Zhiwen Fan

Myocardin related transcription factor A programs epigenetic activation of hepatic stellate cells

BACKGROUND & AIMS Activation of hepatic stellate cells (HSCs) represents a key process in liver injury and, in the absence of intervention, leads to irreversible cirrhosis contributing significantly to the mortality of patients with liver disease. A missing link in the current understanding of HSC activation is the involvement of the epigenetic machinery. We investigated the role of the myocardin related transcription factor A (MRTF-A) in HSC activation. METHODS Liver fibrosis was induced in wild type (WT) and MRTF-A deficient (KO) mice by CCl4 injection. Expression of mRNA and protein was measured by real-time PCR, Western blotting, and immunohistochemistry. Protein binding to DNA was assayed by chromatin immunoprecipitation (ChIP). Knockdown of endogenous proteins was mediated by either small interfering RNA (siRNA) or short hairpin RNA (shRNA), carried by lentiviral particles. RESULTS KO mice exhibited resistance to CCl4-induced liver fibrosis compared to WT littermates. The expression of activated HSC signature genes was suppressed in the absence of MRTF-A. ChIP assays revealed that MRTF-A deficiency led to the erasure of key histone modifications, associated with transcriptional activation, such as H3K4 di- and tri-methylation, on the promoter regions of fibrogenic genes. Mechanistically, MRTF-A recruited a histone methyltransferase complex (COMPASS) to the promoters of fibrogenic genes to activate transcription. Silencing of individual COMPASS components dampened transactivation of fibrogenic genes in vitro and blocked liver fibrosis in mice. Oestradiol suppressed HSC activation by dampening the expression and binding activity of COMPASS. CONCLUSIONS Our data illustrate a novel mechanism that connects MRTF-A dependent histone H3K4 methylation to HSC activation.

Histone Methyltransferase SET1 Mediates Angiotensin II–Induced Endothelin-1 Transcription and Cardiac Hypertrophy in Mice

Objective— Endothelin-1 is a potent vasoconstrictor derived from vascular endothelium. Elevated endothelin-1 levels are observed in a host of cardiovascular pathologies including cardiomyopathy. The epigenetic mechanism responsible for endothelin-1 induction in these pathological processes remains elusive. Approach and Results— We report here that induction of endothelin-1 expression in endothelial cells by angiotensin II (Ang II) was accompanied by the accumulation of histone H3K4 trimethylation, a preeminent histone modification for transcriptional activation, on the endothelin-1 promoter. In the meantime, Ang II stimulated the expression and the occupancy of Suv, Ez, and Trithorax domain 1 (SET1), a mammalian histone H3K4 trimethyltransferase, on the endothelin-1 promoter, both in vitro and in vivo. SET1 was recruited to the endothelin-1 promoter by activating protein 1 (c-Jun/c-Fos) and synergized with activating protein 1 to activate endothelin-1 transcription in response to Ang II treatment. Knockdown of SET1 in endothelial cells blocked Ang II–induced endothelin-1 synthesis and abrogated hypertrophy of cultured cardiomyocyte. Finally, endothelial-specific depletion of SET1 in mice attenuated Ang II–induced pathological hypertrophy and cardiac fibrosis. Conclusions— Our data suggest that SET1 epigenetically activates endothelin-1 transcription in endothelial cells, thereby contributing to Ang II–induced cardiac hypertrophy. As such, screening of small-molecule compound that inhibits SET1 activity will likely offer a new therapeutic solution to the treatment of cardiomyopathy.

MKL1 is an epigenetic modulator of TGF-β induced fibrogenesis.

Transforming growth factor (TGF-β) induced activation of portal fibroblast cells serves as a primary cause for liver fibrosis following cholestatic injury. The underlying epigenetic mechanism is not clear. We studied the role of a transcriptional modulator, megakaryoblastic leukemia 1 (MKL1) in this process. We report here that MKL1 deficiency ameliorated BDL-induced liver fibrosis in mice as assessed by histological stainings and expression levels of pro-fibrogenic genes. MKL1 silencing by small interfering RNA (siRNA) abrogated TGF-β induced transactivation of pro-fibrogenic genes in portal fibroblast cells. TGF-β stimulated the binding of MKL1 on the promoters of pro-fibrogenic genes and promoted the interaction between MKL1 and SMAD3. While SMAD3 was necessary for MKL1 occupancy on the gene promoters, MKL1 depletion impaired SMAD3 binding reciprocally. TGF-β treatment induced the accumulation of trimethylated histone H3K4 on the gene promoters by recruiting a methyltransferase complex. Knockdown of individual members of this complex significantly weakened the binding of SMAD3 and down-regulated the activation of portal fibroblast cells. In conclusion, we have identified an epigenetic pathway that dictates TGF-β induced pro-fibrogenic transcription in portal fibroblast thereby providing novel insights for the development of therapeutic solutions to treat liver fibrosis.

Myocardin-related transcription factor A (MRTF-A) plays an essential role in hepatic stellate cell activation by epigenetically modulating TGF-β signaling

Fibrosis following injury is a common adaptive response in the liver, which can lead to irreparable and life-threatening cirrhosis and hepatocellular carcinoma without effectual intervention. The molecular mechanisms underlying fibrogenic response in the liver remains poorly understood. Here we report that mice with deficiency in myocardin-related transcription factor A (MRTF-A) showed resistance to thioacetamide (TAA)-induced liver fibrosis with significantly reduced expression of pro-fibrogenic genes when compared to wild type littermates. Over-expression of MRTF-A enhanced whereas depletion of MRTF-A alleviated pro-fibrogenic transcription induced by TGF-β, a major pro-fibrogenic factor in hepatic stellate cells (HSCs). Mechanistically, MRTF-A silencing in HSCs impacted the chromatin structure by reducing the deposition of methylated histone H3K4 on the promoters of pro-fibrogenic genes. Further analyses revealed that MRTF-A interacted with and recruited several key epigenetic factors involved in H3K4 methylation, including ASH2, WDR5, and SET1, to the promoters of pro-fibrogenic genes in response to TGF-β treatment. Over-expression of ASH2, WDR5, or SET1 enhanced the transactivation of pro-fibrogenic gene promoters by TGF-β in an MRTF-A-dependent manner. In conclusion, MRTF-A regulates liver fibrosis by epigenetically tuning the TGF-β signaling pathway in HSCs.

HDAC4 mediates IFN-γ induced disruption of energy expenditure-related gene expression by repressing SIRT1 transcription in skeletal muscle cells.

Metabolic homeostasis is achieved through balanced energy storage and output. Impairment of energy expenditure is a hallmark event in patients with obesity and type 2 diabetes. Previously we have shown that the pro-inflammatory cytokine interferon gamma (IFN-γ) disrupts energy expenditure in skeletal muscle cells via hypermethylated in cancer 1 (HIC1)-class II transactivator (CIITA) dependent repression of SIRT1 transcription. Here we report that repression of SIRT1 transcription by IFN-γ paralleled loss of histone acetylation on the SIRT1 promoter region with simultaneous recruitment of histone deacetylase 4 (HDAC4). IFN-γ activated HDAC4 in vitro and in vivo by up-regulating its expression and stimulating its nuclear accumulation. HIC1 and CIITA recruited HDAC4 to the SIRT1 promoter and cooperated with HDAC4 to repress SIRT1 transcription. HDAC4 depletion by small interfering RNA or pharmaceutical inhibition normalized histone acetylation on the SIRT1 promoter and restored SIRT1 expression in the presence of IFN-γ. Over-expression of HDAC4 suppressed the transcription of genes involved in energy expenditure in a SIRT1-dependent manner. In contrast, HDAC4 knockdown/inhibition neutralized the effect of IFN-γ on cellular metabolism by normalizing SIRT1 expression. Therefore, our data reveal a role for HDAC4 in regulating cellular energy output and as such provide insights into rationalized design of novel anti-diabetic therapeutics.

Angiogenic factor with G patch and FHA domains 1 (Aggf1) regulates liver fibrosis by modulating TGF-β signaling

Fibrosis is a common pathophysiological process following liver injury and can lead to, if left unattended to, irreversible end-stage liver disease such as cirrhosis. Hepatic stellate cells (HSCs) are a major contributor to liver fibrosis. Here we investigated the involvement of angiogenic factor with G patch and FHA domains 1 (Aggf1) in HSC activation and the underlying mechanisms. Aggf1 expression was down-regulated in the livers in three different mouse models of liver fibrosis following injury. Aggf1 expression was also suppressed in activated HSCs when compared to quiescent HSCs. Over-expression of Aggf1 alleviated liver fibrosis in mice and in cultured HSCs. RNA-sequencing (RNA-seq) analysis performed in HSCs revealed that Aggf1-dependent transcription regulates several key fibrogenic pathways. Mechanistically, Aggf1 regulated liver fibrogenesis by forming a complex with the inhibitor SMAD protein (SMAD7) thereby leading to diminished SMAD3 binding to the pro-fibrogenic gene promoters. On the contrary, SMAD7 knockdown abrogated the effect of Aggf1 and rescued HSC activation. Aggf1 expression was silenced during HSC activation/liver fibrogenesis as a result of DNA methylation. Treatment with a DNA methyltransferase inhibitor (5-Azacytidine) restored Aggf1 expression and repressed liver fibrosis in an Aggf1-dependent manner. In conclusion, our data illustrate a previously unknown role for Aggf1 and shed light on the development of novel therapeutic solutions against liver fibrosis.

Aggf1 attenuates hepatic inflammation and activation of hepatic stellate cells by repressing Ccl2 transcription.

Liver injury represents a continuum of pathophysiological processes involving a complex interplay between hepatocytes, macrophages, and hepatic stellate cells. The mechanism whereby these intercellular interactions contribute to liver injury and fibrosis is not completely understood. We report here that angiogenic factor with G patch and FHA domains 1 (Aggf1) was downregulated in the livers of cirrhotic patients compared to healthy controls and in primary hepatocytes in response to carbon tetrachloride (CCl4) stimulation. Overexpression of Aggf1 attenuated macrophage chemotaxis. Aggf1 interacted with NF-κB to block its binding to the Ccl2 gene promoter and repressed Ccl2 transcription in hepatocytes. Macrophages cultured in the conditioned media collected from Aggf1-overexpressing hepatocytes antagonized HSC activation. Taken together, our data illustrate a novel role for Aggf1 in regulating hepatic inflammation and provide insights on the development of interventional strategies against cirrhosis.Liver injury represents a continuum of pathophysiological processes involving a complex interplay between hepatocytes, macrophages, and hepatic stellate cells. The mechanism whereby these intercellular interactions contribute to liver injury and fibrosis is not completely understood. We report here that angiogenic factor with G patch and FHA domains 1 (Aggf1) was downregulated in the livers of cirrhotic patients compared to healthy controls and in primary hepatocytes in response to carbon tetrachloride (CCl4) stimulation. Overexpression of Aggf1 attenuated macrophage chemotaxis. Aggf1 interacted with NF-κB to block its binding to the Ccl2 gene promoter and repressed Ccl2 transcription in hepatocytes. Macrophages cultured in the conditioned media collected from Aggf1-overexpressing hepatocytes antagonized HSC activation. Taken together, our data illustrate a novel role for Aggf1 in regulating hepatic inflammation and provide insights on the development of interventional strategies against cirrhosis.

Transcriptional repression of SIRT1 by protein inhibitor of activated STAT 4 (PIAS4) in hepatic stellate cells contributes to liver fibrosis

Interstitial fibrosis represents a key pathological process in non-alcoholic steatohepatitis (NASH). In the liver, fibrogenesis is primarily mediated by activated hepatic stellate cells (HSCs) transitioning from a quiescent state in response to a host of stimuli. The molecular mechanism underlying HSC activation is not completely understood. Here we report that there was a simultaneous up-regulation of PIAS4 expression and down-regulation of SIRT1 expression accompanying increased hepatic fibrogenesis in an MCD-diet induced mouse model of NASH. In cultured primary mouse HSCs, stimulation with high glucose activated PIAS4 while at the same time repressed SIRT1. Over-expression of PIAS4 directly repressed SIRT1 promoter activity. In contrast, depletion of PIAS4 restored SIRT1 expression in HSCs treated with high glucose. Estrogen, a known NASH-protective hormone, antagonized HSC activation by targeting PIAS4. Lentivirus-mediated delivery of short hairpin RNA (shRNA) targeting PIAS4 in mice ameliorated MCD diet induced liver fibrosis by normalizing SIRT1 expression in vivo. PIAS4 promoted HSC activation in a SIRT1-dependent manner in vitro. Mechanistically, PIAS4 mediated SIRT1 repression led to SMAD3 hyperacetylation and enhanced SMAD3 binding to fibrogenic gene promoters. Taken together, our data suggest SIRT1 trans-repression by PIAS4 plays an important role in HSC activation and liver fibrosis.

The arginine methyltransferase PRMT5 regulates CIITA-dependent MHC II transcription

Class II major histocompatibility complex (MHC II) dependent antigen presentation serves as a key step in mammalian adaptive immunity and host defense. In antigen presenting cells (e.g., macrophages), MHC II transcription can be activated by interferon gamma (IFN-γ) and mediated by class II transactivator (CIITA). The underlying epigenetic mechanism, however, is not completely understood. Here we report that following IFN-γ stimulation, symmetrically dimethylated histone H3 arginine 2 (H3R2Me2s) accumulated on the MHC II promoter along with CIITA. IFN-γ augmented expression, nuclear translocation, and promoter binding of the protein arginine methyltransferase PRMT5 in macrophages. Over-expression of PRMT5 potentiated IFN-γ induced activation of MHC II transcription in an enzyme activity-dependent manner. In contrast, PRMT5 silencing or inhibition of PRMT5 activity by methylthioadenosine (MTA) suppressed MHC II transactivation by IFN-γ. CIITA interacted with and recruited PRMT5 to the MHC II promoter and mediated the synergy between PRMT5 and ASH2/WDR5 to activate MHC II transcription. PRMT5 expression was down-regulated in senescent and H2O2-treated macrophages rendering ineffectual induction of MHC II transcription by IFN-γ. Taken together, our data reveal a pathophysiologically relevant role for PRMT5 in MHC II transactivation in macrophages.

Protein inhibitor of activated STAT 4 (PIAS4) regulates liver fibrosis through modulating SMAD3 activity

Abstract Excessive fibrogenesis disrupts normal liver structure, impairs liver function, and precipitates the development of cirrhosis, an irreversible end-stage liver disease. A host of factors including nutrition surplus contribute to liver fibrosis but the underlying mechanism is not fully understood. In the present study, we investigated the involvement of protein inhibitor for activated stat 4 (PIAS4) in liver fibrosis in a mouse model of non-alcoholic steatohepatitis (NASH). We report that PIAS4 silencing using short hairpin RNA (shRNA) attenuated high-fat high-carbohydrate (HFHC) diet induced liver fibrosis in mice. Quantitative PCR and Western blotting analyses confirmed that PIAS4 knockdown downregulated a panel of pro-fibrogenic genes including type I and type III collagens, smooth muscle actin, and tissue inhibitors of metalloproteinase. Mechanistically, PIAS4 silencing blocked the recruitment of SMAD3, a potent pro-fibrogenic transcription factor, to the promoter regions of pro-fibrogenic genes and dampened SMAD3 acetylation likely by upregulating SIRT1 expression. In conclusion, PIAS4 may contribute to liver fibrosis by modulating SIRT1-dependent SMAD3 acetylation.