title: Smad3-mSin3A-HDAC1 Complex is Required for TGF-β1-Induced Transcriptional Inhibition of PPARγ in Mouse Cardiac Fibroblasts.
abstract: BACKGROUND
  We have recently demonstrated that activated transforming growth factor-β (TGF-β) signaling suppresses myocardial peroxisome proliferator-activated receptor γ (PPARγ) expression in the pressure overloaded heart. In this study, we aim to further define the molecular mechanisms that underlie TGF-β-induced PPARγ transcriptional inhibition.
  
  
  METHODS
  Adult mouse cardiac fibroblasts were isolated and cultured. PPARγ promoter activity was measured by the dual-Luciferase reporter assay. Interactions between transcription factors and the target gene were identified.
  
  
  RESULTS
  In cultured cardiac fibroblasts transfected with a plasmid containing a human PPARγ promoter, co-transfection of Smad3 and Smad4, but not Smad2, plasmids significantly enhanced TGF-β1-induced inhibition of PPARγ promoter activity. Promoter deletion analysis and site-directed mutagenesis assays defined two Smad binding elements on the promoter of the PPARγ gene. Utilizing chromatin immunoprecipitation analysis and DNA-affinity precipitation methods, we demonstrated that the transcriptional regulatory complex consisting of Smad3, mSin3A and HDAC1 bound to the promoter of the PPARγ gene in cardiac fibroblasts in response to TGF-β1 stimulation. Either silencing endogenous mSin3A expression by Lentivirus-mediated transduction of mSin3A shRNA or pretreatment with the specific HDAC1 inhibitor MS-275 effectively attenuated TGF-β-induced transcriptional suppression of PPARγ.
  
  
  CONCLUSIONS
  These results suggest that TGF-β1-induced inhibition of PPARγ transcription depends on formation of a functional transcriptional regulatory complex that includes Smad3, mSin3A and HDAC1 at the PPARγ promoter.
authors.affiliation: Department of Cardiology, the Affiliated Hospital of Yangzhou University, Yangzhou, China.
authors.forename: Kaizheng
authors.initials: K
authors.lastname: Gong
authors.forename: Mingxing
authors.initials: M
authors.lastname: Chen
authors.forename: Rujun
authors.initials: R
authors.lastname: Li
authors.forename: Yanghong
authors.initials: Y
authors.lastname: He
authors.forename: Huajiang
authors.initials: H
authors.lastname: Zhu
authors.forename: Dan
authors.initials: D
authors.lastname: Yao
authors.forename: Suzanne
authors.initials: S
authors.lastname: Oparil
authors.forename: Zhengang
authors.initials: Z
authors.lastname: Zhang
mesh_terms: D000818:Animals; D001665:Binding Sites; D002478:Cells, Cultured; D015536:Down-Regulation / Q000187:drug effects; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D020868:Gene Silencing / Q000187:drug effects; D056284:Histone Deacetylase 1 / Q000378:metabolism*; D006801:Humans; D008297:Male; D008810:Mice, Inbred C57BL; D046912:Multiprotein Complexes / Q000378:metabolism; D009206:Myocardium / Q000166:cytology*; D047495:PPAR gamma / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding / Q000187:drug effects; D012097:Repressor Proteins / Q000378:metabolism*; D056264:Sin3 Histone Deacetylase and Corepressor Complex; D051899:Smad2 Protein / Q000378:metabolism; D051900:Smad3 Protein / Q000378:metabolism*; D051901:Smad4 Protein / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects*; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology*
chemical_list: D046912:Multiprotein Complexes; D047495:PPAR gamma; D012097:Repressor Proteins; C099683:SIN3A transcription factor; D051899:Smad2 Protein; D051900:Smad3 Protein; D051901:Smad4 Protein; D053773:Transforming Growth Factor beta1; C484389:Hdac1 protein, mouse; D056284:Histone Deacetylase 1; D056264:Sin3 Histone Deacetylase and Corepressor Complex