Reardon Colin, McKay Derek M
Gastrointestinal Research Group, Department of Physiology and Biophysics, University of Calgary, 3330 Hospital Drive Northwest, Calgary, Alberta, Canada.
J Immunol. 2007 Apr 1;178(7):4284-95. doi: 10.4049/jimmunol.178.7.4284.
IFN-gamma and TGF-beta are important regulators of mucosal immunity, typically functioning in opposition to each other. In this study, we assessed whether TGF-beta could modulate IFN-gamma-induced STAT1 signaling. Model epithelial cell lines (HEp-2, HT-29, and T84) or monocytes/macrophages (THP-1 cell line, human blood mononuclear cells) were pretreated with TGF-beta (1 ng/ml; 5-60 min), followed by IFN-gamma exposure (20 ng/ml; 30 min), and then STAT1 transcriptional activity, DNA-binding activity, phosphorylation, and methylation were assessed. Some epithelia were transfected with an expression plasmid encoding SMAD7 to block TGF-beta-SMAD signaling. Epithelia, but not macrophages, pretreated with TGF-beta were hyporesponsive to IFN-gamma stimulation as indicated by reduced expression of four STAT1-regulated genes and reduced STAT1 DNA binding on EMSA. However, STAT1 Tyr(701)-, Ser(727) phosphorylation, and nuclear recruitment of STAT1 were not significantly different in IFN-gamma with or without TGF-beta-treated cells, indicating that the effects of TGF-beta are downstream of IFN-gammaR-JAK-STAT1 interaction. The TGF-beta effect was not dependent on ERK1/2, p38, or JNK activation but was prevented by overexpression of the inhibitory SMAD7 protein. Additional studies suggest that TGF-beta blockade of IFN-gamma activity in epithelia is via enhanced sequestering of STAT1 by pre-existing protein inhibitor of activated STAT1. These results demonstrate that TGF-beta rapidly suppresses IFN-gamma-driven STAT1 signaling by reducing DNA binding via promotion of STAT1-protein inhibitor of activated STAT1 interactions and not inhibition of STAT1 activation; an event that may be specific to epithelia and represent a novel mode of action of TGF-beta.
干扰素-γ（IFN-γ）和转化生长因子-β（TGF-β）是黏膜免疫的重要调节因子，通常发挥相互拮抗的作用。在本研究中，我们评估了TGF-β是否能够调节IFN-γ诱导的信号转导及转录激活因子1（STAT1）信号通路。用TGF-β（1纳克/毫升；5 - 60分钟）预处理模型上皮细胞系（人喉表皮样癌细胞系（HEp-2）、人结肠癌细胞系（HT-29）和人小肠上皮细胞系（T84））或单核细胞/巨噬细胞（人单核细胞白血病细胞系（THP-1）、人血单个核细胞），随后用IFN-γ（20纳克/毫升；30分钟）处理，然后评估STAT1的转录活性、DNA结合活性、磷酸化和甲基化情况。一些上皮细胞用编码SMAD7的表达质粒转染，以阻断TGF-β - SMAD信号通路。如通过四个STAT1调节基因表达降低以及电泳迁移率变动分析（EMSA）中STAT1 DNA结合减少所示，用TGF-β预处理的上皮细胞而非巨噬细胞对IFN-γ刺激反应低下。然而，在有或没有TGF-β处理的细胞中，IFN-γ刺激下STAT1酪氨酸（Tyr）701位点、丝氨酸（Ser）727位点的磷酸化以及STAT1的核募集没有显著差异，这表明TGF-β的作用在IFN-γ受体 - Janus激酶（JAK） - STAT1相互作用的下游。TGF-β的作用不依赖于细胞外信号调节激酶1/2（ERK1/2）、p38或应激活化蛋白激酶（JNK）的激活，但被抑制性SMAD7蛋白的过表达所阻断。进一步的研究表明，TGF-β对上皮细胞中IFN-γ活性的阻断是通过已存在的活化STAT1蛋白抑制剂增强对STAT1的隔离实现的。这些结果表明，TGF-β通过促进STAT1与活化STAT1蛋白抑制剂的相互作用来减少DNA结合，从而迅速抑制IFN-γ驱动的STAT1信号通路，而非抑制STAT1的激活；这一事件可能是上皮细胞特有的，代表了TGF-β的一种新作用模式。
