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Epigenetic mechanism of stellate cell trans-differentiation
Journal of Hepatology 46 (2007) 352–353 www.elsevier.com/locate/jhep
Journal Club Special Section Editors: Peter R. Galle, Peter L.M. Jansen, Francesco Negro
Epigenetic mechanism of stellate cell trans-differentiation Hide Tsukamoto* Department of Pathology, Research Center for Alcoholic Liver and Pancreatic Diseases and Cirrhosis, University of Southern California, Los Angeles, CA, USA
Regulation of myofibroblast transdifferentiation by DNA methylation and MeCP2: implications for wound healing and fibrogenesis. Mann J, Oakley F, Akiboye F, Elsharkawy A, Thorne AW, Mann DA. Myofibroblasts are critical cellular elements of wound healing generated at sites of injury by transdifferentiation of resident cells. A paradigm for this process is conversion of hepatic stellate cells (HSC) into hepatic myofibroblasts. Treatment of HSC with DNA methylation inhibitor 5-aza-2 0 -deoxycytidine (5-azadC) blocked transdifferentiation. 5-azadC also prevented loss of IjBa and PPARc expression that occurs during transdifferentiation to allow acquisition of proinflammatory and profibrogenic characteristics. ChIP analysis revealed IjBa promoter is associated with transcriptionally repressed chromatin that converts to an active state with 5-azadC treatment. The methyl-CpG-binding protein MeCP2 which promotes repressed chromatin structure is selectively detected in myofibroblasts of diseased liver. siRNA knockdown of MeCP2 elevated IjBa promoter activity, mRNA and protein expression in myofibroblasts. MeCP2 interacts with IjBa promoter via a methyl-CpG-dependent mechanism and recruitment into a CBF1 corepression complex. We conclude that MeCP2 and DNA methylation exert epigenetic control over hepatic wound healing and fibrogenesis. [Abstract reproduced by permission of Cell Death Differ 2006; doi:10.1038/sj.cdd.4401979; Epub ahead of print]
*
Tel.: +1 323 442 5107; fax: +1 323 442 3126. E-mail address: [email protected]
Myofibroblastic trans-differentiation (MTD) of hepatic stellate cells (HSC) is a pivotal cellular event in liver fibrogenesis. In MTD that is commonly called ‘‘activation’’, quiescent HSC lose their ability to store vitamin A and acquire a myofibroblastic phenotype typified by expression of a-smooth muscle actin and increased contractility. Research during the past two decades has identified phenotypic and transcriptional alterations that characterize MTD as well as mediators that are implicated in inducing these alterations. Yet, fundamental questions still remain as to what this MTD signifies in terms of the cell identity of HSC and how MTD is achieved by global mechanisms. A publication by Mann, J, et al. [1] addresses the latter question and their results link epigenetic regulation to the former question. Differentiation, de-differentiation, and trans-differentiation require coordinated regulation of a group of genes to facilitate a drastic phenotypic switch. Epigenetic regulation is known to be ideal for this requirement. Mann et al. present the first evidence of epigenetic regulation that underlies MTD of HSC. They demonstrate that the treatment of cultured HSC with DNA methylation inhibitor 5-aza-2-deoxycytidine (5-azadC) prevents MTD and loss of IjBa and PPARc expression that are both signature molecular changes and causal events for MTD. The methyl-CpG binding protein MeCP2 is induced in MTD and detectable in myofibroblastic cells of fibrotic human livers. Chromatin immunoprecipitation detects the binding of MeCP2 to IjBa promoter in ‘‘activated’’ HSC. Furthermore, knockdown of MeCP2 expression with siRNA restores IjBa expression and IjBa promoter activity. In order for MeCP2 to cause inhibitory epigenetic regulation via chromatin modification, a corepressor complex has to be recruited to the IjBa promoter including histone deacetylases (HDACs)
0168-8278/$32.00 Ó 2006 Published by Elsevier B.V. on behalf of the European Association for the Study of the Liver. doi:10.1016/j.jhep.2006.11.002
H. Tsukamoto / Journal of Hepatology 46 (2007) 352–353
and histone methyltransferases (HMTs). Although recruitment of HDACs and HMTs to the promoter is not directly demonstrated, ‘‘activated’’ HSC are shown to have increased levels of dimethylated lysine 9 (K9) and lack of acetylation in the tail of histone 3 as indication of transcriptionally repressed chromatin. Further, the 5-azadC treatment causes loss of the repressive dimethyl-K9 and enrichment of the transciptionally active acetylated histone 3. They also demonstrate an interaction of MeCP2 with CBF1, the co-repressor that was shown in their previous study to repress IjBa transcription in ‘‘activated’’ HSC. In essence, this study demonstrated the evidence of epigenetic regulation involving MeCP2 in MTD of HSC by using IjBa promoter as a model. How about PPARc? PPARc is considered as a potentially important therapeutic target for liver fibrosis [2]. Is there any evidence for its epigenetic regulation? The fact that the DNA methyltransferase inhibitor 5-azadC restored PPARc expression in Mann’s study suggests this notion. Indeed, evidence for epigenetic regulation of PPARc has recently emerged. Cyclin D1 down-regulates the activity of PPARc [3]. This effect is mediated in part via recruitment of HDAC 1 and 3 and HMT SUV39H1 to a PPAR response element as demonstrated for the lipoprotein lipase promoter [4]. This epigenetic regulation is apparently sufficient to inhibit adipocyte differentiation of murine embryonic fibroblasts induced by a PPARc ligand. The retinoblastoma protein (Rb) attenuates the activity of PPARc to drive adipocyte specific genes and adipocyte differentiation by directly interacting with the transcription factor and recruiting HDAC3 [5]. Dissociation of the PPARc-Rb-HDAC3 complex by phosphorylation of Rb or inhibition of HDAC activity promotes adipocyte differentiation [5]. More recently, the effects of global histone modifications on adipocyte differentiation have been reported [6]. This study demonstrates that histone hyperacetylation is selectively induced at the promoter regions of adipogenic genes including PPARc in differentiating adipocyte and that these changes are closely associated with uniformly suppressed expression of HDACs. Further, HDAC1 knockdown with siRNA enhances acetylation of K9 of histone 3; K8 and K12 of histone 4 while suppressing methylation of K9 of histone 3 and more importantly promotes adipocyte differentiation of 3T3L1 cells. These results suggest that HDAC expression
353
may be an essential regulatory event required for suppression of a panel of adipogenic transcription factors and maintenance of the preadipocyte phenotype and that down regulation of HDAC lifts this global inhibition of adipogenesis. Recent studies highlight the importance of adipogenic regulation in maintenance of HSC quiescence and loss of this regulation in HSC MTD [7–9]. Therefore, it is attractive to speculate that epigenetic regulation demonstrated for preadipocyte-adipocyte differentiation may also exist for determination of HSC MTD. In this respect, Mann’s study is not only seminal but also touches upon a fundamental question on the role of epigenetic regulation in mesenchymal cell plasticity and trans-differentiation. References [1] Mann J, Oakley F, Akiboye F, Elsharkawy A, Thorne AW, Mann DA. Regulation of Myofibroblast transdifferentiation by DNA methylation and MeCP2: implications for would healing and fibrogenesis. Cell Death Differ 2006. doi:10.1038/sj.cdd.4401979, [Epub ahead of print]. [2] Galli A, Crabb DW, Ceni E, Salzano R, Mello T, Svegliati-Baroni G, et al. Antidiabetic thiazolidinediones inhibit collagen synthesis and hepatic stellate cell activation in vivo and in vitro. Gastroenterology 2002;122:1924–1940. [3] Wang C, Pattabiraman N, Zhou JN, Fu M, Sakamaki T, Albanese C, et al. Cyclin D1 repression of peroxisome proliferator-activated receptor gamma expression and transactivation. Mol Cell Biol 2003;23:6159–6173. [4] Fu M, Rao M, Bouras T, Wang C, Wu K, Zhang X, et al. Cyclin D1 inhibits peroxisome proliferator-activated receptor gammamediated adipogenesis through histone deacetylase recruitment. J Biol Chem 2005;280:16934–16941. [5] Fajas L, Egler V, Reiter R, Hansen J, Kristiansen K, Debril MB, et al. The retinoblastoma-histone deacetylase 3 complex inhibits PPARgamma and adipocyte differentiation. Dev Cell 2002;3:903–910. [6] Yoo EJ, Chung JJ, Choe SS, Kim KH, Kim JB. Down-regulation of histone deacetylases stimulates adipocyte differentiation. J Biol Chem 2006;281:6608–6615. [7] Miyahara T, Schrum L, Rippe R, Xiong S, Yee Jr HF, Motomura K, et al. Peroxisome proliferator-activated receptors and hepatic stellate cell activation. J Biol Chem 2000;275:35715–35722. [8] Hazra S, Xiong S, Wang J, Rippe RA, Krishna V, Chatterjee K, et al. Peroxisome proliferator-activated receptor gamma induces a phenotypic switch from activated to quiescent hepatic stellate cells. J Biol Chem 2004;279:11392–11401. [9] She H, Xiong S, Hazra S, Tsukamoto H. Adipogenic transcriptional regulation of hepatic stellate cells. J Biol Chem 2005;280:4959–4967.
Journal Club Special Section Editors: Peter R. Galle, Peter L.M. Jansen, Francesco Negro
Epigenetic mechanism of stellate cell trans-differentiation Hide Tsukamoto* Department of Pathology, Research Center for Alcoholic Liver and Pancreatic Diseases and Cirrhosis, University of Southern California, Los Angeles, CA, USA
Regulation of myofibroblast transdifferentiation by DNA methylation and MeCP2: implications for wound healing and fibrogenesis. Mann J, Oakley F, Akiboye F, Elsharkawy A, Thorne AW, Mann DA. Myofibroblasts are critical cellular elements of wound healing generated at sites of injury by transdifferentiation of resident cells. A paradigm for this process is conversion of hepatic stellate cells (HSC) into hepatic myofibroblasts. Treatment of HSC with DNA methylation inhibitor 5-aza-2 0 -deoxycytidine (5-azadC) blocked transdifferentiation. 5-azadC also prevented loss of IjBa and PPARc expression that occurs during transdifferentiation to allow acquisition of proinflammatory and profibrogenic characteristics. ChIP analysis revealed IjBa promoter is associated with transcriptionally repressed chromatin that converts to an active state with 5-azadC treatment. The methyl-CpG-binding protein MeCP2 which promotes repressed chromatin structure is selectively detected in myofibroblasts of diseased liver. siRNA knockdown of MeCP2 elevated IjBa promoter activity, mRNA and protein expression in myofibroblasts. MeCP2 interacts with IjBa promoter via a methyl-CpG-dependent mechanism and recruitment into a CBF1 corepression complex. We conclude that MeCP2 and DNA methylation exert epigenetic control over hepatic wound healing and fibrogenesis. [Abstract reproduced by permission of Cell Death Differ 2006; doi:10.1038/sj.cdd.4401979; Epub ahead of print]
*
Tel.: +1 323 442 5107; fax: +1 323 442 3126. E-mail address: [email protected]
Myofibroblastic trans-differentiation (MTD) of hepatic stellate cells (HSC) is a pivotal cellular event in liver fibrogenesis. In MTD that is commonly called ‘‘activation’’, quiescent HSC lose their ability to store vitamin A and acquire a myofibroblastic phenotype typified by expression of a-smooth muscle actin and increased contractility. Research during the past two decades has identified phenotypic and transcriptional alterations that characterize MTD as well as mediators that are implicated in inducing these alterations. Yet, fundamental questions still remain as to what this MTD signifies in terms of the cell identity of HSC and how MTD is achieved by global mechanisms. A publication by Mann, J, et al. [1] addresses the latter question and their results link epigenetic regulation to the former question. Differentiation, de-differentiation, and trans-differentiation require coordinated regulation of a group of genes to facilitate a drastic phenotypic switch. Epigenetic regulation is known to be ideal for this requirement. Mann et al. present the first evidence of epigenetic regulation that underlies MTD of HSC. They demonstrate that the treatment of cultured HSC with DNA methylation inhibitor 5-aza-2-deoxycytidine (5-azadC) prevents MTD and loss of IjBa and PPARc expression that are both signature molecular changes and causal events for MTD. The methyl-CpG binding protein MeCP2 is induced in MTD and detectable in myofibroblastic cells of fibrotic human livers. Chromatin immunoprecipitation detects the binding of MeCP2 to IjBa promoter in ‘‘activated’’ HSC. Furthermore, knockdown of MeCP2 expression with siRNA restores IjBa expression and IjBa promoter activity. In order for MeCP2 to cause inhibitory epigenetic regulation via chromatin modification, a corepressor complex has to be recruited to the IjBa promoter including histone deacetylases (HDACs)
0168-8278/$32.00 Ó 2006 Published by Elsevier B.V. on behalf of the European Association for the Study of the Liver. doi:10.1016/j.jhep.2006.11.002
H. Tsukamoto / Journal of Hepatology 46 (2007) 352–353
and histone methyltransferases (HMTs). Although recruitment of HDACs and HMTs to the promoter is not directly demonstrated, ‘‘activated’’ HSC are shown to have increased levels of dimethylated lysine 9 (K9) and lack of acetylation in the tail of histone 3 as indication of transcriptionally repressed chromatin. Further, the 5-azadC treatment causes loss of the repressive dimethyl-K9 and enrichment of the transciptionally active acetylated histone 3. They also demonstrate an interaction of MeCP2 with CBF1, the co-repressor that was shown in their previous study to repress IjBa transcription in ‘‘activated’’ HSC. In essence, this study demonstrated the evidence of epigenetic regulation involving MeCP2 in MTD of HSC by using IjBa promoter as a model. How about PPARc? PPARc is considered as a potentially important therapeutic target for liver fibrosis [2]. Is there any evidence for its epigenetic regulation? The fact that the DNA methyltransferase inhibitor 5-azadC restored PPARc expression in Mann’s study suggests this notion. Indeed, evidence for epigenetic regulation of PPARc has recently emerged. Cyclin D1 down-regulates the activity of PPARc [3]. This effect is mediated in part via recruitment of HDAC 1 and 3 and HMT SUV39H1 to a PPAR response element as demonstrated for the lipoprotein lipase promoter [4]. This epigenetic regulation is apparently sufficient to inhibit adipocyte differentiation of murine embryonic fibroblasts induced by a PPARc ligand. The retinoblastoma protein (Rb) attenuates the activity of PPARc to drive adipocyte specific genes and adipocyte differentiation by directly interacting with the transcription factor and recruiting HDAC3 [5]. Dissociation of the PPARc-Rb-HDAC3 complex by phosphorylation of Rb or inhibition of HDAC activity promotes adipocyte differentiation [5]. More recently, the effects of global histone modifications on adipocyte differentiation have been reported [6]. This study demonstrates that histone hyperacetylation is selectively induced at the promoter regions of adipogenic genes including PPARc in differentiating adipocyte and that these changes are closely associated with uniformly suppressed expression of HDACs. Further, HDAC1 knockdown with siRNA enhances acetylation of K9 of histone 3; K8 and K12 of histone 4 while suppressing methylation of K9 of histone 3 and more importantly promotes adipocyte differentiation of 3T3L1 cells. These results suggest that HDAC expression
353
may be an essential regulatory event required for suppression of a panel of adipogenic transcription factors and maintenance of the preadipocyte phenotype and that down regulation of HDAC lifts this global inhibition of adipogenesis. Recent studies highlight the importance of adipogenic regulation in maintenance of HSC quiescence and loss of this regulation in HSC MTD [7–9]. Therefore, it is attractive to speculate that epigenetic regulation demonstrated for preadipocyte-adipocyte differentiation may also exist for determination of HSC MTD. In this respect, Mann’s study is not only seminal but also touches upon a fundamental question on the role of epigenetic regulation in mesenchymal cell plasticity and trans-differentiation. References [1] Mann J, Oakley F, Akiboye F, Elsharkawy A, Thorne AW, Mann DA. Regulation of Myofibroblast transdifferentiation by DNA methylation and MeCP2: implications for would healing and fibrogenesis. Cell Death Differ 2006. doi:10.1038/sj.cdd.4401979, [Epub ahead of print]. [2] Galli A, Crabb DW, Ceni E, Salzano R, Mello T, Svegliati-Baroni G, et al. Antidiabetic thiazolidinediones inhibit collagen synthesis and hepatic stellate cell activation in vivo and in vitro. Gastroenterology 2002;122:1924–1940. [3] Wang C, Pattabiraman N, Zhou JN, Fu M, Sakamaki T, Albanese C, et al. Cyclin D1 repression of peroxisome proliferator-activated receptor gamma expression and transactivation. Mol Cell Biol 2003;23:6159–6173. [4] Fu M, Rao M, Bouras T, Wang C, Wu K, Zhang X, et al. Cyclin D1 inhibits peroxisome proliferator-activated receptor gammamediated adipogenesis through histone deacetylase recruitment. J Biol Chem 2005;280:16934–16941. [5] Fajas L, Egler V, Reiter R, Hansen J, Kristiansen K, Debril MB, et al. The retinoblastoma-histone deacetylase 3 complex inhibits PPARgamma and adipocyte differentiation. Dev Cell 2002;3:903–910. [6] Yoo EJ, Chung JJ, Choe SS, Kim KH, Kim JB. Down-regulation of histone deacetylases stimulates adipocyte differentiation. J Biol Chem 2006;281:6608–6615. [7] Miyahara T, Schrum L, Rippe R, Xiong S, Yee Jr HF, Motomura K, et al. Peroxisome proliferator-activated receptors and hepatic stellate cell activation. J Biol Chem 2000;275:35715–35722. [8] Hazra S, Xiong S, Wang J, Rippe RA, Krishna V, Chatterjee K, et al. Peroxisome proliferator-activated receptor gamma induces a phenotypic switch from activated to quiescent hepatic stellate cells. J Biol Chem 2004;279:11392–11401. [9] She H, Xiong S, Hazra S, Tsukamoto H. Adipogenic transcriptional regulation of hepatic stellate cells. J Biol Chem 2005;280:4959–4967.