantly reduced the ND of DPSC. Inversely, KLF2 overexpression accelerated the molecular phenomenon of DPSC's commitment towards ND, indicating the crucial role of KLF2 in neurogenesis. Moreover, we found that the KLF2 positively regulated autophagy, mitophagy, and the Wnt5a signaling pathway during neurogenesis. Seahorse XFe24 analysis revealed that the ECAR and OCR parameters were significantly increased during ND, and inhibition of KLF2 marginally reversed them towards DPSC's cellular bioenergetics. However, KLF2 overexpression shifted the cellular energy metabolism toward the quiescent stage. CONCLUSION Collectively, our findings provide the first evidence that the KLF2 critically regulates the neurogenesis of DPSC by inducing autophagy and mitophagy.
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Affiliation(s)
- Prateeksha Prateeksha
- Department of Pharmaceutical Sciences, Jerry H. Hodge School of Pharmacy, Texas Tech University Health Sciences Center, ARB Suite 2116, 1406 South Coulter Street, Amarillo, TX, 79106, USA
| | - Prathyusha Naidu
- Department of Pharmaceutical Sciences, Jerry H. Hodge School of Pharmacy, Texas Tech University Health Sciences Center, ARB Suite 2116, 1406 South Coulter Street, Amarillo, TX, 79106, USA
| | - Manjusri Das
- Department of Pharmaceutical Sciences, Jerry H. Hodge School of Pharmacy, Texas Tech University Health Sciences Center, ARB Suite 2116, 1406 South Coulter Street, Amarillo, TX, 79106, USA
| | - Derek Barthels
- Department of Pharmaceutical Sciences, Jerry H. Hodge School of Pharmacy, Texas Tech University Health Sciences Center, ARB Suite 2116, 1406 South Coulter Street, Amarillo, TX, 79106, USA
| | - Hiranmoy Das
- Department of Pharmaceutical Sciences, Jerry H. Hodge School of Pharmacy, Texas Tech University Health Sciences Center, ARB Suite 2116, 1406 South Coulter Street, Amarillo, TX, 79106, USA.
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24
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Agriesti F, Landini F, Tamma M, Pacelli C, Mazzoccoli C, Calice G, Ruggieri V, Capitanio G, Mori G, Piccoli C, Capitanio N. Bioenergetic profile and redox tone modulate in vitro osteogenesis of human dental pulp stem cells: new perspectives for bone regeneration and repair. Stem Cell Res Ther 2023; 14:215. [PMID: 37608350 PMCID: PMC10463344 DOI: 10.1186/s13287-023-03447-9] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/29/2023] [Accepted: 08/10/2023] [Indexed: 08/24/2023] Open
Abstract
BACKGROUND Redox signaling and energy metabolism are known to be involved in controlling the balance between self-renewal and proliferation/differentiation of stem cells. In this study we investigated metabolic and redox changes occurring during in vitro human dental pulp stem cells (hDPSCs) osteoblastic (OB) differentiation and tested on them the impact of the reactive oxygen species (ROS) signaling. METHODS hDPSCs were isolated from dental pulp and subjected to alkaline phosphatase and alizarin red staining, q-RT-PCR, and western blotting analysis of differentiation markers to assess achievement of osteogenic/odontogenic differentiation. Moreover, a combination of metabolic flux analysis and confocal cyto-imaging was used to profile the metabolic phenotype and to evaluate the redox tone of hDPSCs. RESULTS In differentiating hDPSCs we observed the down-regulation of the mitochondrial respiratory chain complexes expression since the early phase of the process, confirmed by metabolic flux analysis, and a reduction of the basal intracellular peroxide level in its later phase. In addition, dampened glycolysis was observed, thereby indicating a lower energy-generating phenotype in differentiating hDPSCs. Treatment with the ROS scavenger Trolox, applied in the early-middle phases of the process, markedly delayed OB differentiation of hDPSCs assessed as ALP activity, Runx2 expression, mineralization capacity, expression of stemness and osteoblast marker genes (Nanog, Lin28, Dspp, Ocn) and activation of ERK1/2. In addition, the antioxidant partly prevented the inhibitory effect on cell metabolism observed following osteogenic induction. CONCLUSIONS Altogether these results provided evidence that redox signaling, likely mediated by peroxide species, influenced the stepwise osteogenic expansion/differentiation of hDPSCs and contributed to shape its accompanying metabolic phenotype changes thus improving their efficiency in bone regeneration and repair.
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Affiliation(s)
- Francesca Agriesti
- Department of Clinical and Experimental Medicine, University of Foggia, 71122 Foggia, Italy
- Laboratory of Pre-Clinical and Translational Research, IRCCS-CROB, Referral Cancer Center of Basilicata, 85028 Rionero in Vulture, Italy
| | - Francesca Landini
- Department of Clinical and Experimental Medicine, University of Foggia, 71122 Foggia, Italy
| | - Mirko Tamma
- Department of Clinical and Experimental Medicine, University of Foggia, 71122 Foggia, Italy
| | - Consiglia Pacelli
- Department of Clinical and Experimental Medicine, University of Foggia, 71122 Foggia, Italy
| | - Carmela Mazzoccoli
- Laboratory of Pre-Clinical and Translational Research, IRCCS-CROB, Referral Cancer Center of Basilicata, 85028 Rionero in Vulture, Italy
| | - Giovanni Calice
- Laboratory of Pre-Clinical and Translational Research, IRCCS-CROB, Referral Cancer Center of Basilicata, 85028 Rionero in Vulture, Italy
| | - Vitalba Ruggieri
- Laboratory of Pre-Clinical and Translational Research, IRCCS-CROB, Referral Cancer Center of Basilicata, 85028 Rionero in Vulture, Italy
- Clinical Pathology Unit, “Madonna delle Grazie’’ Hospital, Matera, Italy
| | - Giuseppe Capitanio
- Department of Translational Biomedicine and Neuroscience “DiBraiN”, University of Bari “Aldo Moro”, 70124 Bari, Italy
| | - Giorgio Mori
- Department of Clinical and Experimental Medicine, University of Foggia, 71122 Foggia, Italy
| | - Claudia Piccoli
- Department of Clinical and Experimental Medicine, University of Foggia, 71122 Foggia, Italy
| | - Nazzareno Capitanio
- Department of Clinical and Experimental Medicine, University of Foggia, 71122 Foggia, Italy
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Sugier HR, Bellebon L, Aider JL, Larghero J, Peltzer J, Martinaud C. Feasibility of an acoustophoresis-based system for a high-throughput cell washing: application to bioproduction. Cytotherapy 2023; 25:891-899. [PMID: 37269272 DOI: 10.1016/j.jcyt.2023.05.003] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/28/2022] [Revised: 04/12/2023] [Accepted: 05/08/2023] [Indexed: 06/05/2023]
Abstract
BACKGROUND AIMS These last decades have seen the emergence and development of cell-based therapies, notably those based on mesenchymal stromal cells (MSCs). The advancement of these promising treatments requires increasing the throughput of processed cell for industrialization in order to reduce production costs. Among the various bioproduction challenges, downstream processing, including medium exchange, cell washing, cell harvesting and volume reduction, remains a critical step for which improvements are needed. Typically, these processes are performed by centrifugation. However, this approach limits the automation, especially in small batch productions where it is performed manually in open system. METHODS An acoustophoresis-based system was developed for cell washing. The cells were transferred from one stream to another via the acoustic forces and were collected in a different medium. The optimal flow rates of the different streams were assessed using red blood cells suspended in an albumin solution. Finally, the impact of acoustic washing on adipose tissue-derived MSCs (AD-MSCs) transcriptome was investigated by RNA-sequencing. RESULTS With a single passage through the acoustic device at input flow rate of 45 mL/h, the albumin removal was up to 90% while recovering 99% of RBCs. To further increase the protein removal, a loop washing in two steps was performed and has allowed an albumin removal ≥99% and a red blood cell/AD-MSCs recovery of 99%. After loop washing of AD-MSCs, only two genes, HES4 and MIR-3648-1, were differently expressed compared with the input. CONCLUSIONS In this study, we developed a continuous cell-washing system based on acoustophoresis. The process allows a theoretically high cell throughput while inducing little gene expression changes. These results indicate that cell washing based on acoustophoresis is a relevant and promising solution for numerous applications in cell manufacturing.
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Affiliation(s)
- Hugo R Sugier
- Aenitis Technologies, Paris, France; Institut André Lwoff, INSERM UMR-MD 1197, Villejuif, France.
| | - Ludovic Bellebon
- Laboratoire PMMH, UMR7636 CNRS, ESPCI Paris - PSL, Paris Sciences Lettres, Sorbonne Université, Paris, France
| | - Jean-Luc Aider
- Laboratoire PMMH, UMR7636 CNRS, ESPCI Paris - PSL, Paris Sciences Lettres, Sorbonne Université, Paris, France
| | - Jérôme Larghero
- Université de Paris, Assistance Publique-Hôpitaux de Paris, Hôpital Saint-Louis, Paris, France; Unité de Thérapie Cellulaire, INSERM U976, Centre d'investigation clinique de Biothérapies CBT501, Paris, France
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Barthels D, Prateeksha P, Nozohouri S, Villalba H, Zhang Y, Sharma S, Anderson S, Howlader MSI, Nambiar A, Abbruscato TJ, Das H. Dental Pulp-Derived Stem Cells Preserve Astrocyte Health During Induced Gliosis by Modulating Mitochondrial Activity and Functions. Cell Mol Neurobiol 2023; 43:2105-2127. [PMID: 36201091 PMCID: PMC11412198 DOI: 10.1007/s10571-022-01291-8] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2022] [Accepted: 09/27/2022] [Indexed: 11/03/2022]
Abstract
Astrocytes have been implicated in the onset and complication of various central nervous system (CNS) injuries and disorders. Uncontrolled astrogliosis (gliosis), while a necessary process for recovery after CNS trauma, also causes impairments in CNS performance and functions. The ability to preserve astrocyte health and better regulate the gliosis process could play a major role in controlling damage in the aftermath of acute insults and during chronic dysfunction. Here in, we demonstrate the ability of dental pulp-derived stem cells (DPSCs) in protecting the health of astrocytes during induced gliosis. First of all, we have characterized the expression of genes in primary astrocytes that are relevant to the pathological conditions of CNS by inducing gliosis. Subsequently, we found that astrocytes co-cultured with DPSCs reduced ROS production, NRF2 and GCLM expressions, mitochondrial membrane potential, and mitochondrial functions compared to the astrocytes that were not co-cultured with DPSCs in gliosis condition. In addition, hyperactive autophagy was also decreased in astrocytes that were co-cultured with DPSCs compared to the astrocytes that were not co-cultured with DPSCs during gliosis. This reversal and mitigation of gliosis in astrocytes were partly due to induction of neurogenesis in DPSCs through enhanced expressions of the neuronal genes like GFAP, NeuN, and Synapsin in DPSCs and by secretion of higher amounts of neurotropic factors, such as BDNF, GDNF, and TIMP-2. Protein-Protein docking analysis suggested that BDNF and GDNF can bind with CSPG4 and block the downstream signaling. Together these findings demonstrate novel functions of DPSCs to preserve astrocyte health during gliosis.
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Affiliation(s)
- Derek Barthels
- Department of Pharmaceutical Sciences, Jerry H. Hodge School of Pharmacy, Texas Tech University Health Sciences Center, ARB Suite 2116, 1406 South Coulter Street, Amarillo, TX, 79106, USA
| | - Prateeksha Prateeksha
- Department of Pharmaceutical Sciences, Jerry H. Hodge School of Pharmacy, Texas Tech University Health Sciences Center, ARB Suite 2116, 1406 South Coulter Street, Amarillo, TX, 79106, USA
| | - Saeideh Nozohouri
- Department of Pharmaceutical Sciences, Jerry H. Hodge School of Pharmacy, Texas Tech University Health Sciences Center, ARB Suite 2116, 1406 South Coulter Street, Amarillo, TX, 79106, USA
| | - Heidi Villalba
- Department of Pharmaceutical Sciences, Jerry H. Hodge School of Pharmacy, Texas Tech University Health Sciences Center, ARB Suite 2116, 1406 South Coulter Street, Amarillo, TX, 79106, USA
| | - Yong Zhang
- Department of Pharmaceutical Sciences, Jerry H. Hodge School of Pharmacy, Texas Tech University Health Sciences Center, ARB Suite 2116, 1406 South Coulter Street, Amarillo, TX, 79106, USA
| | - Sejal Sharma
- Department of Pharmaceutical Sciences, Jerry H. Hodge School of Pharmacy, Texas Tech University Health Sciences Center, ARB Suite 2116, 1406 South Coulter Street, Amarillo, TX, 79106, USA
| | - Sarah Anderson
- Department of Pharmaceutical Sciences, Jerry H. Hodge School of Pharmacy, Texas Tech University Health Sciences Center, ARB Suite 2116, 1406 South Coulter Street, Amarillo, TX, 79106, USA
| | - Md Sariful Islam Howlader
- Department of Pharmaceutical Sciences, Jerry H. Hodge School of Pharmacy, Texas Tech University Health Sciences Center, ARB Suite 2116, 1406 South Coulter Street, Amarillo, TX, 79106, USA
| | - Adarsh Nambiar
- Department of Pharmaceutical Sciences, Jerry H. Hodge School of Pharmacy, Texas Tech University Health Sciences Center, ARB Suite 2116, 1406 South Coulter Street, Amarillo, TX, 79106, USA
| | - Thomas J Abbruscato
- Department of Pharmaceutical Sciences, Jerry H. Hodge School of Pharmacy, Texas Tech University Health Sciences Center, ARB Suite 2116, 1406 South Coulter Street, Amarillo, TX, 79106, USA
| | - Hiranmoy Das
- Department of Pharmaceutical Sciences, Jerry H. Hodge School of Pharmacy, Texas Tech University Health Sciences Center, ARB Suite 2116, 1406 South Coulter Street, Amarillo, TX, 79106, USA.
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27
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Huang W, Huang Q, He H, Huang F. PER2 Promotes Odontoblastic/Osteogenic Differentiation of Dental Pulp Stem Cells by Modulating Mitochondrial Metabolism. Int J Mol Sci 2023; 24:10661. [PMID: 37445839 PMCID: PMC10341716 DOI: 10.3390/ijms241310661] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/07/2023] [Revised: 06/21/2023] [Accepted: 06/22/2023] [Indexed: 07/15/2023] Open
Abstract
Human dental pulp stem cells (hDPSCs) possess remarkable self-renewal and multilineage differentiation ability. PER2, an essential circadian molecule, regulates various physiological processes. Evidence suggests that circadian rhythm and PER2 participate in physiological functions of DPSCs. However, the influence of PER2 on DPSCs' differentiation remains largely unknown. This study aimed to explore the effect and potential mechanism of PER2 on hDPSCs' differentiation. Dental pulp tissues were extracted, and hDPSCs were cultured for in vitro and in vivo experiments. Dorsal subcutaneous transplantation was performed in 6-week-old male BALB/c mice. The hDPSCs' odontoblastic/osteogenic differentiation was assessed, and mitochondrial metabolism was evaluated. The results indicated PER2 expression increasing during hDPSCs' odontoblastic/osteogenic differentiation. Gain- and loss-of function studies confirmed that PER2 promoted alkaline phosphatase (ALP) activity, mineralized nodules deposition, mRNA expression of DSPP, DMP1, COL1A1 and protein expression of DSPP and DMP1 in hDPSCs. Furthermore, PER2 enhanced collagen deposition, osteodentine-like tissue formation and DSPP expression in vivo. Mitochondrial metabolic evaluation aimed to investigate the mechanism of PER2-mediated hDPSC odontoblastic/osteogenic differentiation, which showed that PER2 increased ATP synthesis, elevated mitochondrial membrane potential and changed expression of proteins regulating mitochondrial dynamics. This study demonstrated that PER2 promoted hDPSCs' odontoblastic/osteogenic differentiation, which involved mitochondrial metabolic change.
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Affiliation(s)
- Wushuang Huang
- Hospital of Stomatology, Sun Yat-sen University, Guangzhou 510055, China; (W.H.)
- Guangdong Provincial Key Laboratory of Stomatology, Guangzhou 510055, China
- Institute of Stomatology, Sun Yat-sen University, Guangzhou 510055, China
| | - Qi Huang
- Hospital of Stomatology, Sun Yat-sen University, Guangzhou 510055, China; (W.H.)
- Guangdong Provincial Key Laboratory of Stomatology, Guangzhou 510055, China
- Institute of Stomatology, Sun Yat-sen University, Guangzhou 510055, China
| | - Hongwen He
- Hospital of Stomatology, Sun Yat-sen University, Guangzhou 510055, China; (W.H.)
- Guangdong Provincial Key Laboratory of Stomatology, Guangzhou 510055, China
- Institute of Stomatology, Sun Yat-sen University, Guangzhou 510055, China
| | - Fang Huang
- Hospital of Stomatology, Sun Yat-sen University, Guangzhou 510055, China; (W.H.)
- Guangdong Provincial Key Laboratory of Stomatology, Guangzhou 510055, China
- Institute of Stomatology, Sun Yat-sen University, Guangzhou 510055, China
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Liu W, Zhang F, Liang W, Huang K, Jia C, Zhang J, Li X, Wei W, Gong R, Chen J. Integrated insight into the molecular mechanisms of selenium-modulated, MPP +-induced cytotoxicity in a Parkinson's disease model. J Trace Elem Med Biol 2023; 79:127208. [PMID: 37269647 DOI: 10.1016/j.jtemb.2023.127208] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/03/2021] [Revised: 06/13/2022] [Accepted: 05/17/2023] [Indexed: 06/05/2023]
Abstract
OBJECTIVE Parkinson's disease (PD) is a neurodegenerative disease that is associated with oxidative stress. Due to the anti-inflammatory and antioxidant functions of Selenium (Se), this molecule may have neuroprotective functions in PD; however, the involvement of Se in such a protective function is unclear. METHODS 1-methyl-4-phenylpyridinium (MPP+), which inhibits mitochondrial respiration, is generally used to produce a reliable cellular model of PD. In this study, a MPP+-induced PD model was used to test if Se could modulate cytotoxicity, and we further capture gene expression profiles following PC12 cell treatment with MPP+ with or without Se by genome wide high-throughput sequencing. RESULTS We identified 351 differentially expressed genes (DEGs) and 14 differentially expressed long non-coding RNAs (DELs) in MPP+-treated cells when compared to controls. We further document 244 DEGs and 27 DELs in cells treated with MPP+ and Se vs. cells treated with MPP+ only. Functional annotation analysis of DEGs and DELs revealed that these groups were enriched in genes that respond to reactive oxygen species (ROS), metabolic processes, and mitochondrial control of apoptosis. Thioredoxin reductase 1 (Txnrd1) was also identified as a biomarker of Se treatment. CONCLUSIONS Our data suggests that the DEGs Txnrd1, Siglec1 and Klf2, and the DEL AABR07044454.1 which we hypothesize to function in cis on the target gene Cdkn1a, may modulate the underlying neurodegenerative process, and act a protective function in the PC12 cell PD model. This study further systematically demonstrated that mRNAs and lncRNAs induced by Se are involved in neuroprotection in PD, and provides novel insight into how Se modulates cytotoxicity in the MPP+-induced PD model.
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Affiliation(s)
- Wen Liu
- Department of Neurosurgery, Zhongnan Hospital, Wuhan University, Donghu Road 169, Wuhan 430071, China
| | - Feiyang Zhang
- Department of Neurosurgery, Zhongnan Hospital, Wuhan University, Donghu Road 169, Wuhan 430071, China
| | - Wu Liang
- Department of Neurosurgery, Minda Hospital of Hubei Minzu University, Enshi 445000, China
| | - Kaixin Huang
- Department of Neurosurgery, Zhongnan Hospital, Wuhan University, Donghu Road 169, Wuhan 430071, China
| | - Chenguang Jia
- Department of Neurosurgery, Zhongnan Hospital, Wuhan University, Donghu Road 169, Wuhan 430071, China
| | - Jie Zhang
- Department of Neurosurgery, Zhongnan Hospital, Wuhan University, Donghu Road 169, Wuhan 430071, China
| | - Xiang Li
- Department of Neurosurgery, Zhongnan Hospital, Wuhan University, Donghu Road 169, Wuhan 430071, China; Neuroepigenetic Research Lab, Medical Research Institute, Wuhan University, Donghu Road 115, Wuhan 430071, China
| | - Wei Wei
- Department of Neurosurgery, Zhongnan Hospital, Wuhan University, Donghu Road 169, Wuhan 430071, China; Neuroepigenetic Research Lab, Medical Research Institute, Wuhan University, Donghu Road 115, Wuhan 430071, China.
| | - Rui Gong
- Department of Neurosurgery, Zhongnan Hospital, Wuhan University, Donghu Road 169, Wuhan 430071, China.
| | - Jincao Chen
- Department of Neurosurgery, Zhongnan Hospital, Wuhan University, Donghu Road 169, Wuhan 430071, China.
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He Z, Du J, Zhang Y, Xu Y, Huang Q, Zhou Q, Wu M, Li Y, Zhang X, Zhang H, Cai Y, Ye K, Wang X, Zhang Y, Han Q, Xiao J. Kruppel-like factor 2 contributes to blood-spinal cord barrier integrity and functional recovery from spinal cord injury by augmenting autophagic flux. Theranostics 2023; 13:849-866. [PMID: 36632224 PMCID: PMC9830435 DOI: 10.7150/thno.74324] [Citation(s) in RCA: 16] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/01/2022] [Accepted: 11/01/2022] [Indexed: 01/06/2023] Open
Abstract
Background: Increasing evidence suggests that acute traumatic spinal cord injury (SCI)-induced defects in autophagy and autophagy-lysosomal pathway (ALP) may contribute to endothelial barrier disruption following injury. Recently, Kruppel-like factor 2 (KLF2) was reported as a key molecular switch on regulating autophagy. Whether KLF2 coordinates endothelial endothelial ALP in SCI is not known. Methods: Genetic manipulations of KLF2 were performed in bEnd.3 cells and SCI model. Western blot, qRT-PCR, immunofluorescence staining and Lyso-Tracker Red staining, Evans blue dye extravasation, behavioral assessment via Basso mouse scale (BMS), electrophysiology and footprint analysis were performed. Results: In SCI, autophagy flux disruption in endothelial cells contributes to TJ proteins degradation, leading to blood-spinal cord barrier (BSCB) impairment. Furthermore, the KLF2 level was decreased in SCI, overexpression of which alleviated TJ proteins loss and BSCB damage, which improve motor function recovery in SCI mice, while knockdown of KLF2 displayed the opposite effects. At the molecular level, KLF2 overexpression alleviated the TJ proteins degradation and the endothelial permeability by tuning the ALP dysfunction caused by SCI and oxygen glucose deprivation (OGD). Conclusions: Endothelial KLF2 as one of the key contributors to SCI-mediated ALP dysfunction and BSCB disruption. KLF2 could be a promising pharmacological target for the management and treatment of SCI.
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Affiliation(s)
- Zili He
- Oujiang Laboratory (Zhejiang Lab for Regenerative Medicine, Vision and Brain Health), Department of Orthopaedics Surgery, The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University, Wenzhou, 325035, China.,Department of Orthopaedics, Affiliated Pingyang Hospital and School of Pharmaceutical Science, Wenzhou Medical University, Wenzhou, 325000, China
| | - Jiqing Du
- Department of Orthopaedics, Affiliated Pingyang Hospital and School of Pharmaceutical Science, Wenzhou Medical University, Wenzhou, 325000, China
| | - Yu Zhang
- Department of Orthopaedics, Affiliated Pingyang Hospital and School of Pharmaceutical Science, Wenzhou Medical University, Wenzhou, 325000, China
| | - Yitie Xu
- Department of Orthopaedics, Affiliated Pingyang Hospital and School of Pharmaceutical Science, Wenzhou Medical University, Wenzhou, 325000, China
| | - Qian Huang
- Department of Orthopaedics, Affiliated Pingyang Hospital and School of Pharmaceutical Science, Wenzhou Medical University, Wenzhou, 325000, China
| | - Qingwei Zhou
- Department of Orthopaedics, Affiliated Pingyang Hospital and School of Pharmaceutical Science, Wenzhou Medical University, Wenzhou, 325000, China
| | - Min Wu
- Department of Orthopaedics, Affiliated Pingyang Hospital and School of Pharmaceutical Science, Wenzhou Medical University, Wenzhou, 325000, China
| | - Yao Li
- Oujiang Laboratory (Zhejiang Lab for Regenerative Medicine, Vision and Brain Health), Department of Orthopaedics Surgery, The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University, Wenzhou, 325035, China
| | - Xie Zhang
- Department of Pharmacy, Ningbo Medical Treatment Center Li Huili Hospital, Ningbo, 315040, China
| | - Hongyu Zhang
- Department of Orthopaedics, Affiliated Pingyang Hospital and School of Pharmaceutical Science, Wenzhou Medical University, Wenzhou, 325000, China
| | - Yuepiao Cai
- Oujiang Laboratory (Zhejiang Lab for Regenerative Medicine, Vision and Brain Health), Department of Orthopaedics Surgery, The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University, Wenzhou, 325035, China.,Department of Orthopaedics, Affiliated Pingyang Hospital and School of Pharmaceutical Science, Wenzhou Medical University, Wenzhou, 325000, China
| | - Keyong Ye
- Department of Orthopaedics, Affiliated Pingyang Hospital and School of Pharmaceutical Science, Wenzhou Medical University, Wenzhou, 325000, China
| | - Xiangyang Wang
- Oujiang Laboratory (Zhejiang Lab for Regenerative Medicine, Vision and Brain Health), Department of Orthopaedics Surgery, The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University, Wenzhou, 325035, China
| | - Yingze Zhang
- Oujiang Laboratory (Zhejiang Lab for Regenerative Medicine, Vision and Brain Health), Department of Orthopaedics Surgery, The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University, Wenzhou, 325035, China.,Department of Orthopaedic Surgery, The Third Hospital of Hebei Medical University, Shijiazhuang, 050051 China
| | - Qi Han
- Department of Anatomy and Physiology, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China
| | - Jian Xiao
- Oujiang Laboratory (Zhejiang Lab for Regenerative Medicine, Vision and Brain Health), Department of Orthopaedics Surgery, The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University, Wenzhou, 325035, China.,Department of Orthopaedics, Affiliated Pingyang Hospital and School of Pharmaceutical Science, Wenzhou Medical University, Wenzhou, 325000, China
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Jobst M, Kiss E, Gerner C, Marko D, Del Favero G. Activation of autophagy triggers mitochondrial loss and changes acetylation profile relevant for mechanotransduction in bladder cancer cells. Arch Toxicol 2023; 97:217-233. [PMID: 36214828 PMCID: PMC9816236 DOI: 10.1007/s00204-022-03375-2] [Citation(s) in RCA: 16] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/21/2022] [Accepted: 09/05/2022] [Indexed: 01/19/2023]
Abstract
Bladder cells are constantly exposed to multiple xenobiotics and bioactive metabolites. In addition to this challenging chemical environment, they are also exposed to shear stress originating from urine and interstitial fluids. Hence, physiological function of bladder cells relies on a high biochemical and biomechanical adaptive competence, which, in turn, is largely supported via autophagy-related mechanisms. As a negative side of this plasticity, bladder cancer cells are known to adapt readily to chemotherapeutic programs. At the molecular level, autophagy was described to support resistance against pharmacological treatments and to contribute to the maintenance of cell structure and metabolic competence. In this study, we enhanced autophagy with rapamycin (1-100 nM) and assessed its effects on the motility of bladder cells, as well as the capability to respond to shear stress. We observed that rapamycin reduced cell migration and the mechanical-induced translocation potential of Krüppel-like transcription factor 2 (KLF2). These effects were accompanied by a rearrangement of cytoskeletal elements and mitochondrial loss. In parallel, intracellular acetylation levels were decreased. Mechanistically, inhibition of the NAD + -dependent deacetylase sirtuin-1 (SIRT1) with nicotinamide (NAM; 0.1-5 mM) restored acetylation levels hampered by rapamycin and cell motility. Taken together, we described the effects of rapamycin on cytoskeletal elements crucial for mechanotransduction and the dependency of these changes on the mitochondrial turnover caused by autophagy activation. Additionally, we could show that targeted metabolic intervention could revert the outcome of autophagy activation, reinforcing the idea that bladder cells can easily adapt to multiple xenobiotics and circumvent in this way the effects of single chemicals.
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Affiliation(s)
- Maximilian Jobst
- Department of Food Chemistry and Toxicology, Faculty of Chemistry, University of Vienna, Währingerstr. 38-40, 1090 Vienna, Austria
| | - Endre Kiss
- Core Facility Multimodal, Imaging, Faculty of Chemistry, University of Vienna, Währingerstr. 38-40, 1090 Vienna, Austria
| | - Christopher Gerner
- Department of Analytical Chemistry, Faculty of Chemistry, University of Vienna, Währingerstr. 38-40, 1090 Vienna, Austria ,Joint Metabolome Facility, University of Vienna and Medical University of Vienna, Vienna, Austria
| | - Doris Marko
- Department of Food Chemistry and Toxicology, Faculty of Chemistry, University of Vienna, Währingerstr. 38-40, 1090 Vienna, Austria
| | - Giorgia Del Favero
- Department of Food Chemistry and Toxicology, Faculty of Chemistry, University of Vienna, Währingerstr. 38-40, 1090 Vienna, Austria ,Core Facility Multimodal, Imaging, Faculty of Chemistry, University of Vienna, Währingerstr. 38-40, 1090 Vienna, Austria
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31
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Sarkar J, Das M, Howlader MSI, Prateeksha P, Barthels D, Das H. Epigallocatechin-3-gallate inhibits osteoclastic differentiation by modulating mitophagy and mitochondrial functions. Cell Death Dis 2022; 13:908. [PMID: 36307395 PMCID: PMC9616829 DOI: 10.1038/s41419-022-05343-1] [Citation(s) in RCA: 32] [Impact Index Per Article: 10.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/23/2022] [Revised: 10/04/2022] [Accepted: 10/12/2022] [Indexed: 01/23/2023]
Abstract
A natural plant product, epigallocatechin-3-gallate (EGCG), was evaluated for its effectiveness in the regulation of osteoclastogenesis. We found that EGCG inhibited the osteoclast (OC) differentiation in vitro, and in primary bone marrow cells in a dose-dependent manner. Quantitative RT-PCR studies showed that the EGCG reduced the expression of OC differentiation markers. DCFDA, MitoSOX, and JC-1 staining revealed that the EGCG attenuated the reactive oxygen species (ROS), and mitochondrial membrane potential; and flux analysis corroborated the effect of EGCG. We further found that the EGCG inhibited mRNA and protein expressions of mitophagy-related molecules. We confirmed that the OC differentiation was inhibited by EGCG by modulating mitophagy through AKT and p38MAPK pathways. Furthermore, in silico analysis revealed that the binding of RANK and RANKL was blocked by EGCG. Overall, we defined the mechanisms of osteoclastogenesis during arthritis for developing a new therapy using a natural compound besides the existing therapeutics.
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Affiliation(s)
- Jaganmay Sarkar
- grid.416992.10000 0001 2179 3554Department of Pharmaceutical Sciences, Jerry H. Hodge School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX USA
| | - Manjusri Das
- grid.416992.10000 0001 2179 3554Department of Pharmaceutical Sciences, Jerry H. Hodge School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX USA
| | - Md Sariful Islam Howlader
- grid.416992.10000 0001 2179 3554Department of Pharmaceutical Sciences, Jerry H. Hodge School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX USA
| | - Prateeksha Prateeksha
- grid.416992.10000 0001 2179 3554Department of Pharmaceutical Sciences, Jerry H. Hodge School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX USA
| | - Derek Barthels
- grid.416992.10000 0001 2179 3554Department of Pharmaceutical Sciences, Jerry H. Hodge School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX USA
| | - Hiranmoy Das
- grid.416992.10000 0001 2179 3554Department of Pharmaceutical Sciences, Jerry H. Hodge School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX USA
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Epigenetic Alterations under Oxidative Stress in Stem Cells. OXIDATIVE MEDICINE AND CELLULAR LONGEVITY 2022; 2022:6439097. [PMID: 36071870 PMCID: PMC9444469 DOI: 10.1155/2022/6439097] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 04/15/2022] [Revised: 07/16/2022] [Accepted: 07/27/2022] [Indexed: 11/18/2022]
Abstract
Epigenetic regulation of gene expression, including DNA methylation and histone modifications, provides finely tuned responses for cells that undergo cellular environment changes. Abundant evidences have demonstrated the detrimental role of oxidative stress in various human pathogenesis since oxidative stress results from the imbalance between reactive oxygen species (ROS) accumulation and antioxidant defense system. Stem cells can self-renew themselves and meanwhile have the potential to differentiate into many other cell types. As some studies have described the effects of oxidative stress on homeostasis and cell fate decision of stem cells, epigenetic alterations have emerged crucial for mediating the stem cell behaviours under oxidative stress. Here, we review recent findings on the oxidative effects on DNA and histone modifications in stem cells. We propose that epigenetic alterations and oxidative stress may influence each other in stem cells.
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DPSC Products Accelerate Wound Healing in Diabetic Mice through Induction of SMAD Molecules. Cells 2022; 11:cells11152409. [PMID: 35954256 PMCID: PMC9368341 DOI: 10.3390/cells11152409] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/22/2022] [Revised: 07/28/2022] [Accepted: 07/30/2022] [Indexed: 12/17/2022] Open
Abstract
Despite advances in diabetic wound care, many amputations are still needed each year due to their diabetic wounds, so a more effective therapy is warranted. Herein, we show that the dental pulp-derived stem cell (DPSC) products are effective in wound healing in diabetic NOD/SCID mice. Our results showed that the topical application of DPSC secretory products accelerated wound closure by inducing faster re-epithelialization, angiogenesis, and recellularization. In addition, the number of neutrophils producing myeloperoxidase, which mediates persisting inflammation, was also reduced. NFκB and its downstream effector molecules like IL-6 cause sustained pro-inflammatory activity and were reduced after the application of DPSC products in the experimental wounds. Moreover, the DPSC products also inhibited the activation of NFκB, and its translocation to the nucleus, by which it initiates the inflammation. Furthermore, the levels of TGF-β, and IL-10, potent anti-inflammatory molecules, were also increased after the addition of DPSC products. Mechanistically, we showed that this wound-healing process was mediated by the upregulation and activation of Smad 1 and 2 molecules. In sum, we have defined the cellular and molecular mechanisms by which DPSC products accelerated diabetic wound closure, which can be used to treat diabetic wounds in the near future.
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The Role of Epigenetic in Dental and Oral Regenerative Medicine by Different Types of Dental Stem Cells: A Comprehensive Overview. Stem Cells Int 2022; 2022:5304860. [PMID: 35721599 PMCID: PMC9203206 DOI: 10.1155/2022/5304860] [Citation(s) in RCA: 54] [Impact Index Per Article: 18.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/19/2022] [Revised: 05/17/2022] [Accepted: 05/27/2022] [Indexed: 12/13/2022] Open
Abstract
Postnatal teeth, wisdom teeth, and exfoliated deciduous teeth can be harvested for dental stem cell (DSC) researches. These mesenchymal stem cells (MSCs) can differentiate and also consider as promising candidates for dental and oral regeneration. Thus, the development of DSC therapies can be considered a suitable but challenging target for tissue regeneration. Epigenetics describes changes in gene expression rather than changes in DNA and broadly happens in bone homeostasis, embryogenesis, stem cell fate, and disease development. The epigenetic regulation of gene expression and the regulation of cell fate is mainly governed by deoxyribonucleic acid (DNA) methylation, histone modification, and noncoding RNAs (ncRNAs). Tissue engineering utilizes DSCs as a target. Tissue engineering therapies are based on the multipotent regenerative potential of DSCs. It is believed that epigenetic factors are essential for maintaining the multipotency of DSCs. A wide range of host and environmental factors influence stem cell differentiation and differentiation commitment, of which epigenetic regulation is critical. Several lines of evidence have shown that epigenetic modification of DNA and DNA-correlated histones are necessary for determining cells' phenotypes and regulating stem cells' pluripotency and renewal capacity. It is increasingly recognized that nuclear enzyme activities, such as histone deacetylases, can be used pharmacologically to induce stem cell differentiation and dedifferentiation. In this review, the role of epigenetic in dental and oral regenerative medicine by different types of dental stem cells is discussed in two new and promising areas of medical and biological researches in recent studies (2010-2022).
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Maity J, Barthels D, Sarkar J, Prateeksha P, Deb M, Rolph D, Das H. Ferutinin induces osteoblast differentiation of DPSCs via induction of KLF2 and autophagy/mitophagy. Cell Death Dis 2022; 13:452. [PMID: 35552354 PMCID: PMC9098908 DOI: 10.1038/s41419-022-04903-9] [Citation(s) in RCA: 22] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/22/2021] [Accepted: 04/29/2022] [Indexed: 01/18/2023]
Abstract
Osteoblast differentiation is critically reduced in various bone-related pathogenesis, including arthritis and osteoporosis. For future development of effective regenerative therapeutics, herein, we reveal the involved molecular mechanisms of a phytoestrogen, ferutinin-induced initiation of osteoblast differentiation from dental pulp-derived stem cell (DPSC). We demonstrate the significantly increased expression level of a transcription factor, Kruppel-like factor 2 (KLF2) along with autophagy-related molecules in DPSCs after induction with ferutinin. The loss-of-function and the gain-of-function approaches of KLF2 confirmed that the ferutinin-induced KLF2 modulated autophagic and OB differentiation-related molecules. Further, knockdown of the autophagic molecule (ATG7 or BECN1) from DPSC resulted not only in a decreased level of KLF2 but also in the reduced levels of OB differentiation-related molecules. Moreover, mitochondrial membrane potential-related molecules were increased and induction of mitophagy was observed in DPSCs after the addition of ferutinin. The reduction of mitochondrial as well as total ROS generations; and induction of intracellular Ca2+ production were also observed in ferutinin-treated DPSCs. To test the mitochondrial respiration in DPSCs, we found that the cells treated with ferutinin showed a reduced extracellular acidification rate (ECAR) than that of their vehicle-treated counterparts. Furthermore, mechanistically, chromatin immunoprecipitation (ChIP) analysis revealed that the addition of ferutinin in DPSCs not only induced the level of KLF2, but also induced the transcriptionally active epigenetic marks (H3K27Ac and H3K4me3) on the promoter region of the autophagic molecule ATG7. These results provide strong evidence that ferutinin stimulates OB differentiation via induction of KLF2-mediated autophagy/mitophagy.
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Affiliation(s)
- Jyotirindra Maity
- grid.416992.10000 0001 2179 3554Department of Pharmaceutical Sciences, Jerry H. Hodge School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX USA
| | - Derek Barthels
- grid.416992.10000 0001 2179 3554Department of Pharmaceutical Sciences, Jerry H. Hodge School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX USA
| | - Jaganmay Sarkar
- grid.416992.10000 0001 2179 3554Department of Pharmaceutical Sciences, Jerry H. Hodge School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX USA
| | - Prateeksha Prateeksha
- grid.416992.10000 0001 2179 3554Department of Pharmaceutical Sciences, Jerry H. Hodge School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX USA
| | - Moonmoon Deb
- grid.416992.10000 0001 2179 3554Department of Pharmaceutical Sciences, Jerry H. Hodge School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX USA
| | - Daniela Rolph
- grid.416992.10000 0001 2179 3554Department of Pharmaceutical Sciences, Jerry H. Hodge School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX USA
| | - Hiranmoy Das
- grid.416992.10000 0001 2179 3554Department of Pharmaceutical Sciences, Jerry H. Hodge School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX USA
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Metabolic Remodeling Impacts the Epigenetic Landscape of Dental Mesenchymal Stem Cells. Stem Cells Int 2022; 2022:3490433. [PMID: 35422867 PMCID: PMC9005295 DOI: 10.1155/2022/3490433] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2021] [Accepted: 03/12/2022] [Indexed: 02/05/2023] Open
Abstract
Epigenetic regulation can dynamically adjust the gene expression program of cell fate decision according to the cellular microenvironment. Emerging studies have shown that metabolic activities provide fundamental components for epigenetic modifications and these metabolic-sensitive epigenetic events dramatically impact the cellular function of stem cells. Dental mesenchymal stem cells are promising adult stem cell resource for in situ injury repair and tissue engineering. In this review, we discuss the impact of metabolic fluctuations on epigenetic modifications in the oral and maxillofacial regions. The principles of the metabolic link to epigenetic modifications and the interaction between metabolite substrates and canonical epigenetic events in dental mesenchymal stem cells are summarized. The coordination between metabolic pathways and epigenetic events plays an important role in cellular progresses including differentiation, inflammatory responses, and aging. The metabolic-epigenetic network is critical for expanding our current understanding of tissue homeostasis and cell fate decision and for guiding potential therapeutic approaches in dental regeneration and infectious diseases.
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37
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Wang FF, Zhang JL, Ji Y, Yan XJ, Sun L, Zhu Y, Jin H. KLF2 mediates the suppressive effect of BDNF on diabetic intimal calcification by inhibiting HK1 induced endothelial-to-mesenchymal transition. Cell Signal 2022; 94:110324. [PMID: 35364229 DOI: 10.1016/j.cellsig.2022.110324] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/25/2022] [Revised: 03/19/2022] [Accepted: 03/24/2022] [Indexed: 12/19/2022]
Abstract
Diabetic vascular calcification in the arterial intima is closely associated with endothelial-to-mesenchymal transition (EndMT). Glucose metabolism reprogramming is involved in EndMT. Although brain-derived neurotrophic factor (BDNF) and Krüppel-like family of transcription factor 2 (KLF2) play protective roles in the physiological activity of the vascular endothelium, the underlying mechanisms are unclear. Human umbilical vein endothelial cells (HUVECs) were incubated with diabetic osteogenic medium (DOM) to induce EndMT and accelerate osteogenic differentiation. Glycolysis in HUVECs was assessed by monitoring glucose uptake, lactate production, extracellular acidification rate and expression of key glycolytic enzymes. DOM induced EndMT and accelerated osteo-induction in HUVECs, which was alleviated by BDNF/tropomyosin receptor kinase B (TrkB) pathway. Mechanistically, DOM caused hyperactivation of glycolysis in HUVECs and inhibition of the BDNF/TrkB pathway. BDNF preserved KLF2 and downregulated hexokinase 1 (HK1) in HUVECs after DOM treatment. Furthermore, KLF2 interacted with HK1. Increased KLF2 alleviated HK1-mediated glucose metabolism abnormality. HK1 knockdown or a targeted glycolysis inhibitor suppressed EndMT, apoptosis, inflammation and vascular calcification of HUVECs after DOM exposure. This study suggests that KLF2 mediates the suppressive effect of BDNF on diabetic intimal calcification by inhibiting HK1-induced glucose metabolism reprogramming and the EndMT process.
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Affiliation(s)
- Fang-Fang Wang
- Department of Cardiology, The Affiliated Changzhou No.2 People's Hospital of Nanjing Medical University, Changzhou 213000, PR China
| | - Jia-Li Zhang
- Department of Gastroenterology Centre, The Affiliated Changzhou No.2 People's Hospital of Nanjing Medical University, Changzhou 213000, PR China
| | - Yuan Ji
- Department of Cardiology, The Affiliated Changzhou No.2 People's Hospital of Nanjing Medical University, Changzhou 213000, PR China
| | - Xue-Jiao Yan
- Department of Cardiology, The Affiliated Changzhou No.2 People's Hospital of Nanjing Medical University, Changzhou 213000, PR China
| | - Ling Sun
- Department of Cardiology, The Affiliated Changzhou No.2 People's Hospital of Nanjing Medical University, Changzhou 213000, PR China
| | - Yi Zhu
- Department of Cardiology, The Affiliated Changzhou No.2 People's Hospital of Nanjing Medical University, Changzhou 213000, PR China.
| | - Hong Jin
- Department of Cardiology, Zhongda Hospital, School of Medicine, Southeast University, Nanjing 210009, PR China.
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Zakeri S, Aminian H, Sadeghi S, Esmaeilzadeh-Gharehdaghi E, Razmara E. Krüppel-like factors in bone biology. Cell Signal 2022; 93:110308. [PMID: 35301064 DOI: 10.1016/j.cellsig.2022.110308] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/12/2022] [Revised: 03/07/2022] [Accepted: 03/09/2022] [Indexed: 12/27/2022]
Abstract
The krüppel-like factor (KLF) family is a group of zinc finger transcription factors and contributes to different cellular processes such as differentiation, proliferation, migration, and apoptosis. While different studies show the roles of this family in skeletal development-specifically in chondrocyte and osteocyte development and bone homeostasis-there are few reviews summarizing their importance. To fill this gap, this review discusses current knowledge on different functions of the KLF family during skeletal development, including their roles in stem cell maintenance and differentiation, cell apoptosis, and cell cycle. To understand the importance of the KLF family, we also review genotype-phenotype correlations in different animal models. We also discuss how KLF proteins function through different signaling pathways and display their paramount importance in skeletal development. To highlight their roles in cartilage- or bone-related cells, we also use single-cell RNA sequencing publicly available data on mouse hindlimb. We also challenge our knowledge of how the KLF family is epigenetically regulated-e.g., using DNA methylation, histone modifications, and noncoding RNAs-during chondrocyte and osteocyte development.
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Affiliation(s)
- Sina Zakeri
- Department of Veterinary Science, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran
| | - Hesam Aminian
- Department of Biology, Faculty of Sciences, Nour Danesh Institute of Higher Education, Meymeh, Isfahan, Iran
| | - Soheila Sadeghi
- Department of Biology, Faculty of Basic Sciences, Sanandaj Branch, Islamic Azad University, Kurdistan, Iran
| | | | - Ehsan Razmara
- Department of Medical Genetics, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
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Li N, Liu H, Xue Y, Chen J, Kong X, Zhang Y. Upregulation of Neogenin-1 by a CREB1-BAF47 Complex in Vascular Endothelial Cells is Implicated in Atherogenesis. Front Cell Dev Biol 2022; 10:803029. [PMID: 35186922 PMCID: PMC8851423 DOI: 10.3389/fcell.2022.803029] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/27/2021] [Accepted: 01/10/2022] [Indexed: 12/29/2022] Open
Abstract
Atherosclerosis is generally considered a human pathology of chronic inflammation, to which endothelial dysfunction plays an important role. Here we investigated the role of neogenin 1 (Neo-1) in oxidized low-density lipoprotein (oxLDL) induced endothelial dysfunction focusing on its transcriptional regulation. We report that Neo-1 expression was upregulated by oxLDL in both immortalized vascular endothelial cells and primary aortic endothelial cells. Neo-1 knockdown attenuated whereas Neo-1 over-expression enhanced oxLDL-induced leukocyte adhesion to endothelial cells. Neo-1 regulated endothelial-leukocyte interaction by modulating nuclear factor kappa B (NF-κB) activity to alter the expression of adhesion molecules. Neo-1 blockade with a blocking antibody ameliorated atherogenesis in Apoe−/− mice fed a Western diet. Ingenuity pathway analysis combined with validation assays confirmed that cAMP response element binding protein 1 (CREB1) and Brg1-associated factor 47 (BAF47) mediated oxLDL induced Neo-1 upregulation. CREB1 interacted with BAF47 and recruited BAF47 to the proximal Neo-1 promoter leading to Neo-1 trans-activation. In conclusion, our data delineate a novel transcriptional mechanism underlying Neo-1 activation in vascular endothelial cells that might contribute to endothelial dysfunction and atherosclerosis.
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Affiliation(s)
- Nan Li
- Department of Human Anatomy, Nanjing Medical University, Nanjing, China
- Key Laboratory of Targeted Intervention of Cardiovascular Disease and Collaborative Innovation Center for Cardiovascular Translational Medicine, Nanjing Medical University, Nanjing, China
| | - Hong Liu
- Key Laboratory of Targeted Intervention of Cardiovascular Disease and Collaborative Innovation Center for Cardiovascular Translational Medicine, Nanjing Medical University, Nanjing, China
| | - Yujia Xue
- Key Laboratory of Targeted Intervention of Cardiovascular Disease and Collaborative Innovation Center for Cardiovascular Translational Medicine, Nanjing Medical University, Nanjing, China
| | - Junliang Chen
- Department of Pathophysiology, Wuxi Medical School, Jiangnan University, Wuxi, China
| | - Xiaocen Kong
- Department of Endocrinology, Nanjing First Hospital, Nanjing Medical University, Nanjing, China
- Institute of Biomedical Research, Liaocheng Univeristy, Liaocheng, China
- *Correspondence: Xiaocen Kong, ; Yuanyuan Zhang,
| | - Yuanyuan Zhang
- Hainan Provincial Key Laboratory for Tropical Cardiovascular Diseases Research, Key Laboratory of Emergency and Trauma of Ministry of Education, Department of Cardiology, The First Affiliated Hospital of Hainan Medical University, Haikou, China
- *Correspondence: Xiaocen Kong, ; Yuanyuan Zhang,
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40
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The Role of KLF2 in the Regulation of Atherosclerosis Development and Potential Use of KLF2-Targeted Therapy. Biomedicines 2022; 10:biomedicines10020254. [PMID: 35203463 PMCID: PMC8869605 DOI: 10.3390/biomedicines10020254] [Citation(s) in RCA: 31] [Impact Index Per Article: 10.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2022] [Revised: 01/21/2022] [Accepted: 01/24/2022] [Indexed: 12/26/2022] Open
Abstract
Kruppel like factor 2 (KLF2) is a mechanosensitive transcription factor participating in the regulation of vascular endothelial cells metabolism. Activating KLF2 in endothelial cells induces eNOS (endothelial nitric oxide synthase) expression, subsequent NO (nitric oxide) release, and vasodilatory effect. In addition, many KLF2-regulated genes participate in the anti-thrombotic, antioxidant, and anti-inflammatory activities, thereby preventing atherosclerosis development and progression. In this review, we summarise recent evidence suggesting that KLF2 plays a major role in regulating atheroprotective effects in endothelial cells. We also discuss several recently identified repurposed drugs and natural plant-based bioactive compounds with KLF2-mediated atheroprotective activities. Herein, we present a comprehensive overview of the role of KLF2 in atherosclerosis and as a pharmacological target for different drugs and natural compounds and highlight the potential application of these phytochemicals for the treatment of atherosclerosis.
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41
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Gao G, Hu S, Zhang K, Wang H, Xie Y, Zhang C, Wu R, Zhao X, Zhang H, Wang Q. Genome-Wide Gene Expression Profiles Reveal Distinct Molecular Characteristics of the Goose Granulosa Cells. Front Genet 2021; 12:786287. [PMID: 34992633 PMCID: PMC8725158 DOI: 10.3389/fgene.2021.786287] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/01/2021] [Accepted: 11/30/2021] [Indexed: 11/24/2022] Open
Abstract
Granulosa cells (GCs) are decisive players in follicular development. In this study, the follicle tissues and GCs were isolated from the goose during the peak-laying period to perform hematoxylin-eosin staining and RNA-seq, respectively. Moreover, the dynamic mRNA and lncRNA expression profiles and mRNA-lncRNA network analysis were integrated to identify the important genes and lncRNAs. The morphological analysis showed that the size of the GCs did not significantly change, but the thickness of the granulosa layer cells differed significantly across the developmental stages. Subsequently, 14,286 mRNAs, 3,956 lncRNAs, and 1,329 TUCPs (transcripts with unknown coding potential) were detected in the GCs. We identified 37 common DEGs in the pre-hierarchical and hierarchical follicle stages, respectively, which might be critical for follicle development. Moreover, 3,089 significant time-course DEGs (Differentially expressed genes) and 13 core genes in 4 clusters were screened during goose GCs development. Finally, the network lncRNA G8399 with CADH5 and KLF2, and lncRNA G8399 with LARP6 and EOMES were found to be important for follicular development in GCs. Thus, the results would provide a rich resource for elucidating the reproductive biology of geese and accelerate the improvement of the egg-laying performance of geese.
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Affiliation(s)
- Guangliang Gao
- Chongqing Academy of Animal Sciences, Chongqing, China
- Institute of Animal Genetics and Breeding, College of Animal Science and Technology, Sichuan Agricultural University, Chengdu, China
- Chongqing Engineering Research Center of Goose Genetic Improvement, Chongqing, China
- *Correspondence: Guangliang Gao, ; Hongmei Zhang, ; Qigui Wang,
| | - Silu Hu
- Institute of Animal Genetics and Breeding, College of Animal Science and Technology, Sichuan Agricultural University, Chengdu, China
| | - Keshan Zhang
- Chongqing Academy of Animal Sciences, Chongqing, China
- Chongqing Engineering Research Center of Goose Genetic Improvement, Chongqing, China
| | - Haiwei Wang
- Chongqing Academy of Animal Sciences, Chongqing, China
- Chongqing Engineering Research Center of Goose Genetic Improvement, Chongqing, China
| | - Youhui Xie
- Chongqing Academy of Animal Sciences, Chongqing, China
- Chongqing Engineering Research Center of Goose Genetic Improvement, Chongqing, China
| | - Changlian Zhang
- Chongqing Academy of Animal Sciences, Chongqing, China
- Chongqing Engineering Research Center of Goose Genetic Improvement, Chongqing, China
| | - Rui Wu
- Chongqing Academy of Animal Sciences, Chongqing, China
| | - Xianzhi Zhao
- Chongqing Academy of Animal Sciences, Chongqing, China
- Chongqing Engineering Research Center of Goose Genetic Improvement, Chongqing, China
| | - Hongmei Zhang
- Department of Cardiovascular Ultrasound and Non-invasive Cardiology, Sichuan Academy of Medical Sciences and Sichuan Provincial People’s Hospital, Chengdu, China
- Ultrasound in Cardiac Electrophysiology and Biomechanics Key Laboratory of Sichuan Province, Chengdu, China
- *Correspondence: Guangliang Gao, ; Hongmei Zhang, ; Qigui Wang,
| | - Qigui Wang
- Chongqing Academy of Animal Sciences, Chongqing, China
- Chongqing Engineering Research Center of Goose Genetic Improvement, Chongqing, China
- *Correspondence: Guangliang Gao, ; Hongmei Zhang, ; Qigui Wang,
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42
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Cai W, Ji Y, Han L, Zhang J, Ni Y, Cheng Y, Zhang Y. METTL3-Dependent Glycolysis Regulates Dental Pulp Stem Cell Differentiation. J Dent Res 2021; 101:580-589. [PMID: 34796755 DOI: 10.1177/00220345211051594] [Citation(s) in RCA: 31] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023] Open
Abstract
N6-methyladenosine (m6A) is a eukaryotic messenger RNA modification catalyzed by methyltransferase-like 3 (METTL3), which is involved in various developmental and disease processes. However, the connection between the epigenetic modification of m6A and glucose metabolism during osteogenesis is still unclear. Here, we show that interference with METTL3 in dental pulp stem cells (DPSCs) inhibits cell proliferation and osteogenic differentiation. Moreover, transcriptome sequencing and metabolic testing were used to explore the mechanism between glucose metabolism and m6A modification in METTL3-knockdown DPSCs. Methylated RNA immunoprecipitation-quantitative polymerase chain reaction and RNA stability assays were used to determine the target genes of METTL3. Mechanistically, METTL3 directly interacts with ATP citrate lyase (ACLY) and a mitochondrial citrate transporter (SLC25A1) and then further affects the glycolytic pathway. M6A-mediated ACLY and SLC25A1 stability depends on the m6A readers IGF2BP2 and IGF2BP2/3, respectively. Our experiments uncovered the potential molecular mechanism of epigenetic modification in osteogenic differentiation, providing new ideas for the clinical application of stem cells and the intervention of metabolic bone diseases.
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Affiliation(s)
- W Cai
- State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory of Oral Biomedicine, Ministry of Education, School and Hospital of Stomatology, Wuhan University, Wuhan, China
| | - Y Ji
- State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory of Oral Biomedicine, Ministry of Education, School and Hospital of Stomatology, Wuhan University, Wuhan, China
| | - L Han
- State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory of Oral Biomedicine, Ministry of Education, School and Hospital of Stomatology, Wuhan University, Wuhan, China
| | - J Zhang
- State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory of Oral Biomedicine, Ministry of Education, School and Hospital of Stomatology, Wuhan University, Wuhan, China
| | - Y Ni
- State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory of Oral Biomedicine, Ministry of Education, School and Hospital of Stomatology, Wuhan University, Wuhan, China
| | - Y Cheng
- State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory of Oral Biomedicine, Ministry of Education, School and Hospital of Stomatology, Wuhan University, Wuhan, China
| | - Y Zhang
- State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory of Oral Biomedicine, Ministry of Education, School and Hospital of Stomatology, Wuhan University, Wuhan, China
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43
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Ma C, Wu H, Yang G, Xiang J, Feng K, Zhang J, Hua Y, Kang L, Fan G, Yang S. Calycosin ameliorates atherosclerosis by enhancing autophagy via regulating the interaction between KLF2 and MLKL in apoE -/- mice. Br J Pharmacol 2021; 179:252-269. [PMID: 34713437 DOI: 10.1111/bph.15720] [Citation(s) in RCA: 28] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2021] [Revised: 09/23/2021] [Accepted: 10/06/2021] [Indexed: 10/20/2022] Open
Abstract
BACKGROUND AND PURPOSE Atherosclerosis is one of the underlying causes of cardiovascular disease. Formation of foam cells and necrotic core in the plaque is a hallmark of atherosclerosis, which results from lipid deposition, apoptosis, and inflammation in macrophage. Macrophage autophagy is a critical anti-atherogenic process and defective autophagy aggravates atherosclerosis by enhancing foam cell formation, apoptosis, and inflammation. Hence, enhancing autophagy can be a strategy for atherosclerosis treatment. Calycosin, a flavonoid from Astragali Radix, displays antioxidant and anti-inflammatory activities, and therefore is potential to reduce the risk of cardiovascular disease. However, the antiatherogenic effect of calycosin and the involved mechanism remains unclear. In this study, we assessed the potential benefits of calycosin on autophagy and atherosclerosis, and revealed the underlying mechanism. EXPERIMENTAL APPROACH In this study, apoE-/- mice were fed high-fat diet for 16 weeks in presence of calycosin and/or autophagy inhibitor chloroquine, which was followed by determination of atherosclerosis development, autophagy activity, and the involved mechanisms. KEY RESULTS Calycosin protected against atherosclerosis and enhanced plaque stability via promoting autophagy. Calycosin inhibited foam cells formation, inflammation, and apoptosis by enhancing autophagy. MLKL was demonstrated as a new autophagy regulator, which can be negatively regulated by KLF2. Mechanistically, inhibitory effects of calycosin on atherogenesis were via improving autophagy through modulating KLF2-MLKL signaling pathway. CONCLUSIONS AND IMPLICATIONS This study demonstrated the atheroprotective effect of calycosin was through upregulating KLF2-MLKL-mediated autophagy, which not only proposed novel mechanistic insights into the atherogenesis but also identified calycosin as a potential drug candidate for atherosclerosis treatment.
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Affiliation(s)
- Chuanrui Ma
- First Teaching Hospital of Tianjin University of Traditional Chinese Medicine, Tianjin, China.,National Clinical Research Center for Chinese Medicine Acupuncture and Moxibustion
| | - Han Wu
- Department of Endocrinology, Shenzhen People's Hospital, The Second Clinical Medical College of Jinan University & The First Affiliated Hospital of Southern University of Science and Technology, Shenzhen, Guangdong, China
| | - Guangyan Yang
- Department of Endocrinology, Shenzhen People's Hospital, The Second Clinical Medical College of Jinan University & The First Affiliated Hospital of Southern University of Science and Technology, Shenzhen, Guangdong, China
| | - Jiaqing Xiang
- Department of Endocrinology, Shenzhen People's Hospital, The Second Clinical Medical College of Jinan University & The First Affiliated Hospital of Southern University of Science and Technology, Shenzhen, Guangdong, China
| | - Ke Feng
- College of Life Sciences, Nankai University, Tianjin, China
| | - Jing Zhang
- First Teaching Hospital of Tianjin University of Traditional Chinese Medicine, Tianjin, China.,National Clinical Research Center for Chinese Medicine Acupuncture and Moxibustion
| | - Yunqing Hua
- First Teaching Hospital of Tianjin University of Traditional Chinese Medicine, Tianjin, China.,National Clinical Research Center for Chinese Medicine Acupuncture and Moxibustion
| | - Lin Kang
- Department of Endocrinology, Shenzhen People's Hospital, The Second Clinical Medical College of Jinan University & The First Affiliated Hospital of Southern University of Science and Technology, Shenzhen, Guangdong, China.,The Biobank of National Innovation Center for Advanced Medical Devices, Shenzhen People's Hospital, Shenzhen, Guangdong, China
| | - Guanwei Fan
- First Teaching Hospital of Tianjin University of Traditional Chinese Medicine, Tianjin, China.,National Clinical Research Center for Chinese Medicine Acupuncture and Moxibustion
| | - Shu Yang
- Department of Endocrinology, Shenzhen People's Hospital, The Second Clinical Medical College of Jinan University & The First Affiliated Hospital of Southern University of Science and Technology, Shenzhen, Guangdong, China.,Integrated Chinese and Western Medicine Postdoctoral Research Station, Jinan University, Guangzhou, China
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HES5-mediated repression of LIGHT transcription may contribute to apoptosis in hepatocytes. Cell Death Discov 2021; 7:308. [PMID: 34689159 PMCID: PMC8542050 DOI: 10.1038/s41420-021-00707-6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/07/2021] [Revised: 10/04/2021] [Accepted: 10/06/2021] [Indexed: 11/26/2022] Open
Abstract
Non-alcoholic fatty liver disease (NAFLD) is prototypical form of metabolic syndrome and has become a global pandemic. Hepatocytes undergo apoptosis in the pathogenesis of NAFLD. We report that the lymphokine LIGHT/TNFSF14 was upregulated in the murine NAFLD livers and in hepatocytes treated with free fatty acids (palmitate, PA). LIGHT knockdown or neutralization attenuated PA-induced apoptosis of hepatocytes. Similarly, knockdown or blockade of LTβR, the receptor for LIGHT, ameliorated apoptosis in hepatocytes exposed to PA. Ingenuity pathway analysis (IPA) revealed several Notch-related transcription factors as upstream regulators of LIGHT, of which HES5 expression was downregulated paralleling LIGHT induction in the pathogenesis of NAFLD. HES5 knockdown enhanced whereas HES5 over-expression weakened LIGHT induction in hepatocytes. HES5 was found to directly bind to the LIGHT promoter and repress LIGHT transcription. Mechanistically, HES5 interacted with SIRT1 to deacetylate histone H3/H4 on the LIGHT promoter to repress LIGHT transcription. SIRT1 knockdown or inhibition offset the effect of HES5 over-expression on LIGHT transcription and hepatocyte apoptosis. In conclusion, our data unveil a novel mechanism that might contribute to excessive apoptosis in hepatocyte exposed to free fatty acids.
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45
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Lv F, Shao T, Xue Y, Miao X, Guo Y, Wang Y, Xu Y. Dual Regulation of Tank Binding Kinase 1 by BRG1 in Hepatocytes Contributes to Reactive Oxygen Species Production. Front Cell Dev Biol 2021; 9:745985. [PMID: 34660604 PMCID: PMC8517266 DOI: 10.3389/fcell.2021.745985] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/23/2021] [Accepted: 09/15/2021] [Indexed: 01/14/2023] Open
Abstract
Excessive accumulation of reactive oxygen species (ROS) is considered a major culprit for the pathogenesis of non-alcoholic fatty liver disease (NAFLD). We have previously shown that deletion of Brahma related gene 1 (BRG1) mitigated NAFLD in mice in part by attenuating ROS production in hepatocyte. Here we report that BRG1 deletion led to simultaneous down-regulation in expression and phosphorylation of tank binding kinase 1 (TBK1) in vivo and in vitro. On the one hand, BRG1 interacted with AP-1 to bind to the TBK1 promoter and directly activated TBK1 transcription in hepatocytes. On the other hand, BRG1 interacted with Sp1 to activate the transcription of c-SRC, a tyrosine kinase essential for TBK1 phosphorylation. Over-expression of c-SRC and TBK1 corrected the deficiency in ROS production in BRG1-null hepatocytes whereas depletion of TBK1 or c-SRC attenuated ROS production. In conclusion, our data suggest that dual regulation of TBK1 activity, at the transcription level and the post-transcriptional level, by BRG1 may constitute an important mechanism underlying excessive ROS production in hepatocytes.
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Affiliation(s)
- Fangqiao Lv
- Department of Cell Biology, Municipal Laboratory for Liver Protection and Regulation of Regeneration, School of Basic Medical Sciences, Capital Medical University, Beijing, China
| | - Tinghui Shao
- Key Laboratory of Targeted Intervention of Cardiovascular Disease and Collaborative Innovation Center for Cardiovascular Translational Medicine, Department of Pathophysiology, Nanjing Medical University, Nanjing, China
| | - Yujia Xue
- Key Laboratory of Targeted Intervention of Cardiovascular Disease and Collaborative Innovation Center for Cardiovascular Translational Medicine, Department of Pathophysiology, Nanjing Medical University, Nanjing, China
| | - Xiulian Miao
- College of Life Sciences and Institute of Biomedical Research, Liaocheng University, Liaocheng, China
| | - Yan Guo
- College of Life Sciences and Institute of Biomedical Research, Liaocheng University, Liaocheng, China
| | - Yutong Wang
- Department of Cell Biology, Municipal Laboratory for Liver Protection and Regulation of Regeneration, School of Basic Medical Sciences, Capital Medical University, Beijing, China
| | - Yong Xu
- Key Laboratory of Targeted Intervention of Cardiovascular Disease and Collaborative Innovation Center for Cardiovascular Translational Medicine, Department of Pathophysiology, Nanjing Medical University, Nanjing, China.,College of Life Sciences and Institute of Biomedical Research, Liaocheng University, Liaocheng, China
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46
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SETD2-mediated epigenetic regulation of noncanonical Wnt5A during osteoclastogenesis. Clin Epigenetics 2021; 13:192. [PMID: 34663428 PMCID: PMC8522097 DOI: 10.1186/s13148-021-01125-2] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/08/2021] [Accepted: 06/28/2021] [Indexed: 01/17/2023] Open
Abstract
To define the role of SETD2 in the WNT5a signaling in the context of osteoclastogenesis, we exploited two different models: in vitro osteoclast differentiation, and K/BxN serum-induced arthritis model. We found that SETD2 and WNT5a were upregulated during osteoclast differentiation and after induction of arthritis. Using gain- and loss-of-function approaches in the myeloid cell, we confirmed that SETD2 regulated the osteoclast markers, and WNT5a via modulating active histone marks by enriching H3K36me3, and by reducing repressive H3K27me3 mark. Additionally, during osteoclastic differentiation, the transcription of Wnt5a was also associated with the active histone H3K9 and H4K8 acetylations. Mechanistically, SETD2 directed induction of NF-κβ expression facilitated the recruitment of H3K9Ac and H4K8Ac around the TSS region of the Wnt5a gene, thereby, assisting osteoclast differentiation. Together these findings for the first time revealed that SETD2 mediated epigenetic regulation of Wnt5a plays a critical role in osteoclastogenesis and induced arthritis. Model for the Role of SETD2 dependent regulation of osteoclastic differentiation. A In monocyte cells SETD2-dependent H3K36 trimethylation help to create open chromatin region along with active enhancer mark, H3K27Ac. This chromatin state facilitated the loss of a suppressive H3K27me3 mark. B Additionally, SETD2 mediated induction of NF-κβ expression leads to the recruitment of histone acetyl transferases, P300/PCAF, to the Wnt5a gene and establish H3K9Ac and H4K8Ac marks. Along with other activation marks, these acetylation marks help in Wnt5a transcription which leads to osteoclastogenesis.
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47
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Chen B, Dong W, Shao T, Miao X, Guo Y, Liu X, Feng Y. A KDM4-DBC1-SIRT1 Axis Contributes to TGF-b Induced Mesenchymal Transition of Intestinal Epithelial Cells. Front Cell Dev Biol 2021; 9:697614. [PMID: 34631698 PMCID: PMC8493255 DOI: 10.3389/fcell.2021.697614] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/20/2021] [Accepted: 08/31/2021] [Indexed: 12/13/2022] Open
Abstract
Intestinal fibrosis is one of the common pathophysiological processes in inflammatory bowel diseases (IBDs). Previously it has been demonstrated that epithelial-mesenchymal transition (EMT) can contribute to the development of intestinal fibrosis. Here we report that conditional ablation of SIRT1, a class III lysine deacetylase, in intestinal epithelial cells exacerbated 2, 4, 6-trinitro-benzene sulfonic acid (TNBS) induced intestinal fibrosis in mice. SIRT1 activity, but not SIRT1 expression, was down-regulated during EMT likely due to up-regulation of its inhibitor deleted in breast cancer 1 (DBC1). TGF-β augmented the recruitment of KDM4A, a histone H3K9 demethylase, to the DBC1 promoter in cultured intestinal epithelial cells (IEC-6) leading to DBC1 trans-activation. KDM4A depletion or inhibition abrogated DBC1 induction by TGF-β and normalized SIRT1 activity. In addition, KDM4A deficiency attenuated TGF-β induced EMT in IEC-6 cells. In conclusion, our data identify a KDM4-DBC1-SIRT1 pathway that regulates EMT to contribute to intestinal fibrosis.
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Affiliation(s)
- Baoyu Chen
- Department of Pathophysiology, Nanjing Medical University, Nanjing, China
| | - Wenhui Dong
- Department of Pathophysiology, Nanjing Medical University, Nanjing, China
| | - Tinghui Shao
- Department of Pathophysiology, Nanjing Medical University, Nanjing, China
| | - Xiulian Miao
- College of Life Sciences and Institute of Biomedical Research, Liaocheng University, Liaocheng, China
| | - Yan Guo
- College of Life Sciences and Institute of Biomedical Research, Liaocheng University, Liaocheng, China
| | - Xingyu Liu
- College of Life Sciences and Institute of Biomedical Research, Liaocheng University, Liaocheng, China
| | - Yifei Feng
- Department of General Surgery, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China.,The First School of Clinical Medicine, Nanjing Medical University, Nanjing, China
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Wu X, Dong W, Kong M, Ren H, Wang J, Shang L, Zhu Z, Zhu W, Shi X. Down-Regulation of CXXC5 De-Represses MYCL1 to Promote Hepatic Stellate Cell Activation. Front Cell Dev Biol 2021; 9:680344. [PMID: 34621736 PMCID: PMC8490686 DOI: 10.3389/fcell.2021.680344] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/14/2021] [Accepted: 08/24/2021] [Indexed: 12/23/2022] Open
Abstract
Liver fibrosis is mediated by myofibroblasts, a specialized cell type involved in wound healing and extracellular matrix production. Hepatic stellate cells (HSC) are the major source of myofibroblasts in the fibrotic livers. In the present study we investigated the involvement of CXXC-type zinc-finger protein 5 (CXXC5) in HSC activation and the underlying mechanism. Down-regulation of CXXC5 was observed in activated HSCs compared to quiescent HSCs both in vivo and in vitro. In accordance, over-expression of CXXC5 suppressed HSC activation. RNA-seq analysis revealed that CXXC5 influenced multiple signaling pathways to regulate HSC activation. The proto-oncogene MYCL1 was identified as a novel target for CXXC5. CXXC5 bound to the proximal MYCL1 promoter to repress MYCL1 transcription in quiescent HSCs. Loss of CXXC5 expression during HSC activation led to the removal of CpG methylation and acquisition of acetylated histone H3K9/H3K27 on the MYCL1 promoter resulting in MYCL1 trans-activation. Finally, MYCL1 knockdown attenuated HSC activation whereas MYCL1 over-expression partially relieved the blockade of HSC activation by CXXC5. In conclusion, our data unveil a novel transcriptional mechanism contributing to HSC activation and liver fibrosis.
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Affiliation(s)
- Xiaoyan Wu
- Department of Hepatobiliary Surgery, Affiliated Nanjing Drum Tower Hospital of Nanjing University Medical School, Nanjing, China
- Hepatobiliary Institute of Nanjing University, Nanjing, China
- Institute of Biomedical Research, Liaocheng University, Liaocheng, China
- Key Laboratory of Targeted Intervention of Cardiovascular Disease, Collaborative Innovation Center for Cardiovascular Translational Medicine, and Center for Experimental Medicine, Department of Pathophysiology, Nanjing Medical University, Nanjing, China
| | - Wenhui Dong
- Key Laboratory of Targeted Intervention of Cardiovascular Disease, Collaborative Innovation Center for Cardiovascular Translational Medicine, and Center for Experimental Medicine, Department of Pathophysiology, Nanjing Medical University, Nanjing, China
| | - Ming Kong
- Key Laboratory of Targeted Intervention of Cardiovascular Disease, Collaborative Innovation Center for Cardiovascular Translational Medicine, and Center for Experimental Medicine, Department of Pathophysiology, Nanjing Medical University, Nanjing, China
| | - Haozhen Ren
- Department of Hepatobiliary Surgery, Affiliated Nanjing Drum Tower Hospital of Nanjing University Medical School, Nanjing, China
- Hepatobiliary Institute of Nanjing University, Nanjing, China
| | - Jinglin Wang
- Department of Hepatobiliary Surgery, Affiliated Nanjing Drum Tower Hospital of Nanjing University Medical School, Nanjing, China
- Hepatobiliary Institute of Nanjing University, Nanjing, China
| | - Longcheng Shang
- Department of Hepatobiliary Surgery, Affiliated Nanjing Drum Tower Hospital of Nanjing University Medical School, Nanjing, China
| | - Zhengyi Zhu
- Department of Hepatobiliary Surgery, Affiliated Nanjing Drum Tower Hospital of Nanjing University Medical School, Nanjing, China
| | - Wei Zhu
- Department of Anesthesiology, The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing, China
| | - Xiaolei Shi
- Department of Hepatobiliary Surgery, Affiliated Nanjing Drum Tower Hospital of Nanjing University Medical School, Nanjing, China
- Hepatobiliary Institute of Nanjing University, Nanjing, China
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Kong M, Dong W, Xu H, Fan Z, Miao X, Guo Y, Li C, Ye Q, Wang Y, Xu Y. Choline Kinase Alpha Is a Novel Transcriptional Target of the Brg1 in Hepatocyte: Implication in Liver Regeneration. Front Cell Dev Biol 2021; 9:705302. [PMID: 34422825 PMCID: PMC8377418 DOI: 10.3389/fcell.2021.705302] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/05/2021] [Accepted: 07/20/2021] [Indexed: 12/14/2022] Open
Abstract
Liver regeneration is a key compensatory process in response to liver injury serving to contain damages and to rescue liver functions. Hepatocytes, having temporarily exited the cell cycle after embryogenesis, resume proliferation to regenerate the injured liver parenchyma. In the present study we investigated the transcriptional regulation of choline kinase alpha (Chka) in hepatocytes in the context of liver regeneration. We report that Chka expression was significantly up-regulated in the regenerating livers in the partial hepatectomy (PHx) model and the acetaminophen (APAP) injection model. In addition, treatment with hepatocyte growth factor (HGF), a strong pro-proliferative cue, stimulated Chka expression in primary hepatocytes. Chka depletion attenuated HGF-induced proliferation of hepatocytes as evidenced by quantitative PCR and Western blotting measurements of pro-proliferative genes as well as EdU incorporation into replicating DNA. Of interest, deletion of Brahma-related gene 1 (Brg1), a chromatin remodeling protein, attenuated Chka induction in the regenerating livers in mice and in cultured hepatocytes. Further analysis revealed that Brg1 interacted with hypoxia-inducible factor 1 alpha (HIF-1α) to directly bind to the Chka promoter and activate Chka transcription. Finally, examination of human acute liver failure (ALF) specimens identified a positive correlation between Chka expression and Brg1 expression. In conclusion, our data suggest that Brg1-dependent trans-activation of Chka expression may contribute to liver regeneration.
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Affiliation(s)
- Ming Kong
- Key Laboratory of Targeted Intervention of Cardiovascular Disease, Department of Pathophysiology, Collaborative Innovation Center for Cardiovascular Translational Medicine, Nanjing Medical University, Nanjing, China
| | - Wenhui Dong
- Key Laboratory of Targeted Intervention of Cardiovascular Disease, Department of Pathophysiology, Collaborative Innovation Center for Cardiovascular Translational Medicine, Nanjing Medical University, Nanjing, China
| | - Huihui Xu
- Key Laboratory of Targeted Intervention of Cardiovascular Disease, Department of Pathophysiology, Collaborative Innovation Center for Cardiovascular Translational Medicine, Nanjing Medical University, Nanjing, China
| | - Zhiwen Fan
- Institute of Biomedical Research, Liaocheng University, Liaocheng, China.,Department of Pathology, Nanjing Drum Tower Hospital Affiliated with Nanjing University School of Medicine, Nanjing, China
| | - Xiulian Miao
- Institute of Biomedical Research, Liaocheng University, Liaocheng, China.,College of Life Sciences, Liaocheng University, Liaocheng, China
| | - Yan Guo
- Institute of Biomedical Research, Liaocheng University, Liaocheng, China.,College of Life Sciences, Liaocheng University, Liaocheng, China
| | - Chengping Li
- Institute of Biomedical Research, Liaocheng University, Liaocheng, China.,College of Life Sciences, Liaocheng University, Liaocheng, China
| | - Qing Ye
- Division of Life Sciences and Medicine, Department of Pathology, The First Affiliated Hospital, University of Science and Technology of China, Hefei, China.,Division of Life Sciences and Medicine, Intelligent Pathology Institute, University of Science and Technology of China, Hefei, China
| | - Yutong Wang
- Department of Cell Biology, The Municipal Laboratory of Liver Protection and Regulation of Regeneration, School of Basic Medical Sciences, Beijing, China
| | - Yong Xu
- Key Laboratory of Targeted Intervention of Cardiovascular Disease, Department of Pathophysiology, Collaborative Innovation Center for Cardiovascular Translational Medicine, Nanjing Medical University, Nanjing, China.,Institute of Biomedical Research, Liaocheng University, Liaocheng, China
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50
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Li L, Wang H, Chen X, Li X, Wang G, Jie Z, Zhao X, Sun X, Huang H, Fan S, Xie Z, Wang J. Oxidative Stress-Induced Hypermethylation of KLF5 Promoter Mediated by DNMT3B Impairs Osteogenesis by Diminishing the Interaction with β-Catenin. Antioxid Redox Signal 2021; 35:1-20. [PMID: 33588625 DOI: 10.1089/ars.2020.8200] [Citation(s) in RCA: 23] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
Aims: Emerging evidence suggests that the pathogenesis of osteoporosis, characterized by impaired osteogenesis, is shifting from estrogen centric to oxidative stress. Our previous studies have shown that the zinc-finger transcription factor krüppel-like factor 5 (KLF5) plays a key role in the degeneration of nucleus pulposus and cartilage. However, its role in osteoporosis remains unknown. We aimed to investigate the effect and mechanism of KLF5 on osteogenesis under oxidative stress. Results: First, KLF5 was required for osteogenesis and stimulated osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). KLF5 was hypermethylated and downregulated in ovariectomy-induced osteoporosis mice and in BMSCs treated with H2O2. Interestingly, DNA methyltransferases 3B (DNMT3B) upregulation mediated the hypermethylation of KLF5 induced by oxidative stress, thereby impairing osteogenic differentiation. The inhibition of KLF5 hypermethylation using DNMT3B siRNA or 5-AZA-2-deoxycytidine (5-AZA) protected osteogenic differentiation of BMSCs from oxidative stress. Regarding the downstream mechanism, KLF5 induced β-catenin expression. More importantly, KLF5 promoted the nuclear translocation of β-catenin, which was mediated by the armadillo repeat region of β-catenin. Consistently, oxidative stress-induced KLF5 hypermethylation inhibited osteogenic differentiation by reducing the expression and nuclear translocation of β-catenin. Innovation: We describe the novel effect and mechanism of KLF5 on osteogenesis under oxidative stress, which is linked to osteoporosis for the first time. Conclusion: Our results suggested that oxidative stress-induced hypermethylation of KLF5 mediated by DNMT3B impairs osteogenesis by diminishing the interaction with β-catenin, which is likely to contribute to osteoporosis. Targeting the hypermethylation of KLF5 might be a new strategy for the treatment of osteoporosis. Antioxid. Redox Signal. 35, 1-20.
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Affiliation(s)
- Liangping Li
- Department of Orthopaedics, Medical College of Zhejiang University, Sir Run Run Shaw Hospital, Hangzhou, People's Republic of China
- Key Laboratory of Musculoskeletal System Degeneration and Regeneration Translational Research of Zhejiang Province, Hangzhou, People's Republic of China
- Department of Surgery, The Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, People's Republic of China
| | - Haoming Wang
- Department of Orthopaedics, Medical College of Zhejiang University, Sir Run Run Shaw Hospital, Hangzhou, People's Republic of China
- Key Laboratory of Musculoskeletal System Degeneration and Regeneration Translational Research of Zhejiang Province, Hangzhou, People's Republic of China
| | - Xiaoying Chen
- Department of Emergency, The Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, People's Republic of China
| | - Xiang Li
- Department of Orthopaedics, Medical College of Zhejiang University, Sir Run Run Shaw Hospital, Hangzhou, People's Republic of China
- Key Laboratory of Musculoskeletal System Degeneration and Regeneration Translational Research of Zhejiang Province, Hangzhou, People's Republic of China
| | - Gangliang Wang
- Department of Orthopaedics, Medical College of Zhejiang University, Sir Run Run Shaw Hospital, Hangzhou, People's Republic of China
- Key Laboratory of Musculoskeletal System Degeneration and Regeneration Translational Research of Zhejiang Province, Hangzhou, People's Republic of China
| | - Zhiwei Jie
- Department of Orthopaedics, Medical College of Zhejiang University, Sir Run Run Shaw Hospital, Hangzhou, People's Republic of China
- Key Laboratory of Musculoskeletal System Degeneration and Regeneration Translational Research of Zhejiang Province, Hangzhou, People's Republic of China
| | - Xiangde Zhao
- Department of Orthopaedics, Medical College of Zhejiang University, Sir Run Run Shaw Hospital, Hangzhou, People's Republic of China
- Key Laboratory of Musculoskeletal System Degeneration and Regeneration Translational Research of Zhejiang Province, Hangzhou, People's Republic of China
| | - Xuewu Sun
- Department of Orthopaedics, Medical College of Zhejiang University, Sir Run Run Shaw Hospital, Hangzhou, People's Republic of China
- Key Laboratory of Musculoskeletal System Degeneration and Regeneration Translational Research of Zhejiang Province, Hangzhou, People's Republic of China
| | - Hai Huang
- Department of Orthopaedics, Medical College of Zhejiang University, Sir Run Run Shaw Hospital, Hangzhou, People's Republic of China
- Key Laboratory of Musculoskeletal System Degeneration and Regeneration Translational Research of Zhejiang Province, Hangzhou, People's Republic of China
| | - Shunwu Fan
- Department of Orthopaedics, Medical College of Zhejiang University, Sir Run Run Shaw Hospital, Hangzhou, People's Republic of China
- Key Laboratory of Musculoskeletal System Degeneration and Regeneration Translational Research of Zhejiang Province, Hangzhou, People's Republic of China
| | - Ziang Xie
- Department of Orthopaedics, Medical College of Zhejiang University, Sir Run Run Shaw Hospital, Hangzhou, People's Republic of China
- Key Laboratory of Musculoskeletal System Degeneration and Regeneration Translational Research of Zhejiang Province, Hangzhou, People's Republic of China
| | - Jian Wang
- Department of Orthopaedics, Medical College of Zhejiang University, Sir Run Run Shaw Hospital, Hangzhou, People's Republic of China
- Key Laboratory of Musculoskeletal System Degeneration and Regeneration Translational Research of Zhejiang Province, Hangzhou, People's Republic of China
- Department of Orthopaedics, Tongde Hospital of Zhejiang Province, Hangzhou, People's Republic of China
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