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Xie H, Xiong T, Guan J, Han Y, Feng H, Xu F, Chen S, Li J, Xie Z, Liu D, Chen R. Induction of mitochondrial damage via the CREB3L1/miR-34c/COX1 axis by porcine epidemic diarrhea virus infection facilitates pathogenicity. J Virol 2025; 99:e0059124. [PMID: 40071922 PMCID: PMC11998543 DOI: 10.1128/jvi.00591-24] [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: 07/05/2024] [Accepted: 12/23/2024] [Indexed: 03/26/2025] Open
Abstract
Porcine epidemic diarrhea virus (PEDV) is a primary cause of viral diarrhea in neonatal piglets, leading to substantial economic losses in the swine industry globally. It primarily targets epithelial cells of the small intestine, compromising intestinal function and resulting in the death of affected animals. As mitochondria are essential for maintaining gut health, this study investigates the effects of PEDV infection on mitochondrial function in small intestinal epithelial cells and its subsequent impacts. Using small RNA sequencing, fluorescence in situ hybridization, dual luciferase reporter assay, gene overexpression, and silencing experiments, we investigated the mitochondrial structural and functional impairments induced by PEDV infection in jejunum epithelial cells of piglets and characterized the regulatory pattern of miRNAs in mitochondria of jejunum epithelial cells during PEDV infection. The results indicate that PEDV infection leads to the upregulation and mitochondrial localization of the nuclear-encoded microRNA, miR-34c, which in turn suppresses COX1 expression. The activation of the miR-34c/COX1 axis diminishes mitochondrial complex III, IV, and V activities, depletes ATP, lowers mitochondrial oxygen consumption, induces mitochondrial depolarization, increases the accumulation of mitochondrial reactive oxygen species (mtROS), and stimulates mitophagy. Furthermore, we confirm that CREB3L1 acts as an upstream transcription factor regulating the miR-34c/COX1 axis during PEDV infection, modulating mitochondrial damage in the epithelial cells of the jejunum. These findings demonstrate for the first time that PEDV infection activates the miR-34c/COX1 axis via the transcription factor CREB3L1 and regulates the nuclear-mitochondrial communication and mitochondrial fate, providing a new perspective on the pathogenesis of PEDV.IMPORTANCEThis study reveals the mechanism by which the porcine epidemic diarrhea virus (PEDV) disrupts mitochondrial function in piglets, enhancing viral pathogenicity. By demonstrating how PEDV infection upregulates miR-34c, leading to COX1 suppression and subsequent mitochondrial dysfunction, the research highlights a novel aspect of viral manipulation of host cellular mechanisms. These findings provide a deeper understanding of the PEDV pathogenesis and identify potential targets for therapeutic intervention, advancing efforts to mitigate the economic impact of PEDV on the swine industry.
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Affiliation(s)
- Hangao Xie
- College of Veterinary Medicine, South China Agricultural University, Guangzhou, China
- Zhaoqing Branch Centre of Guangdong Laboratory for Lingnan Modern Agricultural Science and Technology, Zhaoqing, China
| | - Ting Xiong
- College of Veterinary Medicine, South China Agricultural University, Guangzhou, China
- Zhaoqing Branch Centre of Guangdong Laboratory for Lingnan Modern Agricultural Science and Technology, Zhaoqing, China
| | - Jinlian Guan
- College of Veterinary Medicine, South China Agricultural University, Guangzhou, China
- Zhaoqing Branch Centre of Guangdong Laboratory for Lingnan Modern Agricultural Science and Technology, Zhaoqing, China
| | - Yin Han
- College of Veterinary Medicine, South China Agricultural University, Guangzhou, China
- Zhaoqing Branch Centre of Guangdong Laboratory for Lingnan Modern Agricultural Science and Technology, Zhaoqing, China
| | - Haixia Feng
- College of Veterinary Medicine, South China Agricultural University, Guangzhou, China
| | - Fei Xu
- College of Veterinary Medicine, South China Agricultural University, Guangzhou, China
| | - Sixuan Chen
- College of Veterinary Medicine, South China Agricultural University, Guangzhou, China
| | - Jiahui Li
- College of Veterinary Medicine, South China Agricultural University, Guangzhou, China
| | - Ziwei Xie
- College of Veterinary Medicine, South China Agricultural University, Guangzhou, China
- Zhaoqing Branch Centre of Guangdong Laboratory for Lingnan Modern Agricultural Science and Technology, Zhaoqing, China
| | - Dingxiang Liu
- College of Veterinary Medicine, South China Agricultural University, Guangzhou, China
- Zhaoqing Branch Centre of Guangdong Laboratory for Lingnan Modern Agricultural Science and Technology, Zhaoqing, China
- Integrative Microbiology Research Centre, South China Agricultural University Integrative Microbiology Research Centre, Guangzhou, China
| | - Ruiai Chen
- College of Veterinary Medicine, South China Agricultural University, Guangzhou, China
- Zhaoqing Branch Centre of Guangdong Laboratory for Lingnan Modern Agricultural Science and Technology, Zhaoqing, China
- Key Laboratory of Manufacture Technology of Veterinary Bioproducts, Ministry of Agriculture and Rural Affairs, Beijing, China
- Guangdong Enterprise Key Laboratory of Biotechnology R&D of Veterinary Biologics, Zhaoqing, China
- Zhaoqing Dahuanong Biology Medicine Co. Ltd., Zhaoqing, China
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Kuang Y, Ke M, Liu W, Xu F. FBXW2 inhibits the progression of gastric cancer via promoting β-catenin ubiquitylation. Int J Med Sci 2025; 22:1936-1943. [PMID: 40225854 PMCID: PMC11983310 DOI: 10.7150/ijms.108501] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/10/2024] [Accepted: 02/26/2025] [Indexed: 04/15/2025] Open
Abstract
Background: F-box and WD-repeat-containing protein 2 (FBXW2), an E3 ubiquitin ligase, may play a crucial role in tumorigenesis. However, its function in gastric cancer remains unknown. Methods: The expression levels of FBXW2 and β-catenin in gastric cancer samples were analyzed using RT-PCR and immunohistochemistry, with Pearson correlation analysis to assess their relationship. AGS and HGC-27 gastric cancer cells were transfected with sh-FBXW2, and their viability was evaluated using the CCK8 assay, while invasion ability was assessed via the transwell assay. Western blotting was performed to measure the expression levels of FBXW2, β-catenin, GSK3β, and Axin2 in AGS cells. Additionally, a ubiquitination assay was conducted to examine the effect of sh-FBXW2 on β-catenin ubiquitination. Immunoprecipitation was used to determine the potential interaction between FBXW2 and β-catenin. Results: FBXW2 expression was downregulated, whereas β-catenin expression was upregulated in gastric cancer tissues compared to adjacent normal tissues, showing a significant negative correlation (r = -0.52, P < 0.001). Knockdown of FBXW2 (sh-FBXW2) promoted gastric cancer cell viability and invasion while increasing β-catenin expression and reducing GSK3β and Axin2 levels. Furthermore, FBXW2 was found to bind β-catenin and facilitate its ubiquitination, leading to enhanced nuclear translocation of β-catenin. Conclusions: FBXW2 suppresses gastric cancer progression by promoting β-catenin ubiquitination, highlighting its potential as a therapeutic target.
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Affiliation(s)
- Yanshen Kuang
- Department of General Surgery, the First Medical Center, Chinese PLA General Hospital, Haidian District, No.28, Fuxing Road, Beijing 100853, China
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Wang Y, Zhang FQ, Fan ZP, Zhu XL, Yan WH, Zhang XL. WDR36 inhibits the osteogenic differentiation and migration of periodontal ligament stem cells. World J Stem Cells 2025; 17:99132. [PMID: 40061266 PMCID: PMC11885943 DOI: 10.4252/wjsc.v17.i2.99132] [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: 07/23/2024] [Revised: 11/23/2024] [Accepted: 01/16/2025] [Indexed: 02/24/2025] Open
Abstract
BACKGROUND Periodontitis is an inflammatory disease caused by the host's immune response and various interactions between pathogens, which lead to the loss of connective tissue and bone. In recent years, mesenchymal stem cell (SC) transplantation technology has become a research hotspot, which can form periodontal ligament, cementum, and alveolar bone through proliferation and differentiation. AIM To elucidate the regulatory effects of WD repeat-containing protein 36 (WDR36) on the senescence, migration, and osteogenic differentiation of periodontal ligament SCs (PDLSCs). METHODS The migration and chemotaxis of PDLSCs were detected by the scratch-wound migration test and transwell chemotaxis test. Alkaline phosphatase (ALP) activity, Alizarin red staining, calcium content, and real-time reverse transcription polymerase chain reaction (RT-qPCR) of key transcription factors were used to detect the osteogenic differentiation function of PDLSCs. Cell senescence was determined by senescence-associated β-galactosidase staining. RESULTS The 24-hour and 48-hour scratch-wound migration test and 48-hour transwell chemotaxis test showed that overexpression of WDR36 inhibited the migration/chemotaxis of PDLSCs. Simultaneously, WDR36 depletion promoted the migration/chemotaxis of PDLSCs. The results of ALP activity, Alizarin red staining, calcium content, and RT-qPCR showed that overexpression of WDR36 inhibited the osteogenic differentiation of PDLSCs, and WDR36 depletion promoted the osteogenic differentiation of PDLSCs. Senescence-associated β-galactosidase staining showed that 0.1 μg/mL icariin (ICA) and overexpression of WDR36 inhibited the senescence of PDLSCs, and WDR36 depletion promoted the osteogenic differentiation of PDLSCs. CONCLUSION WDR36 inhibits the migration and chemotaxis, osteogenic differentiation, and senescence of PDLSCs; 0.1 μg/mL ICA inhibits the senescence of PDLSCs. Therefore, WDR36 might serve as a target for periodontal tissue regeneration and the treatment of periodontitis.
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Affiliation(s)
- Yi Wang
- Department of Wangfujing General School of Stomatology, Capital Medical University, Beijing 100070, China
| | - Feng-Qiu Zhang
- Department of Periodontics School of Stomatology, Capital Medical University, Beijing 100070, China.
| | - Zhi-Peng Fan
- Key Laboratory of Tooth Regeneration and Function Reconstruction, Beijing Stomatological Hospital, School of Stomatology, Beijing 100070, China
- Beijing Laboratory of Oral Health, Capital Medical University, Beijing 100070, China
- Research Unit of Tooth Development and Regeneration, Chinese Academy of Medical Sciences, Beijing 100070, China
| | - Xin-Ling Zhu
- Department of Periodontology, Hangzhou Stomatology Hospital, Hangzhou 310006, Zhejiang Province, China
| | - Wan-Hao Yan
- Laboratory of Molecular Signaling and Stem Cells Therapy, Beijing Key Laboratory of Tooth Regeneration and Function Reconstruction, Capital Medical University School of Stomatology, Beijing 100070, China
| | - Xiu-Li Zhang
- Department of Stomatology, Civil Aviation General Hospital, Beijing 100123, China
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Zeng X, Cao J, Xu J, Zhou Z, Long C, Zhou Y, Tang J. SKP1-CUL1-F-box: Key molecular targets affecting disease progression. FASEB J 2025; 39:e70326. [PMID: 39812503 PMCID: PMC11734646 DOI: 10.1096/fj.202402816rr] [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: 11/11/2024] [Revised: 01/01/2025] [Accepted: 01/06/2025] [Indexed: 01/16/2025]
Abstract
The correct synthesis and degradation of proteins are vital for numerous biological processes in the human body, with protein degradation primarily facilitated by the ubiquitin-proteasome system. The SKP1-CUL1-F-box (SCF) E3 ubiquitin ligase, a member of the Cullin-RING E3 ubiquitin ligase (CRL) family, plays a crucial role in mediating protein ubiquitination and subsequent 26S proteasome degradation during normal cellular metabolism. Notably, SCF is intricately linked to the pathogenesis of various diseases, including malignant tumors. This paper provides a comprehensive overview of the functional characteristics of SCF complexes, encompassing their assembly, disassembly, and regulatory factors. Furthermore, we discuss the diverse effects of SCF on crucial cellular processes such as cell cycle progression, DNA replication, oxidative stress response, cell proliferation, apoptosis, cell differentiation, maintenance of stem cell characteristics, tissue development, circadian rhythm regulation, and immune response modulation. Additionally, we summarize the associations between SCF and the onset, progression, and prognosis of malignant tumors. By synthesizing current knowledge, this review aims to offer a novel perspective for a holistic and systematic understanding of SCF complexes and their multifaceted functions in cellular physiology and disease pathogenesis.
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Affiliation(s)
- Xiangrong Zeng
- Department of GeriatricsThe Second Xiangya Hospital of Central South UniversityChangshaHunanChina
- Cancer Research Institute, Basic School of Medicine, Central South UniversityChangshaHunanChina
| | - Jiaying Cao
- Cancer Research Institute, Basic School of Medicine, Central South UniversityChangshaHunanChina
| | - Juan Xu
- Department of Critical Care MedicinThe Affiliated Cancer Hospital of Xiangya School of Medicine Central South University, Hunan Cancer HospitalChangshaHunanChina
| | - Zihua Zhou
- Department of OncologyLoudi Central HospitalLoudiChina
| | - Chen Long
- Department of General SurgeryThe Second Xiangya Hospital of Central South UniversityChangshaHunanChina
| | - Yanhong Zhou
- Cancer Research Institute, Basic School of Medicine, Central South UniversityChangshaHunanChina
| | - Jingqiong Tang
- Department of GeriatricsThe Second Xiangya Hospital of Central South UniversityChangshaHunanChina
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Sato Y, Habara M, Hanaki S, Masaki T, Tomiyasu H, Miki Y, Sakurai M, Morimoto M, Kobayashi D, Miyamoto T, Shimada M. Calcineurin-mediated dephosphorylation stabilizes E2F1 protein by suppressing binding of the FBXW7 ubiquitin ligase subunit. Proc Natl Acad Sci U S A 2024; 121:e2414618121. [PMID: 39361641 PMCID: PMC11474076 DOI: 10.1073/pnas.2414618121] [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: 07/24/2024] [Accepted: 08/26/2024] [Indexed: 10/05/2024] Open
Abstract
The transcription factor E2F1 serves as a regulator of the cell cycle and promotes cell proliferation. It is highly expressed in cancer tissues and contributes to their malignant transformation. Degradation by the ubiquitin-proteasome system may help to prevent such overexpression of E2F1 and thereby to suppress carcinogenesis. A detailed understanding of the mechanisms underlying E2F1 degradation may therefore inform the development of new cancer treatments. We here identified SCFFBXW7 as a ubiquitin ligase for E2F1 by comprehensive analysis. We found that phosphorylation of E2F1 at serine-403 promotes its binding to FBXW7 (F-box/WD repeat-containing protein 7) followed by its ubiquitination and degradation. Furthermore, calcineurin, a Ca2+/calmodulin-dependent serine-threonine phosphatase, was shown to stabilize E2F1 by mediating its dephosphorylation at serine-403 and thereby preventing FBXW7 binding. Treatment of cells with Ca2+ channel blockers resulted in downregulation of both E2F1 protein and the expression of E2F1 target genes, whereas treatment with the Ca2+ ionophore ionomycin induced upregulation of E2F1. Finally, the calcineurin inhibitor FK506 attenuated xenograft tumor growth in mice in association with downregulation of E2F1 in the tumor tissue. Impairment of the balance between the opposing actions of FBXW7 and calcineurin in the regulation of E2F1 abundance may therefore play an important role in carcinogenesis.
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Affiliation(s)
- Yuki Sato
- Department of Veterinary Biochemistry, Yamaguchi University, Yamaguchi753-8511, Japan
| | - Makoto Habara
- Department of Veterinary Biochemistry, Yamaguchi University, Yamaguchi753-8511, Japan
| | - Shunsuke Hanaki
- Department of Veterinary Biochemistry, Yamaguchi University, Yamaguchi753-8511, Japan
| | - Takahiro Masaki
- Department of Veterinary Biochemistry, Yamaguchi University, Yamaguchi753-8511, Japan
| | - Haruki Tomiyasu
- Department of Veterinary Biochemistry, Yamaguchi University, Yamaguchi753-8511, Japan
| | - Yosei Miki
- Department of Veterinary Biochemistry, Yamaguchi University, Yamaguchi753-8511, Japan
| | - Masashi Sakurai
- Department of Veterinary Pathology, Yamaguchi University, Yamaguchi753-8511, Japan
| | - Masahiro Morimoto
- Department of Veterinary Pathology, Yamaguchi University, Yamaguchi753-8511, Japan
| | - Daigo Kobayashi
- Department of Molecular and Cellular Physiology, Graduate School of Medicine, Yamaguchi University, Ube, Yamaguchi 755-8505, Japan
| | - Tatsuo Miyamoto
- Department of Molecular and Cellular Physiology, Graduate School of Medicine, Yamaguchi University, Ube, Yamaguchi 755-8505, Japan
- Division of Advanced Genome Editing Therapy Research, Research Institute for Cell Design Medical Science, Yamaguchi University, Ube, Yamaguchi 755-8505, Japan
| | - Midori Shimada
- Department of Veterinary Biochemistry, Yamaguchi University, Yamaguchi753-8511, Japan
- Department of Molecular Biology, Graduate School of Medicine, Nagoya University, Nagoya, Aichi 466-8550, Japan
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Zhao Y, Yu Z, Song Y, Fan L, Lei T, He Y, Hu S. The Regulatory Network of CREB3L1 and Its Roles in Physiological and Pathological Conditions. Int J Med Sci 2024; 21:123-136. [PMID: 38164349 PMCID: PMC10750332 DOI: 10.7150/ijms.90189] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/15/2023] [Accepted: 10/26/2023] [Indexed: 01/03/2024] Open
Abstract
CREB3 subfamily belongs to the bZIP transcription factor family and comprises five members. Normally they are located on the endoplasmic reticulum (ER) membranes and proteolytically activated through RIP (regulated intramembrane proteolysis) on Golgi apparatus to liberate the N-terminus to serve as transcription factors. CREB3L1 acting as one of them transcriptionally regulates the expressions of target genes and exhibits distinct functions from the other members of CREB3 family in eukaryotes. Physiologically, CREB3L1 involves in the regulation of bone morphogenesis, neurogenesis, neuroendocrine, secretory cell differentiation, and angiogenesis. Pathologically, CREB3L1 implicates in the modulation of osteogenesis imperfecta, low grade fibro myxoid sarcoma (LGFMS), sclerosing epithelioid fibrosarcoma (SEF), glioma, breast cancer, thyroid cancer, and tissue fibrosis. This review summarizes the upstream and downstream regulatory network of CREB3L1 and thoroughly presents our current understanding of CREB3L1 research progress in both physiological and pathological conditions with special focus on the novel findings of CREB3L1 in cancers.
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Affiliation(s)
- Ying Zhao
- Department of Anesthesiology and Perioperative Medicine, Xi'an People's Hospital (Xi'an Fourth Hospital), Northwest University, Xi'an, Shaanxi Province, China
| | - Zhou Yu
- Department of Plastic Surgery, Xijing Hospital, Fourth Military Medical University, Xi'an, Shaanxi Province, China
| | - Yajuan Song
- Department of Plastic Surgery, Xijing Hospital, Fourth Military Medical University, Xi'an, Shaanxi Province, China
| | - Liumeizi Fan
- Department of Anesthesiology and Perioperative Medicine, Xi'an People's Hospital (Xi'an Fourth Hospital), Northwest University, Xi'an, Shaanxi Province, China
| | - Ting Lei
- Department of Anesthesiology and Perioperative Medicine, Xi'an People's Hospital (Xi'an Fourth Hospital), Northwest University, Xi'an, Shaanxi Province, China
| | - Yinbin He
- Department of Anesthesiology and Perioperative Medicine, Xi'an People's Hospital (Xi'an Fourth Hospital), Northwest University, Xi'an, Shaanxi Province, China
| | - Sheng Hu
- Department of Anesthesiology and Perioperative Medicine, Xi'an People's Hospital (Xi'an Fourth Hospital), Northwest University, Xi'an, Shaanxi Province, China
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Yuxiong W, Faping L, Bin L, Yanghe Z, Yao L, Yunkuo L, Yishu W, Honglan Z. Regulatory mechanisms of the cAMP-responsive element binding protein 3 (CREB3) family in cancers. Biomed Pharmacother 2023; 166:115335. [PMID: 37595431 DOI: 10.1016/j.biopha.2023.115335] [Citation(s) in RCA: 8] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/05/2023] [Revised: 08/13/2023] [Accepted: 08/14/2023] [Indexed: 08/20/2023] Open
Abstract
The CREB3 family of proteins, encompassing CREB3 and its four homologs (CREB3L1, CREB3L2, CREB3L3, and CREB3L4), exerts pivotal control over cellular protein metabolism in response to unfolded protein reactions. Under conditions of endoplasmic reticulum stress, activation of the CREB3 family occurs through regulated intramembrane proteolysis within the endoplasmic reticulum membrane. Perturbations in the function and expression of the CREB3 family have been closely associated with the development of diverse diseases, with a particular emphasis on cancer. Recent investigations have shed light on the indispensable role played by CREB3 family members in modulating the onset and progression of various human cancers. This comprehensive review endeavors to provide an in-depth examination of the involvement of CREB3 family members in distinct human cancer types, accentuating their significance in the pathogenesis of cancer and the manifestation of malignant phenotypes.
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Affiliation(s)
- Wang Yuxiong
- Department of Urology II, The First Hospital of Jilin University, Changchun 130011, China
| | - Li Faping
- Department of Urology II, The First Hospital of Jilin University, Changchun 130011, China
| | - Liu Bin
- Department of Urology II, The First Hospital of Jilin University, Changchun 130011, China
| | - Zhang Yanghe
- Key Laboratory of Pathobiology, Ministry of Education, Jilin University, Changchun 130011, China
| | - Li Yao
- Key Laboratory of Pathobiology, Ministry of Education, Jilin University, Changchun 130011, China
| | - Li Yunkuo
- Department of Urology II, The First Hospital of Jilin University, Changchun 130011, China
| | - Wang Yishu
- Key Laboratory of Pathobiology, Ministry of Education, Jilin University, Changchun 130011, China.
| | - Zhou Honglan
- Department of Urology II, The First Hospital of Jilin University, Changchun 130011, China,.
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Yumimoto K, Sugiyama S, Motomura S, Takahashi D, Nakayama KI. Molecular evolution of Keap1 was essential for adaptation of vertebrates to terrestrial life. SCIENCE ADVANCES 2023; 9:eadg2379. [PMID: 37205751 DOI: 10.1126/sciadv.adg2379] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/10/2022] [Accepted: 04/14/2023] [Indexed: 05/21/2023]
Abstract
Reactive oxygen species (ROS) posed a risk for the transition of vertebrates from aquatic to terrestrial life. How ancestral organisms adapted to such ROS exposure has remained a mystery. Here, we show that attenuation of the activity of the ubiquitin ligase CRL3Keap1 for the transcription factor Nrf2 during evolution was key to development of an efficient response to ROS exposure. The Keap1 gene was duplicated in fish to give rise to Keap1A and the only remaining mammalian paralog Keap1B, the latter of which shows a lower affinity for Cul3 and contributes to robust Nrf2 induction in response to ROS exposure. Mutation of mammalian Keap1 to resemble zebrafish Keap1A resulted in an attenuated Nrf2 response, and most knock-in mice expressing such a Keap1 mutant died on exposure as neonates to sunlight-level ultraviolet radiation. Our results suggest that molecular evolution of Keap1 was essential for adaptation to terrestrial life.
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Affiliation(s)
- Kanae Yumimoto
- Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, Japan
| | - Shigeaki Sugiyama
- Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, Japan
| | - Saori Motomura
- Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, Japan
| | - Daisuke Takahashi
- Department of Protein Structure, Function, and Design, Graduate School of Pharmaceutical Science, Kyushu University, Fukuoka 812-8582, Japan
| | - Keiichi I Nakayama
- Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, Japan
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Khired ZA, Kattan SW, Alzahrani AK, Milebary AJ, Hussein MH, Qusti SY, Alshammari EM, Toraih EA, Fawzy MS. Analysis of MIR27A (rs11671784) Variant Association with Systemic Lupus Erythematous. Life (Basel) 2023; 13:701. [PMID: 36983856 PMCID: PMC10058767 DOI: 10.3390/life13030701] [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: 01/29/2023] [Revised: 02/23/2023] [Accepted: 03/02/2023] [Indexed: 03/08/2023] Open
Abstract
Multiple microRNAs (miRs) are associated with systemic autoimmune disease susceptibility/phenotype, including systemic lupus erythematosus (SLE). With this work, we aimed to unravel the association of the miR-27a gene (MIR27A) rs11671784G/A variant with SLE risk/severity. One-hundred sixty-three adult patients with SLE and matched controls were included. A TaqMan allelic discrimination assay was applied for MIR27A genotyping. Logistic regression models were run to test the association with SLE susceptibility/risk. Genotyping of 326 participants revealed that the heterozygote form was the most common genotype among the study cohort, accounting for 72% of the population (n = 234), while A/A and G/G represented 15% (n = 49) and 13% (n = 43), respectively. Similarly, the most prevalent genotype among cases was the A/G genotype, which was present in approximately 93.3% of cases (n = 152). In contrast, only eight and three patients had A/A and G/G genotypes, respectively. The MIR27A rs11671784 variant conferred protection against the development of SLE in several genetic models, including heterozygous (G/A vs. A/A; OR = 0.10, 95% CI = 0.05-0.23), dominant (G/A + G/G vs. AA; OR = 0.15, 95% CI = 0.07-0.34), and overdominant (G/A vs. A/A + G/G; OR = 0.07, 95% CI = 0.04-0.14) models. However, the G/G genotype was associated with increased SLE risk in the recessive model (G/G vs. A/A+ G/G; OR = 17.34, 95% CI = 5.24-57.38). Furthermore, the variant showed significant associations with musculoskeletal and mucocutaneous manifestations in the patient cohort (p = 0.035 and 0.009, respectively) and platelet and white blood cell counts (p = 0.034 and 0.049, respectively). In conclusion, the MIR27A rs11671784 variant showed a potentially significant association with SLE susceptibility/risk in the studied population. Larger-scale studies on multiethnic populations are recommended to verify the results.
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Affiliation(s)
- Zenat Ahmed Khired
- Department of Surgery, College of Medicine, Jazan University, Jazan 45142, Saudi Arabia
| | - Shahad W. Kattan
- Department of Medical Laboratory, College of Applied Medical Sciences, Taibah University, Yanbu 46423, Saudi Arabia
| | - Ahmad Khuzaim Alzahrani
- Medical Laboratory Technology, Faculty of Applied Medical Sciences, Northern Border University, Arar 91431, Saudi Arabia
| | - Ahmad J. Milebary
- Department of Medical Laboratory, King Fahad Armed Forces Hospital, Jeddah 23311, Saudi Arabia
| | - Mohammad H. Hussein
- Division of Endocrine and Oncologic Surgery, Department of Surgery, Tulane University School of Medicine, New Orleans, LA 70112, USA
| | - Safaa Y. Qusti
- Department of Biochemistry, Faculty of Science, King Abdulaziz University, Jeddah 21589, Saudi Arabia
| | - Eida M. Alshammari
- Department of Chemistry, College of Sciences, University of Ha’il, Ha’il 2440, Saudi Arabia
| | - Eman A. Toraih
- Division of Endocrine and Oncologic Surgery, Department of Surgery, Tulane University School of Medicine, New Orleans, LA 70112, USA
- Medical Genetics Unit, Department of Histology and Cell Biology, Suez Canal University, Ismailia 41522, Egypt
| | - Manal S. Fawzy
- Department of Medical Biochemistry and Molecular Biology, Faculty of Medicine, Suez Canal University, Ismailia 41522, Egypt
- Department of Biochemistry, Faculty of Medicine, Northern Border University, Arar 1321, Saudi Arabia
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Regulon active landscape reveals cell development and functional state changes of human primary osteoblasts in vivo. Hum Genomics 2023; 17:11. [PMID: 36793138 PMCID: PMC9930257 DOI: 10.1186/s40246-022-00448-2] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/10/2022] [Accepted: 12/20/2022] [Indexed: 02/17/2023] Open
Abstract
BACKGROUND While transcription factor (TF) regulation is known to play an important role in osteoblast development, differentiation, and bone metabolism, the molecular features of TFs in human osteoblasts at the single-cell resolution level have not yet been characterized. Here, we identified modules (regulons) of co-regulated genes by applying single-cell regulatory network inference and clustering to the single-cell RNA sequencing profiles of human osteoblasts. We also performed cell-specific network (CSN) analysis, reconstructed regulon activity-based osteoblast development trajectories, and validated the functions of important regulons both in vivo and in vitro. RESULTS We identified four cell clusters: preosteoblast-S1, preosteoblast-S2, intermediate osteoblasts, and mature osteoblasts. CSN analysis results and regulon activity-based osteoblast development trajectories revealed cell development and functional state changes of osteoblasts. CREM and FOSL2 regulons were mainly active in preosteoblast-S1, FOXC2 regulons were mainly active in intermediate osteoblast, and RUNX2 and CREB3L1 regulons were most active in mature osteoblasts. CONCLUSIONS This is the first study to describe the unique features of human osteoblasts in vivo based on cellular regulon active landscapes. Functional state changes of CREM, FOSL2, FOXC2, RUNX2, and CREB3L1 regulons regarding immunity, cell proliferation, and differentiation identified the important cell stages or subtypes that may be predominantly affected by bone metabolism disorders. These findings may lead to a deeper understanding of the mechanisms underlying bone metabolism and associated diseases.
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11
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Murase R, Yamamoto A, Hirata Y, Oh-Hashi K. Expression analysis and functional characterization of thioredoxin domain-containing protein 11. Mol Biol Rep 2022; 49:10541-10556. [PMID: 36152228 DOI: 10.1007/s11033-022-07932-x] [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: 07/23/2022] [Accepted: 09/06/2022] [Indexed: 11/24/2022]
Abstract
BACKGROUNDS The endoplasmic reticulum (ER) is a crucial organelle that regulates both the folding, modification and transport of many proteins and senses certain stimuli inside and outside of cells. ER-associated degradation (ERAD), including SEL1L is a crucial mechanism to maintain homeostasis. In this study, we performed comparative proteome analysis in wild-type (wt) and SEL1L-deficient cells. METHODS AND RESULTS We found constitutively high expression of thioredoxin domain-containing protein 11 (TXNDC11) mRNA and protein in our SEL1L-deficient HEK293 cells by RT-PCR and Western blot analysis. The TXNDC11 gene possesses a well-conserved unfolded protein response element (UPRE) around its transcription start site, and ER stress increased TXNDC11 mRNA and luciferase reporter activity via this putative UPRE in HEK293 cells. The amounts of TXNDC11 protein in wild-type and SEL1L-deficient cells with or without thapsigargin (Tg) treatment were parallel to their mRNAs in these cells, which was almost proportional to spliced XBP1 (sXBP1) mRNA expression. The establishment and characterization of TXNDC11-deficient HEK293 cells revealed that the expression of three different ER resident stress sensors, ATF6α, CREB3 and CREB3L2, is regulated by TXNDC11. The rate of disappearance of the three proteins by CHX treatment in wt cells was remarkably different, and the full-length CREB3L2 protein was almost completely degraded within 15 min after CHX treatment. TXNDC11 deficiency increased the expression of each full-length form under resting conditions and delayed their disappearance by CHX treatment. Interestingly, the degree of increase in full-length CREB3/CREB3L2 by TXNDC11 deficiency was apparently higher than that in full-length ATF6α. The increase in these proteins by TXNDC11 deficiency was hardly correlated with the expression of each mRNA. Treatment with ER stress inducers influenced each full-length mature form, and the difference in each full-length form observed in wt and TXNDC11-deficient cells was smaller. CONCLUSION This study demonstrated that TXNDC11 is an ER stress-inducible gene regulated by the IRE1-sXBP1 pathway. In addition, TXNDC11 is involved in the regulation of ATF6α, CREB3 and CREB3L2 protein expression, although the contribution to the stability of these proteins is quite variable. Therefore, its further characterization will provide new insights for understanding protein homeostasis in ER physiology and pathology.
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Affiliation(s)
- Ryoichi Murase
- Graduate School of Natural Science and Technology, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan
| | - Ayumi Yamamoto
- Department of Chemistry and Biomolecular Science, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan
| | - Yoko Hirata
- Graduate School of Natural Science and Technology, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan.,Department of Chemistry and Biomolecular Science, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan.,United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan
| | - Kentaro Oh-Hashi
- Graduate School of Natural Science and Technology, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan. .,Department of Chemistry and Biomolecular Science, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan. .,United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan.
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12
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Ren YZ, Ding SS, Jiang YP, Wen H, Li T. Application of exosome-derived noncoding RNAs in bone regeneration: Opportunities and challenges. World J Stem Cells 2022; 14:473-489. [PMID: 36157529 PMCID: PMC9350624 DOI: 10.4252/wjsc.v14.i7.473] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/19/2022] [Revised: 05/15/2022] [Accepted: 07/11/2022] [Indexed: 02/06/2023] Open
Abstract
With advances in the fields of regenerative medicine, cell-free therapy has received increased attention. Exosomes have a variety of endogenous properties that provide stability for molecular transport across biological barriers to cells, as a form of cell-to-cell communication that regulates function and phenotype. In addition, exosomes are an important component of paracrine signaling in stem-cell-based therapy and can be used as a stand-alone therapy or as a drug delivery system. The remarkable potential of exosomes has paved the pathway for cell-free treatment in bone regeneration. Exosomes are enriched in distinct noncoding RNAs (ncRNAs), including microRNAs, long ncRNAs and circular RNAs. Different ncRNAs have multiple functions. Altered expression of ncRNA in exosomes is associated with the regenerative potential and development of various diseases, such as femoral head osteonecrosis, myocardial infarction, and cancer. Although there is increasing evidence that exosome-derived ncRNAs (exo-ncRNAs) have the potential for bone regeneration, the detailed mechanisms are not fully understood. Here, we review the biogenesis of exo-ncRNA and the effects of ncRNAs on angiogenesis and osteoblast- and osteoclast-related pathways in different diseases. However, there are still many unsolved problems and challenges in the clinical application of ncRNA; for instance, production, storage, targeted delivery and therapeutic potency assessment. Advancements in exo-ncRNA methods and design will promote the development of therapeutics, revolutionizing the present landscape.
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Affiliation(s)
- Yuan-Zhong Ren
- Department of Emergency Trauma Surgery, Luoyang Central Hospital Affiliated to Zhengzhou University, Luoyang 471000, Henan Province, China
| | - Shan-Shan Ding
- Department of Geriatrics, Luoyang Central Hospital Affiliated to Zhengzhou University, Luoyang 471000, Henan Province, China
| | - Ya-Ping Jiang
- Department of Oral Implantology, The Affiliated Hospital of Qingdao University, Qingdao 266000, Shandong Province, China
| | - Hui Wen
- Department of Emergency Trauma Surgery, Luoyang Central Hospital Affiliated to Zhengzhou University, Luoyang 471000, Henan Province, China
| | - Tao Li
- Department of Joint Surgery, The Affiliated Hospital of Qingdao University, Qingdao 266003, Shandong Province, China
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13
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Wong WK, Yin B, Lam CYK, Huang Y, Yan J, Tan Z, Wong SHD. The Interplay Between Epigenetic Regulation and CD8 + T Cell Differentiation/Exhaustion for T Cell Immunotherapy. Front Cell Dev Biol 2022; 9:783227. [PMID: 35087832 PMCID: PMC8787221 DOI: 10.3389/fcell.2021.783227] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/25/2021] [Accepted: 12/20/2021] [Indexed: 12/15/2022] Open
Abstract
Effective immunotherapy treats cancers by eradicating tumourigenic cells by activated tumour antigen-specific and bystander CD8+ T-cells. However, T-cells can gradually lose cytotoxicity in the tumour microenvironment, known as exhaustion. Recently, DNA methylation, histone modification, and chromatin architecture have provided novel insights into epigenetic regulations of T-cell differentiation/exhaustion, thereby controlling the translational potential of the T-cells. Thus, developing strategies to govern epigenetic switches of T-cells dynamically is critical to maintaining the effector function of antigen-specific T-cells. In this mini-review, we 1) describe the correlation between epigenetic states and T cell phenotypes; 2) discuss the enzymatic factors and intracellular/extracellular microRNA imprinting T-cell epigenomes that drive T-cell exhaustion; 3) highlight recent advances in epigenetic interventions to rescue CD8+ T-cell functions from exhaustion. Finally, we express our perspective that regulating the interplay between epigenetic changes and transcriptional programs provides translational implications of current immunotherapy for cancer treatments.
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Affiliation(s)
- Wai Ki Wong
- Department of Bioengineering, Imperial College London, London, United Kingdom
| | - Bohan Yin
- Department of Biomedical Engineering, The Hong Kong Polytechnic University, Kowloon, Hong Kong, China
| | - Ching Ying Katherine Lam
- Department of Biomedical Engineering, The Hong Kong Polytechnic University, Kowloon, Hong Kong, China
| | - Yingying Huang
- Department of Biomedical Engineering, The Hong Kong Polytechnic University, Kowloon, Hong Kong, China
| | - Jiaxiang Yan
- Department of Biomedical Engineering, The Hong Kong Polytechnic University, Kowloon, Hong Kong, China
| | - Zhiwu Tan
- AIDS Institute and Department of Microbiology, The University of Hong Kong, Pokfulam, Hong Kong, China
| | - Siu Hong Dexter Wong
- Department of Biomedical Engineering, The Hong Kong Polytechnic University, Kowloon, Hong Kong, China
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14
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Lan H, Sun Y. Tumor Suppressor FBXW7 and Its Regulation of DNA Damage Response and Repair. Front Cell Dev Biol 2021; 9:751574. [PMID: 34760892 PMCID: PMC8573206 DOI: 10.3389/fcell.2021.751574] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/01/2021] [Accepted: 10/05/2021] [Indexed: 12/21/2022] Open
Abstract
The proper DNA damage response (DDR) and repair are the central molecular mechanisms for the maintenance of cellular homeostasis and genomic integrity. The abnormality in this process is frequently observed in human cancers, and is an important contributing factor to cancer development. FBXW7 is an F-box protein serving as the substrate recognition component of SCF (SKP1-CUL1-F-box protein) E3 ubiquitin ligase. By selectively targeting many oncoproteins for proteasome-mediated degradation, FBXW7 acts as a typical tumor suppressor. Recent studies have demonstrated that FBXW7 also plays critical roles in the process of DDR and repair. In this review, we first briefly introduce the processes of protein ubiquitylation by SCFFBXW7 and DDR/repair, then provide an overview of the molecular characteristics of FBXW7. We next discuss how FBXW7 regulates the process of DDR and repair, and its translational implication. Finally, we propose few future perspectives to further elucidate the role of FBXW7 in regulation of a variety of biological processes and tumorigenesis, and to design a number of approaches for FBXW7 reactivation in a subset of human cancers for potential anticancer therapy.
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Affiliation(s)
- Huiyin Lan
- Department of Thoracic Radiation Oncology, Zhejiang Cancer Hospital, Cancer Hospital of University of Chinese Academy of Sciences, Hangzhou, China.,Institute of Cancer and Basic Medicine, Chinese Academy of Sciences, Hangzhou, China
| | - Yi Sun
- Cancer Institute of the Second Affiliated Hospital, Institute of Translational Medicine, Zhejiang University School of Medicine, Hangzhou, China.,Cancer Center, Zhejiang University, Hangzhou, China
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15
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Zhang E, Chen S, Tang H, Fei C, Yuan Z, Mu X, Qin Y, Liu H, Fan Y, Tan M, Wang X. CDK1/FBXW7 facilitates degradation and ubiquitination of MLST8 to inhibit progression of renal cell carcinoma. Cancer Sci 2021; 113:91-108. [PMID: 34741373 PMCID: PMC8748215 DOI: 10.1111/cas.15188] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/10/2021] [Revised: 10/06/2021] [Accepted: 10/28/2021] [Indexed: 11/29/2022] Open
Abstract
Recent studies have reported that MLST8 is upregulated in many malignant tumors. Nevertheless, the underlying molecular mechanism is still unclear. The aim of this work was to investigate how MLST8 contributes to the development and progression of clear cell renal cell carcinoma (ccRCC). MLST8 is an oncogenic protein in the TCGA database and ccRCC clinical specimens. We also ascertain that MLST8 interacts with FBXW7, which was universally regarded as an E3 ubiquitin ligase. MLST8 can be degraded and ubiquitinated by tumor suppressor FBXW7. FBXW7 recognizes a consensus motif (T/S) PXX (S/T/D/E) of MLST8 and triggers MLST8 degradation via the ubiquitin‐proteasome pathway. Strikingly, the activated cyclin dependent kinase 1 (CDK1) kinase engages in the MLST8 phosphorylation required for FBXW7‐mediated degradation. In vitro, we further prove that MLST8 is an essential mediator of FBXW7 inactivation‐induced tumor growth, migration, and invasion. Furthermore, the MLST8 and FBXW7 proteins are negatively correlated in human renal cancer specimens. Our findings suggest that MLST8 is a putative oncogene that functions via interaction with FBXW7, and inhibition MLST8 could be a potential future target in ccRCC treatment.
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Affiliation(s)
- Encheng Zhang
- Department of Urology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Siteng Chen
- Department of Urology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Heting Tang
- Department of Urology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Cheng Fei
- Department of Urology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Zhihao Yuan
- Department of Urology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Xingyu Mu
- Department of Urology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Yan Qin
- Department of Urology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Haixia Liu
- State Key Laboratory of Genetic Engineering, Collaborative Innovation Center for Genetics and Development, School of Life Sciences, Fudan University, Shanghai, China
| | - Yu Fan
- Department of Renal Transplantation, Xiang'an Hospital of Xiamen University, Xiamen, China
| | - Mingyue Tan
- Department of Urology, Shuguang Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, China
| | - Xiang Wang
- Department of Urology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
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16
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Li Y, Wang J, Ma Y, Du W, Feng K, Wang S. miR-101-loaded exosomes secreted by bone marrow mesenchymal stem cells requires the FBXW7/HIF1α/FOXP3 axis, facilitating osteogenic differentiation. J Cell Physiol 2021; 236:4258-4272. [PMID: 33438204 DOI: 10.1002/jcp.30027] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/19/2020] [Revised: 08/12/2020] [Accepted: 08/16/2020] [Indexed: 12/20/2022]
Abstract
Exosomes derived from mesenchymal stem cells (MSCs) have emerged as significant mediators of intercellular communication, with studies highlighting their role in the transmission of biological signals between cells. Dominant microRNA (miRNA)-mediated translational repression of messenger RNAs has been extensively investigated in regard to its influence in orchestrating osteogenic differentiation. In the current study, we sought to ascertain the contributory role of miRNA-101 (miR-101) encapsulated in the process of bone marrow mesenchymal stem cell (BMSC)-derived exosomes in osteogenic differentiation. Exosomes were initially extracted from BMSCs at Days 0, 3, 12, and 21 of osteogenic differentiation by ultracentrifugation. Artificial modulation of miR-101 and FBXW7 (silencing and overexpression) were performed in the BMSCs to identify its effects on osteogenic factors, alkaline phosphatase activity, and osteogenic differentiation. Mechanistic exploration was performed to evaluate the binding affinity between miR-101 and FBXW7, the FBXW7-mediated HIF1α ubiquitination, and the HIF1α enrichment in the FOXP3 promoter region. Exosomes from MSCs in the late stage of osteogenic differentiation exhibited enhanced osteogenic differentiation. Upregulated miR-101 in MSC-derived exosomes was detected during osteogenic differentiation, while diminished levels of FBXW7 expression was noted. Importantly, miR-101 was found to specifically bind to the 3'-untranslated region of FBXW7. Meanwhile, data was obtained indicating that FBXW7 could ubiquitinate and degrade HIF1α to repress its upregulation during osteogenic differentiation. HIF1α bound to the promoter region of FOXP3 to facilitate osteogenic differentiation. Ultimately, the findings of the current study demonstrate that BMSC-derived exosomal miR-101 augments osteogenic differentiation in MSCs by inhibiting FBXW7 to regulate the HIF1α/FOXP3 axis.
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Affiliation(s)
- Yanhong Li
- Department of Orthopaedics, Lanzhou University Second Hospital, Lanzhou, Gansu, China
| | - Jing Wang
- Department of Orthopaedics, Lanzhou University Second Hospital, Lanzhou, Gansu, China
| | - Yanchao Ma
- Department of Orthopaedics, Lanzhou University Second Hospital, Lanzhou, Gansu, China
| | - Wenjia Du
- Department of Orthopaedics, Lanzhou University Second Hospital, Lanzhou, Gansu, China
| | - Kai Feng
- Department of Orthopaedics, Lanzhou University Second Hospital, Lanzhou, Gansu, China
| | - Shuanke Wang
- Department of Orthopaedics, Lanzhou University Second Hospital, Lanzhou, Gansu, China
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17
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Comparative Analysis of CREB3 and CREB3L2 Protein Expression in HEK293 Cells. Int J Mol Sci 2021; 22:ijms22052767. [PMID: 33803345 PMCID: PMC7967177 DOI: 10.3390/ijms22052767] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/20/2021] [Revised: 03/01/2021] [Accepted: 03/03/2021] [Indexed: 12/13/2022] Open
Abstract
We performed a comparative analysis of two ER-resident CREB3 family proteins, CREB3 and CREB3L2, in HEK293 cells using pharmacological and genome editing approaches and identified several differences between the two. Treatment with brefeldin A (BFA) and monensin induced the cleavage of full-length CREB3 and CREB3L2; however, the level of the full-length CREB3 protein, but not CREB3L2 protein, was not noticeably reduced by the monensin treatment. On the other hand, treatment with tunicamycin (Tm) shifted the molecular weight of the full-length CREB3L2 protein downward but abolished CREB3 protein expression. Thapsigargin (Tg) significantly increased the expression of only full-length CREB3L2 protein concomitant with a slight increase in the level of its cleaved form. Treatment with cycloheximide and MG132 revealed that both endogenous CREB3 and CREB3L2 are proteasome substrates. In addition, kifunensine, an α-mannosidase inhibitor, significantly increased the levels of both full-length forms. Consistent with these findings, cells lacking SEL1L, a crucial ER-associated protein degradation (ERAD) component, showed increased expression of both full-length CREB3 and CREB3L2; however, cycloheximide treatment downregulated full-length CREB3L2 protein expression more rapidly in SEL1L-deficient cells than the full-length CREB3 protein. Finally, we investigated the induction of the expression of several CREB3 and CREB3L2 target genes by Tg and BFA treatments and SEL1L deficiency. In conclusion, this study suggests that both endogenous full-length CREB3 and CREB3L2 are substrates for ER-associated protein degradation but are partially regulated by distinct mechanisms, each of which contributes to unique cellular responses that are distinct from canonical ER signals.
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18
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Hoshikawa S, Shimizu K, Watahiki A, Chiba M, Saito K, Wei W, Fukumoto S, Inuzuka H. Phosphorylation-dependent osterix degradation negatively regulates osteoblast differentiation. FASEB J 2020; 34:14930-14945. [PMID: 32931083 DOI: 10.1096/fj.202001340r] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/26/2020] [Revised: 08/15/2020] [Accepted: 08/25/2020] [Indexed: 01/05/2023]
Abstract
Proteasome inhibitors exert an anabolic effect on bone formation with elevated levels of osteoblast markers. These findings suggest the important role of the proteasomal degradation of osteogenic regulators, while the underlying molecular mechanisms are not fully understood. Here, we report that the proteasome inhibitors bortezomib and ixazomib markedly increased protein levels of the osteoblastic key transcription factor osterix/Sp7 (Osx). Furthermore, we revealed that Osx was targeted by p38 and Fbw7 for proteasomal degradation. Mechanistically, p38-mediated Osx phosphorylation at S73/77 facilitated Fbw7 interaction to trigger subsequent Osx ubiquitination. Consistent with these findings, p38 knockdown or pharmacological p38 inhibition resulted in Osx protein stabilization. Treatment with p38 inhibitors following osteogenic stimulation efficiently induced osteoblast differentiation through Osx stabilization. Conversely, pretreatment of p38 inhibitor followed by osteogenic challenge impaired osteoblastogenesis via suppressing Osx expression, suggesting that p38 exerts dual but opposite effects in the regulation of Osx level to fine-tune its activity during osteoblast differentiation. Furthermore, Fbw7-depleted human mesenchymal stem cells and primary mouse calvarial cells resulted in increased osteogenic capacity. Together, our findings unveil the molecular mechanisms underlying the Osx protein stability control and suggest that targeting the Osx degradation pathway could help enhance efficient osteogenesis and bone matrix regeneration.
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Affiliation(s)
- Seira Hoshikawa
- Division of Pediatric Dentistry, Department of Oral Health and Development Sciences, Tohoku University Graduate School of Dentistry, Sendai, Japan.,Center for Advanced Stem Cell and Regenerative Research, Tohoku University Graduate School of Dentistry, Sendai, Japan
| | - Kouhei Shimizu
- Center for Advanced Stem Cell and Regenerative Research, Tohoku University Graduate School of Dentistry, Sendai, Japan
| | - Asami Watahiki
- Center for Advanced Stem Cell and Regenerative Research, Tohoku University Graduate School of Dentistry, Sendai, Japan
| | - Mitsuki Chiba
- Division of Pediatric Dentistry, Department of Oral Health and Development Sciences, Tohoku University Graduate School of Dentistry, Sendai, Japan.,Center for Advanced Stem Cell and Regenerative Research, Tohoku University Graduate School of Dentistry, Sendai, Japan
| | - Kan Saito
- Division of Pediatric Dentistry, Department of Oral Health and Development Sciences, Tohoku University Graduate School of Dentistry, Sendai, Japan
| | - Wenyi Wei
- Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA
| | - Satoshi Fukumoto
- Division of Pediatric Dentistry, Department of Oral Health and Development Sciences, Tohoku University Graduate School of Dentistry, Sendai, Japan.,Center for Advanced Stem Cell and Regenerative Research, Tohoku University Graduate School of Dentistry, Sendai, Japan.,Department of Pediatric Dentistry, Kyushu University Graduate School of Dental Science, Fukuoka, Japan
| | - Hiroyuki Inuzuka
- Center for Advanced Stem Cell and Regenerative Research, Tohoku University Graduate School of Dentistry, Sendai, Japan
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19
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Myeloid FBW7 deficiency disrupts redox homeostasis and aggravates dietary-induced insulin resistance. Redox Biol 2020; 37:101688. [PMID: 32853822 PMCID: PMC7451763 DOI: 10.1016/j.redox.2020.101688] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/25/2020] [Revised: 08/10/2020] [Accepted: 08/13/2020] [Indexed: 02/07/2023] Open
Abstract
The E3 ubiquitin ligase FBW7 plays critical roles in multiple pathological and physiological processes. Here, we report that after high-fat diet (HFD) feeding for 16 weeks, myeloid-specific FBW7-deficient mice demonstrate increased redox stress, inflammatory responses and insulin resistance. Macrophages activation under FBW7 deficiency decreases substrate flux through the pentose phosphate pathway (PPP) to produce less equivalents (NADPH and GSH) and aggravate the generation of intracellular reactive oxygen species (ROS) in macrophages, thereby over-activating proinflammatory reaction. Mechanistically, we identify that pyruvate kinase muscle isozyme M2 (PKM2) is a new bona fide ubiquitin substrate of SCFFBW7. While challenged with HFD stress, pharmacological inhibition of PKM2 protects FBW7-deficient macrophages against production of ROS, proinflammatory reaction and insulin resistance. Intriguingly, we further find an inverse correlation between FBW7 level and relative higher H2O2 level and the severity of obesity-related diabetes. Overall, the results suggest that FBW7 can play a crucial role in modulating inflammatory response through maintaining the intracellular redox homeostasis during HFD insults.
Myeloid FBW7 deficiency aggravates HFD-induced oxidative stress, inflammation and insulin resistance. PKM2 is a new bona fide ubiquitin substrate of SCFFBW7. FBW7 divert glycolysis to combat oxidative stress via PKM2 in macrophages. FBW7 expression inversely correlates with ROS level to govern obesity-related metabolic disorder.
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20
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Yumimoto K, Yamauchi Y, Nakayama KI. F-Box Proteins and Cancer. Cancers (Basel) 2020; 12:cancers12051249. [PMID: 32429232 PMCID: PMC7281081 DOI: 10.3390/cancers12051249] [Citation(s) in RCA: 31] [Impact Index Per Article: 6.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/15/2020] [Revised: 05/09/2020] [Accepted: 05/12/2020] [Indexed: 12/20/2022] Open
Abstract
Controlled protein degradation is essential for the operation of a variety of cellular processes including cell division, growth, and differentiation. Identification of the relations between ubiquitin ligases and their substrates is key to understanding the molecular basis of cancer development and to the discovery of novel targets for cancer therapeutics. F-box proteins function as the substrate recognition subunits of S-phase kinase-associated protein 1 (SKP1)−Cullin1 (CUL1)−F-box protein (SCF) ubiquitin ligase complexes. Here, we summarize the roles of specific F-box proteins that have been shown to function as tumor promoters or suppressors. We also highlight proto-oncoproteins that are targeted for ubiquitylation by multiple F-box proteins, and discuss how these F-box proteins are deployed to regulate their cognate substrates in various situations.
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21
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Yumimoto K, Nakayama KI. Recent insight into the role of FBXW7 as a tumor suppressor. Semin Cancer Biol 2020; 67:1-15. [PMID: 32113998 DOI: 10.1016/j.semcancer.2020.02.017] [Citation(s) in RCA: 68] [Impact Index Per Article: 13.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/28/2019] [Revised: 02/15/2020] [Accepted: 02/26/2020] [Indexed: 12/12/2022]
Abstract
FBXW7 (also known as Fbw7, Sel10, hCDC4, or hAgo) is a tumor suppressor and the most frequently mutated member of the F-box protein family in human cancers. FBXW7 functions as the substrate recognition component of an SCF-type E3 ubiquitin ligase. It specifically controls the proteasome-mediated degradation of many oncoproteins such as c-MYC, NOTCH, KLF5, cyclin E, c-JUN, and MCL1. In this review, we summarize the molecular and biological features of FBXW7 and its substrates as well as the impact of mutations of FBXW7 on cancer development. We also address the clinical potential of anticancer therapy targeting FBXW7.
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Affiliation(s)
- Kanae Yumimoto
- Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Fukuoka, 812-8582, Japan
| | - Keiichi I Nakayama
- Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Fukuoka, 812-8582, Japan.
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22
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Sampieri L, Di Giusto P, Alvarez C. CREB3 Transcription Factors: ER-Golgi Stress Transducers as Hubs for Cellular Homeostasis. Front Cell Dev Biol 2019; 7:123. [PMID: 31334233 PMCID: PMC6616197 DOI: 10.3389/fcell.2019.00123] [Citation(s) in RCA: 90] [Impact Index Per Article: 15.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/15/2019] [Accepted: 06/17/2019] [Indexed: 12/21/2022] Open
Abstract
CREB3 family of transcription factors are ER localized proteins that belong to the bZIP family. They are transported from the ER to the Golgi, cleaved by S1P and S2P proteases and the released N-terminal domains act as transcription factors. CREB3 family members regulate the expression of a large variety of genes and according to their tissue-specific expression profiles they play, among others, roles in acute phase response, lipid metabolism, development, survival, differentiation, organelle autoregulation, and protein secretion. They have been implicated in the ER and Golgi stress responses as regulators of the cell secretory capacity and cell specific cargos. In this review we provide an overview of the diverse functions of each member of the family (CREB3, CREB3L1, CREB3L2, CREB3L3, CREB3L4) with special focus on their role in the central nervous system.
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Affiliation(s)
- Luciana Sampieri
- Centro de Investigaciones en Bioquímica Clínica e Inmunología (CIBICI-CONICET), Córdoba, Argentina.,Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba, Argentina
| | - Pablo Di Giusto
- Centro de Investigaciones en Bioquímica Clínica e Inmunología (CIBICI-CONICET), Córdoba, Argentina.,Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba, Argentina
| | - Cecilia Alvarez
- Centro de Investigaciones en Bioquímica Clínica e Inmunología (CIBICI-CONICET), Córdoba, Argentina.,Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba, Argentina
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23
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He J, Song Y, Li G, Xiao P, Liu Y, Xue Y, Cao Q, Tu X, Pan T, Jiang Z, Cao X, Lai L, Wang Q. Fbxw7 increases CCL2/7 in CX3CR1hi macrophages to promote intestinal inflammation. J Clin Invest 2019; 129:3877-3893. [PMID: 31246581 DOI: 10.1172/jci123374] [Citation(s) in RCA: 104] [Impact Index Per Article: 17.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
Resident and inflammatory mononuclear phagocytes (MPh) with functional plasticity in the intestine are critically involved in the pathology of Inflammatory Bowel Diseases (IBD), in which the mechanism remains incompletely understood. In the present study, we found that increased expression of E3 ligase FBXW7 in the inflamed intestine was significantly correlated to IBD severity in both human diseases and mice model. Myeloid-Fbxw7 deficiency protected mice from dextran sodium sulfate (DSS) and 2,6,4-trinitrobenzene sulfonic acid (TNBS) induced colitis. Fbxw7 deficiency resulted in decreased production of chemokines CCL2 and CCL7 by colonic CX3CR1hi resident macrophages and reduced accumulation of CX3CR1int pro-inflammatory MPh in colitis colon tissue. Mice received AAV-shFbxw7 administration showed significantly improved survival rate and alleviated colitis. Mechanisms screening demonstrated that FBXW7 suppresses H3K27me3 modification and promotes Ccl2 and Ccl7 expression via degradation of histone-lysine N-methyltransferase EZH2 in macrophages. Taken together, our results indicate that FBXW7 degrades EZH2 and increases Ccl2/Ccl7 in CX3CR1hi macrophages, which promotes the recruiting CX3CR1int pro-inflammatory MPh into local colon tissues with colitis. Targeting FBXW7 might represent a potential therapeutic approach for intestine inflammation intervention.
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Affiliation(s)
- Jia He
- Institute of Immunology, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
| | - Yinjing Song
- Institute of Immunology, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
| | - Gaopeng Li
- Institute of Immunology, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
| | | | - Yang Liu
- Institute of Immunology, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
| | - Yue Xue
- Institute of Immunology, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
| | - Qian Cao
- Department of Gastroenterology and
| | - Xintao Tu
- Institute of Immunology, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
| | - Ting Pan
- Institute of Immunology, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
| | - Zhinong Jiang
- Department of Pathology, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
| | - Xuetao Cao
- Institute of Immunology, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China.,National Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, China
| | - Lihua Lai
- Institute of Immunology, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
| | - Qingqing Wang
- Institute of Immunology, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
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24
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Sugiyama S, Yumimoto K, Inoue I, Nakayama KI. SCF
Fbxw7
ubiquitylates KLF7 for degradation in a manner dependent on GSK‐3‐mediated phosphorylation. Genes Cells 2019; 24:354-365. [DOI: 10.1111/gtc.12680] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/16/2019] [Revised: 03/01/2019] [Accepted: 03/03/2019] [Indexed: 12/11/2022]
Affiliation(s)
- Shigeaki Sugiyama
- Department of Molecular and Cellular Biology Medical Institute of Bioregulation, Kyushu University Fukuoka Japan
| | - Kanae Yumimoto
- Department of Molecular and Cellular Biology Medical Institute of Bioregulation, Kyushu University Fukuoka Japan
| | - Ippei Inoue
- Department of Molecular and Cellular Biology Medical Institute of Bioregulation, Kyushu University Fukuoka Japan
| | - Keiichi I. Nakayama
- Department of Molecular and Cellular Biology Medical Institute of Bioregulation, Kyushu University Fukuoka Japan
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25
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Feng Y, Fu X, Lou X. Notch pathway deactivation mediated by F-box/WD repeat domain-containing 7 ameliorates hydrogen peroxide-induced apoptosis in rat periodontal ligament stem cells. Arch Oral Biol 2019; 100:93-99. [PMID: 30822705 DOI: 10.1016/j.archoralbio.2019.02.010] [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: 08/21/2018] [Revised: 02/11/2019] [Accepted: 02/18/2019] [Indexed: 11/25/2022]
Abstract
OBJECTIVE To investigate the protective role of F-box/WD repeat domain-containing 7 in rat periodontal ligament stem cells under oxidative stress. MATERIALS AND METHODS The apoptosis of rat periodontal ligament stem cells was induced by exposure to various concentrations of hydrogen peroxide for 24 h, after which cell viability and the cleaved caspase-3 and -9 levels were determined. The levels of proteins in the Notch signaling pathway were determined by western blotting. RESULTS The overexpression of F-box/WD repeat domain-containing 7 increased cell viability following hydrogen peroxide administration and suppressed the activation of caspases-3 and -9. The overexpression of F-box/WD repeat domain-containing 7 inhibited Notch signaling. Furthermore, the protective effect of F-box/WD repeat domain-containing 7 could be resumed by PF-03084014, a Notch-specific inhibitor. CONCLUSIONS These observations suggest a protective role of F-box/WD repeat domain-containing 7 against hydrogen peroxide-induced oxidative stress in rat periodontal ligament stem cells. These findings will facilitate the in vitro culturing of periodontal ligament stem cell for clinical usage and promote stem cell-based therapy for periodontal tissue regeneration.
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Affiliation(s)
- Yimiao Feng
- Department of Orthodontics, Second Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, China
| | - Xiaohui Fu
- Department of General Dentistry, Second Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, China
| | - Xintian Lou
- Department of Stomatology, Punan Hospital of Pudong New District, Shanghai, China.
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26
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Liu L, Liu K, Yan Y, Chu Z, Tang Y, Tang C. Two Transcripts of FBXO5 Promote Migration and Osteogenic Differentiation of Human Periodontal Ligament Mesenchymal Stem Cells. BIOMED RESEARCH INTERNATIONAL 2018; 2018:7849294. [PMID: 29850565 PMCID: PMC5933072 DOI: 10.1155/2018/7849294] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 01/09/2018] [Revised: 03/03/2018] [Accepted: 03/13/2018] [Indexed: 12/15/2022]
Abstract
OBJECTIVES Enhanced migration and osteogenic differentiation of mesenchymal stem cells (MSCs) are beneficial for MSC-mediated periodontal tissue regeneration, a promising method for periodontitis treatment. FBXO5, a member of the F-box protein family, is involved in the osteogenic differentiation of MSCs. Here, we investigated the effect of FBXO5 on human periodontal ligament stem cells (hPDLSCs). MATERIALS AND METHODS hPDLSCs were isolated from periodontal ligament tissue. Lentivirus FBXO5 shRNA was used to silence FBXO5 expression. Two transcripts of FBXO5 were overexpressed and transduced into hPDLSCs via retroviral infection. Migration and osteogenic differentiation of hPDLSCs were evaluated using the scratch migration assay, alkaline phosphatase (ALP) activity, ALP staining, alizarin red staining, western blotting, and real-time polymerase chain reaction. RESULTS The expression of FBXO5 was upregulated after osteogenic induction in hPDLSCs. FBXO5 knockdown attenuated migration, inhibited ALP activity and mineralization, and decreased RUNX2, OSX, and OCN expression, while the overexpression of two transcript isoforms significantly accelerated migration, enhanced ALP activity and mineralization, and increased RUNX2, OSX, and OCN expression in hPDLSCs. CONCLUSIONS Both isoforms of FBXO5 promoted the migration and osteogenic differentiation potential of hPDLSCs, which identified a potential target for improving periodontal tissue regeneration.
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Affiliation(s)
- Lin Liu
- Jiangsu Key Laboratory of Oral Diseases, Nanjing Medical University, Nanjing, Jiangsu, China
| | - Kun Liu
- Jiangsu Key Laboratory of Oral Diseases, Nanjing Medical University, Nanjing, Jiangsu, China
| | - Yanzhe Yan
- Jiangsu Key Laboratory of Oral Diseases, Nanjing Medical University, Nanjing, Jiangsu, China
| | - Zhuangzhuang Chu
- Jiangsu Key Laboratory of Oral Diseases, Nanjing Medical University, Nanjing, Jiangsu, China
| | - Yi Tang
- Jiangsu Key Laboratory of Oral Diseases, Nanjing Medical University, Nanjing, Jiangsu, China
| | - Chunbo Tang
- Department of Dental Implantology, Affiliated Hospital of Stomatology, Nanjing Medical University, Nanjing, Jiangsu, China
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27
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Nakayama S, Yumimoto K, Kawamura A, Nakayama KI. Degradation of the endoplasmic reticulum-anchored transcription factor MyRF by the ubiquitin ligase SCF Fbxw7 in a manner dependent on the kinase GSK-3. J Biol Chem 2018; 293:5705-5714. [PMID: 29472293 DOI: 10.1074/jbc.ra117.000741] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/01/2017] [Revised: 02/14/2018] [Indexed: 12/16/2022] Open
Abstract
The ubiquitin-proteasome system regulates the abundance of many cellular proteins by mediating their targeted degradation. We previously developed a method-differential proteomics-based identification of ubiquitylation substrates (DiPIUS)-for the comprehensive identification of substrates for a given F-box protein subunit of SCF-type ubiquitin ligases. We have now applied DiPIUS to the F-box protein Fbxw7 in three cell lines (mHepa, Neuro2A, and C2C12) and thereby identified myelin regulatory factor (MyRF), an endoplasmic reticulum-anchored transcription factor that is essential for myelination of nerves in the central nervous system, as a candidate substrate of Fbxw7 specifically in mHepa cells. Co-immunoprecipitation analysis confirmed that the NH2-terminal cytoplasmic domain of MyRF interacted with Fbxw7 in these cells. Furthermore, an in vitro ubiquitylation assay revealed that MyRF undergoes polyubiquitylation in the presence of purified recombinant SCFFbxw7 In addition, the stability of MyRF in mHepa cells was increased by mutation of a putative phosphodegron sequence or by exposure of the cells to an inhibitor of glycogen synthase kinase-3 (GSK-3). We found that MyRF mRNA is not restricted to the central nervous system but is instead distributed widely among mouse tissues. Furthermore, with the use of RNA sequencing in mHepa cells overexpressing or depleted of MyRF, we identified many novel potential target genes of MyRF. Our results thus suggest that Fbxw7 controls the transcription of MyRF target genes in various tissues through regulation of MyRF protein stability in a manner dependent on MyRF phosphorylation by GSK-3.
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Affiliation(s)
- Shogo Nakayama
- From the Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Fukuoka 812-8582, Japan
| | - Kanae Yumimoto
- From the Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Fukuoka 812-8582, Japan
| | - Atsuki Kawamura
- From the Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Fukuoka 812-8582, Japan
| | - Keiichi I Nakayama
- From the Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Fukuoka 812-8582, Japan
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28
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Fukushima H, Shimizu K, Watahiki A, Hoshikawa S, Kosho T, Oba D, Sakano S, Arakaki M, Yamada A, Nagashima K, Okabe K, Fukumoto S, Jimi E, Bigas A, Nakayama KI, Nakayama K, Aoki Y, Wei W, Inuzuka H. NOTCH2 Hajdu-Cheney Mutations Escape SCF FBW7-Dependent Proteolysis to Promote Osteoporosis. Mol Cell 2017; 68:645-658.e5. [PMID: 29149593 DOI: 10.1016/j.molcel.2017.10.018] [Citation(s) in RCA: 26] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2017] [Revised: 09/20/2017] [Accepted: 10/17/2017] [Indexed: 12/11/2022]
Abstract
Hajdu-Cheney syndrome (HCS), a rare autosomal disorder caused by heterozygous mutations in NOTCH2, is clinically characterized by acro-osteolysis, severe osteoporosis, short stature, neurological symptoms, cardiovascular defects, and polycystic kidneys. Recent studies identified that aberrant NOTCH2 signaling and consequent osteoclast hyperactivity are closely associated with the bone-related disorder pathogenesis, but the exact molecular mechanisms remain unclear. Here, we demonstrate that sustained osteoclast activity is largely due to accumulation of NOTCH2 carrying a truncated C terminus that escapes FBW7-mediated ubiquitination and degradation. Mice with osteoclast-specific Fbw7 ablation revealed osteoporotic phenotypes reminiscent of HCS, due to elevated Notch2 signaling. Importantly, administration of Notch inhibitors in Fbw7 conditional knockout mice alleviated progressive bone resorption. These findings highlight the molecular basis of HCS pathogenesis and provide clinical insights into potential targeted therapeutic strategies for skeletal disorders associated with the aberrant FBW7/NOTCH2 pathway as observed in patients with HCS.
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Affiliation(s)
- Hidefumi Fukushima
- Center for Advanced Stem Cell and Regenerative Research, Graduate School of Dentistry, Tohoku University, Sendai 980-8575, Japan.
| | - Kouhei Shimizu
- Center for Advanced Stem Cell and Regenerative Research, Graduate School of Dentistry, Tohoku University, Sendai 980-8575, Japan; Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA
| | - Asami Watahiki
- Center for Advanced Stem Cell and Regenerative Research, Graduate School of Dentistry, Tohoku University, Sendai 980-8575, Japan
| | - Seira Hoshikawa
- Center for Advanced Stem Cell and Regenerative Research, Graduate School of Dentistry, Tohoku University, Sendai 980-8575, Japan; Division of Pediatric Dentistry, Department of Oral Health and Development Sciences, Graduate School of Dentistry, Tohoku University, Sendai 980-8575, Japan
| | - Tomoki Kosho
- Department of Medical Genetics, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto 390-8621, Japan
| | - Daiju Oba
- Department of Medical Genetics, Tohoku University School of Medicine, 1-1 Seiryo-machi, Sendai 980-8574, Japan
| | - Seiji Sakano
- Corporate R&D, Asahi Kasei Corporation, 2-1 Samejima, Fuji-shi, Shizuoka 416-8501, Japan
| | - Makiko Arakaki
- Center for Advanced Stem Cell and Regenerative Research, Graduate School of Dentistry, Tohoku University, Sendai 980-8575, Japan; Division of Pediatric Dentistry, Department of Oral Health and Development Sciences, Graduate School of Dentistry, Tohoku University, Sendai 980-8575, Japan
| | - Aya Yamada
- Division of Pediatric Dentistry, Department of Oral Health and Development Sciences, Graduate School of Dentistry, Tohoku University, Sendai 980-8575, Japan
| | - Katsuyuki Nagashima
- Department of Physiological Sciences and Molecular Biology, Fukuoka Dental College, Fukuoka 814-0193, Japan
| | - Koji Okabe
- Department of Physiological Sciences and Molecular Biology, Fukuoka Dental College, Fukuoka 814-0193, Japan
| | - Satoshi Fukumoto
- Center for Advanced Stem Cell and Regenerative Research, Graduate School of Dentistry, Tohoku University, Sendai 980-8575, Japan; Division of Pediatric Dentistry, Department of Oral Health and Development Sciences, Graduate School of Dentistry, Tohoku University, Sendai 980-8575, Japan
| | - Eijiro Jimi
- Department of Dental Science, Graduate School of Dentistry, Kyushu University, Fukuoka 812-8582, Japan
| | - Anna Bigas
- Institut Hospital del Mar d'Investigacions Mèdiques, CIBERONC, Dr Aiguader 88, 08003 Barcelona, Spain
| | - Keiichi I Nakayama
- Division of Cell Regulation Systems, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, Japan
| | - Keiko Nakayama
- Division of Cell Proliferation, ART, Graduate School of Medicine, Tohoku University, Sendai 980-8575, Japan
| | - Yoko Aoki
- Department of Medical Genetics, Tohoku University School of Medicine, 1-1 Seiryo-machi, Sendai 980-8574, Japan
| | - Wenyi Wei
- Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA.
| | - Hiroyuki Inuzuka
- Center for Advanced Stem Cell and Regenerative Research, Graduate School of Dentistry, Tohoku University, Sendai 980-8575, Japan.
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29
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He R, Li X, Zhong M, Yan J, Ji R, Li X, Wang Q, Wu D, Sun M, Tang D, Lin J, Li H, Liu B, Liu H, Liu X, Zhao X, Lin C. A photo-responsive F-box protein FOF2 regulates floral initiation by promoting FLC expression in Arabidopsis. THE PLANT JOURNAL : FOR CELL AND MOLECULAR BIOLOGY 2017; 91:788-801. [PMID: 28608936 DOI: 10.1111/tpj.13607] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/10/2017] [Revised: 04/08/2017] [Accepted: 05/13/2017] [Indexed: 05/09/2023]
Abstract
Floral initiation is regulated by various genetic pathways in response to light, temperature, hormones and developmental status; however, the molecular mechanisms underlying the interactions between different genetic pathways are not fully understood. Here, we show that the photoresponsive gene FOF2 (F-box of flowering 2) negatively regulates flowering. FOF2 encodes a putative F-box protein that interacts specifically with ASK14, and its overexpression results in later flowering under both long-day and short-day photoperiods. Conversely, transgenic plants expressing the F-box domain deletion mutant of FOF2 (FOF2ΔF), or double loss of function mutant of FOF2 and FOL1 (FOF2-LIKE 1) present early flowering phenotypes. The late flowering phenotype of the FOF2 overexpression lines is suppressed by the flc-3 loss-of-function mutation. Furthermore, FOF2 mRNA expression is regulated by autonomous pathway gene FCA, and the repressive effect of FOF2 in flowering can be overcome by vernalization. Interestingly, FOF2 expression is regulated by light. The protein level of FOF2 accumulates in response to light, whereas it is degraded under dark conditions via the 26S proteasome pathway. Our findings suggest a possible mechanistic link between light conditions and the autonomous floral promotion pathway in Arabidopsis.
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Affiliation(s)
- Reqing He
- Hunan Province Key Laboratory of Plant Functional Genomics and Developmental Regulation, State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Biology, Hunan University, Changsha, 410082, China
| | - Xinmei Li
- Hunan Province Key Laboratory of Plant Functional Genomics and Developmental Regulation, State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Biology, Hunan University, Changsha, 410082, China
| | - Ming Zhong
- Hunan Province Key Laboratory of Plant Functional Genomics and Developmental Regulation, State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Biology, Hunan University, Changsha, 410082, China
| | - Jindong Yan
- Hunan Province Key Laboratory of Plant Functional Genomics and Developmental Regulation, State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Biology, Hunan University, Changsha, 410082, China
| | - Ronghuan Ji
- Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing, 100081, China
| | - Xu Li
- National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200032, China
| | - Qin Wang
- Department of Molecular, Cell & Developmental Biology, University of California, Los Angeles, CA, 90095, USA
| | - Dan Wu
- Hunan Province Key Laboratory of Plant Functional Genomics and Developmental Regulation, State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Biology, Hunan University, Changsha, 410082, China
| | - Mengsi Sun
- Hunan Province Key Laboratory of Plant Functional Genomics and Developmental Regulation, State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Biology, Hunan University, Changsha, 410082, China
| | - Dongying Tang
- Hunan Province Key Laboratory of Plant Functional Genomics and Developmental Regulation, State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Biology, Hunan University, Changsha, 410082, China
| | - Jianzhong Lin
- Hunan Province Key Laboratory of Plant Functional Genomics and Developmental Regulation, State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Biology, Hunan University, Changsha, 410082, China
| | - Hongyu Li
- Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing, 100081, China
| | - Bin Liu
- Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing, 100081, China
| | - Hongtao Liu
- National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200032, China
| | - Xuanming Liu
- Hunan Province Key Laboratory of Plant Functional Genomics and Developmental Regulation, State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Biology, Hunan University, Changsha, 410082, China
| | - Xiaoying Zhao
- Hunan Province Key Laboratory of Plant Functional Genomics and Developmental Regulation, State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Biology, Hunan University, Changsha, 410082, China
| | - Chentao Lin
- Department of Molecular, Cell & Developmental Biology, University of California, Los Angeles, CA, 90095, USA
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30
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Cheng Y, Gao WW, Tang HMV, Deng JJ, Wong CM, Chan CP, Jin DY. β-TrCP-mediated ubiquitination and degradation of liver-enriched transcription factor CREB-H. Sci Rep 2016; 6:23938. [PMID: 27029215 PMCID: PMC4814919 DOI: 10.1038/srep23938] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/22/2015] [Accepted: 03/16/2016] [Indexed: 12/13/2022] Open
Abstract
CREB-H is an endoplasmic reticulum-resident bZIP transcription factor which critically regulates lipid homeostasis and gluconeogenesis in the liver. CREB-H is proteolytically activated by regulated intramembrane proteolysis to generate a C-terminally truncated form known as CREB-H-ΔTC, which translocates to the nucleus to activate target gene expression. CREB-H-ΔTC is a fast turnover protein but the mechanism governing its destruction was not well understood. In this study, we report on β-TrCP-dependent ubiquitination and proteasomal degradation of CREB-H-ΔTC. The degradation of CREB-H-ΔTC was mediated by lysine 48-linked polyubiquitination and could be inhibited by proteasome inhibitor. CREB-H-ΔTC physically interacted with β-TrCP, a substrate recognition subunit of the SCFβ-TrCP E3 ubiquitin ligase. Forced expression of β-TrCP increased the polyubiquitination and decreased the stability of CREB-H-ΔTC, whereas knockdown of β-TrCP had the opposite effect. An evolutionarily conserved sequence, SDSGIS, was identified in CREB-H-ΔTC, which functioned as the β-TrCP-binding motif. CREB-H-ΔTC lacking this motif was stabilized and resistant to β-TrCP-induced polyubiquitination. This motif was a phosphodegron and its phosphorylation was required for β-TrCP recognition. Furthermore, two inhibitory phosphorylation sites close to the phosphodegron were identified. Taken together, our work revealed a new intracellular signaling pathway that controls ubiquitination and degradation of the active form of CREB-H transcription factor.
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Affiliation(s)
- Yun Cheng
- School of Biomedical Sciences, The University of Hong Kong, Pokfulam, Hong Kong.,State Key Laboratory for Liver Research, The University of Hong Kong, Pokfulam, Hong Kong
| | - Wei-Wei Gao
- School of Biomedical Sciences, The University of Hong Kong, Pokfulam, Hong Kong.,State Key Laboratory for Liver Research, The University of Hong Kong, Pokfulam, Hong Kong
| | - Hei-Man Vincent Tang
- School of Biomedical Sciences, The University of Hong Kong, Pokfulam, Hong Kong.,State Key Laboratory for Liver Research, The University of Hong Kong, Pokfulam, Hong Kong.,Shenzhen Institute of Research and Innovation, The University of Hong Kong, Shenzhen, China
| | - Jian-Jun Deng
- School of Biomedical Sciences, The University of Hong Kong, Pokfulam, Hong Kong.,State Key Laboratory for Liver Research, The University of Hong Kong, Pokfulam, Hong Kong.,Department of Food Science and Engineering, College of Chemical Engineering, Northwestern University, Xi'an 710069, China
| | - Chi-Ming Wong
- Shenzhen Institute of Research and Innovation, The University of Hong Kong, Shenzhen, China.,Department of Medicine and State Key Laboratory of Pharmaceutical Biotechnology, The University of Hong Kong, Pokfulam, Hong Kong
| | - Chi-Ping Chan
- School of Biomedical Sciences, The University of Hong Kong, Pokfulam, Hong Kong.,State Key Laboratory for Liver Research, The University of Hong Kong, Pokfulam, Hong Kong.,Shenzhen Institute of Research and Innovation, The University of Hong Kong, Shenzhen, China
| | - Dong-Yan Jin
- School of Biomedical Sciences, The University of Hong Kong, Pokfulam, Hong Kong.,State Key Laboratory for Liver Research, The University of Hong Kong, Pokfulam, Hong Kong.,Shenzhen Institute of Research and Innovation, The University of Hong Kong, Shenzhen, China
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31
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Kumar Y, Kapoor I, Khan K, Thacker G, Khan MP, Shukla N, Kanaujiya JK, Sanyal S, Chattopadhyay N, Trivedi AK. E3 Ubiquitin Ligase Fbw7 Negatively Regulates Osteoblast Differentiation by Targeting Runx2 for Degradation. J Biol Chem 2015; 290:30975-87. [PMID: 26542806 DOI: 10.1074/jbc.m115.669531] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/05/2015] [Indexed: 11/06/2022] Open
Abstract
Runx2, a master regulator of osteoblast differentiation, is tightly regulated at both transcriptional and post-translational levels. Post-translational modifications such as phosphorylation and ubiquitination have differential effects on Runx2 functions. Here, we show that the reduced expression and functions of Runx2 upon its phosphorylation by GSK3β are mediated by its ubiquitin-mediated degradation through E3 ubiquitin ligase Fbw7α. Fbw7α through its WD domain interacts with Runx2 both in a heterologous (HEK293T cells) system as well as in osteoblasts. GSK3β was also present in the same complex as determined by co-immunoprecipitation. Furthermore, overexpression of either Fbw7α or GSK3β was sufficient to down-regulate endogenous Runx2 expression and function; however, both failed to inhibit endogenous Runx2 when either of them was depleted in osteoblasts. Fbw7α-mediated inhibition of Runx2 expression also led to reduced Runx2 transactivation and osteoblast differentiation. In contrast, inhibition of Fbw7α restored Runx2 levels and promoted osteoblast differentiation. We also observed reciprocal expression levels of Runx2 and Fbw7α in models of bone loss such as lactating (physiological bone loss condition) and ovariectomized (induction of surgical menopause) animals that show reduced Runx2 and enhanced Fbw7α, whereas this was reversed in the estrogen-treated ovariectomized animals. In addition, methylprednisolone (a synthetic glucocorticoid) treatment to neonatal rats showed a temporal decrease in Runx2 with a reciprocal increase in Fbw7 in their calvarium. Taken together, these data demonstrate that Fbw7α negatively regulates osteogenesis by targeting Runx2 for ubiquitin-mediated degradation in a GSK3β-dependent manner and thus provides a plausible explanation for GSK3β-mediated bone loss as described before.
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Affiliation(s)
- Yogesh Kumar
- From the Biochemistry Division, CSIR-Central Drug Research Institute (CSIR-CDRI), Sector-10, Jankipuram Extension, Lucknow, 226031 Uttar Pradesh and
| | - Isha Kapoor
- From the Biochemistry Division, CSIR-Central Drug Research Institute (CSIR-CDRI), Sector-10, Jankipuram Extension, Lucknow, 226031 Uttar Pradesh and
| | - Kainat Khan
- the Division of Endocrinology and Center for Research in Anabolic Skeletal Targets in Health and Illness (ASTHI), CSIR-Central Drug Research Institute (CSIR-CDRI), Sector-10, Jankipuram Extension, Lucknow, 226031 Uttar Pradesh, India
| | - Gatha Thacker
- From the Biochemistry Division, CSIR-Central Drug Research Institute (CSIR-CDRI), Sector-10, Jankipuram Extension, Lucknow, 226031 Uttar Pradesh and
| | - Mohd Parvez Khan
- the Division of Endocrinology and Center for Research in Anabolic Skeletal Targets in Health and Illness (ASTHI), CSIR-Central Drug Research Institute (CSIR-CDRI), Sector-10, Jankipuram Extension, Lucknow, 226031 Uttar Pradesh, India
| | - Nidhi Shukla
- From the Biochemistry Division, CSIR-Central Drug Research Institute (CSIR-CDRI), Sector-10, Jankipuram Extension, Lucknow, 226031 Uttar Pradesh and
| | - Jitendra Kumar Kanaujiya
- From the Biochemistry Division, CSIR-Central Drug Research Institute (CSIR-CDRI), Sector-10, Jankipuram Extension, Lucknow, 226031 Uttar Pradesh and
| | - Sabyasachi Sanyal
- From the Biochemistry Division, CSIR-Central Drug Research Institute (CSIR-CDRI), Sector-10, Jankipuram Extension, Lucknow, 226031 Uttar Pradesh and
| | - Naibedya Chattopadhyay
- the Division of Endocrinology and Center for Research in Anabolic Skeletal Targets in Health and Illness (ASTHI), CSIR-Central Drug Research Institute (CSIR-CDRI), Sector-10, Jankipuram Extension, Lucknow, 226031 Uttar Pradesh, India
| | - Arun Kumar Trivedi
- From the Biochemistry Division, CSIR-Central Drug Research Institute (CSIR-CDRI), Sector-10, Jankipuram Extension, Lucknow, 226031 Uttar Pradesh and
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Zhou Z, He C, Wang J. Regulation mechanism of Fbxw7-related signaling pathways (Review). Oncol Rep 2015; 34:2215-24. [PMID: 26324296 DOI: 10.3892/or.2015.4227] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/25/2015] [Accepted: 05/29/2015] [Indexed: 11/05/2022] Open
Abstract
F-box and WD repeat domain-containing 7 (Fbxw7), the substrate-recognition component of SCFFbxw7 complex, is thought to be a tumor suppressor involved in cell growth, proliferation, differentiation and survival. Although an increasing number of ubiquitin substrates of Fbxw7 have been identified, the best characterized substrates are cyclin E and c-Myc. Fbxw7/cyclin E and Fbxw7/c-Myc pathways are tightly regulated by multiple regulators. Fbxw7 has been identified as a tumor suppressor in hepatocellular carcinoma. This review focused on the regulation of Fbxw7/cyclin E and Fbxw7/c-Myc pathways and discussed findings to gain a better understanding of the role of Fbxw7 in hepatocellular carcinoma.
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Affiliation(s)
- Zhenyu Zhou
- Department of Hepatobiliary Surgery, Sun Yat-sen Memorial Hospital, Sun Yat‑sen University, Guangzhou, Guangdong 510120, P.R. China
| | - Chuanchao He
- Department of Hepatobiliary Surgery, Sun Yat-sen Memorial Hospital, Sun Yat‑sen University, Guangzhou, Guangdong 510120, P.R. China
| | - Jie Wang
- Department of Hepatobiliary Surgery, Sun Yat-sen Memorial Hospital, Sun Yat‑sen University, Guangzhou, Guangdong 510120, P.R. China
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Shibata A, Machida J, Yamaguchi S, Kimura M, Tatematsu T, Miyachi H, Matsushita M, Kitoh H, Ishiguro N, Nakayama A, Higashi Y, Shimozato K, Tokita Y. Characterisation of novel RUNX2 mutation with alanine tract expansion from Japanese cleidocranial dysplasia patient. Mutagenesis 2015. [PMID: 26220009 DOI: 10.1093/mutage/gev057] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022] Open
Abstract
Cleidocranial dysplasia (CCD; MIM 119600) is an autosomal dominant skeletal dysplasia characterised by hypopalstic and/or aplastic clavicles, midface hypoplasia, absent or delayed closure of cranial sutures, moderately short stature, delayed eruption of permanent dentition and supernumerary teeth. The molecular pathogenesis can be explained in about two-thirds of CCD patients by haploinsufficiency of the RUNX2 gene. In our current study, we identified a novel and rare variant of the RUNX2 gene (c.181_189dupGCGGCGGCT) in a Japanese patient with phenotypic features of CCD. The insertion led an alanine tripeptide expansion (+3Ala) in the polyalanine tract. To date, a RUNX2 variant with alanine decapeptide expansion (+10Ala) is the only example of a causative variant of RUNX2 with polyalanine tract expansion to be reported, whilst RUNX2 (+1Ala) has been isolated from the healthy population. Thus, precise analyses of the RUNX2 (+3Ala) variant were needed to clarify whether the tripeptide expanded RUNX2 is a second disease-causing mutant with alanine tract expansion. We therefore investigated the biochemical properties of the mutant RUNX2 (+3Ala), which contains 20 alanine residues in the polyalanine tract. When transfected in COS7 cells, RUNX2 (+3Ala) formed intracellular ubiquitinated aggregates after 24h, and exerted a dominant negative effect in vitro. At 24h after gene transfection, whereas slight reduction was observed in RUNX2 (+10Ala), all of these mutants significantly activated osteoblast-specific element-2, a cis-acting sequence in the promoter of the RUNX2 target gene osteocalcin. The aggregation growth of RUNX2 (+3Ala) was clearly lower and slower than that of RUNX2 (+10Ala). Furthermore, we investigated several other RUNX2 variants with various alanine tract lengths, and found that the threshold for aggregation may be RUNX2 (+3Ala). We conclude that RUNX2 (+3Ala) is the cause of CCD in our current case, and that the accumulation of intracellular aggregates in vitro is related to the length of the alanine tract.
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Affiliation(s)
- Akio Shibata
- Department of Maxillofacial Surgery, Aichi-Gakuin University School of Dentistry, Nagoya 464-8651, Japan, Department of Perinatology, Institute for Developmental Research, Aichi Human Service Center, Kasugai 480-0392, Japan, Department of Oral and Maxillofacial Surgery, Ogaki Municipal Hospital, Ogaki 503-0864, Japan
| | - Junichiro Machida
- Department of Maxillofacial Surgery, Aichi-Gakuin University School of Dentistry, Nagoya 464-8651, Japan, Department of Oral and Maxillofacial Surgery, Toyota Memorial Hospital, Toyota 471-0821, Japan
| | - Seishi Yamaguchi
- Department of Maxillofacial Surgery, Aichi-Gakuin University School of Dentistry, Nagoya 464-8651, Japan, Department of Dentistry and Oral Surgery, Aichi Children's Health and Medical Center, Obu 474-8710, Japan
| | - Masashi Kimura
- Department of Maxillofacial Surgery, Aichi-Gakuin University School of Dentistry, Nagoya 464-8651, Japan, Department of Oral and Maxillofacial Surgery, Ogaki Municipal Hospital, Ogaki 503-0864, Japan
| | - Tadashi Tatematsu
- Department of Maxillofacial Surgery, Aichi-Gakuin University School of Dentistry, Nagoya 464-8651, Japan, Department of Perinatology, Institute for Developmental Research, Aichi Human Service Center, Kasugai 480-0392, Japan
| | - Hitoshi Miyachi
- Department of Maxillofacial Surgery, Aichi-Gakuin University School of Dentistry, Nagoya 464-8651, Japan
| | - Masaki Matsushita
- Department of Orthopaedic Surgery, Nagoya University, Graduate School of Medicine, Nagoya 466-8550, Aichi, Japan
| | - Hiroshi Kitoh
- Department of Orthopaedic Surgery, Nagoya University, Graduate School of Medicine, Nagoya 466-8550, Aichi, Japan
| | - Naoki Ishiguro
- Department of Orthopaedic Surgery, Nagoya University, Graduate School of Medicine, Nagoya 466-8550, Aichi, Japan
| | - Atsuo Nakayama
- Department of Embryology, Institute for Developmental Research, Aichi Human Service Center, Kasugai 480-0392, Japan
| | - Yujiro Higashi
- Department of Perinatology, Institute for Developmental Research, Aichi Human Service Center, Kasugai 480-0392, Japan
| | - Kazuo Shimozato
- Department of Maxillofacial Surgery, Aichi-Gakuin University School of Dentistry, Nagoya 464-8651, Japan
| | - Yoshihito Tokita
- Department of Maxillofacial Surgery, Aichi-Gakuin University School of Dentistry, Nagoya 464-8651, Japan, Department of Perinatology, Institute for Developmental Research, Aichi Human Service Center, Kasugai 480-0392, Japan,
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Watanabe K, Yumimoto K, Nakayama KI. FBXO21 mediates the ubiquitylation and proteasomal degradation of EID1. Genes Cells 2015; 20:667-74. [PMID: 26085330 DOI: 10.1111/gtc.12260] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/28/2015] [Accepted: 05/12/2015] [Indexed: 12/22/2022]
Abstract
Although identification of substrates for ubiquitin ligase (E3) is important for understanding its biological functions, detection of the interaction between an E3 and its substrates has remained challenging. We recently developed a new approach, termed differential proteomics-based identification of ubiquitylation substrates (DiPIUS), for the discovery of substrates of a given E3 ligase. We have now applied this approach to an uncharacterized human F-box protein, FBXO21, which serves as the substrate-recognition subunit of a SKP1-CUL1-F-box protein (SCF)-type E3, thereby identifying EID1 (EP300-interacting inhibitor of differentiation 1) as a candidate substrate. The central and COOH-terminal portion of FBXO21 was found to interact with the COOH-terminal region of EID1 in transfected cells. Over-expression of FBXO21 resulted in the down-regulation of EID1, whereas disruption of the FBXO21 gene with the CRISPR/Cas9 system stabilized EID1 and led to its accumulation in both the cytoplasm and nucleus. An in vitro ubiquitylation assay showed that EID1 is a direct substrate of SCF(FBXO)(21). Collectively, our results suggest that EID1 is a bona fide substrate of FBXO21 and that the control of EID1 abundance by SCF(FBXO)(21) might affect the transcriptional repression activity of EID1.
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Affiliation(s)
- Koki Watanabe
- Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan
| | - Kanae Yumimoto
- Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan
| | - Keiichi I Nakayama
- Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan
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Demasi M, Simões V, Bonatto D. Cross-talk between redox regulation and the ubiquitin-proteasome system in mammalian cell differentiation. Biochim Biophys Acta Gen Subj 2014; 1850:1594-606. [PMID: 25450485 DOI: 10.1016/j.bbagen.2014.10.031] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/25/2014] [Revised: 10/24/2014] [Accepted: 10/28/2014] [Indexed: 12/15/2022]
Abstract
BACKGROUND Embryogenesis and stem cell differentiation are complex and orchestrated signaling processes. Reactive oxygen species (ROS) act as essential signal transducers in cellular differentiation, as has been shown through recent discoveries. On the other hand, the ubiquitin-proteasome system (UPS) has long been known to play an important role in all cellular regulated processes, including differentiation. SCOPE OF REVIEW In the present review, we focus on findings that highlight the interplay between redox signaling and the UPS regarding cell differentiation. Through systems biology analyses, we highlight major routes during cardiomyocyte differentiation based on redox signaling and UPS modulation. MAJOR CONCLUSION Oxygen availability and redox signaling are fundamental regulators of cell fate upon differentiation. The UPS plays an important role in the maintenance of pluripotency and the triggering of differentiation. GENERAL SIGNIFICANCE Cellular differentiation has been a matter of intense investigation mainly because of its potential therapeutic applications. Understanding regulatory mechanisms underlying cell differentiation is an important issue. Correspondingly, the role of UPS and regulation of redox processes have been emerged as essential factors to control the fate of cells upon differentiation. This article is part of a Special Issue entitled Redox regulation of differentiation and de-differentiation.
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Affiliation(s)
- Marilene Demasi
- Laboratory of Biochemistry and Biophysics, Instituto Butantan, São Paulo, SP, Brazil.
| | - Vanessa Simões
- Department of Genetics and Evolutive Biology, IB, Universidade de São Paulo, São Paulo, Brazil
| | - Diego Bonatto
- Center of Biotechnology, Universidade Federal do Rio Grande do Sul., Porto Alegre, RS, Brazil.
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Characterization of novel MSX1 mutations identified in Japanese patients with nonsyndromic tooth agenesis. PLoS One 2014; 9:e102944. [PMID: 25101640 PMCID: PMC4125152 DOI: 10.1371/journal.pone.0102944] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/30/2014] [Accepted: 06/24/2014] [Indexed: 11/19/2022] Open
Abstract
Since MSX1 and PAX9 are linked to the pathogenesis of nonsyndromic tooth agenesis, we performed detailed mutational analysis of these two genes sampled from Japanese patients. We identified two novel MSX1 variants with an amino acid substitution within the homeodomain; Thr174Ile (T174I) from a sporadic hypodontia case and Leu205Arg (L205R) from a familial oligodontia case. Both the Thr174 and Leu205 residues in the MSX1 homeodomain are highly conserved among different species. To define possible roles of mutations at these amino acids in the pathogenesis of nonsyndromic tooth agenesis, we performed several functional analyses. It has been demonstrated that MSX1 plays a pivotal role in hard tissue development as a suppressor for mesenchymal cell differentiation. To evaluate the suppression activity of the variants in mesenchymal cells, we used the myoD-promoter, which is one of convenient reporter assay system for MSX1. Although the gene products of these MSX1 variants are stable and capable of normal nuclear localization, they do not suppress myoD-promoter activity in differentiated C2C12 cells. To clarify the molecular mechanisms underlying our results, we performed further analyses including electrophoretic mobility shift assays, and co-immunoprecipitation assays to survey the molecular interactions between the mutant MSX1 proteins and the oligonucleotide DNA with MSX1 consensus binding motif or EZH2 methyltransferase. Since EZH2 is reported to interact with MSX1 and regulate MSX1 mediated gene suppression, we hypothesized that the T174I and L205R substitutions would impair this interaction. We conclude from the results of our experiments that the DNA binding ability of MSX1 is abolished by these two amino acid substitutions. This illustrates a causative role of the T174I and L205R MSX1 homeodomain mutations in tooth agenesis, and suggests that they may influence cell proliferation and differentiation resulting in lesser tooth germ formation in vivo.
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Luan Y, Wang P. FBW7-mediated ubiquitination and degradation of KLF5. World J Biol Chem 2014; 5:216-223. [PMID: 24921010 PMCID: PMC4050114 DOI: 10.4331/wjbc.v5.i2.216] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/14/2013] [Revised: 01/15/2014] [Accepted: 03/18/2014] [Indexed: 02/05/2023] Open
Abstract
Krüppel-like factor (KLF) family proteins are transcription factors that regulate numerous cellular functions, such as cell proliferation, differentiation, and cell death. Posttranslational modification of KLF proteins is important for their transcriptional activities and biological functions. One KLF family member with important roles in cell proliferation and tumorigenesis is KLF5. The function of KLF5 is tightly controlled by post-translational modifications, including SUMOylation, phosphorylation, and ubiquitination. Recent studies from our lab and others’ have demonstrated that the tumor suppressor FBW7 is an essential E3 ubiquitin ligase that targets KLF5 for ubiquitination and degradation. KLF5 contains functional Cdc4 phospho-degrons (CPDs), which are required for its interaction with FBW7. Mutation of CPDs in KLF5 blocks the ubiquitination and degradation of KLF5 by FBW7. The protein kinase Glycogen synthase kinase 3β is involved in the phosphorylation of KLF5 CPDs. In both cancer cell lines and mouse models, it has been shown that FBW7 regulates the expression of KLF5 target genes through the modulation of KLF5 stability. In this review, we summarize the current progress on delineating FBW7-mediated KLF5 ubiquitination and degradation.
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