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• W2026639078 abstract "DNA methylation is a key mechanism for repression of gene expression, including that of α-smooth muscle actin (α-SMA) gene expression in fibroblasts. However, the trans-acting factors that interact with the methylated α-SMA gene to regulate its expression have not been identified. Using gel shift and chromatin immunoprecipitation (ChIP) assays, methyl CpG binding protein 2 (MeCP2) was shown to bind to the α-SMA gene. Suppression of MeCP2 gene expression by siRNA or its deficiency in lung fibroblasts isolated from MeCP2 knockout mice caused significant reduction of α-SMA gene expression. In contrast, transient transfection of MeCP2 expression plasmid into fibroblasts enhanced α-SMA gene expression. Moreover, in vivo studies revealed that compared to their wild type littermates, MeCP2-deficient mice exhibited significantly decreased alveolar wall thickness, inflammatory cell infiltration, interstitial collagen deposition, and myofibroblast differentiation in response to endotracheal injection of bleomycin. Thus, MeCP2 is essential for myofibroblast differentiation and pulmonary fibrosis. DNA methylation is a key mechanism for repression of gene expression, including that of α-smooth muscle actin (α-SMA) gene expression in fibroblasts. However, the trans-acting factors that interact with the methylated α-SMA gene to regulate its expression have not been identified. Using gel shift and chromatin immunoprecipitation (ChIP) assays, methyl CpG binding protein 2 (MeCP2) was shown to bind to the α-SMA gene. Suppression of MeCP2 gene expression by siRNA or its deficiency in lung fibroblasts isolated from MeCP2 knockout mice caused significant reduction of α-SMA gene expression. In contrast, transient transfection of MeCP2 expression plasmid into fibroblasts enhanced α-SMA gene expression. Moreover, in vivo studies revealed that compared to their wild type littermates, MeCP2-deficient mice exhibited significantly decreased alveolar wall thickness, inflammatory cell infiltration, interstitial collagen deposition, and myofibroblast differentiation in response to endotracheal injection of bleomycin. Thus, MeCP2 is essential for myofibroblast differentiation and pulmonary fibrosis. Induction of myofibroblast differentiation is a key feature of wound healing, tissue repair, and remodeling or fibrosis.1Phan S.H. The myofibroblast in pulmonary fibrosis.Chest. 2002; 122: 286S-289SCrossref PubMed Scopus (408) Google Scholar, 2Scotton C.J. Chambers R.C. Molecular targets in pulmonary fibrosis: the myofibroblast in focus.Chest. 2007; 132 (1311-1121)Crossref PubMed Scopus (437) Google Scholar, 3Darby I.A. Hewitson T.D. Fibroblast differentiation in wound healing and fibrosis.Int Rev Cytol. 2007; 257: 143-179Crossref PubMed Scopus (391) Google Scholar, 4Thannickal V.J. Toews G.B. White E.S. Lynch 3rd, J.P. Martinez F.J. Mechanisms of pulmonary fibrosis.Annu Rev Med. 2004; 55: 395-417Crossref PubMed Scopus (530) Google Scholar, 5Gharaee-Kermani M. Hu B. Phan S.H. Gyetko M.R. Recent advances in molecular targets and treatment of idiopathic pulmonary fibrosis: focus on TGFbeta signaling and the myofibroblast.Curr Med Chem. 2009; 16: 1400-1417Crossref PubMed Scopus (118) Google Scholar, 6Pardo A. Selman M. Idiopathic pulmonary fibrosis: new insights in its pathogenesis.Int J Biochem Cell Biol. 2002; 34: 1534-1538Crossref PubMed Scopus (116) Google Scholar The myofibroblast arises de novo at these sites of tissue repair, and is a characteristic cellular component of active tissue remodeling, such as that in the fibroblastic foci of affected lungs from patients with idiopathic pulmonary fibrosis.1Phan S.H. The myofibroblast in pulmonary fibrosis.Chest. 2002; 122: 286S-289SCrossref PubMed Scopus (408) Google Scholar, 2Scotton C.J. Chambers R.C. Molecular targets in pulmonary fibrosis: the myofibroblast in focus.Chest. 2007; 132 (1311-1121)Crossref PubMed Scopus (437) Google Scholar, 3Darby I.A. Hewitson T.D. Fibroblast differentiation in wound healing and fibrosis.Int Rev Cytol. 2007; 257: 143-179Crossref PubMed Scopus (391) Google Scholar, 4Thannickal V.J. Toews G.B. White E.S. Lynch 3rd, J.P. Martinez F.J. Mechanisms of pulmonary fibrosis.Annu Rev Med. 2004; 55: 395-417Crossref PubMed Scopus (530) Google Scholar, 5Gharaee-Kermani M. Hu B. Phan S.H. Gyetko M.R. Recent advances in molecular targets and treatment of idiopathic pulmonary fibrosis: focus on TGFbeta signaling and the myofibroblast.Curr Med Chem. 2009; 16: 1400-1417Crossref PubMed Scopus (118) Google Scholar, 6Pardo A. Selman M. Idiopathic pulmonary fibrosis: new insights in its pathogenesis.Int J Biochem Cell Biol. 2002; 34: 1534-1538Crossref PubMed Scopus (116) Google Scholar The myofibroblast is a major source of the extracellular matrix that is found in these areas of remodeling, as well as cytokines, such as transforming growth factor β (TGFβ).1Phan S.H. The myofibroblast in pulmonary fibrosis.Chest. 2002; 122: 286S-289SCrossref PubMed Scopus (408) Google Scholar, 2Scotton C.J. Chambers R.C. Molecular targets in pulmonary fibrosis: the myofibroblast in focus.Chest. 2007; 132 (1311-1121)Crossref PubMed Scopus (437) Google Scholar, 3Darby I.A. Hewitson T.D. Fibroblast differentiation in wound healing and fibrosis.Int Rev Cytol. 2007; 257: 143-179Crossref PubMed Scopus (391) Google Scholar, 4Thannickal V.J. Toews G.B. White E.S. Lynch 3rd, J.P. Martinez F.J. Mechanisms of pulmonary fibrosis.Annu Rev Med. 2004; 55: 395-417Crossref PubMed Scopus (530) Google Scholar, 5Gharaee-Kermani M. Hu B. Phan S.H. Gyetko M.R. Recent advances in molecular targets and treatment of idiopathic pulmonary fibrosis: focus on TGFbeta signaling and the myofibroblast.Curr Med Chem. 2009; 16: 1400-1417Crossref PubMed Scopus (118) Google Scholar, 6Pardo A. Selman M. Idiopathic pulmonary fibrosis: new insights in its pathogenesis.Int J Biochem Cell Biol. 2002; 34: 1534-1538Crossref PubMed Scopus (116) Google Scholar The myofibroblast's characteristic expression of α-SMA is widely used as a marker for its identification as well as differentiation from precursor cells, such as fibroblasts.1Phan S.H. The myofibroblast in pulmonary fibrosis.Chest. 2002; 122: 286S-289SCrossref PubMed Scopus (408) Google Scholar In the lung the myofibroblast is found to be essential for alveolar development,7Boström H. Willetts K. Pekny M. Levéen P. Lindahl P. Hedstrand H. Pekna M. Hellström M. Gebre-Medhin S. Schalling M. Nilsson M. Kurland S. Törnell J. Heath b.J.K. Betsholtz C. PDGF-A signaling is a critical event in lung alveolar myofibroblast development and alveogenesis.Cell. 1996; 85: 863-873Abstract Full Text Full Text PDF PubMed Scopus (693) Google Scholar but it is also implicated in pathogenesis of chronic fibrotic lung diseases.6Pardo A. Selman M. Idiopathic pulmonary fibrosis: new insights in its pathogenesis.Int J Biochem Cell Biol. 2002; 34: 1534-1538Crossref PubMed Scopus (116) Google Scholar The myofibroblast is thought to promote fibrosis by engaging in cross talk with adjacent alveolar epithelial cells resulting in heightened production of fibrogenic cytokines and extracellular matrix components, with consequent distortion of normal lung architecture and mechanical properties.1Phan S.H. The myofibroblast in pulmonary fibrosis.Chest. 2002; 122: 286S-289SCrossref PubMed Scopus (408) Google Scholar, 2Scotton C.J. Chambers R.C. Molecular targets in pulmonary fibrosis: the myofibroblast in focus.Chest. 2007; 132 (1311-1121)Crossref PubMed Scopus (437) Google Scholar, 3Darby I.A. Hewitson T.D. Fibroblast differentiation in wound healing and fibrosis.Int Rev Cytol. 2007; 257: 143-179Crossref PubMed Scopus (391) Google Scholar, 4Thannickal V.J. Toews G.B. White E.S. Lynch 3rd, J.P. Martinez F.J. Mechanisms of pulmonary fibrosis.Annu Rev Med. 2004; 55: 395-417Crossref PubMed Scopus (530) Google Scholar, 5Gharaee-Kermani M. Hu B. Phan S.H. Gyetko M.R. Recent advances in molecular targets and treatment of idiopathic pulmonary fibrosis: focus on TGFbeta signaling and the myofibroblast.Curr Med Chem. 2009; 16: 1400-1417Crossref PubMed Scopus (118) Google Scholar, 6Pardo A. Selman M. Idiopathic pulmonary fibrosis: new insights in its pathogenesis.Int J Biochem Cell Biol. 2002; 34: 1534-1538Crossref PubMed Scopus (116) Google Scholar Given these significant developmental and pathogenic roles of the myofibroblast, obtaining insight into the mechanism of its differentiation from precursor cells would be of considerable interest. Recent studies have focused on signaling and complex/combinatorial transcriptional, regulatory mechanisms of the marker gene, α-SMA expression,8Hu B. Wu Z. Phan S.H. Smad3 mediates transforming growth factor-beta-induced alpha-SMA expression.Am J Respir Cell Mol Biol. 2003; 29: 397-404Crossref PubMed Scopus (270) Google Scholar, 9Hu B. Wu Z. Liu T. Ullenbruch M.R. Jin H. Phan S.H. Gut-enriched Krüppel-like factor interaction with Smad3 inhibits myofibroblast differentiation.Am J Respir Cell Mol Biol. 2007; 36: 78-84Crossref PubMed Scopus (51) Google Scholar, 10Hu B. Wu Z. Jin H. Hashimoto N. Liu T. Phan S.H. CCAAT/enhancer-binding protein beta isoforms and the regulation of alpha-SMA gene expression by IL-1 beta.J Immunol. 2004; 173: 4661-4668Crossref PubMed Scopus (45) Google Scholar, 11Hu B. Ullenbruch M.R. Jin H. Gharaee-Kermani M. Phan S.H. An essential role for CCAAT/enhancer binding protein beta in bleomycin-induced pulmonary fibrosis.J Pathol. 2007; 211: 455-462Crossref PubMed Scopus (31) Google Scholar, 12Cogan J.G. Subramanian S.V. Polikandriotis J.A. Kelm Jr., R.J. Strauch A.R. Vascular smooth muscle alpha-actin gene transcription during myofibroblast differentiation requires Sp1/3 protein binding proximal to the MCAT enhancer.J Biol Chem. 2002; 277: 36433-36442Crossref PubMed Scopus (60) Google Scholar, 13Qiu P. Feng X.H. Li L. Interaction of Smad3 and SRF-associated complex mediates TGF-beta1 signals to regulate SM22 transcription during myofibroblast differentiation.J Mol Cell Cardiol. 2003; 35: 1407-1420Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 14Buck M. Kim D.J. Houglum K. Hassanein T. Chojkier M. c-Myb modulates transcription of the alpha-SMA gene in activated hepatic stellate cells.Am J Physiol Gastrointest Liver Physiol. 2000; 278: G321-G328PubMed Google Scholar, 15Liu T. Hu B. Choi Y.Y. Chung M. Ullenbruch M. Yu H. Lowe J.B. Phan S.H. Notch1 signaling in FIZZ1 induction of myofibroblast differentiation.Am J Pathol. 2009; 174: 1745-1755Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 16Noseda M. Fu Y. Niessen K. Wong F. Chang L. McLean G. Karsan A. Smooth muscle α-actin is a direct target of Notch/CSL.Circ Res. 2006; 98: 1468-1470Crossref PubMed Scopus (146) Google Scholar, 17Hinz B. Phan S.H. Thannickal V.J. Galli A. Bochaton-Piallat M.L. Gabbiani G. The myofibroblast: one function, multiple origins.Am J Pathol. 2007; 170: 1807-1816Abstract Full Text Full Text PDF PubMed Scopus (1600) Google Scholar but remain incompletely understood especially at the epigenetic level. Epigenetic regulation of gene expression commonly occurs at two primary levels, namely DNA methylation and modified histone interaction with DNA.18Jaenisch R. Bird A. Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals.Nat Genet. 2003; 33: 245-254Crossref PubMed Scopus (4663) Google Scholar There is evidence that both modes of epigenetic regulation affect myofibroblast differentiation.19Hu B. Gharaee-Kermani M. Wu Z. Phan S.H. Epigenetic regulation of myofibroblast differentiation by DNA methylation.Am J Pathol. 2010; 177: 21-28Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 20Niki T. Rombouts K. De Bleser P. De Smet K. Rogiers V. Schuppan D. Yoshida M. Gabbiani G. Geerts A. A histone deacetylase inhibitor, trichostatin A, suppresses myofibroblastic differentiation of rat hepatic stellate cells in primary culture.Hepatology. 1999; 29: 858-867Crossref PubMed Scopus (177) Google Scholar, 21Glenisson W. Castronovo V. Waltregny D. Histone deacetylase 4 is required for TGFbeta1-induced myofibroblastic differentiation.Biochim Biophys Acta. 2007; 1773: 1572-1582Crossref PubMed Scopus (134) Google Scholar, 22Guo W. Shan B. Klingsberg R.C. Qin X. Lasky J.A. Abrogation of TGF-beta1-induced fibroblast-myofibroblast differentiation by histone deacetylase inhibition.Am J Physiol Lung Cell Mol Physiol. 2009; 297: L864-L870Crossref PubMed Scopus (176) Google Scholar Although inhibition of histone deacetylase is known to suppress myofibroblast differentiation, the molecular mechanism is unclear, especially vis-à-vis regulation of α-SMA gene expression.20Niki T. Rombouts K. De Bleser P. De Smet K. Rogiers V. Schuppan D. Yoshida M. Gabbiani G. Geerts A. A histone deacetylase inhibitor, trichostatin A, suppresses myofibroblastic differentiation of rat hepatic stellate cells in primary culture.Hepatology. 1999; 29: 858-867Crossref PubMed Scopus (177) Google Scholar, 21Glenisson W. Castronovo V. Waltregny D. Histone deacetylase 4 is required for TGFbeta1-induced myofibroblastic differentiation.Biochim Biophys Acta. 2007; 1773: 1572-1582Crossref PubMed Scopus (134) Google Scholar, 22Guo W. Shan B. Klingsberg R.C. Qin X. Lasky J.A. Abrogation of TGF-beta1-induced fibroblast-myofibroblast differentiation by histone deacetylase inhibition.Am J Physiol Lung Cell Mol Physiol. 2009; 297: L864-L870Crossref PubMed Scopus (176) Google Scholar With respect to DNA methylation, there is recent evidence of indirect mechanisms affecting expression of the α-SMA gene.23Mann J. Chu D.C. Maxwell A. Oakley F. Zhu N.L. Tsukamoto H. Mann D.A. MeCP2 controls an epigenetic pathway that promotes myofibroblast transdifferentiation and fibrosis.Gastroenterology. 2010; 138: 705-714Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar, 24Mann J. Oakley F. Akiboye F. Elsharkawy A. Thorne A.W. Mann D.A. Regulation of myofibroblast transdifferentiation by DNA methylation and MeCP2: implications for wound healing and fibrogenesis.Cell Death Differ. 2007; 14: 275-285Crossref PubMed Scopus (194) Google Scholar These studies implicate a role for the methyl CpG binding protein 2 (MeCP2), a key member of the methyl-DNA binding protein family of proteins.25Lewis J.D. Meehan R.R. Henzel W.J. Maurer-Fogy I. Jeppesen P. Klein F. Bird Al Purification, sequence, and cellular localization of a novel chromosomal protein that binds to methylated DNA.Cell. 1992; 69: 905-914Abstract Full Text PDF PubMed Scopus (1076) Google Scholar, 26Yasui D.H. Peddada S. Bieda M.C. Vallero R.O. Hogart A. Nagarajan R.P. Thatcher K.N. Farnham P.J. Lasalle J.M. Integrated epigenomic analyses of neuronal MeCP2 reveal a role for long-range interaction with active genes.Proc Natl Acad Sci USA. 2007; 104: 19416-19421Crossref PubMed Scopus (320) Google Scholar, 27Chahrour M. Jung S.Y. Shaw C. Zhou X. Wong S.T. Qin J. Zoghbi H.Y. MeCP2, a key contributor to neurological disease, activates and represses transcription.Science. 2008; 320: 1224-1229Crossref PubMed Scopus (1365) Google Scholar, 28Nikitina T. Shi X. Ghosh R.P. Horowitz-Scherer R.A. Hansen J.C. Woodcock C.L. Multiple modes of interaction between the methylated DNA binding protein MeCP2 and chromatin.Mol Cell Biol. 2007; 27: 864-877Crossref PubMed Scopus (143) Google Scholar, 29Georgel P.T. Horowitz-Scherer R.A. Adkins N. Woodcock C.L. Wade P.A. Hansen J.C. Chromatin compaction by human MeCP2 Assembly of novel secondary chromatin structures in the absence of DNA methylation.J Biol Chem. 2003; 278: 32181-32188Crossref PubMed Scopus (251) Google Scholar While MeCP2 can bind to unmethylated DNA,29Georgel P.T. Horowitz-Scherer R.A. Adkins N. Woodcock C.L. Wade P.A. Hansen J.C. Chromatin compaction by human MeCP2 Assembly of novel secondary chromatin structures in the absence of DNA methylation.J Biol Chem. 2003; 278: 32181-32188Crossref PubMed Scopus (251) Google Scholar preferentially it binds methylated DNA at the 5′-CpG residues.30Gregory R.I. Randall T.E. Johnson C.A. Khosla S. Hatada I. O'Neill L.P. Turner B.M. Feil R. DNA methylation is linked to deacetylation of histone H3, but not H4, on the imprinted genes Snrpn and U2af1-rs1.Mol Cell Biol. 2001; 21: 5426-5436Crossref PubMed Scopus (117) Google Scholar, 31Nan X. Tate P. Li E. Bird A. DNA methylation specifies chromosomal localization of MeCP.Mol Cell Biol. 1996; 16: 414-421Crossref PubMed Scopus (284) Google Scholar MeCP2 is originally considered to be a transcriptional repressor in conjunction with Sin3A and histone deacetylase, but was found later to also have a significant role as a transcriptional activator, as well as in the regulation of chromatin architecture and RNA splicing.32Shahbazian M. Young J. Yuva-Paylor L. Spencer C. Antalffy B. Noebels J. Armstrong D. Paylor R. Zoghbi H. Mice with truncated MeCP2 recapitulate many Rett syndrome features and display hyperacetylation of histone H3.Neuron. 2002; 35: 243-254Abstract Full Text Full Text PDF PubMed Scopus (635) Google Scholar, 33Hite K.C. Adams V.H. Hansen J.C. Recent advances in MeCP2 structure and function.Biochem Cell Biol. 2009; 87: 219-227Crossref PubMed Scopus (97) Google Scholar, 34Cohen S. Zhou Z. Greenberg M.E. Medicine: activating a repressor.Science. 2008; 320: 1172-1173Crossref PubMed Scopus (34) Google Scholar Thus, there is considerable complexity in the possible mechanisms by which MeCP2 can regulate gene expression. The presence of three CpG islands in the α-SMA gene is recently reported and their methylation is associated with suppression of α-SMA gene expression and thus, the undifferentiated state of the precursor fibroblasts.19Hu B. Gharaee-Kermani M. Wu Z. Phan S.H. Epigenetic regulation of myofibroblast differentiation by DNA methylation.Am J Pathol. 2010; 177: 21-28Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar In cells that do not express α-SMA, such as the alveolar epithelial type II cell, intronic regions are also highly methylated.19Hu B. Gharaee-Kermani M. Wu Z. Phan S.H. Epigenetic regulation of myofibroblast differentiation by DNA methylation.Am J Pathol. 2010; 177: 21-28Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar This is in contrast to the situation in fibroblasts with the potential to express α-SMA, wherein only the promoter regions were significantly methylated.19Hu B. Gharaee-Kermani M. Wu Z. Phan S.H. Epigenetic regulation of myofibroblast differentiation by DNA methylation.Am J Pathol. 2010; 177: 21-28Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar Moreover in fibroblasts, inhibition of DNA methyltransferase (Dnmt) activity or induced deficiency of Dnmt 1, 3a, and/or 3b is sufficient to induce α-SMA expression.19Hu B. Gharaee-Kermani M. Wu Z. Phan S.H. Epigenetic regulation of myofibroblast differentiation by DNA methylation.Am J Pathol. 2010; 177: 21-28Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar TGFβ-induced myofibroblast differentiation is associated with reduction in Dnmt1 and Dnmt3a expression, while induced over-expression of all three Dnmts suppress α-SMA expression without complete suppression of TGFβ inducibility.19Hu B. Gharaee-Kermani M. Wu Z. Phan S.H. Epigenetic regulation of myofibroblast differentiation by DNA methylation.Am J Pathol. 2010; 177: 21-28Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar Thus, DNA methylation appears to be a key mechanism for maintenance of the undifferentiated state, but the regulatory mechanism associated with gene repression is not clear. Given that MeCP2 is implicated in mechanisms regulating gene sequences with methylated DNA and the previous identification of methylated DNA in the α-SMA gene, the objective of this study was to investigate the potential role of MeCP2 in the regulation of α-SMA gene expression in lung fibroblasts. The results indicated that MeCP2 preferentially bound the methylated α-SMA gene promoter and enhanced α-SMA gene expression. Suppression of MeCP2 gene expression in lung fibroblasts by siRNA or its deficiency in cells from MeCP2 knock out mice resulted in reduced α-SMA gene expression. Further studies indicated that MeCP2 deficient mice, compared to their wild-type littermates, exhibited significantly decreased alveolar wall thickness, inflammatory cell infiltration, interstitial collagen deposition, and myofibroblast differentiation on endotracheal injection of bleomycin. Thus, an essential role of MeCP2 in myofibroblast differentiation and pulmonary fibrosis was suggested. All animal care was in accordance with the National Institutes of Health ethics, procedures, and regulations. Pathogen-free female Fisher 344 rats (7 to 8 weeks old) were purchased from Charles River Breeding Laboratories, Inc. (Wilmington, MA). The MeCP2 deficient mice and their wild-type littermates were purchased from the Jackson Laboratory (Bar Harbor, ME).32Shahbazian M. Young J. Yuva-Paylor L. Spencer C. Antalffy B. Noebels J. Armstrong D. Paylor R. Zoghbi H. Mice with truncated MeCP2 recapitulate many Rett syndrome features and display hyperacetylation of histone H3.Neuron. 2002; 35: 243-254Abstract Full Text Full Text PDF PubMed Scopus (635) Google Scholar Fibroblasts were isolated from mouse and rat lungs by enzymatic digestion and then maintained in Dulbecco's modified Eagle's medium, supplemented with 10% plasma-derived serum (Cocalico Biologicals, Inc., Reamstown, PA), antibiotics; 1% insulin, transferrin, and selenium (Sigma Chemicals, St. Louis, MO); 5 ng/mL platelet-derived growth factor (R&D Systems, Minneapolis, MN); and 10 ng/mL EGF (R&D Systems) as before.8Hu B. Wu Z. Phan S.H. Smad3 mediates transforming growth factor-beta-induced alpha-SMA expression.Am J Respir Cell Mol Biol. 2003; 29: 397-404Crossref PubMed Scopus (270) Google Scholar The adherent cells were then trypsinized and passaged for at least three times before use. Bleomycin-induced pulmonary fibrosis was induced as previously described.11Hu B. Ullenbruch M.R. Jin H. Gharaee-Kermani M. Phan S.H. An essential role for CCAAT/enhancer binding protein beta in bleomycin-induced pulmonary fibrosis.J Pathol. 2007; 211: 455-462Crossref PubMed Scopus (31) Google Scholar The control group received the same volume of sterile phosphate-buffered saline only (saline treated). At 7 or 21 days after bleomycin injection, the mice were sacrificed and the lungs of some of the mice were removed and extracted for total mRNA and total protein, while the remainder of lungs was formalin-fixed and stained for routine histopathology. The human MeCP2 expression cDNA construct was purchased from B-Bridge International, Inc. (Mt. View, CA). The lentivirus-based siRNA construct specific for rat MeCP2 and the corresponding negative control siRNA construct were purchased from Thermo Scientific (Huntsville, AL). The −2880 to +2803 (numbered from transcription start site, including promoter and first intron) rat α-SMA gene promoter was previously amplified by PCR and cloned into promoterless pGL3-basic vector to form α-SMApro-intron-Luc construct,19Hu B. Gharaee-Kermani M. Wu Z. Phan S.H. Epigenetic regulation of myofibroblast differentiation by DNA methylation.Am J Pathol. 2010; 177: 21-28Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar wherein the luciferase reporter gene expression was driven by this promoter. Escherichia coli CpG methyltransferase (M. SssI) was purchased from New England Biolabs, Inc. (Ipswich, MA). Electrophoretic mobility shift assay was done as previously described.8Hu B. Wu Z. Phan S.H. Smad3 mediates transforming growth factor-beta-induced alpha-SMA expression.Am J Respir Cell Mol Biol. 2003; 29: 397-404Crossref PubMed Scopus (270) Google Scholar Briefly, methylated and unmethylated double-stranded oligonucleotide DNA probe corresponding to the CpG islands in the indicated α-SMA promoter and intronic regions were synthesized and labeled with 32P by T4 polynucleotide kinase. These probes corresponded to the following regions: i) −735 to −646 (numbered from the transcriptional start site) with methylated sequence 5′-AAAAGAmCGGTCCTTAAGCATGATATCAAGGGTCAGmCGATAAACCAACAACATGCAmCGTGGACTGTACCTAAGGGTTAAmCGCAGTTACAGT-3′, ii) site 1 in the first intronic region from +191 to +280 of the α-SMA promoter with methylated sequence 5′-ATGATTTmCGTATCTAAAmCGGGACTAAAAATGAATmCGTGGTTTACTGGCAAAGGAGATGGAGAGGAAATTAAAGTTTGTTCATGmCGTGGCA-3′, and iii) site 2 in the first intronic region from +444 to +533 of the α-SMA promoter with methylated sequence 5′-ACmCGAGTACAGCmCGGGTTAACTGGAAGTGGATGTCAGGAGTGAACTGGmCGmCGGTTGCCTGmCGCTCTGGTTTTGGCTGAGTGGACTGmCGTT-3′. They were individually incubated with MeCP2 (Abnova, Walnut, CA) at the indicated concentrations, 100 ng of Poly dI-dC and 0.1 μg poly-L-lysine in a final volume of 25 μl in Dignam's Buffer C (20 mmol/L HEPES, pH 7.9, 0.42 M NaCl, 1 mmol/L EDTA, 0.1 mmol/L EGTA, 1.0 mmol/L DTT, 100 μmol/L sodium orthovanadate and protease inhibitors). Samples were then analyzed by electrophoresis on 4% nondenaturing polyacrylamide gels at 150 V in 1 × Tris-borate-EDTA buffer (90 mM Tris, 90 mM boric acid, 2 mM EDTA, pH. 8.0) for about 2 hours. Following electrophoresis, the gels were dried and exposed to X-ray film for 24 to 48 hours. Chromatin immunoprecipitation (ChIP) assay was performed using a kit from Millipore Co. (Billerica, MA) following the manufacturer's protocol as previously described.9Hu B. Wu Z. Liu T. Ullenbruch M.R. Jin H. Phan S.H. Gut-enriched Krüppel-like factor interaction with Smad3 inhibits myofibroblast differentiation.Am J Respir Cell Mol Biol. 2007; 36: 78-84Crossref PubMed Scopus (51) Google Scholar Lung fibroblasts were treated with TGFβ or buffer only for 24 hours, fixed with 1% formaldehyde in Dulbecco's modified Eagle's medium, washed twice with PBS, and then lysed in SDS lysis buffer. After sonication to shear the DNA to an average of 1000 bp, the lysate was centrifuged and the supernatant was collected. After preclearing with protein A agarose, each sample was aliquoted (20 μl) separately for use as “input DNA” in PCR analysis. The remainder of each sample was then divided equally into three aliquots for incubation with: i) anti-MeCP2 antibody (anti-MeCP2 bound DNA fraction), ii) nonimmune rabbit IgG (nonspecific antibody background DNA fraction), or iii) PBS-buffer (no antibody background fraction). Any immune complexes formed were affinity-adsorbed with protein A agarose and collected by centrifugation. The bound DNA was washed extensively and eluted from the protein A agarose with freshly made elution buffer (1% SDS, 0.1 M NaHCO3). The eluates and the original “input DNA” sample were incubated at 65°C for 4 hours to reverse the crosslink and then used as templates for PCR analysis along with the oligonucleotide primer pairs A (5′-CGTTGACTGTCCATTGAAGC-3′) and B (5′-TGTAGTCTGGAGTCTGTGTG-3′), C (5′-CAGTCGCCATCAGGGTAAGT-3′) and D (5′-CAACACCTAAGTAGAAACAA-3′), and E (5′-TTGTTTCTACTTAGGTGTTG-3′) and F (5′-CATAGGTTTGAATCGTAAGG-3′). These primers were designed to amplify the CpG islands in the α-SMA promoter region from −917 to −531, site 1 in first intronic region from +17 to +390 and site 2 in first intronic region from +371 to +600 of the rat α-SMA gene, respectively. The PCR products were then analyzed by gel electrophoresis in 1.3% agarose along with 100 bp DNA ladder from New England Biolabs, Inc. All transient transfections were performed using the FuGENE6 reagent (Roche Applied Science, Indianapolis, IN) according to the manufacturer's instructions as before.8Hu B. Wu Z. Phan S.H. Smad3 mediates transforming growth factor-beta-induced alpha-SMA expression.Am J Respir Cell Mol Biol. 2003; 29: 397-404Crossref PubMed Scopus (270) Google Scholar Supercoiled DNA was isolated with an endotoxin-free Qiagen column kit (Qiagen Inc., Valencia, CA). Unless otherwise indicated, cells were seeded in six-well plates at a density of 105 per well in Dulbecco's modified Eagle's medium containing 10% plasma-derived serum, and incubated at 37°C overnight. In all, 2 μg DNA of interest were transfected per culture in Dulbecco's modified Eagle's medium containing 0.5% plasma-derived serum with or without 4 ng/mL TGFβ for the indicated time. To test the effect of MeCP2 on α-SMA promoter activity, 2 μg pLenti6-MeCP2 or the control empty vector pLenti6-v5 were co-transfected with the rat α-SMA promoter luciferase construct, α-SMA pro-intron-Luc, or plasmid pRL-TK control vector (used for normalization), respectively. After 48 hours, the cells were harvested and the activity of firefly or Renilla luciferase was measured using the dual luciferase assay system from Promega Co. (Madison, WI). The relative luciferase activity was calculated by normalizing firefly luciferase activity to that of Renilla luciferase to correct for transfection efficiency. Experiments with each construct were repeated 2 to 4 times, and the relative light units were expressed as mean ± SE. This was undertaken to assess gene expression using a GeneAmp 7500 Sequence Detection System (Applied Biosystems, Foster City, CA) as before.8Hu B. Wu Z. Phan S.H. Smad3 mediates transforming growth factor-beta-induced alpha-SMA expression.Am J Respir Cell Mol Biol. 2003; 29: 397-404Crossref PubMed Scopus (270) Google Scholar, 9Hu B. Wu Z. Liu T. Ullenbruch M.R. Jin H. Phan S.H. Gut-enriched Krüppel-like factor interaction with Smad3 inhibits myofibroblast differentiation.Am J Respir Cell Mol Biol. 2007; 36: 78-84Crossref PubMed Scopus (51) Google Scholar, 10Hu B. Wu Z. Jin H. Hashimoto N. Liu T. Phan S.H. CCAAT/enhancer-binding protein beta isoforms and the regulation of alpha-SMA gene expression by IL-1 beta.J Immunol. 2004; 173: 4661-4668Crossref PubMed Scopus (45) Google Scholar, 11Hu B. Ullenbruch M.R. Jin H. Gharaee-Kermani M. Phan S.H. An essential role for CCAAT/enhancer binding protein beta in bleomycin-induce" @default.
• W2026639078 created "2016-06-24" @default.
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