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-γ (IFN-γ) and transforming growth factor-β1 (TGF-β1). We investigated the effect of TGF-β1 on IFN-γ-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-γ 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-γ were down-regulated by TGF-β1. IFN-γ increased phosphorylation of STAT1 and this activation was not inhibited by TGF-β1. IFN-γ increased binding of p-STAT1 to p300, while TGF-β1 increased binding of Smad3 to p300. TGF-β1-induced Smad3 binding to p300 was inhibited by IFN-γ, whereas IFN-γ-induced p-STAT1 binding to p300 was not inhibited by TGF-β1. IFN-γ 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-β1 did not inhibit IFN-γ-induced STAT1 binding to DNA. Conclusions: TGF-β1 down-regulated IFN-γ-induced CIITA and HLA-DR expression in HGECs. Though there was an antagonism between IFN-γ and TGF-β1, the competition for p300 between p-STAT1 and Smad3 was not the mechanism for it.

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