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MKL1 Is an Epigenetic Modulator of TGF-Β Induced Fibrogenesis

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MKL1 Is an Epigenetic Modulator of TGF-Β Induced Fibrogenesis Biochimica et Biophysica Acta 1849 (2015) 1219–1228 Contents lists available at ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbagrm MKL1 is an epigenetic modulator of TGF-β induced fibrogenesis Zhiwen Fan a, Chenzhi Hao a,MinLia, Xin Dai a, Hao Qin a,JianfeiLia,HuihuiXua, Xiaoyan Wu a, Liping Zhang b,MingmingFanga,c, Bisheng Zhou a, Wenfang Tian a,YongXua,⁎ a Key Laboratory of Cardiovascular Disease, Department of Pathophysiology, Nanjing Medical University, Nanjing, China b Department of Biochemistry, Xinjiang Medical University, Urumqi, China c Department of Nursing, Jiangsu Jiankang Vocational University, Nanjing, China article info abstract Article history: Transforming growth factor (TGF-β) induced activation of portal fibroblast cells serves as a primary cause for Received 16 May 2015 liver fibrosis following cholestatic injury. The underlying epigenetic mechanism is not clear. We studied the Received in revised form 13 July 2015 role of a transcriptional modulator, megakaryoblastic leukemia 1 (MKL1) in this process. We report here that Accepted 31 July 2015 MKL1 deficiency ameliorated BDL-induced liver fibrosis in mice as assessed by histological stainings and expres- Available online 1 August 2015 sion 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 Keywords: fi Epigenetics promoters of pro- brogenic genes and promoted the interaction between MKL1 and SMAD3. While SMAD3 Transcriptional regulation was necessary for MKL1 occupancy on the gene promoters, MKL1 depletion impaired SMAD3 binding reciprocal- Portal fibroblast ly. TGF-β treatment induced the accumulation of trimethylated histone H3K4 on the gene promoters by Liver fibrosis recruiting a methyltransferase complex. Knockdown of individual members of this complex significantly weak- TGF-β ened the binding of SMAD3 and down-regulated the activation of portal fibroblast cells. In conclusion, we have Histone methylation 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. © 2015 Elsevier B.V. All rights reserved. 1. Introduction primarily derived from portal fibroblasts [6]. A host of growth factors and metabolites have been shown to activate portal fibroblasts, among Fibrogenesis is an adaptive response to liver injury, which can lead which TGF-β is by far the most extensively studied [7–9].Itisunclear, to, if left unattended to, irreversible tissue scar formation, cirrhosis however, how the epigenetic machinery modulates TGF-β signaling and and hepatocellular carcinoma [1]. In the process of liver fibrosis, there pro-fibrogenic transcriptional program in portal fibroblast cells. is accelerated synthesis and deposition of extracellular matrix (ECM) Megakaryoblastic leukemia 1 (MKL1), also termed myocardin- proteins such as collagen type I, collagen type III, and fibronectin. related transcription factor A (MRTF-A), is a transcriptional modulator The mechanism underlying transactivation of these so-called “pro- that plays a redundant role in smooth muscle cell differentiation [10]. fibrogenic” genes remains incompletely understood. Independent investigations in our laboratory as well as several It is generally believed that hepatic stellate cells (HSCs), transitioning other laboratories have recently uncovered a role for MKL1 in tissue from a quiescent lipid-storing state to a proliferative ECM-producing fibrogenesis in a number of different settings including ischemia- state, primarily contribute to liver fibrosis following hepatotoxic injuries induced myocardial fibrosis [11], bleomycin-induced pulmonary fibro- (e.g., chemicals, viruses) [2]. On the other hand, there has been much con- sis [12], and diabetes-induced renal fibrosis [13]. Previous investigations troversy regarding the source of cells for liver fibrogenesis as a result of have also revealed that MKL1 regulates transcription by bridging the cholestatic injury (e.g., bile duct obstruction); several different types of epigenetic machinery to various intrinsic and extrinsic stress cues cells, including HSCs and hepatocytes, have been suggested to play a [14–18].Buildingonthesefindings, we sought to clarify the role of role in this process [3–5]. By using a series of delicate lineage tracing MKL1 in TGF-β induced activation of portal fibroblast cells and the experiments, Iwaisako et al have recently demonstrated that the pro- underlying epigenetic mechanism in the present study. fibrogenic myofibroblasts observed in cholestatic liver injury are 2. Materials and methods Abbreviations: TGF-β, transforming growth factor; MKL1, megakaryoblastic leukemia 2.1. Animals 1; BDL, bile duct ligation; ChIP, chromatin immunoprecipitation; COMPASS, complex proteins associated with SET; TF, transcription factor fi ⁎ Corresponding author at: Nanjing Medical University, Nanjing, Jiangsu 210029, China. MKL1 de cient (KO) mice were obtained from Dr. Steve Morris at E-mail address: [email protected] (Y. Xu). St. Jude Children's Hospital [19]. To induce liver injury, 8-week old, KO http://dx.doi.org/10.1016/j.bbagrm.2015.07.013 1874-9399/© 2015 Elsevier B.V. All rights reserved. 1220 Z. Fan et al. / Biochimica et Biophysica Acta 1849 (2015) 1219–1228 male mice and wild type (WT) littermates were anesthetized with Col1a2 promoter, 5′-GACATGCTCAAGTGCTGAGTCAC-3′ and 5′- ketamine. The common bile duct was ligated twice with silk sutures. AGATTGCACAATGTGACGTCG-3′; Acta2 promoter, 5′-CATGCACGTG Bile duct ligation (BDL) and sham-operated mice were sacrificed two GACTGTACCT-3′ and 5′-AAAGATGCTTGGGTCACCTG-3′; Gapdh pro- weeks following the surgical procedure. All animal experiments have moter, 5′-ATCACTGCCACCCAGAAGACTGTGGA-3′ and 5′-C TCATAC been performed following guidelines by the intramural Committee on CAGGAAATGAGCTTGACAAA-3′. All experiments were repeated at Ethical Conduct of Animal Studies. least three times. 2.2. Cell culture and treatment 2.7. Histology Primary rat portal fibroblast cells were isolated and maintained as Histological analyses were performed essentially as described before previously described [20]. The identity of primary portal fibroblast [27,30]. Briefly, paraffin sections were stained with hematoxylin and cells were verified by immunostaining as previously described [21]. eosin (Sigma), picrosirius red (Sigma), or Masson's trichrome (Sigma) Mouse embryonic fibroblast cells were isolated from wild type and according to standard procedures. Alternatively, the sections were MKL1 deficient mice [19].TGF-β was purchased from R&D. Prior to blocked with 10% normal goat serum for 1 h at room temperature and treatment, cells were starved in .4% DMEM overnight. then incubated with anti-collagen type I or anti-α-SMA antibodies. Staining was visualized by incubation with an appropriate biotinylated 2.3. Plasmids, siRNAs, and transient transfection 2° antibody and developed with a streptavidin-horseradish peroxidase kit (Pierce) for 20 min. Pictures were taken using an Olympus IX-70 mi- Col1a1, Col1a2,andActa2 promoter–luciferase fusion constructs [22] croscope. For quantification, positive stainings were counted using as well as small interfering RNA (siRNA) sequences for rat ASH2, WDR5, Image Pro (Media Cybernetics). At least 3 slides were included for and SET1 [23] have been have been previously described. FLAG-tagged each mouse and for each slide at least 5 fields were counted. Data are full-length MKL1 expression construct in a pCMV backbone was a gift expressed as relative staining compared to the control group which is from Dr. Ron Prywes [24]. Transient transfections were performed arbitrarily set as 1. with Lipofectamine 2000 (Invitrogen). Luciferase activities were assayed 24–48 h after transfection using a luciferase reporter assay 2.8. Statistical analysis system (Promega). For retrovirus production, the FLAG-tagged MKL1 was amplified from its original vector by PCR and sub-cloned into a One-way ANOVA with post-hoc Scheffe analyses were performed pBabe-puro vector [25]. Retroviral stocks were produced in 293FT using an SPSS package. Unless otherwise specified, p values smaller cells as previously described [26] and used to infect MEF cells in the than .05 were considered statistically significant (*). presence of polybrene (Sigma). All experiments were repeated at least three times. 3. Results 2.4. Protein extraction and Western blotting 3.1. MKL1 deficient mice are resistant to bile duct ligation (BDL) induced liver fibrosis Whole-cell lysates were obtained by re-suspending cell pellets in RIPA buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1% Triton X-100) with We first examined the involvement of MKL1 in TGF-β induced liver freshly added protease inhibitor (Roche). Nuclear proteins were ex- fibrogenesis in a murine BDL model, in which TGF-β has been shown to tracted using a NE-PER kit (Thermo Fisher) as previously described play a key role in driving the synthesis of multiple ECM proteins [27]. Western blot analyses were performed with anti-collagen type I [31–33]. BDL-induced liver fibrosis differs from chemical/toxin- (Rockland, 600-401-103), anti-α-SMA (Sigma, A2547), anti-β-actin induced liver fibrosis in that portal fibroblast cell plays a central role (Sigma, A1978), anti-α-tubulin (Sigma, T9026), anti-MKL1 (Santa in the former model whereas hepatic stellate cell plays a dominant Cruz, SC-32909),
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