Xu Yong, Wang Lin, Buttice Giovanna, Sengupta Pritam K, Smith Barbara D
Department of Biochemistry, Boston University School of Medicine and the Veterans Affairs Boston Healthcare System, Boston, Massachusetts 02118, USA.
J Biol Chem. 2003 Dec 5;278(49):49134-44. doi: 10.1074/jbc.M309003200. Epub 2003 Sep 10.
Interferon gamma (IFN-gamma) plays an important physiological role during inflammation by down-regulating collagen gene expression and activating major histocompatibility II (MHC-II) complex. The activation of MHC-II by IFN-gamma requires activation of a trimeric DNA binding transcriptional complex, RFX5 complex, containing RFXB (also called RFXANK or Tvl-1), RFXAP, as well as RFX5 protein. Previously, we demonstrated that RFX5 binds to the collagen transcription start site and represses collagen gene expression (Sengupta, P. K., Fargo, J., Smith, B. D. (2002) J. Biol. Chem. 277, 24926-24937). In this report, we have examined the role of RFXB and RFXAP proteins within the RFX5 complex to regulate collagen gene expression. The data show that all three RFX5 complex proteins are required for maximum repression. Expression of proteins with mutations known to be important for RFX5 complex formation does not repress collagen promoter activity. Two mutated forms of RFX5 act as dominant negative proteins activating collagen expression and reversing IFN-gamma down-regulation of collagen expression in human lung fibroblasts. IFN-gamma increases expression and nuclear translocation of RFX5. RFXB has a naturally occurring splice variant isoform (RFX SV). Interferon increases expression of the long form of RFXB and decreases expression of RFX SV with the same kinetics as collagen gene expression. Overexpression of the splice variant form reverses the IFN-gamma induced collagen repression in human lung fibroblasts. Finally, all three RFX5 complex proteins increase at the collagen transcription start site with IFN-gamma treatment using chromatin immunoprecipitation analysis. Thus, these studies suggest an important role for RFX5 complex in collagen repression.
干扰素γ（IFN-γ）在炎症过程中通过下调胶原蛋白基因表达和激活主要组织相容性复合体II（MHC-II）发挥重要的生理作用。IFN-γ对MHC-II的激活需要激活一个三聚体DNA结合转录复合体，即RFX5复合体，它包含RFXB（也称为RFXANK或Tvl-1）、RFXAP以及RFX5蛋白。此前，我们证明RFX5与胶原蛋白转录起始位点结合并抑制胶原蛋白基因表达（Sengupta, P. K., Fargo, J., Smith, B. D. (2002) J. Biol. Chem. 277, 24926 - 24937）。在本报告中，我们研究了RFX5复合体内RFXB和RFXAP蛋白在调节胶原蛋白基因表达中的作用。数据表明，RFX5复合体的所有三种蛋白对于最大程度的抑制都是必需的。已知对RFX5复合体形成重要的突变蛋白的表达并不能抑制胶原蛋白启动子活性。两种突变形式的RFX5作为显性负性蛋白激活胶原蛋白表达，并逆转IFN-γ对人肺成纤维细胞中胶原蛋白表达的下调作用。IFN-γ增加RFX5的表达和核转位。RFXB有一种天然存在的剪接变异体异构体（RFX SV）。干扰素以与胶原蛋白基因表达相同的动力学增加RFXB长形式的表达并降低RFX SV的表达。剪接变异体形式的过表达逆转了IFN-γ诱导的人肺成纤维细胞中胶原蛋白的抑制作用。最后，使用染色质免疫沉淀分析表明，经IFN-γ处理后，所有三种RFX5复合体蛋白在胶原蛋白转录起始位点处均增加。因此，这些研究表明RFX5复合体在胶原蛋白抑制中起重要作用。
