title: STAT1-independent down-regulation of interferon-gamma-induced class II transactivator and HLA-DR expression by transforming growth factor beta-1 in human glomerular endothelial cells.
abstract: BACKGROUND
  The competition between STAT1 and Smad3 for a limiting amount of the nuclear protein p300, a transcriptional coactivator, was suggested to be a mechanism for the antagonism between interferon-gamma (IFN-gamma) and transforming growth factor-beta1 (TGF-beta1). We investigated the effect of TGF-beta1 on IFN-gamma-induced HLA-DR production in cultured human glomerular endothelial cells (HGECs), and the involvement of p300 in this process.
  
  
  METHODS
  Cell surface expression of HLA-DR and mRNA levels of HLA-DR and class II transactivator (CIITA), the master regulator of HLA-DR gene transcription, were measured by cellular ELISA and Northern blot, respectively. The levels of STAT1 and Smad3 protein were analyzed by Western blot. Nuclear binding activity of STAT1 was assessed by electrophoretic mobility shift assay.
  
  
  RESULTS
  IFN-gamma increased the cell surface expression of HLA-DR along with increases in the mRNA levels of CIITA and HLA-DR, while these stimulatory effects of IFN-gamma were down-regulated by TGF-beta1. IFN-gamma increased phosphorylation of STAT1 and this activation was not inhibited by TGF-beta1. IFN-gamma increased binding of p-STAT1 to p300, while TGF-beta1 increased binding of Smad3 to p300. TGF-beta1-induced Smad3 binding to p300 was inhibited by IFN-gamma, whereas IFN-gamma-induced p-STAT1 binding to p300 was not inhibited by TGF-beta1. IFN-gamma increased DNA binding activity of STAT1. Inhibition of interaction between STAT1 and p300 by addition of anti-p300 antibody to nuclear extract down-regulated DNA binding activity of STAT1. In contrast, TGF-beta1 did not inhibit IFN-gamma-induced STAT1 binding to DNA.
  
  
  CONCLUSIONS
  TGF-beta1 down-regulated IFN-gamma-induced CIITA and HLA-DR expression in HGECs. Though there was an antagonism between IFN-gamma and TGF-beta1, the competition for p300 between p-STAT1 and Smad3 was not the mechanism for it.
authors.affiliation: Department of Internal Medicine, Asan Medical Center, College of Medicine, University of Ulsan, Seoul, Korea.
authors.forename: Won Seok
authors.initials: WS
authors.lastname: Yang
authors.forename: Nam Jeong
authors.initials: NJ
authors.lastname: Han
authors.forename: Choung-Soo
authors.initials: CS
authors.lastname: Kim
authors.forename: Hanjong
authors.initials: H
authors.lastname: Ahn
authors.forename: Sang Koo
authors.initials: SK
authors.lastname: Lee
authors.forename: Ki-Up
authors.initials: KU
authors.lastname: Lee
authors.forename: Su-Kil
authors.initials: SK
authors.lastname: Park
mesh_terms: D015152:Blotting, Northern; D015153:Blotting, Western; D002292:Carcinoma, Renal Cell / Q000473:pathology*; D015536:Down-Regulation; D050881:E1A-Associated p300 Protein / Q000502:physiology; D024202:Electrophoretic Mobility Shift Assay; D042783:Endothelial Cells / Q000502:physiology; D004797:Enzyme-Linked Immunosorbent Assay; D006684:HLA-DR Antigens / Q000096:biosynthesis*; D006801:Humans; D007150:Immunohistochemistry; D007371:Interferon-gamma / Q000502:physiology*; D007674:Kidney Diseases / Q000276:immunology* / Q000503:physiopathology; D007678:Kidney Glomerulus / Q000502:physiology*; D007680:Kidney Neoplasms / Q000473:pathology; D009687:Nuclear Proteins / Q000096:biosynthesis; D050794:STAT1 Transcription Factor / Q000502:physiology*; D051900:Smad3 Protein / Q000502:physiology; D015534:Trans-Activators / Q000096:biosynthesis; D016212:Transforming Growth Factor beta / Q000502:physiology*; D053773:Transforming Growth Factor beta1; D014407:Tumor Cells, Cultured
chemical_list: D006684:HLA-DR Antigens; C083728:MHC class II transactivator protein; D009687:Nuclear Proteins; C497790:SMAD3 protein, human; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; D051900:Smad3 Protein; C508960:TGFB1 protein, human; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D007371:Interferon-gamma; D050881:E1A-Associated p300 Protein; C494360:EP300 protein, human