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Egea V, Lutterberg K, Steinritz D, Rothmiller S, Steinestel K, Caca J, Nerlich A, Blum H, Reschke S, Khani S, Bartelt A, Worek F, Thiermann H, Weber C, Ries C. Targeting miR-497-5p rescues human keratinocyte dysfunction upon skin exposure to sulfur mustard. Cell Death Dis 2024; 15:585. [PMID: 39127703 PMCID: PMC11316827 DOI: 10.1038/s41419-024-06974-2] [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: 01/04/2024] [Revised: 07/30/2024] [Accepted: 08/05/2024] [Indexed: 08/12/2024]
Abstract
Sulfur mustard (SM) is a highly toxic chemical warfare agent. Exposure to SM results in various pathologies including skin lesions with subsequent impaired wound healing. To date, there are no effective treatments available. Here we discover a SM-triggered pathomechanism involving miR-497-5p and its target survivin which contributes to keratinocyte dysfunction. Transcriptome analysis using RNA-seq in normal human epidermal keratinocytes (NHEK) revealed that SM evoked differential expression of 1896 mRNAs and 25 miRNAs with many of these RNAs known to be involved in keratinocyte function and wound healing. We demonstrated that keratinocyte differentiation and proliferation were efficiently regulated by miRNAs induced in skin cells after exposure to SM. The inhibition of miR-497-5p counteracted SM-induced premature differentiation and stimulated proliferation of NHEK. In addition, we showed that microneedle-mediated transdermal application of lipid-nanoparticles containing miR-497-5p inhibitor restored survivin biosynthesis and cellular functionality upon exposure to SM using human skin biopsies. Our findings expand the current understanding of SM-associated molecular toxicology in keratinocytes and highlight miR-497-5p as feasible clinical target for specific skin therapy in SM-exposed patients and beyond.
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Affiliation(s)
- Virginia Egea
- Institute for Cardiovascular Prevention (IPEK), Ludwig-Maximilians-University (LMU) in Munich, Munich, Germany.
- DZHK (German Centre for Cardiovascular Research), partner site Munich Heart Alliance, Munich, Germany.
| | - Karina Lutterberg
- Institute for Cardiovascular Prevention (IPEK), Ludwig-Maximilians-University (LMU) in Munich, Munich, Germany
| | - Dirk Steinritz
- Bundeswehr Institute of Pharmacology and Toxicology, Munich, Germany
| | - Simone Rothmiller
- Bundeswehr Institute of Pharmacology and Toxicology, Munich, Germany
| | - Konrad Steinestel
- Institute of Pathology and Molecular Pathology, Bundeswehrkrankenhaus Ulm, Ulm, Germany
| | - Jan Caca
- Institute for Cardiovascular Prevention (IPEK), Ludwig-Maximilians-University (LMU) in Munich, Munich, Germany
| | - Andreas Nerlich
- Institute of Pathology, Academic Clinic Munich-Bogenhausen, Munich, Germany
| | - Helmut Blum
- Laboratory for Functional Genome Analysis (LAFUGA), Gene Center, LMU Munich, Munich, Germany
| | - Sarah Reschke
- Laboratory for Functional Genome Analysis (LAFUGA), Gene Center, LMU Munich, Munich, Germany
| | - Sajjad Khani
- Institute for Cardiovascular Prevention (IPEK), Ludwig-Maximilians-University (LMU) in Munich, Munich, Germany
- DZHK (German Centre for Cardiovascular Research), partner site Munich Heart Alliance, Munich, Germany
| | - Alexander Bartelt
- Institute for Cardiovascular Prevention (IPEK), Ludwig-Maximilians-University (LMU) in Munich, Munich, Germany
- DZHK (German Centre for Cardiovascular Research), partner site Munich Heart Alliance, Munich, Germany
| | - Franz Worek
- Bundeswehr Institute of Pharmacology and Toxicology, Munich, Germany
| | - Horst Thiermann
- Bundeswehr Institute of Pharmacology and Toxicology, Munich, Germany
| | - Christian Weber
- Institute for Cardiovascular Prevention (IPEK), Ludwig-Maximilians-University (LMU) in Munich, Munich, Germany
- DZHK (German Centre for Cardiovascular Research), partner site Munich Heart Alliance, Munich, Germany
- Laboratory for Functional Genome Analysis (LAFUGA), Gene Center, LMU Munich, Munich, Germany
- Department of Pathology, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University Medical Centre, Maastricht, The Netherlands
- Munich Cluster for Systems Neurology (SyNergy), Munich, Germany
| | - Christian Ries
- Institute for Cardiovascular Prevention (IPEK), Ludwig-Maximilians-University (LMU) in Munich, Munich, Germany.
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Cui HS, Joo SY, Lee SY, Cho YS, Kim DH, Seo CH. Effect of Hypertrophic Scar Fibroblast-Derived Exosomes on Keratinocytes of Normal Human Skin. Int J Mol Sci 2023; 24:ijms24076132. [PMID: 37047109 PMCID: PMC10094451 DOI: 10.3390/ijms24076132] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/06/2022] [Revised: 02/16/2023] [Accepted: 03/22/2023] [Indexed: 04/14/2023] Open
Abstract
Epidermal keratinocytes are highly activated, hyper-proliferated, and abnormally differentiated in the post-burn hypertrophic scar (HTS); however, the effects of scar fibroblasts (SFs) on keratinocytes through cell-cell interaction in HTS remain unknown. Here, we investigated the effects of HTSF-derived exosomes on the proliferation and differentiation of normal human keratinocytes (NHKs) compared with normal fibroblasts (NFs) and their possible mechanism to provide a reference for clinical intervention of HTS. Fibroblasts were isolated and cultured from HTS and normal skin. Both HTSF-exosomes and NF-exosomes were extracted via a column-based method from the cell culture supernatant. NHKs were treated for 24 or 48 h with 100 μg/mL of cell-derived exosomes. The expression of proliferation markers (Ki-67 and keratin 14), activation markers (keratins 6, 16, and 17), differentiation markers (keratins 1 and 10), apoptosis factors (Bax, Bcl2, caspase 14, and ASK1), proliferation/differentiation regulators (p21 and p27), and epithelial-mesenchymal transition (EMT) markers (E-cadherin, N-cadherin, and vimentin) was investigated. Compared with NF-exosomes, HTSF-exosomes altered the molecular pattern of proliferation, activation, differentiation, and apoptosis, proliferation/differentiation regulators of NHKs, and EMT markers differently. In conclusion, our findings indicate that HTSF-derived exosomes may play a role in the epidermal pathological development of HTS.
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Affiliation(s)
- Hui Song Cui
- Burn Institute, Department of Rehabilitation Medicine, Hangang Sacred Heart Hospital, College of Medicine, Hallym University, Seoul 07247, Republic of Korea
| | - So Young Joo
- Department of Rehabilitation Medicine, Hangang Sacred Heart Hospital, College of Medicine, Hallym University, Seoul 07247, Republic of Korea
| | - Seung Yeol Lee
- Department of Physical Medicine and Rehabilitation, College of Medicine, Soonchunhyang University Hospital, Bucheon 14158, Republic of Korea
| | - Yoon Soo Cho
- Department of Rehabilitation Medicine, Hangang Sacred Heart Hospital, College of Medicine, Hallym University, Seoul 07247, Republic of Korea
| | - Dong Hyun Kim
- Department of Rehabilitation Medicine, Kangdong Sacred Heart Hospital, College of Medicine, Hallym University, Seoul 05355, Republic of Korea
| | - Cheong Hoon Seo
- Department of Rehabilitation Medicine, Hangang Sacred Heart Hospital, College of Medicine, Hallym University, Seoul 07247, Republic of Korea
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Henriet E, Abdallah F, Laurent Y, Guimpied C, Clement E, Simon M, Pichon C, Baril P. Targeting TGF-β1/miR-21 pathway in keratinocytes reveals protective effects of silymarin on imiquimod-induced psoriasis mouse model. JID INNOVATIONS 2022; 3:100175. [PMID: 36968096 PMCID: PMC10034514 DOI: 10.1016/j.xjidi.2022.100175] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2022] [Revised: 11/10/2022] [Accepted: 11/11/2022] [Indexed: 12/23/2022] Open
Abstract
Epidermal cells integrate multiple signals that activate the signaling pathways involved in skin homeostasis. TGF-β1 signaling pathway upregulates microRNA (miR)-21-5p in keratinocytes and is often deregulated in skin diseases. To identify the bioactive compounds that enable to modulate the TGF-β1/miR-21-5p signaling pathway, we screened a library of medicinal plant extracts using our miR-ON RILES luciferase reporter system placed under the control of the miR-21-5p in keratinocytes treated with TGF-β1. We identified silymarin, a mixture of flavonolignans extracted from Silybum marianum (L.) Gaertn., as the most potent regulator of miR-21-5p expression. Using Argonaute 2 immunoprecipitation and RT-qPCR, we showed that silymarin regulates the expression of miR-21-5p through a noncanonical TGF-β1 signaling pathway, whereas RNA-sequencing analysis revealed three unexpected transcriptomic signatures associated with keratinocyte differentiation, cell cycle, and lipid metabolism. Mechanistically, we demonstrated that SM blocks cell cycle progression, inhibits keratinocyte differentiation through repression of Notch3 expression, stimulates lipid synthesis via activation of PPARγ signaling and inhibits inflammatory responses by suppressing the transcriptional activity of NF-κB. We finally showed that topical application of silymarin alleviates the development of imiquimod-induced psoriasiform lesions in mice by abrogating the altered expression levels of markers involved in inflammation, proliferation, differentiation, and lipid metabolism.
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Yamaguchi H, Shen J, Little DR, Li M, Sozen S, Suzuki K, Mishina Y, Komatsu Y. Enhanced BMP signaling through ALK2 attenuates keratinocyte differentiation. Biochem Biophys Res Commun 2022; 629:101-105. [PMID: 36116371 DOI: 10.1016/j.bbrc.2022.09.014] [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: 07/25/2022] [Revised: 07/29/2022] [Accepted: 09/02/2022] [Indexed: 11/02/2022]
Abstract
Accumulated studies have suggested that bone morphogenetic proteins (BMPs) are critical for skin development. However, it remains elusive how BMP signaling via ALK2 (aka ACVR1), one of the important BMP type I receptors, regulates keratinocyte differentiation. To address this question, we utilized a genetic system that enhances BMP signaling via ALK2 in an epidermis-specific manner in mice (hereafter ca-Alk2:K14-Cre). Ca-Alk2:K14-Cre mice displayed a sticky and hairless skin phenotype with a thinner epidermis incapable of differentiating. Although cellular proliferation and survival were comparable between wild-type and ca-Alk2:K14-Cre mice, skin differentiation was severely hampered in ca-Alk2:K14-Cre mice. To uncover the mechanism of altered keratinocyte differentiation, we performed a transcriptome analysis. As a result, we found that the expression levels of cell cycle inhibitor p21 were increased in ca-Alk2:K14-Cre mice. Our findings suggest that aberrant BMP signaling via ALK2 positively regulates p21 expression that attenuates keratinocyte differentiation, and further highlights the critical role of BMP signaling in skin development.
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Affiliation(s)
- Hiroyuki Yamaguchi
- Department of Pediatrics, McGovern Medical School, The University of Texas Health Science Center at Houston, Houston, TX, 77030, USA
| | - Jingling Shen
- Institute of Life Sciences, College of Life and Environmental Sciences, Wenzhou University, Wenzhou, 325035, China.
| | - Danielle R Little
- Department of Pediatrics, McGovern Medical School, The University of Texas Health Science Center at Houston, Houston, TX, 77030, USA; Department of Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, TN, 38105, USA
| | - Margaret Li
- Department of Pediatrics, McGovern Medical School, The University of Texas Health Science Center at Houston, Houston, TX, 77030, USA; Department of Kinesiology, Rice University Wiess School of Natural Science, Houston, TX, 77005, USA
| | - Serra Sozen
- Department of Pediatrics, McGovern Medical School, The University of Texas Health Science Center at Houston, Houston, TX, 77030, USA; Department of Medicine, The Robert Larner, M.D. College of Medicine, University of Vermont, Burlington, VT, 05405, USA
| | - Kentaro Suzuki
- Faculty of Life and Environmental Sciences, University of Yamanashi, Yamanashi, 400-8510, Japan
| | - Yuji Mishina
- Department of Biologic and Materials Sciences & Prosthodontics, School of Dentistry, University of Michigan, Ann Arbor, MI, 48109, USA
| | - Yoshihiro Komatsu
- Department of Pediatrics, McGovern Medical School, The University of Texas Health Science Center at Houston, Houston, TX, 77030, USA; Graduate Program in Genes and Development, The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, TX, 77030, USA.
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Molecular Mechanisms of Cutaneous Squamous Cell Carcinoma. Int J Mol Sci 2022; 23:ijms23073478. [PMID: 35408839 PMCID: PMC8998533 DOI: 10.3390/ijms23073478] [Citation(s) in RCA: 40] [Impact Index Per Article: 13.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2022] [Revised: 03/18/2022] [Accepted: 03/18/2022] [Indexed: 12/25/2022] Open
Abstract
Non-melanoma skin cancers are cutaneous malignancies representing the most common form of cancer in the United States. They are comprised predominantly of basal cell carcinomas and squamous cell carcinomas (cSCC). The incidence of cSCC is increasing, resulting in substantial morbidity and ever higher treatment costs; currently in excess of one billion dollars, per annum. Here, we review research defining the molecular basis and development of cSCC that aims to provide new insights into pathogenesis and drive the development of novel, cost and morbidity saving therapies.
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Reedich EJ, Kalski M, Armijo N, Cox GA, DiDonato CJ. Spinal motor neuron loss occurs through a p53-and-p21-independent mechanism in the Smn 2B/- mouse model of spinal muscular atrophy. Exp Neurol 2020; 337:113587. [PMID: 33382987 DOI: 10.1016/j.expneurol.2020.113587] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/23/2020] [Revised: 12/12/2020] [Accepted: 12/23/2020] [Indexed: 12/22/2022]
Abstract
Spinal muscular atrophy (SMA) is a pediatric neuromuscular disease caused by genetic deficiency of the survival motor neuron (SMN) protein. Pathological hallmarks of SMA are spinal motor neuron loss and skeletal muscle atrophy. The molecular mechanisms that elicit and drive preferential motor neuron degeneration and death in SMA remain unclear. Transcriptomic studies consistently report p53 pathway activation in motor neurons and spinal cord tissue of SMA mice. Recent work has identified p53 as an inducer of spinal motor neuron loss in severe Δ7 SMA mice. Additionally, the cyclin-dependent kinase inhibitor P21 (Cdkn1a), an inducer of cell cycle arrest and mediator of skeletal muscle atrophy, is consistently increased in motor neurons, spinal cords, and other tissues of various SMA models. p21 is a p53 transcriptional target but can be independently induced by cellular stressors. To ascertain whether p53 and p21 signaling pathways mediate spinal motor neuron death in milder SMA mice, and how they affect the overall SMA phenotype, we introduced Trp53 and P21 null alleles onto the Smn2B/- background. We found that p53 and p21 depletion did not modulate the timing or degree of Smn2B/- motor neuron loss as evaluated using electrophysiological and immunohistochemical methods. Moreover, we determined that Trp53 and P21 knockout differentially affected Smn2B/- mouse lifespan: p53 ablation impaired survival while p21 ablation extended survival through Smn-independent mechanisms. These results demonstrate that p53 and p21 are not primary drivers of spinal motor neuron death in Smn2B/- mice, a milder SMA mouse model, as motor neuron loss is not alleviated by their ablation.
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Affiliation(s)
- Emily J Reedich
- Human Molecular Genetics and Physiology Program, Stanley Manne Children's Research Institute at Ann & Robert H. Lurie Children's Hospital, Chicago, IL, USA; Department of Pediatrics, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA
| | - Martin Kalski
- Human Molecular Genetics and Physiology Program, Stanley Manne Children's Research Institute at Ann & Robert H. Lurie Children's Hospital, Chicago, IL, USA
| | - Nicholas Armijo
- Human Molecular Genetics and Physiology Program, Stanley Manne Children's Research Institute at Ann & Robert H. Lurie Children's Hospital, Chicago, IL, USA
| | - Gregory A Cox
- The Jackson Laboratory, Bar Harbor, ME, USA; Graduate School of Biomedical Sciences and Engineering, University of Maine, Orono, ME, USA
| | - Christine J DiDonato
- Human Molecular Genetics and Physiology Program, Stanley Manne Children's Research Institute at Ann & Robert H. Lurie Children's Hospital, Chicago, IL, USA; Department of Pediatrics, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA.
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Y08197 is a novel and selective CBP/EP300 bromodomain inhibitor for the treatment of prostate cancer. Acta Pharmacol Sin 2019; 40:1436-1447. [PMID: 31097763 DOI: 10.1038/s41401-019-0237-5] [Citation(s) in RCA: 34] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/13/2018] [Accepted: 04/11/2019] [Indexed: 12/21/2022]
Abstract
In advanced prostate cancer, CREB (cAMP-responsive element-binding protein) binding protein (CBP) and its homolog EP300 are highly expressed; targeting the bromodomain of CBP is a new strategy for the treatment of prostate cancer. In the current study we identified Y08197, a novel 1-(indolizin-3-yl) ethanone derivative, as a selective inhibitor of CBP/EP300 bromodomain and explored its antitumor activity against prostate cancer cell lines in vitro. In the AlphaScreen assay, we demonstrated that Y08197 dose-dependently inhibited the CBP bromodomain with an IC50 value at 100.67 ± 3.30 nM. Y08197 also exhibited high selectivity for CBP/EP300 over other bromodomain-containing proteins. In LNCaP, 22Rv1 and VCaP prostate cancer cells, treatment with Y08197 (1, 5 μM) strongly affected downstream signaling transduction, thus markedly inhibiting the expression of androgen receptor (AR)-regulated genes PSA, KLK2, TMPRSS2, and oncogenes C-MYC and ERG. Notably, Y08197 potently inhibited cell growth in several AR-positive prostate cancer cell lines including LNCaP, 22Rv1, VCaP, and C4-2B. In 22Rv1 prostate cancer cells, treatment with Y08197 (1, 4, 16 μM) dose-dependently induced G0/G1 phase arrest and apoptosis. Furthermore, treatment with Y08197 (5 μM) significantly decreased ERG-induced invasive capacity of 22Rv1 prostate cancer cells detected in wound-healing assay and cell migration assay. Taken together, CBP/EP300 inhibitor Y08197 represents a promising lead compound for development as new therapeutics for the treatment of castration-resistant prostate cancer.
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Kreis NN, Louwen F, Yuan J. The Multifaceted p21 (Cip1/Waf1/ CDKN1A) in Cell Differentiation, Migration and Cancer Therapy. Cancers (Basel) 2019; 11:cancers11091220. [PMID: 31438587 PMCID: PMC6770903 DOI: 10.3390/cancers11091220] [Citation(s) in RCA: 176] [Impact Index Per Article: 29.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/19/2019] [Revised: 08/15/2019] [Accepted: 08/17/2019] [Indexed: 12/12/2022] Open
Abstract
Loss of cell cycle control is characteristic of tumorigenesis. The protein p21 is the founding member of cyclin-dependent kinase inhibitors and an important versatile cell cycle protein. p21 is transcriptionally controlled by p53 and p53-independent pathways. Its expression is increased in response to various intra- and extracellular stimuli to arrest the cell cycle ensuring genomic stability. Apart from its roles in cell cycle regulation including mitosis, p21 is involved in differentiation, cell migration, cytoskeletal dynamics, apoptosis, transcription, DNA repair, reprogramming of induced pluripotent stem cells, autophagy and the onset of senescence. p21 acts either as a tumor suppressor or as an oncogene depending largely on the cellular context, its subcellular localization and posttranslational modifications. In the present review, we briefly mention the general functions of p21 and summarize its roles in differentiation, migration and invasion in detail. Finally, regarding its dual role as tumor suppressor and oncogene, we highlight the potential, difficulties and risks of using p21 as a biomarker as well as a therapeutic target.
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Affiliation(s)
- Nina-Naomi Kreis
- Department of Gynecology and Obstetrics, University Hospital, J. W. Goethe-University, Theodor-Stern-Kai 7, D-60590 Frankfurt, Germany.
| | - Frank Louwen
- Department of Gynecology and Obstetrics, University Hospital, J. W. Goethe-University, Theodor-Stern-Kai 7, D-60590 Frankfurt, Germany
| | - Juping Yuan
- Department of Gynecology and Obstetrics, University Hospital, J. W. Goethe-University, Theodor-Stern-Kai 7, D-60590 Frankfurt, Germany
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Transcriptomic and proteomic analyses reveal new insights into the regulation of immune pathways during adenovirus type 2 infection. BMC Microbiol 2019; 19:15. [PMID: 30642258 PMCID: PMC6332865 DOI: 10.1186/s12866-018-1375-5] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/16/2018] [Accepted: 12/16/2018] [Indexed: 12/27/2022] Open
Abstract
Background Human adenovirus (Ad) infection leads to the changes of host cell gene expression and biosynthetic processes. Transcriptomics in adenovirus type 2 (Ad2)-infected lung fibroblasts (IMR-90) cells has previously been studied using RNA sequencing. However, this study included only two time points (12 and 24 hpi) using constrained 76 bp long sequencing reads. Therefore, a more detailed study of transcription at different phases of infection using an up-graded sequencing technique is recalled. Furthermore, the correlation between transcription and protein expression needs to be addressed. Results In total, 3556 unique cellular genes were identified as differentially expressed at the transcriptional level with more than 2-fold changes in Ad2-infected cells as compared to non-infected cells by using paired-end sequencing. Based on the kinetics of the gene expression changes at different times after infection, these RNAs fell into 20 clusters. Among them, cellular genes involved in immune response were highly up-regulated in the early phase before becoming down-regulated in the late phase. Comparison of differentially expressed genes at transcriptional and posttranscriptional levels revealed low correlation. Particularly genes involved in cellular immune pathways showed a negative correlation. Here, we highlight the genes which expose inconsistent expression profiles with an emphasis on key factors in cellular immune pathways including NFκB, JAK/STAT, caspases and MAVS. Different from their transcriptional profiles with up- and down-regulation in the early and late phase, respectively, these proteins were up-regulated in the early phase and were sustained in the late phase. A surprising finding was that the target genes of the sustained activators failed to show response. Conclusion There were features common to genes which play important roles in cellular immune pathways. Their expression was stimulated at both RNA and protein levels during the early phase. In the late phase however, their transcription was suppressed while protein levels remained stable. These results indicate that Ad2 and the host cell use different strategies to regulate cellular immune pathways. A control mechanism at the post-translational level must thus exist which is under the control of Ad2. Electronic supplementary material The online version of this article (10.1186/s12866-018-1375-5) contains supplementary material, which is available to authorized users.
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Reichrath J, Saternus R, Vogt T. Challenge and perspective: the relevance of ultraviolet (UV) radiation and the vitamin D endocrine system (VDES) for psoriasis and other inflammatory skin diseases. Photochem Photobiol Sci 2018; 16:433-444. [PMID: 28054069 DOI: 10.1039/c6pp00280c] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/01/2023]
Abstract
During evolution, the ability of many organisms to synthesize vitamin D photochemically represented, and still represents, a major driving factor for the development of life on earth. In humans because not more than 10-20% of the requirement of vitamin D can be satisfied by the diet (under most living conditions in the US and Europe), the remaining 80-90% need to be photochemically synthesized in the skin through the action of solar or artificial ultraviolet-B (UV-B) radiation. The skin is a key organ of the human body's vitamin D endocrine system (VDES), representing both the site of vitamin D synthesis and a target tissue for biologically active vitamin D metabolites. Human keratinocytes contain the enzymatic machinery (CYP27B1) for the synthesis of the biologically most active natural vitamin D metabolite 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), representing an autonomous vitamin D3 pathway. Cutaneous production of 1,25(OH)2D3 may mediate intracrine, autocrine and paracrine effects on keratinocytes and on neighboring cells. Many skin cells (including keratinocytes, sebocytes, fibroblasts, melanocytes, macrophages and other skin immune cells) express the vitamin D receptor (VDR), an absolute pre-requisite for exerting genomic effects of 1,25(OH)2D3 and analogs. The VDR is a member of the superfamily of trans-acting transcriptional regulatory factors, which also contains the steroid and thyroid hormone receptors as well as the retinoid-X receptors (RXR) and retinoic acid receptors (RAR). A large body of evidence, including cDNA microarray analyses of mRNAs, indicates that as many as 500-1000 genes may be controlled by VDR ligands that regulate a broad variety of cellular functions including growth, differentiation, and apoptosis. Clinical and laboratory investigations, including the observation that 1,25(OH)2D3 is very effective in inducing the terminal differentiation and in inhibiting the proliferation of cultured human keratinocytes have resulted in the use of 1,25(OH)2D3 and analogs for the treatment of psoriasis. Focussing on the UV-induced cutaneous synthesis of vitamin D, this review gives an update on the relevance of the VDES and of UV radiation for the management of psoriasis and other inflammatory skin diseases.
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Affiliation(s)
- Jörg Reichrath
- Center for Clinical and Experimental Photo-Dermatology, The Saarland University Hospital, 66421 Homburg, Germany and Department of Dermatology, The Saarland University Hospital, 66421 Homburg, Germany.
| | - Roman Saternus
- Center for Clinical and Experimental Photo-Dermatology, The Saarland University Hospital, 66421 Homburg, Germany and Department of Dermatology, The Saarland University Hospital, 66421 Homburg, Germany.
| | - Thomas Vogt
- Center for Clinical and Experimental Photo-Dermatology, The Saarland University Hospital, 66421 Homburg, Germany and Department of Dermatology, The Saarland University Hospital, 66421 Homburg, Germany.
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Wu X, Tommasi di Vignano A, Zhou Q, Michel-Dziunycz PJ, Bai F, Mi J, Qin J, Zu T, Hofbauer GFL. The ARE-binding protein Tristetraprolin (TTP) is a novel target and mediator of calcineurin tumor suppressing function in the skin. PLoS Genet 2018; 14:e1007366. [PMID: 29723192 PMCID: PMC5953486 DOI: 10.1371/journal.pgen.1007366] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/23/2017] [Revised: 05/15/2018] [Accepted: 04/16/2018] [Indexed: 01/12/2023] Open
Abstract
An increased incidence of skin inflammatory diseases is frequently observed in organtransplanted patients being treated with calcineurin inhibitor-based immunosuppressive agents. The mechanism of increased skin inflammation in this context has however not yet been clarified. Here we report an increased inflammation following inhibition of calcineurin signaling seen in both chemically induced mouse skin tumors and in tumors grafted from H-rasV12 expressing primary human keratinocytes (HKCs). Following UVB or TPA treatment, we specifically found that deletion of the calcineurin gene in mouse keratinocytes (MKCs) resulted in increased inflammation, and this was accompanied by the enhanced production of pro-inflammatory cytokines, such as TNFα, IL-8 and CXCL1. Furthermore, expression of the RNA-binding protein, tristetraprolin (TTP) was down-regulated in response to calcineurin inhibition, wherein TTP was shown to negatively regulate the production of pro-inflammatory cytokines in keratinocytes. The induction of TTP following TPA or UVB treatment was attenuated by calcineurin inhibition in keratinocytes, and correspondingly, disruption of calcineurin signaling down-regulated the amounts of TTP in both clinical and H-rasV12-transformed keratinocyte tumor models. Our results further demonstrated that calcineurin positively controls the stabilization of TTP in keratinocytes through a proteasome-dependent mechanism. Reducing the expression of TTP functionally promoted tumor growth of H-rasV12 expressing HKCs, while stabilizing TTP expression counteracted the tumor-promoting effects of calcineurin inhibition. Collectively these results suggest that calcineurin signaling, acting through TTP protein level stabilization, suppresses keratinocyte tumors by downregulating skin inflammation.
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Affiliation(s)
- Xunwei Wu
- Laboratory for Tissue Engineering and Regeneration and Shandong Provincial Key Laboratory of Oral Tissue Regeneration, School of Stomatology, Shandong University, Jinan, Shandong, China
- Cutaneous Biology Research Centre, Massachusetts General Hospital, Charlestown, MA, United States of America
- * E-mail:
| | - Alice Tommasi di Vignano
- Cutaneous Biology Research Centre, Massachusetts General Hospital, Charlestown, MA, United States of America
| | - Qian Zhou
- Laboratory for Tissue Engineering and Regeneration and Shandong Provincial Key Laboratory of Oral Tissue Regeneration, School of Stomatology, Shandong University, Jinan, Shandong, China
| | | | - Fuxiang Bai
- Laboratory for Tissue Engineering and Regeneration and Shandong Provincial Key Laboratory of Oral Tissue Regeneration, School of Stomatology, Shandong University, Jinan, Shandong, China
| | - Jun Mi
- Laboratory for Tissue Engineering and Regeneration and Shandong Provincial Key Laboratory of Oral Tissue Regeneration, School of Stomatology, Shandong University, Jinan, Shandong, China
| | - Jing Qin
- Laboratory for Tissue Engineering and Regeneration and Shandong Provincial Key Laboratory of Oral Tissue Regeneration, School of Stomatology, Shandong University, Jinan, Shandong, China
| | - Tingjian Zu
- Laboratory for Tissue Engineering and Regeneration and Shandong Provincial Key Laboratory of Oral Tissue Regeneration, School of Stomatology, Shandong University, Jinan, Shandong, China
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Xiang Q, Wang C, Zhang Y, Xue X, Song M, Zhang C, Li C, Wu C, Li K, Hui X, Zhou Y, Smaill JB, Patterson AV, Wu D, Ding K, Xu Y. Discovery and optimization of 1-(1H-indol-1-yl)ethanone derivatives as CBP/EP300 bromodomain inhibitors for the treatment of castration-resistant prostate cancer. Eur J Med Chem 2018; 147:238-252. [PMID: 29448139 DOI: 10.1016/j.ejmech.2018.01.087] [Citation(s) in RCA: 48] [Impact Index Per Article: 6.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/08/2017] [Revised: 01/11/2018] [Accepted: 01/26/2018] [Indexed: 01/08/2023]
Abstract
The CREB (cAMP responsive element binding protein) binding protein (CBP) and its homolog EP300 have emerged as new therapeutic targets for the treatment of cancer and inflammatory diseases. Here we report the identification, optimization and evaluation of 1-(1H-indol-1-yl)ethanone derivatives as CBP/EP300 inhibitors starting from fragment-based virtual screening (FBVS). A cocrystal structure of the inhibitor (22e) in complex with CBP provides a solid structural basis for further optimization. The most potent compound 32h binds to the CBP bromodomain and has an IC50 value of 0.037 μM in the AlphaScreen assay which was 2 times more potent than the reported CBP bromodomain inhibitor SGC-CBP30 in our hands. 32h also exhibit high selectivity for CBP/EP300 over other bromodomain-containing proteins. Notably, the ester derivative (29h) of compound 32h markedly inhibits cell growth in several prostate cancer cell lines including LNCaP, 22Rv1 and LNCaP derived C4-2B. Compound 29h suppresses the mRNA expression of full length AR (AR-FL), AR target genes and other oncogene in LNCaP cells. 29h also reduces the expression of PSA, the biomarker of prostate cancer. CBP/EP300 inhibitor 29h represents a promising lead compound for the development of new therapeutics for the treatment of castration-resistant prostate cancer.
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Affiliation(s)
- Qiuping Xiang
- Guangdong Provincial Key Laboratory of Biocomputing, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou, China; University of Chinese Academy of Sciences, No. 19 Yuquan Road, Beijing 100049, China
| | - Chao Wang
- Guangdong Provincial Key Laboratory of Biocomputing, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou, China; School of Pharmaceutical Sciences, Jilin University, Changchun, China, No. 1266 Fujin Road, Chaoyang District, Changchun, Jilin 130021, China
| | - Yan Zhang
- Guangdong Provincial Key Laboratory of Biocomputing, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou, China; University of Chinese Academy of Sciences, No. 19 Yuquan Road, Beijing 100049, China
| | - Xiaoqian Xue
- Guangdong Provincial Key Laboratory of Biocomputing, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou, China; University of Chinese Academy of Sciences, No. 19 Yuquan Road, Beijing 100049, China
| | - Ming Song
- Guangdong Provincial Key Laboratory of Biocomputing, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou, China
| | - Cheng Zhang
- Guangdong Provincial Key Laboratory of Biocomputing, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou, China; School of Pharmaceutical Sciences, Jilin University, Changchun, China, No. 1266 Fujin Road, Chaoyang District, Changchun, Jilin 130021, China
| | - Chenchang Li
- Guangdong Provincial Key Laboratory of Biocomputing, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou, China; School of Pharmaceutical Sciences, Jilin University, Changchun, China, No. 1266 Fujin Road, Chaoyang District, Changchun, Jilin 130021, China
| | - Chun Wu
- Guangdong Provincial Key Laboratory of Biocomputing, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou, China
| | - Kuai Li
- Guangdong Provincial Key Laboratory of Biocomputing, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou, China
| | - Xiaoyan Hui
- State Key Laboratory of Pharmaceutical Biotechnology, Department of Medicine, The University of Hong Kong, Hong Kong Special Administrative Region
| | - Yulai Zhou
- School of Pharmaceutical Sciences, Jilin University, Changchun, China, No. 1266 Fujin Road, Chaoyang District, Changchun, Jilin 130021, China
| | - Jeff B Smaill
- University of Auckland, Auckland Cancer Society Research Centre, School of Medical Sciences, Private Bag 92019, Auckland, New Zealand
| | - Adam V Patterson
- University of Auckland, Auckland Cancer Society Research Centre, School of Medical Sciences, Private Bag 92019, Auckland, New Zealand
| | - Donghai Wu
- Guangdong Provincial Key Laboratory of Biocomputing, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou, China
| | - Ke Ding
- School of Pharmacy, Jinan University, 601 Huangpu Avenue West, Guangzhou 510632, China
| | - Yong Xu
- Guangdong Provincial Key Laboratory of Biocomputing, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou, China.
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13
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Moody C. Mechanisms by which HPV Induces a Replication Competent Environment in Differentiating Keratinocytes. Viruses 2017; 9:v9090261. [PMID: 28925973 PMCID: PMC5618027 DOI: 10.3390/v9090261] [Citation(s) in RCA: 67] [Impact Index Per Article: 8.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2017] [Revised: 09/14/2017] [Accepted: 09/15/2017] [Indexed: 12/15/2022] Open
Abstract
Human papillomaviruses (HPV) are the causative agents of cervical cancer and are also associated with other genital malignancies, as well as an increasing number of head and neck cancers. HPVs have evolved their life cycle to contend with the different cell states found in the stratified epithelium. Initial infection and viral genome maintenance occurs in the proliferating basal cells of the stratified epithelium, where cellular replication machinery is abundant. However, the productive phase of the viral life cycle, including productive replication, late gene expression and virion production, occurs upon epithelial differentiation, in cells that normally exit the cell cycle. This review outlines how HPV interfaces with specific cellular signaling pathways and factors to provide a replication-competent environment in differentiating cells.
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Affiliation(s)
- Cary Moody
- Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
- Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
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14
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Alonso-Lecue P, de Pedro I, Coulon V, Molinuevo R, Lorz C, Segrelles C, Ceballos L, López-Aventín D, García-Valtuille A, Bernal JM, Mazorra F, Pujol RM, Paramio J, Ramón Sanz J, Freije A, Toll A, Gandarillas A. Inefficient differentiation response to cell cycle stress leads to genomic instability and malignant progression of squamous carcinoma cells. Cell Death Dis 2017; 8:e2901. [PMID: 28661481 PMCID: PMC5520915 DOI: 10.1038/cddis.2017.259] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/16/2016] [Revised: 04/12/2017] [Accepted: 05/04/2017] [Indexed: 12/14/2022]
Abstract
Squamous cell carcinoma (SCC) or epidermoid cancer is a frequent and aggressive malignancy. However in apparent paradox it retains the squamous differentiation phenotype except for very dysplastic lesions. We have shown that cell cycle stress in normal epidermal keratinocytes triggers a squamous differentiation response involving irreversible mitosis block and polyploidisation. Here we show that cutaneous SCC cells conserve a partial squamous DNA damage-induced differentiation response that allows them to overcome the cell division block. The capacity to divide in spite of drug-induced mitotic stress and DNA damage made well-differentiated SCC cells more genomically instable and more malignant in vivo. Consistently, in a series of human biopsies, non-metastatic SCCs displayed a higher degree of chromosomal alterations and higher expression of the S phase regulator Cyclin E and the DNA damage signal γH2AX than the less aggressive, non-squamous, basal cell carcinomas. However, metastatic SCCs lost the γH2AX signal and Cyclin E, or accumulated cytoplasmic Cyclin E. Conversely, inhibition of endogenous Cyclin E in well-differentiated SCC cells interfered with the squamous phenotype. The results suggest a dual role of cell cycle stress-induced differentiation in squamous cancer: the resulting mitotic blocks would impose, when irreversible, a proliferative barrier, when reversible, a source of genomic instability, thus contributing to malignancy.
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Affiliation(s)
- Pilar Alonso-Lecue
- Cell Cycle, Stem Cell Fate and Cancer Laboratory, Instituto de Investigación Marqués de Valdecilla (IDIVAL), Santander, Spain
| | - Isabel de Pedro
- Cell Cycle, Stem Cell Fate and Cancer Laboratory, Instituto de Investigación Marqués de Valdecilla (IDIVAL), Santander, Spain
| | - Vincent Coulon
- Institut de Genétique Moléculaire de Montpellier, CNRS/UM2, Montpellier, France
| | - Rut Molinuevo
- Cell Cycle, Stem Cell Fate and Cancer Laboratory, Instituto de Investigación Marqués de Valdecilla (IDIVAL), Santander, Spain
| | - Corina Lorz
- Molecular Oncology Unit, Department of Basic Research, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT), CIBERONC, Madrid, Spain
| | - Carmen Segrelles
- Molecular Oncology Unit, Department of Basic Research, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT), CIBERONC, Madrid, Spain
| | - Laura Ceballos
- Cell Cycle, Stem Cell Fate and Cancer Laboratory, Instituto de Investigación Marqués de Valdecilla (IDIVAL), Santander, Spain
| | | | | | - José M Bernal
- Cell Cycle, Stem Cell Fate and Cancer Laboratory, Instituto de Investigación Marqués de Valdecilla (IDIVAL), Santander, Spain.,Department of Cardiovascular Surgery, Hospital Universitario Marqués de Valdecilla, Santander, Spain
| | - Francisco Mazorra
- Clínica Mompía, Mompía, Spain.,Department of Pathology, Hospital Universitario Marqués de Valdecilla, Santander, Spain
| | - Ramón M Pujol
- Department of Dermatology, Hospital del Mar, Barcelona, Spain.,Institut Hospital del Mar d'Investigacions Mèdiques, Barcelona, Spain
| | - Jesús Paramio
- Molecular Oncology Unit, Department of Basic Research, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT), CIBERONC, Madrid, Spain
| | - J Ramón Sanz
- Cell Cycle, Stem Cell Fate and Cancer Laboratory, Instituto de Investigación Marqués de Valdecilla (IDIVAL), Santander, Spain.,Clínica Mompía, Mompía, Spain.,Department of Plastic Surgery, Hospital Universitario Marqués de Valdecilla, Santander, Spain
| | - Ana Freije
- Cell Cycle, Stem Cell Fate and Cancer Laboratory, Instituto de Investigación Marqués de Valdecilla (IDIVAL), Santander, Spain
| | - Agustí Toll
- Department of Dermatology, Hospital del Mar, Barcelona, Spain.,Institut Hospital del Mar d'Investigacions Mèdiques, Barcelona, Spain
| | - Alberto Gandarillas
- Cell Cycle, Stem Cell Fate and Cancer Laboratory, Instituto de Investigación Marqués de Valdecilla (IDIVAL), Santander, Spain.,INSERM, Languedoc-Roussillon, Montpellier, France
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15
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Park MH, Lee HJ, Lee HL, Son DJ, Ju JH, Hyun BK, Jung SH, Song JK, Lee DH, Hwang CJ, Han SB, Kim S, Hong JT. Parkin Knockout Inhibits Neuronal Development via Regulation of Proteasomal Degradation of p21. Am J Cancer Res 2017; 7:2033-2045. [PMID: 28656059 PMCID: PMC5485421 DOI: 10.7150/thno.19824] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/26/2017] [Accepted: 03/31/2017] [Indexed: 11/05/2022] Open
Abstract
PARK2 encodes for the E3 ubiquitin ligase parkin and is implicated in the development of Parkinson's disease (PD). Although the neuroprotective role of parkin is well known, the mechanism of PARK2's function in neural stem differentiation has not yet been thoroughly studied. Co-expressions network analysis showed that synaptosomal-associated protein 25 (SNAP-25) and brain-derived neurotrophic factor (BDNF) were positively correlated with parkin, but negatively correlated with p21 in human patient brain. We investigated a link between the ubiquitin E3 ligase parkin and proteasomal degradation of p21 for the control of neural stem cell differentiation. We found that the neurogenesis was lowered in PARK2 knockout (KO) mice compared with non-tg mice. Expression of the marker protein for neural cell differentiation such as class III beta tubulin (TUBBIII), glial fibrillary acidic protein (GFAP) and neurofilament, as well as SNAP25 and BDNF, was down regulated in PARK2 KO mice. Associated with the loss of differentiation function, p21 protein was highly accumulated in the neural stem cells of PARK2 KO mice. We discovered that p21 directly binds with parkin and is ubiquitinated by parkin which resulted in the loss of cell differentiation ability. Introduction of p21 shRNA in PARK2 KO mice significantly rescued the differentiation efficacy as well as SNAP25 and BDNF expression. c-Jun N-terminal kinase (JNK) pathway is implicated in neurogenesis and p21 degradation. We also defined the decreased p21 ubiquitination and differentiation ability were reversed after treatment with JNK inhibitor, SP600125 in PARK2 KO mice derived neural stem cells. Thus, the present study indicated that parkin knockout inhibits neural stem cell differentiation by JNK-dependent proteasomal degradation of p21.
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16
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Reichrath J, Zouboulis CC, Vogt T, Holick MF. Targeting the vitamin D endocrine system (VDES) for the management of inflammatory and malignant skin diseases: An historical view and outlook. Rev Endocr Metab Disord 2016; 17:405-417. [PMID: 27447175 DOI: 10.1007/s11154-016-9353-4] [Citation(s) in RCA: 37] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/29/2022]
Abstract
Vitamin D represents one of the major driving factors for the development of life on earth and for human evolution. While up to 10-20 % of the human organism's requirements in vitamin D can be obtained by the diet (under most living conditions in the USA and Europe), approximately 90 % of all needed vitamin D has to be photosynthesized in the skin through the action of the sun (ultraviolet-B (UV-B)). The skin represents a key organ of the human body's vitamin D endocrine system (VDES), being both the site of vitamin D synthesis and a target tissue for biologically active vitamin D metabolites. It was shown that human keratinocytes possess the enzymatic machinery (CYP27B1) for the synthesis of the biologically most active natural vitamin D metabolite 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), representing an autonomous vitamin D3 pathway. Cutaneous production of 1,25(OH)2D3 may exert intracrine, autocrine, and paracrine effects on keratinocytes and on neighboring cells. Many skin cells (including keratinocytes, sebocytes, fibroblasts, melanocytes, and skin immune cells) express the vitamin D receptor (VDR), an absolute pre-requisite for the mediation of genomic effects of 1,25(OH)2D3 and analogs. VDR belongs to the superfamily of trans-acting transcriptional regulatory factors, which includes the steroid and thyroid hormone receptors as well as the retinoid X receptors (RXR) and retinoic acid receptors (RAR). Numerous studies, including cDNA microarray analyses of messenger RNAs (mRNAs), indicate that as many as 500-1000 genes may be regulated by VDR ligands that control various cellular functions including growth, differentiation, and apoptosis. The observation that 1,25(OH)2D3 is extremely effective in inducing the terminal differentiation and in inhibiting the proliferation of cultured human keratinocytes has resulted in the use of vitamin D analogs for the treatment of psoriasis. This review gives an historical view and summarizes our present knowledge about the relevance of the VDES for the management of inflammatory and malignant skin diseases.
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Affiliation(s)
- Jörg Reichrath
- Center for Clinical and Experimental Photo-Dermatology and Department of Dermatology, The Saarland University Hospital, Kirrbergerstr, 66421, Homburg, Germany.
| | - Christos C Zouboulis
- Departments of Dermatology, Venereology, Allergology and Immunology, Dessau Medical Center, Dessau, Germany
| | - Thomas Vogt
- Center for Clinical and Experimental Photo-Dermatology and Department of Dermatology, The Saarland University Hospital, Kirrbergerstr, 66421, Homburg, Germany
| | - Michael F Holick
- Section of Endocrinology, Diabetes and Nutrition, Department of Medicine, Boston University Medical Center, 85 E Newton St M-1013, Boston, MA, 02118, USA
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17
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Costa MJ, Walls J, Dickerman A, Ames PF, Roth LM, Guinee D. Cell Cycle Control of Ovarian Granulosa Cells in Tumors and Cysts. Int J Surg Pathol 2016. [DOI: 10.1177/106689699600400201] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Ovarian granulosa cell tumors (GCTs) behave unpredictably. Stage I patients suffer recurrences many years after treatment, and histopathologic evaluation of the primary GCT offers few clues. p21IPl/wafl (Waft), the principle downstream effector of the p53-dependent pathway of growth control, inhibits cyclin-dependent kinases responsible for conversion of GI to S in the cell cycle. This study hypothesizes that immunohistochemistry for these proliferation-related markers will help discern granulosa cell growth control and may predict the GCTs' clinical behavior. Paraffin sections from a surgical series of 43 GCTs (34 primary and 9 recurrent: 19 diffuse and 18 typical adult; 6 juvenile type) and 12 benign cysts (8 follicular and 4 corpora luteal) were immunostained for Waft and p53. Ki67 (MIB-1 clone) proliferation index is the percent of nuclei immunoreactive on a count of at least 400. The 43 GCTs stained as follows: 40% Waft + (11 St = < 10%, 4 S2 = 10-50%, and 2 S3 = > 50% of nuclei) and 28% p53 + (2 W = weak, 8 S 1, 2 S2). All p53 + GCTs stained with Waf 1; the number of immunoreactive nuclei correlated (P < .00001). Juvenile more often than adult type GCTs exhibited Waf 1 (6/6) and p53 staining (5/6) (P < .001). No difference in pS3 or Waft staining was present in primary compared with recurrent GCTs. Ki67 proliferation index for GCTs ranged from 1 to 50% (mean, 13.8%; median, 10.9%) and associated with both p53 and Waft (P < .000 1). The 12 benign cysts stained as follows: 100% Waft + (6 S1, 5 S2, 1 S3) and 75% p53+ (4 W, 4 S1, 1 S2). Of the 43 patients, 41 were available for follow-up study: 15 suffered recurrences after 16-133 (mean 59.3, median 55) months, and 26 were disease-free 21-369 (mean 78.2, median 57.5) months after diagnosis. Waft staining of the primary GCT does not help predict recurrence. All GCTs immunoreactive for p53 produce Waft, suggesting detection of an active wild type p53 rather than overproduction of mutant p53. Waft is produced by granulosa cells in all benign functional cysts, suggesting a physiologic role in the ovulation sequence. Waf t's association with proliferation in GCTs suggests possible physiologic feedback. Proliferation (correlated with histopathologic grading) in GCTs may signify appropriate feedback control; thus it is not a predictor of aggressive clinical behavior.
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Affiliation(s)
- Michael J. Costa
- Pathology Department, Building Pat. 1, University of California, Davis, Medical Center, 2315 Stockton Boulevard, Sacramento, CA 95817
| | | | | | - Peter F. Ames
- Pathology Department, University of California, Davis, Davis and Sacramento, California
| | - Lawrence M. Roth
- Pathology Department, Indiana University Medical Center, Indianapolis, Indiana
| | - Donald Guinee
- Pathology Department, University of Utah Medical Center, Salt Lake City, Utah
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18
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Dotto GP, Rustgi AK. Squamous Cell Cancers: A Unified Perspective on Biology and Genetics. Cancer Cell 2016; 29:622-637. [PMID: 27165741 PMCID: PMC4870309 DOI: 10.1016/j.ccell.2016.04.004] [Citation(s) in RCA: 228] [Impact Index Per Article: 25.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/28/2015] [Revised: 01/20/2016] [Accepted: 04/07/2016] [Indexed: 01/11/2023]
Abstract
Squamous cell carcinomas (SCCs) represent the most frequent human solid tumors and are a major cause of cancer mortality. These highly heterogeneous tumors arise from closely interconnected epithelial cell populations with intrinsic self-renewal potential inversely related to the stratified differentiation program. SCCs can also originate from simple or pseudo-stratified epithelia through activation of quiescent cells and/or a switch in cell-fate determination. Here, we focus on specific determinants implicated in the development of SCCs by recent large-scale genomic, genetic, and epigenetic studies, and complementary functional analysis. The evidence indicates that SCCs from various body sites, while clinically treated as separate entities, have common determinants, pointing to a unified perspective of the disease and potential new avenues for prevention and treatment.
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Affiliation(s)
- G Paolo Dotto
- Department of Biochemistry, University of Lausanne, Epalinges 1066, Switzerland; Cutaneous Biology Research Center, Massachusetts General Hospital, Charlestown, MA 02129, USA.
| | - Anil K Rustgi
- Division of Gastroenterology, Departments of Medicine and Genetics, Abramson Cancer Center, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA.
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19
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Recombinant human alpha fetoprotein synergistically potentiates the anti-cancer effects of 1'-S-1'-acetoxychavicol acetate when used as a complex against human tumours harbouring AFP-receptors. Oncotarget 2016; 6:16151-67. [PMID: 26158863 PMCID: PMC4599262 DOI: 10.18632/oncotarget.3951] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/28/2014] [Accepted: 04/08/2015] [Indexed: 01/01/2023] Open
Abstract
Purpose Previous in vitro and in vivo studies have reported that 1′-S-1′-acetoxychavicol acetate (ACA) isolated from rhizomes of the Malaysian ethno-medicinal plant Alpinia conchigera Griff (Zingiberaceae) induces apoptosis-mediated cell death in tumour cells via dysregulation of the NF-κB pathway. However there were some clinical development drawbacks such as poor in vivo solubility, depreciation of biological activity upon exposure to an aqueous environment and non-specific targeting of tumour cells. In the present study, all the problems above were addressed using the novel drug complex formulation involving recombinant human alpha fetoprotein (rhAFP) and ACA. Experimental Design To study the synergistic effect of both agents on human cancer xenografts, athymic nude (Nu/Nu) mice were used and treated with various combination regimes intraperitoneally. Serum levels of tumour markers for carcinoembryonic antigen (CEA) and prostate specific antigen (PSA) were assessed using sandwich ELISA. IHC and Western blotting were also conducted on in vivo tumour biopsies to investigate the involvement of NF-κB regulated genes and inflammatory biomarkers. Quantification and correlation between drug efficacies and AFP-receptors were done using IF-IC and Pearson's correlation analysis. Results Mice exposed to combined treatments displayed higher reductions in tumour volume compared to stand alone agents, consistent with in vitro cytotoxicity assays. Milder signs of systemic toxicity, such as loss in body weight and inflammation of vital organs were also demonstrated compared to stand alone treatments. Tumour marker levels were consistent within all rhAFP/ACA treatment groups where levels of CEA and PSA were initially elevated upon commencement of treatment, and consecutively reduced corresponding to a decrease in tumour bulk volume. Both IHC and Western blotting results indicated that the combined action of rhAFP/ACA was not only able to down-regulate NF-κB activation, but also reduce the expression of NF-κB regulated genes and inflammatory biomarkers. The efficacy of rhAFP/ACA complex was also found to be weakly negatively correlated to the level of surface AFP-receptors between tumour types. Conclusions This drug complex formulation shows great therapeutic potential against AFP-receptor positive tumours, and serves as a basis to overcome insoluble and non-specific anti-neoplastic molecules.
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20
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Suzuki D, Sahu R, Leu NA, Senoo M. The carboxy-terminus of p63 links cell cycle control and the proliferative potential of epidermal progenitor cells. Development 2014; 142:282-90. [PMID: 25503409 DOI: 10.1242/dev.118307] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
The transcription factor p63 (Trp63) plays a key role in homeostasis and regeneration of the skin. The p63 gene is transcribed from dual promoters, generating TAp63 isoforms with growth suppressive functions and dominant-negative ΔNp63 isoforms with opposing properties. p63 also encodes multiple carboxy (C)-terminal variants. Although mutations of C-terminal variants have been linked to the pathogenesis of p63-associated ectodermal disorders, the physiological role of the p63 C-terminus is poorly understood. We report here that deletion of the p63 C-terminus in mice leads to ectodermal malformation and hypoplasia, accompanied by a reduced proliferative capacity of epidermal progenitor cells. Notably, unlike the p63-null condition, we find that p63 C-terminus deficiency promotes expression of the cyclin-dependent kinase inhibitor p21(Waf1/Cip1) (Cdkn1a), a factor associated with reduced proliferative capacity of both hematopoietic and neuronal stem cells. These data suggest that the p63 C-terminus plays a key role in the cell cycle progression required to maintain the proliferative potential of stem cells of many different lineages. Mechanistically, we show that loss of Cα, the predominant C-terminal p63 variant in epithelia, promotes the transcriptional activity of TAp63 and also impairs the dominant-negative activity of ΔNp63, thereby controlling p21(Waf1/Cip1) expression. We propose that the p63 C-terminus links cell cycle control and the proliferative potential of epidermal progenitor cells via mechanisms that equilibrate TAp63 and ΔNp63 isoform function.
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Affiliation(s)
- Daisuke Suzuki
- Department of Animal Biology, University of Pennsylvania School of Veterinary Medicine, Philadelphia, PA 19104, USA Institute for Regenerative Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - Raju Sahu
- Department of Animal Biology, University of Pennsylvania School of Veterinary Medicine, Philadelphia, PA 19104, USA Institute for Regenerative Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - N Adrian Leu
- Department of Animal Biology, University of Pennsylvania School of Veterinary Medicine, Philadelphia, PA 19104, USA
| | - Makoto Senoo
- Department of Animal Biology, University of Pennsylvania School of Veterinary Medicine, Philadelphia, PA 19104, USA Institute for Regenerative Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
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21
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Li XM, Choi DK, Lim SK, Ahn IS, Kim DI, Piao YJ, Kim CD, Lee JH. Stimulation of keratinocyte differentiation by extract of combined medicinal plant. J Biomed Res 2014. [DOI: 10.12729/jbr.2014.15.4.176] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022] Open
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22
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Six1 overexpression at early stages of HPV16-mediated transformation of human keratinocytes promotes differentiation resistance and EMT. Virology 2014; 474:144-53. [PMID: 25463612 DOI: 10.1016/j.virol.2014.10.010] [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: 07/31/2014] [Revised: 08/22/2014] [Accepted: 10/07/2014] [Indexed: 11/20/2022]
Abstract
Previous studies in our laboratory discovered that SIX1 mRNA expression increased during in vitro progression of HPV16-immortalized human keratinocytes (HKc/HPV16) toward a differentiation-resistant (HKc/DR) phenotype. In this study, we explored the role of Six1 at early stages of HPV16-mediated transformation by overexpressing Six1 in HKc/HPV16. We found that Six1 overexpression in HKc/HPV16 increased cell proliferation and promoted cell migration and invasion by inducing epithelial-mesenchymal transition (EMT). Moreover, the overexpression of Six1 in HKc/HPV16 resulted in resistance to serum and calcium-induced differentiation, which is the hallmark of the HKc/DR phenotype. Activation of MAPK in HKc/HPV16 overexpressing Six1 is linked to resistance to calcium-induced differentiation. In conclusion, this study determined that Six1 overexpression resulted in differentiation resistance and promoted EMT at early stages of HPV16-mediated transformation of human keratinocytes.
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23
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Yang CC, Chung A, Ku CY, Brill LM, Williams R, Wolf DA. Systems analysis of the prostate tumor suppressor NKX3.1 supports roles in DNA repair and luminal cell differentiation. F1000Res 2014; 3:115. [PMID: 25177484 PMCID: PMC4141641 DOI: 10.12688/f1000research.3818.2] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Accepted: 12/16/2014] [Indexed: 11/20/2022] Open
Abstract
NKX3.1 is a homeobox transcription factor whose function as a prostate tumor suppressor remains insufficiently understood because neither the transcriptional program governed by NKX3.1, nor its interacting proteins have been fully revealed. Using affinity purification and mass spectrometry, we have established an extensive NKX3.1 interactome which contains the DNA repair proteins Ku70, Ku80, and PARP, thus providing a molecular underpinning to previous reports implicating NKX3.1 in DNA repair. Transcriptomic profiling of NKX3.1-negative prostate epithelial cells acutely expressing NKX3.1 revealed a rapid and complex response that is a near mirror image of the gene expression signature of human prostatic intraepithelial neoplasia (PIN). Pathway and network analyses suggested that NKX3.1 actuates a cellular reprogramming toward luminal cell differentiation characterized by suppression of pro-oncogenic c-MYC and interferon-STAT signaling and activation of tumor suppressor pathways. Consistently, ectopic expression of NKX3.1 conferred a growth arrest depending on TNFα and JNK signaling. We propose that the tumor suppressor function of NKX3.1 entails a transcriptional program that maintains the differentiation state of secretory luminal cells and that disruption of NKX3.1 contributes to prostate tumorigenesis by permitting luminal cell de-differentiation potentially augmented by defects in DNA repair.
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Affiliation(s)
- Chih-Cheng Yang
- Tumor Initiation and Maintenance Program, Sanford-Burnham Medical Research Institute, La Jolla, CA 92037, USA
| | - Alicia Chung
- Genentech Inc., South San Francisco, CA 94080, USA
| | - Chia-Yu Ku
- Tumor Initiation and Maintenance Program, Sanford-Burnham Medical Research Institute, La Jolla, CA 92037, USA
| | - Laurence M Brill
- NCI-designated Cancer Center Proteomics Facility, Sanford-Burnham Medical Research Institute, La Jolla, CA 92037, USA
| | - Roy Williams
- Informatics and Data Management Core, Sanford-Burnham Medical Research Institute, La Jolla, CA 92037, USA
| | - Dieter A Wolf
- Tumor Initiation and Maintenance Program, Sanford-Burnham Medical Research Institute, La Jolla, CA 92037, USA; NCI-designated Cancer Center Proteomics Facility, Sanford-Burnham Medical Research Institute, La Jolla, CA 92037, USA; San Diego Center for Systems Biology, La Jolla, CA 92093-0375, USA
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Borowiec AS, Delcourt P, Dewailly E, Bidaux G. Optimal differentiation of in vitro keratinocytes requires multifactorial external control. PLoS One 2013; 8:e77507. [PMID: 24116231 PMCID: PMC3792032 DOI: 10.1371/journal.pone.0077507] [Citation(s) in RCA: 63] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/17/2013] [Accepted: 09/11/2013] [Indexed: 11/18/2022] Open
Abstract
For almost 30 years, keratinocyte differentiation has been studied in numerous cell models including keratinocyte primary culture with various supplemented culture media. In this respect, it has become quite difficult to draw comparisons between studies using such a variety of culture conditions. Serum-free condition with low calcium has been used to culture basal proliferating cells, though differentiation is induced by various procedures. These latter include the addition of calcium at mM concentration and a concomitant addition of serum and calcium. Lowering the incubation temperature of cells has also been reported to induce a premature differentiation of keratinocytes in organotypic skin culture. This effect of temperature on keratinocyte differentiation has been poorly depicted, although average human skin temperature has been shown to be about 32°C. However, studying differentiation and quantifying shifts in the differentiation rate of a cell population implies to precisely know i) the proportion of differentiated cells in the whole population, and ii) to which extent and to which level of expression, the induction of a gene or a protein might be considered as a marker of differentiation. This lack has rarely been taken into consideration and has surely led to over-interpretations of single protein induction and to consequent extrapolations to real differentiation processes. By means of paralleled analyses with immunocytofluorescence, flow cytometry, and with multiple differentiation markers quantify by qPCR and western-blot, we studied the paradoxical connection between calcium, serum, multilayer culture and incubation temperature on the differentiation of in vitro keratinocytes. Conversely to previous reports, we have shown that calcium switch is indeed a potent model for inducing calcium-dependent genes, but is not an efficient procedure when one wishes to assess the keratinocyte differentiation rate. Moreover, we have demonstrated that a synergic stimulation by calcium, serum, confluence and lower incubation temperature amplified the differentiation rate.
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Affiliation(s)
- Anne-Sophie Borowiec
- Inserm U1003, Equipe Labellisee par la Ligue Nationale Contre le Cancer, Université Lille 1, Villeneuve d’Ascq, France
- Laboratory of Excellence, Ion Channels Science and Therapeutics, Université Lille 1, Villeneuve d’Ascq, France
| | - Philippe Delcourt
- Inserm U1003, Equipe Labellisee par la Ligue Nationale Contre le Cancer, Université Lille 1, Villeneuve d’Ascq, France
- Laboratory of Excellence, Ion Channels Science and Therapeutics, Université Lille 1, Villeneuve d’Ascq, France
| | - Etienne Dewailly
- Inserm U1003, Equipe Labellisee par la Ligue Nationale Contre le Cancer, Université Lille 1, Villeneuve d’Ascq, France
- Laboratory of Excellence, Ion Channels Science and Therapeutics, Université Lille 1, Villeneuve d’Ascq, France
| | - Gabriel Bidaux
- Inserm U1003, Equipe Labellisee par la Ligue Nationale Contre le Cancer, Université Lille 1, Villeneuve d’Ascq, France
- Laboratory of Excellence, Ion Channels Science and Therapeutics, Université Lille 1, Villeneuve d’Ascq, France
- * E-mail:
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25
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Shi G, Sohn KC, Li Z, Choi DK, Park YM, Kim JH, Fan YM, Nam YH, Kim S, Im M, Lee Y, Seo YJ, Kim CD, Lee JH. Expression and functional role of Sox9 in human epidermal keratinocytes. PLoS One 2013; 8:e54355. [PMID: 23349860 PMCID: PMC3548846 DOI: 10.1371/journal.pone.0054355] [Citation(s) in RCA: 48] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/20/2012] [Accepted: 12/11/2012] [Indexed: 01/13/2023] Open
Abstract
In this study, we investigated the expression and putative role of Sox9 in epidermal keratinocyte. Immunohistochemical staining showed that Sox9 is predominantly expressed in the basal layer of normal human skin epidermis, and highly expressed in several skin diseases including psoriasis, basal cell carcinoma, keratoacanthoma and squamous cell carcinoma. In calcium-induced keratinocyte differentiation model, the expression of Sox9 was decreased in a time dependent manner. When Sox9 was overexpressed using a recombinant adenovirus, cell growth was enhanced, while the expression of differentiation-related genes such as loricrin and involucrin was markedly decreased. Similarly, when rat skin was intradermally injected with the adenovirus expressing Sox9, the epidermis was thickened with increase of PCNA positive cells, while the epidermal differentiation was decreased. Finally, UVB irradiation induced Sox9 expression in cultured human epidermal keratinocytes, and keratinocytes are protected from UVB-induced apoptosis by Sox9 overexpression. Together, these results suggest that Sox9 is an important regulator of epidermal keratinocytes with putative pro-proliferation and/or pro-survival functions, and may be related to several cutaneous diseases that are characterized by abnormal differentiation and hyperproliferation.
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Affiliation(s)
- Ge Shi
- Department of Dermatology, The First Affiliated Hospital of Guangdong Medical College, Zhanjiang, China
- Department of Dermatology and Research Institute for Medical Sciences, School of Medicine, Chungnam National University, Daejeon, Korea
| | - Kyung-Cheol Sohn
- Department of Dermatology and Research Institute for Medical Sciences, School of Medicine, Chungnam National University, Daejeon, Korea
| | - Zhengjun Li
- Department of Dermatology and Research Institute for Medical Sciences, School of Medicine, Chungnam National University, Daejeon, Korea
| | - Dae-Kyoung Choi
- Department of Dermatology and Research Institute for Medical Sciences, School of Medicine, Chungnam National University, Daejeon, Korea
| | - Young Min Park
- Department of Dermatology, College of Medicine, Catholic University of Korea, Seoul, Korea
| | | | - Yi-Ming Fan
- Department of Dermatology, The First Affiliated Hospital of Guangdong Medical College, Zhanjiang, China
| | - Yong Hee Nam
- Department of Dermatology and Research Institute for Medical Sciences, School of Medicine, Chungnam National University, Daejeon, Korea
| | - Sooyeon Kim
- Department of Dermatology and Research Institute for Medical Sciences, School of Medicine, Chungnam National University, Daejeon, Korea
| | - Myung Im
- Department of Dermatology and Research Institute for Medical Sciences, School of Medicine, Chungnam National University, Daejeon, Korea
| | - Young Lee
- Department of Dermatology and Research Institute for Medical Sciences, School of Medicine, Chungnam National University, Daejeon, Korea
| | - Young-Joon Seo
- Department of Dermatology and Research Institute for Medical Sciences, School of Medicine, Chungnam National University, Daejeon, Korea
| | - Chang Deok Kim
- Department of Dermatology and Research Institute for Medical Sciences, School of Medicine, Chungnam National University, Daejeon, Korea
| | - Jeung-Hoon Lee
- Department of Dermatology and Research Institute for Medical Sciences, School of Medicine, Chungnam National University, Daejeon, Korea
- * E-mail:
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26
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Walczak K, Turski WA, Rzeski W. Kynurenic acid enhances expression of p21 Waf1/Cip1 in colon cancer HT-29 cells. Pharmacol Rep 2013; 64:745-50. [PMID: 22814028 DOI: 10.1016/s1734-1140(12)70870-8] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/04/2011] [Revised: 12/13/2011] [Indexed: 10/25/2022]
Abstract
BACKGROUND Kynurenic acid (KYNA), a tryptophan metabolite, was found in the mucus of rat small intestine. However, its role in the gastrointestinal tract is still not fully elucidated. METHODS To verify whether KYNA affects cell cycle regulators, the protein expression of cyclin-dependent kinase inhibitor p21 Waf1/Cip1 was investigated in colon adenocarcinoma HT-29 cells exposed to KYNA. MTT, BrdU assay and siRNA technology were used to evaluate the effect of KYNA on cancer cell proliferation. RESULTS KYNA significantly enhanced the expression of p21 Waf1/Cip1. Importantly, the overexpression of this protein was involved in inhibition of proliferation and DNA synthesis in HT-29 cells. CONCLUSIONS KYNA may be considered as a potential chemoprevention agent against colon cancer.
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Affiliation(s)
- Katarzyna Walczak
- Department of Medical Biology, Institute of Rural Health, Jaczewskiego 2, PL 20-950 Lublin, Poland.
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27
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Miyoshi S, Yamazaki S, Uchiumi A, Katagata Y. The Hsp90 inhibitor 17-AAG represses calcium-induced cytokeratin 1 and 10 expression in HaCaT keratinocytes. FEBS Open Bio 2012; 2:47-50. [PMID: 23650580 PMCID: PMC3642114 DOI: 10.1016/j.fob.2012.03.002] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/22/2012] [Accepted: 03/20/2012] [Indexed: 11/28/2022] Open
Abstract
Hsp90 is essential for maintaining the activity of numerous signaling factors, and plays a key role in cellular signal transduction networks. 17-Allylamino-17-demethoxygeldanamycin (17-AAG) is an ansamycin antibiotic that binds to Hsp90 and inhibits its function. HaCaT human keratinocytes were used to investigate the cellular and molecular functions of Hsp90 in keratinocyte differentiation. Inhibition of Hsp90 by 17-AAG leads to downregulation of the differentiation markers cytokeratin 1 and cytokeratin 10 at the protein and mRNA levels.
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Affiliation(s)
- Sadanori Miyoshi
- Department of Biochemistry and Biotechnology, Faculty of Agriculture and Life Science, Hirosaki University, 3 Bunkyo-cho, Hirosaki 036-8561, Japan
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28
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Rezvani HR, Ali N, Nissen LJ, Harfouche G, de Verneuil H, Taïeb A, Mazurier F. HIF-1α in epidermis: oxygen sensing, cutaneous angiogenesis, cancer, and non-cancer disorders. J Invest Dermatol 2011; 131:1793-805. [PMID: 21633368 DOI: 10.1038/jid.2011.141] [Citation(s) in RCA: 105] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
Besides lung, postnatal human epidermis is the only epithelium in direct contact with atmospheric oxygen. Skin epidermal oxygenation occurs mostly through atmospheric oxygen rather than tissue vasculature, resulting in a mildly hypoxic microenvironment that favors increased expression of hypoxia-inducible factor-1α (HIF-1α). Considering the wide spectrum of biological processes, such as angiogenesis, inflammation, bioenergetics, proliferation, motility, and apoptosis, that are regulated by this transcription factor, its high expression level in the epidermis might be important to HIF-1α in skin physiology and pathophysiology. Here, we review the role of HIF-1α in cutaneous angiogenesis, skin tumorigenesis, and several skin disorders.
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29
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McDade SS, Patel D, McCance DJ. p63 maintains keratinocyte proliferative capacity through regulation of Skp2-p130 levels. J Cell Sci 2011; 124:1635-43. [PMID: 21511729 DOI: 10.1242/jcs.084723] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023] Open
Abstract
p63 is a master regulator of proliferation and differentiation in stratifying epithelia, and its expression is frequently altered in carcinogenesis. However, its role in maintaining proliferative capacity remains unclear. Here, we demonstrate that hypoproliferation and loss of differentiation in organotypic raft cultures of primary neonatal human foreskin keratinocytes (HFKs) depleted of the α and β isoforms of p63 result from p53-p21-mediated accumulation of retinoblastoma (Rb) family member p130. Hypoproliferation in p63-depleted HFKs can be rescued by depletion of p53, p21(CIP1) or p130. Furthermore, we identified the gene encoding S-phase kinase-associated protein 2 (Skp2), the recognition component of the SCF(Skp2) E3 ubiquitin ligase, as a novel target of p63, potentially influencing p130 levels. Expression of Skp2 is maintained by p63 binding to a site in intron 2 and mRNA levels are downregulated in p63-depleted cells. Hypoproliferation in p63-depleted cells can be restored by re-expression of Skp2. Taken together, these results indicate that p63 plays a multifaceted role in maintaining proliferation in the mature regenerating epidermis, in addition to being required for differentiation.
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Affiliation(s)
- Simon S McDade
- Centre for Cancer Research and Cell Biology, School of Medicine, Dentistry and Biomedical Science, Queen's University Belfast, Belfast BT9 7BL, UK
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30
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Lee CM, Yang P, Chen LC, Chen CC, Wu SC, Cheng HY, Chang YS. A novel role of RASSF9 in maintaining epidermal homeostasis. PLoS One 2011; 6:e17867. [PMID: 21445300 PMCID: PMC3061870 DOI: 10.1371/journal.pone.0017867] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/06/2010] [Accepted: 02/16/2011] [Indexed: 02/05/2023] Open
Abstract
The physiological role of RASSF9, a member of the Ras-association domain family (RASSF), is currently unclear. Here, we report a mouse line in which an Epstein-Barr virus Latent Membrane Protein 1 (LMP1) transgene insertion has created a 7.2-kb chromosomal deletion, which abolished RASSF9 gene expression. The RASSF9-null mice exhibited interesting phenotypes that resembled human ageing, including growth retardation, short lifespan, less subcutaneous adipose layer and alopecia. In the wild-type mice, RASSF9 is predominantly expressed in the epidermal keratinocytes of skin, as determined by quantitative reverse-transcription PCR, immunofluorescence and in situ hybridization. In contrast, RASSF9-/- mice presented a dramatic change in epithelial organization of skin with increased proliferation and aberrant differentiation as detected by bromodeoxyuridine incorporation assays and immunofluorescence analyses. Furthermore, characteristic functions of RASSF9-/- versus wild type (WT) mouse primary keratinocytes showed significant proliferation linked to a reduction of p21Cip1 expression under growth or early differentiation conditions. Additionally, in RASSF9-/- keratinocytes there was a drastic down-modulation of terminal differentiation markers, which could be rescued by infection with a recombinant adenovirus, Adv/HA-RASSF9. Our results indicate a novel and significant role of RASSF9 in epidermal homeostasis.
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Affiliation(s)
- Chiou-Mei Lee
- Department of Medical Research and Development, Chang Gung Memorial Hospital at Lin-Kou, Taoyuan, Taiwan.
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31
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Weinreb O, Mandel S, Bar-Am O, Amit T. Iron-chelating backbone coupled with monoamine oxidase inhibitory moiety as novel pluripotential therapeutic agents for Alzheimer's disease: a tribute to Moussa Youdim. J Neural Transm (Vienna) 2011; 118:479-92. [PMID: 21360301 DOI: 10.1007/s00702-011-0597-6] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/06/2010] [Accepted: 01/27/2011] [Indexed: 12/13/2022]
Abstract
It is for these authors a great privilege to dedicate this review article to Moussa Youdim, who is one of the most imperative pharmacologists and pioneer investigators in the search and development of novel therapeutics for neurodegenerative diseases. 40 years ago, Moussa Youdim has started studying brain iron, catecholamine receptor and monoamine oxidase (MAO)-A and -B functions. Although Moussa Youdim succeeded in exploring the novel anti-Parkinsonian, selective MAO-B inhibitor drug, rasagiline (Azilect, Teva Pharmaceutical Co.), he did not stop searching for superior therapeutic approaches for neurodegenerative disorders. To date, Moussa Youdim and his research group are designing and synthesizing pluripotential drug candidates possessing diverse pharmacological properties that can act on multiple targets and pathological features ascribed to Parkinson's disease, Alzheimer's disease (AD) and amyotrophic lateral sclerosis. One such example is the multimodal non-toxic, brain-permeable iron-chelating compound, M30 (5-[N-methyl-N-propargylaminomethyl]-8-hydroxyquinoline), which amalgamates the propargyl moiety of rasagiline with the backbone of the potent iron chelator, VK28. This review discusses the multiple effects of several leading compounds of this series, concerning their neuroprotective/neurorestorative molecular mechanisms in vivo and in vitro, with a special focus on the pathological features ascribed to AD, including antioxidant and iron chelating activities, regulation of amyloid precursor protein and amyloid β peptide expression processing, activation of pro-survival signaling pathways and regulation of cell cycle and neurite outgrowth.
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Affiliation(s)
- Orly Weinreb
- Eve Topf Centers of Excellence for Neurodegenerative Diseases Research and Department of Pharmacology, Rappaport Family Research Institute, Technion-Faculty of Medicine, P.O.B. 9697, 31096, Haifa, Israel.
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32
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Shi G, Sohn KC, Choi TY, Choi DK, Lee SS, Ou BS, Kim S, Lee YH, Yoon TJ, Kim SJ, Lee Y, Seo YJ, Lee JH, Kim CD. Expression of paired-like homeodomain transcription factor 2c (PITX2c) in epidermal keratinocytes. Exp Cell Res 2010; 316:3263-71. [PMID: 20875405 DOI: 10.1016/j.yexcr.2010.09.013] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/11/2010] [Revised: 08/23/2010] [Accepted: 09/20/2010] [Indexed: 01/26/2023]
Abstract
Paired-like homeodomain transcription factor 2 (PITX2) has been implicated as one of the genes responsible for Rieger syndrome. It has been also shown to play a central role during development. In this study, we investigated the functional role of PITX2 in keratinocyte differentiation. RT-PCR analysis showed that PITX2c isoform was predominantly expressed in a differentiation-dependent manner. Consistent with, immunohistochemical staining showed that PITX2 expression was increased in the upper layer of epidermis. When PITX2c was overexpressed in cultured keratinocytes by a recombinant adenovirus, the differentiation markers such as involucrin and loricrin were significantly increased at both mRNA and protein levels. In addition, PITX2c overexpression led to the decrease of cell growth, concomitantly with the upregulation of cell cycle-related genes p21. To investigate the effect of PITX2c in vivo, we microinjected PITX2c expression vector into zebrafish embryo. Interestingly, overexpression of PITX2c in zebrafish embryo led to the formation of horn-like structure and thickening of epidermis, together with the increase of keratin 8 (K8) expression. These results suggest that PITX2c has a role in proliferation and differentiation of epidermal keratinocytes.
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Affiliation(s)
- Ge Shi
- Department of Dermatology, School of Medicine, Chungnam National University, Daejeon, 301-747, Republic of Korea
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Weinreb O, Amit T, Mandel S, Kupershmidt L, Youdim MBH. Neuroprotective multifunctional iron chelators: from redox-sensitive process to novel therapeutic opportunities. Antioxid Redox Signal 2010; 13:919-49. [PMID: 20095867 DOI: 10.1089/ars.2009.2929] [Citation(s) in RCA: 61] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
Accumulating evidence suggests that many cytotoxic signals occurring in the neurodegenerative brain can initiate neuronal death processes, including oxidative stress, inflammation, and accumulation of iron at the sites of the neuronal deterioration. Neuroprotection by iron chelators has been widely recognized with respect to their ability to prevent hydroxyl radical formation in the Fenton reaction by sequestering redox-active iron. An additional neuroprotective mechanism of iron chelators is associated with their ability to upregulate or stabilize the transcriptional activator, hypoxia-inducible factor-1alpha (HIF-1alpha). HIF-1alpha stability within the cells is under the control of a class of iron-dependent and oxygen-sensor enzymes, HIF prolyl-4-hydroxylases (PHDs) that target HIF-1alpha for degradation. Thus, an emerging novel target for neuroprotection is associated with the HIF system to promote stabilization of HIF-1alpha and increase transcription of HIF-1-related survival genes, which have been reported to be regulated in patient's brains afflicted with diverse neurodegenerative diseases. In accordance, a new potential therapeutic strategy for neurodegenerative diseases is explored, by which iron chelators would inhibit PHDs, target the HIF-1-signaling pathway and ultimately activate HIF-1-dependent neuroprotective genes. This review discusses two interrelated approaches concerning therapy targets in neurodegeneration, sharing in common the implementation of iron chelation activity: antioxidation and HIF-1-pathway activation.
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Affiliation(s)
- Orly Weinreb
- Eve Topf Centers of Excellence for Neurodegenerative Diseases Research and Department of Pharmacology, Rappaport Family Research Institute, Technion-Faculty of Medicine, Haifa, Israel.
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34
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Pim D, Banks L. Interaction of viral oncoproteins with cellular target molecules: infection with high-risk vs low-risk human papillomaviruses. APMIS 2010; 118:471-93. [PMID: 20553529 DOI: 10.1111/j.1600-0463.2010.02618.x] [Citation(s) in RCA: 97] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Persistent infection by a subgroup of so-called high-risk human papillomaviruses (HPVs) that have a tropism for mucosal epithelia has been defined as the cause of more than 98% of cervical carcinomas as well as a high proportion of other cancers of the anogenital region. Infection of squamous epithelial tissues in the head and neck region by these same high-risk HPVs is also associated with a subset of cancers. Despite the general conservation of genetic structure amongst all HPV types, infection by the low-risk types, whether in genital or head and neck sites, carries a negligible risk of malignant progression, and infections have a markedly different pathology. In this review, we will examine and discuss the interactions that the principal viral oncoproteins of the high-risk mucosotrophic HPVs and their counterparts from the low-risk group make with cellular target proteins, with a view to explaining the differences in their respective pathology.
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Affiliation(s)
- David Pim
- International Centre for Genetic Engineering and Biotechnology, Trieste, Italy. <>
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35
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p300 alters keratinocyte cell growth and differentiation through regulation of p21(Waf1/CIP1). PLoS One 2010; 5:e8369. [PMID: 20084294 PMCID: PMC2805707 DOI: 10.1371/journal.pone.0008369] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2009] [Accepted: 11/20/2009] [Indexed: 11/19/2022] Open
Abstract
BACKGROUND p300 functions as a transcriptional co-activator to regulate many cellular responses such as cell growth, transformation, development and differentiation. It has been shown to affect the transcriptional activity of p53 which regulates p21(Waf1/CIP1) expression, however, the role of p300 in differentiation remains unclear. METHODOLOGY AND PRINCIPAL FINDINGS Knockdown of p300 protein with short hairpin RNA (shRNA) molecules delays human neonatal foreskin keratinocyte (HFKs) differentiation. Moreover, depletion of p300 increases the proliferative capacity of HFKs, extends the life span of cells and allows differentiated HFKs to re-enter the cell cycle. Studies indicate that depletion of p300 down-regulates the acetylation and expression of p53, and chromatin immunoprecipitation (ChIP) analysis shows that induction of p21(Waf1/CIP1) in early differentiation is a result of p300 dependent activation of p53 and that depletion of p21(Waf1/CIP1) results in the delay of differentiation and a phenotype similar to p300 depletion. CONCLUSIONS p300 has a direct role in the control of cell growth and differentiation in primary epithelial cells, and p21(Waf1/CIP1) is an important mediator of these p300 functions.
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36
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Sinitsyna NN, Reznikova TV, Qin Q, Song H, Phillips MA, Rice RH. Arsenite suppression of involucrin transcription through AP1 promoter sites in cultured human keratinocytes. Toxicol Appl Pharmacol 2009; 243:275-82. [PMID: 20006635 DOI: 10.1016/j.taap.2009.12.006] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/06/2009] [Revised: 11/07/2009] [Accepted: 12/05/2009] [Indexed: 02/08/2023]
Abstract
While preserving keratinocyte proliferative ability, arsenite suppresses cellular differentiation markers by preventing utilization of AP1 transcriptional response elements. In present experiments, arsenite had a dramatic effect in electrophoretic mobility supershift analysis of proteins binding to an involucrin promoter AP1 response element. Without arsenite treatment, binding of JunB and Fra1 was readily detected in nuclear extracts from preconfluent cultures and was not detected a week after confluence, while c-Fos was detected only after confluence. By contrast, band shift of nuclear extracts from arsenite treated cultures showed only JunB and Fra1 binding in postconfluent as well as preconfluent cultures. Immunoblotting of cell extracts showed that arsenite treatment prevented the loss of Fra1 and the increase in c-Fos proteins that occurred after confluence in untreated cultures. Chromatin immunoprecipitation assays demonstrated substantial reduction of c-Fos and acetylated histone H3 at the proximal and distal AP1 response elements in the involucrin promoter and of coactivator p300 at the proximal element. Alteration of AP1 transcription factors was also examined in response to treatment with four metal containing compounds (chromate, vanadate, hemin, divalent cadmium) that also suppress involucrin transcription. These agents all influenced transcription at AP1 elements in a transcriptional reporter assay, but exhibited less effect than arsenite on binding activity assessed by mobility shift and chromatin immunoprecipitation and displayed variable effects on AP1 protein levels. These findings help trace a mechanism by which transcriptional effects of arsenite become manifest and help rationalize the unique action of arsenite, compared to the other agents, to preserve proliferative ability.
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Affiliation(s)
- Nadezda N Sinitsyna
- Department of Environmental Toxicology, University of California, Davis, CA 95616-8588, USA
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37
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Stankiewicz E, Kudahetti SC, Prowse DM, Ktori E, Cuzick J, Ambroisine L, Zhang X, Watkin N, Corbishley C, Berney DM. HPV infection and immunochemical detection of cell-cycle markers in verrucous carcinoma of the penis. Mod Pathol 2009; 22:1160-8. [PMID: 19465901 DOI: 10.1038/modpathol.2009.77] [Citation(s) in RCA: 60] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Penile verrucous carcinoma is a rare disease and little is known of its aetiology or pathogenesis. In this study we examined cell-cycle proteins expression and correlation with human papillomavirus infection in a series of 15 pure penile verrucous carcinomas from a single centre. Of 148 penile tumours, 15 (10%) were diagnosed as pure verrucous carcinomas. The expression of the cell-cycle-associated proteins p53, p21, RB, p16(INK4A) and Ki67 were examined by immunohistochemistry. Human papillomavirus infection was determined by polymerase chain reaction to identify a wide range of virus types. The expression of p16(INK4A) and Ki67 was significantly lower in verrucous carcinoma than in usual type squamous cell carcinoma, whereas the expression of p53, p21 and RB was not significantly different. p53 showed basal expression in contrast to usual type squamous cell carcinoma. Human papillomavirus infection was present in only 3 out of 13 verrucous carcinomas. Unique low-risk, high-risk and mixed viral infections were observed in each of the three cases. In conclusion, lower levels of p16(INK4A) and Ki67 expressions differentiate penile verrucous carcinoma from usual type squamous cell carcinoma. The low Ki67 index reflects the slow-growing nature of verrucous tumours. The low level of p16(INK4A) expression and human papillomavirus detection suggests that penile verrucous carcinoma pathogenesis is unrelated to human papillomavirus infection and the oncogenes and tumour suppressor genes classically altered by virus infection.
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Affiliation(s)
- Elzbieta Stankiewicz
- Centre for Molecular Oncology and Imaging, Barts and The London School of Medicine and Dentistry, London, UK
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38
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Legendre F, Heuze A, Boukerrouche K, Leclercq S, Boumediene K, Galera P, Domagala F, Pujol JP, Ficheux H. Rhein, the metabolite of diacerhein, reduces the proliferation of osteoarthritic chondrocytes and synoviocytes without inducing apoptosis. Scand J Rheumatol 2009; 38:104-11. [PMID: 19274517 DOI: 10.1080/03009740802421996] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/22/2023]
Abstract
OBJECTIVE The aim of this study was to determine the effects of pharmacologically relevant concentrations of rhein (1,8-dihydroxy-3-carboxyanthraquinone) on the cell proliferation rate of human chondrocytes and synoviocytes. METHODS Cultures of human osteoarthritic synoviocytes and chondrocytes were incubated with 10(-6), 10(-5), and 10(-4) M rhein. [3H]thymidine incorporation was used to determine rhein proliferative effects after incubation periods of 24 h, 48 h, and 1 week. The cytotoxicity of the drug was assayed with a nonradioactive assay kit. Nuclear extracts were used to detect variations in cell-cycle proteins (p21, p27, and cyclin D1) by Western blotting. The effect of rhein on apoptosis was investigated by measurement of caspase-3/7 activity and DNA fragmentation. RESULTS Rhein was found to downregulate the proliferation rate of both chondrocytes and synoviocytes, two-fold for 10(-5) M rhein and five- to six-fold for 10(-4) M rhein. No cytotoxicity of the drug was observed. Rhein (10(-4) M) decreased caspase-3/7 activity and did not induce DNA fragmentation. Western blots showed that 10(-4) M rhein increased the expression of p21 and/or p27, but not that of cyclin D1. CONCLUSIONS Rhein has previously been shown to reduce the interleukin (IL)-1beta deleterious effects on osteoarthritis (OA) cartilage through inhibition of the expression of degrading enzymes. Here, rhein was also found to inhibit proliferation of both synoviocytes and chondrocytes, suggesting that the drug may decrease the development of the inflammatory synovial tissue that accompanies joint pathologies. Both its anti-catabolic and anti-proliferative effects may explain its beneficial effect in the treatment of joint diseases.
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Affiliation(s)
- F Legendre
- Laboratory of Extracellular Matrix and Pathology, University of Caen
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39
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Dysregulation of CREB binding protein triggers thrombin-induced proliferation of vascular smooth muscle cells. Mol Cell Biochem 2008; 315:123-30. [PMID: 18496732 DOI: 10.1007/s11010-008-9795-4] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/23/2008] [Accepted: 05/06/2008] [Indexed: 10/22/2022]
Abstract
Thrombin is a potent mitogen for vascular smooth muscle cells (VSMCs). CBP has been regarded as a potential therapeutic target on the basis of its ability to affect cell growth. Therefore we hypothesized that CBP mediates thrombin-induced proliferation of VSMCs. We constructed recombinant adenoviral vector that expresses four short hairpin RNA (shRNA) targeting rat CBP mRNA (CBP-shRNA/Ad). VSMCs were infected with CBP-shRNA/Ad and treated with thrombin. CBP level were analyzed by quantitative real-time PCR and Western blot. To evaluate VSMC proliferation, the cell cycle and DNA synthesis were analyzed by flow cytometry and (3)H-thymidine incorporation, respectively. CBP-shRNA/Ad infection inhibited thrombin-induced CBP expression in a dose-dependent manner concomitant with a decrease in the percentage of cells in the S phase and in DNA synthesis. These findings suggest that CBP plays a pivotal role in the S phase progression of VSMCs.
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40
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Valencia-Hernández A, Cuevas-Bennett C, Garrido E. Transcriptional regulation of human papillomavirus type 18 P105 promoter by the co-activator CBP. Intervirology 2008; 50:418-25. [PMID: 18182775 DOI: 10.1159/000112917] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/13/2006] [Accepted: 11/20/2007] [Indexed: 11/19/2022] Open
Abstract
UNLABELLED Human papillomaviruses (HPVs) are the etiological agents of cervical cancer, with HPV-16 and 18 being the representative types of the higher risk group. The expression of the viral genes with transforming activity (E6 and E7) is controlled by the upstream regulatory region (URR), a segment of the viral genome that contains elements recognized by several transcription factors. OBJECTIVE We have analyzed the participation of the cellular co-activator CBP on the transcriptional regulation of the HPV-18 URR. METHODS We generated mutants and 5' end deletion constructs derived from the HPV-18 URR and evaluated their transcriptional activity performing transient co-transfection assays on C-33A cells with a plasmid that over-expresses the co-activator CBP. We also performed quantitative chromatin immunoprecipitation assays to analyze the participation of the co-activator CBP on the HPV-18 P105 promoter. RESULTS Our results demonstrate that in C-33A cells CBP acts as a strong activator of the HPV-18 P105 promoter by a mechanism that depends on the integrity of the SP1-binding site, directly correlating with the acetylation of the histone H3 that is involved in nucleosomal stability. CONCLUSION We propose a mechanism of regulation of the HPV-18 P105 promoter by the cellular co-activator CBP, recruited by the transcription factor SP1.
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Affiliation(s)
- Armando Valencia-Hernández
- Departamento de Genética y Biología Molecular, Centro de Investigación y de Estudios Avanzados del IPN, México, México
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41
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Cho YS, Bae JM, Chun YS, Chung JH, Jeon YK, Kim IS, Kim MS, Park JW. HIF-1alpha controls keratinocyte proliferation by up-regulating p21(WAF1/Cip1). BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH 2007; 1783:323-33. [PMID: 18166158 DOI: 10.1016/j.bbamcr.2007.11.017] [Citation(s) in RCA: 44] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Subscribe] [Scholar Register] [Received: 07/12/2007] [Revised: 11/13/2007] [Accepted: 11/29/2007] [Indexed: 11/19/2022]
Abstract
The cyclin-dependent kinase inhibitor p21(WAF1/Cip1) plays a central role in a spatial and temporal balance of epidermal keratinocyte proliferation and growth arrest. However, what controls p21 expression in keratinocytes remains uncertain. Hypoxia-inducible factor 1alpha (HIF-1alpha) does not only express a variety of genes essential for hypoxic adaptation, but also up-regulates p21 so as to slow down cell cycle under hypoxic conditions. In the present study, we examined the role of HIF-1alpha in p21-mediated growth arrest of keratinocyte. Keratinocyte proliferation was arrested in the G1 phase at a high cell density. p21 was also up-regulated in a cell density-dependent manner and was found to be highly expressed in epidermal keratinocytes of normal human skins. In addition, in the same specimens and cells, we noted robust HIF-1alpha expression. HIF-1alpha siRNAs inhibited p21 expression and released the G1 arrest. In vivo, moreover, the intradermal injection of HIF-1alpha siRNA attenuated p21 expression in rat epidermis and induced skin hyperplasia. Mechanistically, we propose that the production of mitochondrial reactive oxygen species and the activation of the MEK/ERK pathway are involved in the HIF-1alpha stabilization in keratinocytes. These results imply that HIF-1alpha functions as an up-stream player in the p21-mediated growth arrest of keratinocytes.
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Affiliation(s)
- Young-Suk Cho
- Department of Pharmacology, Seoul National University College of Medicine, Seoul, Republic of Korea
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42
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Wang X, Pan L, Feng Y, Wang Y, Han Q, Han L, Han S, Guo J, Huang B, Lu J. P300 plays a role in p16(INK4a) expression and cell cycle arrest. Oncogene 2007; 27:1894-904. [PMID: 17906698 DOI: 10.1038/sj.onc.1210821] [Citation(s) in RCA: 62] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
As a cyclin-dependent kinase inhibitor, p16(INK4a) plays a key role in cell cycle progression and cellular differentiation, and its expression is frequently altered in human cancers through epigenetically mediated transcriptional silencing. In this report, we demonstrate that p300 was able to induce cell cycle arrest, and this process was reversed by p16(INK4a) silencing by RNA interference in HeLa cells. We also show that p300 was involved in activation of p16(INK4a) expression in 293T cells. Specifically, p300 cooperated with Sp1 to stimulate both p16(INK4a) promoter activity and mRNA expression. Co-immunoprecipitation and mammalian two-hybrid assays revealed that p300 and Sp1 formed a complex through interaction between the Q domain of p300 and the N-terminal domain of Sp1. The chromatin immunoprecipitation assays verified that p300 was recruited to p16(INK4a) promoter, and the histone acetyltransferase domain of p300 participated in p16(INK4a) activation through inducing hyperacetylation of histone H4 at p16(INK4a) gene. These data suggest that p300 plays a critical role in transcriptional regulation of p16(INK4a) and in cell cycle arrest.
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Affiliation(s)
- X Wang
- Institute of Genetics and Cytology, Northeast Normal University, Changchun, China
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43
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Hwang-Verslues WW, Sladek FM. Nuclear receptor hepatocyte nuclear factor 4alpha1 competes with oncoprotein c-Myc for control of the p21/WAF1 promoter. Mol Endocrinol 2007; 22:78-90. [PMID: 17885207 PMCID: PMC2194635 DOI: 10.1210/me.2007-0298] [Citation(s) in RCA: 62] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022] Open
Abstract
The dichotomy between cellular differentiation and proliferation is a fundamental aspect of both normal development and tumor progression; however, the molecular basis of this opposition is not well understood. To address this issue, we investigated the mechanism by which the nuclear receptor hepatocyte nuclear factor 4alpha1 (HNF4alpha1) regulates the expression of the human cyclin-dependent kinase inhibitor gene p21/WAF1 (CDKN1A). We found that HNF4alpha1, a transcription factor that plays a central role in differentiation in the liver, pancreas, and intestine, activates the expression of p21 primarily by interacting with promoter-bound Sp1 at both the proximal promoter region and at newly identified sites in a distal region (-2.4 kb). Although HNF4alpha1 also binds two additional regions containing putative HNF4alpha binding sites, HNF4alpha1 mutants deficient in DNA binding activate the p21 promoter to the same extent as wild-type HNF4alpha1, indicating that direct DNA binding by HNF4alpha1 is not necessary for p21 activation. We also observed an in vitro and in vivo interaction between HNF4alpha1 and c-Myc as well as a competition between these two transcription factors for interaction with promoter-bound Sp1 and regulation of p21. Finally, we show that c-Myc competes with HNF4alpha1 for control of apolipoprotein C3 (APOC3), a gene associated with the differentiated hepatic phenotype. These results suggest a general model by which a differentiation factor (HNF4alpha1) and a proliferation factor (c-Myc) may compete for control of genes involved in cell proliferation and differentiation.
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Affiliation(s)
- Wendy W Hwang-Verslues
- Environmental Toxicology Graduate Program, University of California, Riverside, California 92521, USA
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44
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Riuzzi F, Sorci G, Donato R. RAGE expression in rhabdomyosarcoma cells results in myogenic differentiation and reduced proliferation, migration, invasiveness, and tumor growth. THE AMERICAN JOURNAL OF PATHOLOGY 2007; 171:947-61. [PMID: 17640970 PMCID: PMC1959489 DOI: 10.2353/ajpath.2007.070049] [Citation(s) in RCA: 49] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Accepted: 05/30/2007] [Indexed: 11/20/2022]
Abstract
Activation of receptor for advanced glycation end products (RAGE) by its ligand, HMGB1, stimulates myogenesis via a Cdc42-Rac1-MKK6-p38 mitogen-activated protein kinase pathway. In addition, functional inactivation of RAGE in myoblasts results in reduced myogenesis, increased proliferation, and tumor formation in vivo. We show here that TE671 rhabdomyosarcoma cells, which do not express RAGE, can be induced to differentiate on transfection with RAGE (TE671/RAGE cells) but not a signaling-deficient RAGE mutant (RAGEDeltacyto) (TE671/RAGEDeltacyto cells) via activation of a Cdc42-Rac1-MKK6-p38 pathway and that TE671/RAGE cell differentiation depends on RAGE engagement by HMGB1. TE671/RAGE cells also show p38-dependent inactivation of extracellular signal-regulated kinases 1 and 2 and c-Jun NH(2) terminal protein kinase and reduced proliferation, migration, and invasiveness and increased apoptosis, volume, and adhesiveness in vitro; they also grow smaller tumors and show a lower tumor incidence in vivo compared with wild-type cells. Two other rhabdomyosarcoma cell lines that express RAGE, CCA and RMZ-RC2, show an inverse relationship between the level of RAGE expression and invasiveness in vitro and exhibit reduced myogenic potential and enhanced invasive properties in vitro when transfected with RAGEDeltacyto. The rhabdomyosarcoma cell lines used here and C2C12 myoblasts express and release HMGB1, which activates RAGE in an autocrine manner. These data suggest that deregulation of RAGE expression in myoblasts might concur in rhabdomyosarcomagenesis and that increasing RAGE expression in rhabdomyosarcoma cells might reduce their tumor potential.
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Affiliation(s)
- Francesca Riuzzi
- Department of Experimental Medicine and Biochemical Sciences, Section of Anatomy, University of Perugia, Via del Giochetto C.P. 81 Succ. 3, 06122 Perugia, Italy.
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45
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Fukada SI, Uezumi A, Ikemoto M, Masuda S, Segawa M, Tanimura N, Yamamoto H, Miyagoe-Suzuki Y, Takeda S. Molecular signature of quiescent satellite cells in adult skeletal muscle. Stem Cells 2007; 25:2448-59. [PMID: 17600112 DOI: 10.1634/stemcells.2007-0019] [Citation(s) in RCA: 350] [Impact Index Per Article: 19.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
Skeletal muscle satellite cells play key roles in postnatal muscle growth and regeneration. To study molecular regulation of satellite cells, we directly prepared satellite cells from 8- to 12-week-old C57BL/6 mice and performed genome-wide gene expression analysis. Compared with activated/cycling satellite cells, 507 genes were highly upregulated in quiescent satellite cells. These included negative regulators of cell cycle and myogenic inhibitors. Gene set enrichment analysis revealed that quiescent satellite cells preferentially express the genes involved in cell-cell adhesion, regulation of cell growth, formation of extracellular matrix, copper and iron homeostasis, and lipid transportation. Furthermore, reverse transcription-polymerase chain reaction on differentially expressed genes confirmed that calcitonin receptor (CTR) was exclusively expressed in dormant satellite cells but not in activated satellite cells. In addition, CTR mRNA is hardly detected in nonmyogenic cells. Therefore, we next examined the expression of CTR in vivo. CTR was specifically expressed on quiescent satellite cells, but the expression was not found on activated/proliferating satellite cells during muscle regeneration. CTR-positive cells reappeared at the rim of regenerating myofibers in later stages of muscle regeneration. Calcitonin stimulation delayed the activation of quiescent satellite cells. Our data provide roles of CTR in quiescent satellite cells and a solid scaffold to further dissect molecular regulation of satellite cells. Disclosure of potential conflicts of interest is found at the end of this article.
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Affiliation(s)
- So-ichiro Fukada
- Department of Molecular Therapy, National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1 Ogawa-higashi, Kodaira, Tokyo 187-8502, Japan
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46
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You HL, Eng HL, Hsu SF, Chen CM, Ye TC, Liao WT, Huang MY, Baer R, Cheng JT. A PKC-Sp1 signaling pathway induces early differentiation of human keratinocytes through upregulation of TSG101. Cell Signal 2007; 19:1201-11. [PMID: 17321722 DOI: 10.1016/j.cellsig.2007.01.014] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/27/2006] [Revised: 12/18/2006] [Accepted: 01/08/2007] [Indexed: 11/15/2022]
Abstract
The TSG101 protein has been implicated in multiple biological functions including regulation of gene transcription, vesicular trafficking, cellular growth and differentiation. However, the cellular signals that control TSG101 functions are unclear. Here, we demonstrate that TSG101 is upregulated during keratinocyte differentiation in both human foreskin tissue and reconstructed organotypic skin cultures. In addition, we found that TSG101 siRNA inhibits calcium-induced early differentiation of human foreskin keratinocytes, indicating an essential and downstream role for TSG101 in this process. Furthermore, the PKC agonist TPA promotes expression of TSG101 and keratin 10 in keratinocytes under low calcium conditions, while co-treatment with the PKC inhibitor GF 109203X blocks TPA-induced TSG101 and keratin 10 upregulation. Previous work has established that the TSG101 gene is controlled by a TATA-less promoter that harbors a Sp1-binding site. Here we show that both calcium and TPA activate PKC, stimulate phosphorylation of Sp1, and augment the activity of the TSG101 promoter in a manner dependent on its Sp1-binding site. Release of calcium from intracellular stores with thapsigargin, an endoplasmic reticulum Ca2+-ATPase inhibitor that elevates intracellular free Ca2+ without activating PKC, does not affect Sp1 phosphorylation and TSG101 promoter activity. Taken together, these data suggest that an intracellular calcium store independent PKC-Sp1 signaling pathway induces early keratinocyte differentiation through upregulation of TSG101.
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Affiliation(s)
- Huey-Ling You
- Department of Biological Sciences, National Sun Yat-Sen University, 70 Lien Hai Road, and Department of Pathology, Chang Gung Memorial Hospital, Kaohsiung, Taiwan 80833, Republic of China
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47
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Abstract
The epidermis is a stratified epithelium that functions as a barrier protecting the organism from dehydration, mechanical trauma, and microbial insults. This barrier function is established during embryogenesis through a complex and tightly controlled stratification program. Whereas the morphological changes that occur during epidermal development have been extensively studied, the molecular mechanisms that govern this process remain poorly understood. In this review we summarize the current advances that have been made in understanding the molecular mechanisms that regulate epidermal morphogenesis.
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Affiliation(s)
- Maranke I Koster
- Department of Dermatology and Charles C. Gates Program in Regenerative Medicine and Stem Cell Biology, University of Colorado at Denver and Health Sciences Center, Aurora, CO 80010, USA.
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Pajalunga D, Mazzola A, Salzano AM, Biferi MG, De Luca G, Crescenzi M. Critical requirement for cell cycle inhibitors in sustaining nonproliferative states. J Cell Biol 2007; 176:807-18. [PMID: 17353358 PMCID: PMC2064055 DOI: 10.1083/jcb.200608109] [Citation(s) in RCA: 61] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/17/2006] [Accepted: 02/01/2007] [Indexed: 01/02/2023] Open
Abstract
In adult vertebrates, most cells are not in the cell cycle at any one time. Physiological nonproliferation states encompass reversible quiescence and permanent postmitotic conditions such as terminal differentiation and replicative senescence. Although these states appear to be attained and maintained quite differently, they might share a core proliferation-restricting mechanism. Unexpectedly, we found that all sorts of nonproliferating cells can be mitotically reactivated by the sole suppression of histotype-specific cyclin-dependent kinase (cdk) inhibitors (CKIs) in the absence of exogenous mitogens. RNA interference-mediated suppression of appropriate CKIs efficiently triggered DNA synthesis and mitosis in established and primary terminally differentiated skeletal muscle cells (myotubes), quiescent human fibroblasts, and senescent human embryo kidney cells. In serum-starved fibroblasts and myotubes alike, cell cycle reactivation was critically mediated by the derepression of cyclin D-cdk4/6 complexes. Thus, both temporary and permanent growth arrest must be actively maintained by the constant expression of CKIs, whereas the cell cycle-driving cyclins are always present or can be readily elicited. In principle, our findings could find wide application in biotechnology and tissue repair whenever cell proliferation is limiting.
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Affiliation(s)
- Deborah Pajalunga
- Department of Environment and Primary Prevention, Istituto Superiore di Sanità, 00161 Roma, Italy
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Koster MI, Dai D, Marinari B, Sano Y, Costanzo A, Karin M, Roop DR. p63 induces key target genes required for epidermal morphogenesis. Proc Natl Acad Sci U S A 2007; 104:3255-60. [PMID: 17360634 PMCID: PMC1805532 DOI: 10.1073/pnas.0611376104] [Citation(s) in RCA: 190] [Impact Index Per Article: 10.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/06/2006] [Indexed: 01/04/2023] Open
Abstract
Mice lacking p63, a single gene that encodes a group of transcription factors that either contain (TA) or lack (DeltaN) a transactivation domain, fail to develop stratified epithelia as well as epithelial appendages and limbs. DeltaNp63 isoforms are predominantly expressed during late embryonic and postnatal epidermal development, however, the function of these proteins remains elusive. Using an epidermal-specific inducible knockdown mouse model, we demonstrate that DeltaNp63 proteins are essential for maintaining basement membrane integrity and terminal differentiation of keratinocytes. Furthermore, we have identified two DeltaNp63alpha target genes that mediate these processes. We propose that DeltaNp63alpha initially induces expression of the extracellular matrix component Fras1, which is required for maintaining the integrity of the epidermal-dermal interface at the basement membrane. Subsequently, induction of IkappaB kinase-alpha by DeltaNp63alpha initiates epidermal terminal differentiation resulting in the formation of the spinous layer. Our data provide insights into the role of DeltaNp63alpha in epidermal morphogenesis and homeostasis, and may contribute to our understanding of the pathogenic mechanisms underlying disorders caused by p63 mutations.
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Affiliation(s)
| | - Daisy Dai
- Program in Developmental Biology, Baylor College of Medicine, Houston, TX 77030
| | - Barbara Marinari
- Department of Dermatology, University of Rome “Tor Vergata”, 000173 Rome, Italy; and
| | - Yuji Sano
- Department of Pharmacology, University of California at San Diego, La Jolla, CA 92093
| | - Antonio Costanzo
- Department of Dermatology, University of Rome “Tor Vergata”, 000173 Rome, Italy; and
| | - Michael Karin
- Department of Pharmacology, University of California at San Diego, La Jolla, CA 92093
| | - Dennis R. Roop
- Departments of *Molecular and Cellular Biology and
- Dermatology and
- Program in Developmental Biology, Baylor College of Medicine, Houston, TX 77030
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Abstract
Embryonic stem cells have the capacity for unlimited proliferation while retaining their potential to differentiate into a wide variety of cell types. Murine, primate and human embryonic stem cells (ESCs) exhibit a very unusual cell cycle structure, characterized by a short G1 phase and a high proportion of cells in S-phase. In the case of mESCs, this is associated with a unique mechanism of cell cycle regulation, underpinned by the precocious activity of cyclin dependent protein kinase (Cdk) activities. As ES cells differentiate, their cell cycle structure changes dramatically so as to incorporate a significantly longer G1 phase and their mechanism of cell cycle regulation changes to that typically seen in other mammalian cells. The unique cell cycle structure and mechanism of cell cycle control indicates that the cell cycle machinery plays a role in establishment or maintenance of the stem cell state. This idea is supported by the frequent involvement of cell cycle regulatory molecules in cell immortalization.
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Affiliation(s)
- Josephine White
- Department of Molecular Biosciences, University of Adelaide, South Australia, 5005
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