Chen Shu-Jen, Ning Hongyan, Ishida Wataru, Sodin-Semrl Snezna, Takagawa Shinsuke, Mori Yasuji, Varga John
Division of Rheumatology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611.
Division of Rheumatology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611.
J Biol Chem. 2006 Jul 28;281(30):21183-21197. doi: 10.1074/jbc.M603270200. Epub 2006 May 15.
Transforming growth factor-beta (TGF-beta) stimulates collagen synthesis and accumulation, and aberrant TGF-beta signaling is implicated in pathological organ fibrosis. Regulation of type I procollagen gene (COL1A2) transcription by TGF-beta involves the canonical Smad signaling pathway as well as additional protein and lipid kinases, coactivators, and DNA-binding transcription factors that constitute alternate non-Smad pathways. By using Affymetrix microarrays to detect cellular genes whose expression is regulated by Smad3, we identified early growth response factor-1 (EGR-1) as a novel Smad3-inducible gene. Previous studies implicated Egr-1 in cell growth, differentiation, and survival. We found that TGF-beta induced rapid and transient accumulation of Egr-1 protein and mRNA in human skin fibroblasts. In transient transfection assays, TGF-beta stimulated the activity of the Egr-1 gene promoter, as well as that of a minimal Egr-1-responsive reporter construct. Furthermore, TGF-beta enhanced endogenous Egr-1 interaction with a consensus Egr-1-binding site element and with GC-rich DNA sequences of the human COL1A2 promoter in vitro and in vivo. Forced expression of Egr-1 by itself caused dose-dependent up-regulation of COL1A2 promoter activity and further enhanced the stimulation induced by TGF-beta. In contrast, the TGF-beta response was abrogated when the Egr-1-binding sites of the COL1A2 promoter were mutated or deleted. Furthermore, Egr-1-deficient embryonic mouse fibroblasts showed attenuated TGF-beta responses despite intact Smad activation, and forced expression of ectopic EGR-1 in these cells could restore COL1A2 stimulation in a dose-dependent manner. Taken together, these findings identify Egr-1 as a novel intracellular TGF-beta target that is necessary for maximal stimulation of collagen gene expression in fibroblasts. The results therefore implicate Egr-1 in the profibrotic responses elicited by TGF-beta and suggest that Egr-1 may play a new and important role in the pathogenesis of fibrosis.
转化生长因子-β（TGF-β）可刺激胶原蛋白的合成与积累，且异常的TGF-β信号传导与病理性器官纤维化有关。TGF-β对I型前胶原基因（COL1A2）转录的调控涉及经典的Smad信号通路以及其他蛋白质和脂质激酶、共激活因子和构成替代性非Smad通路的DNA结合转录因子。通过使用Affymetrix微阵列检测受Smad3调控表达的细胞基因，我们鉴定出早期生长反应因子-1（EGR-1）是一种新的Smad3诱导基因。先前的研究表明Egr-1参与细胞生长、分化和存活。我们发现TGF-β可诱导人皮肤成纤维细胞中Egr-1蛋白和mRNA快速短暂积累。在瞬时转染实验中，TGF-β刺激了Egr-1基因启动子以及最小的Egr-1反应性报告基因构建体的活性。此外，TGF-β在体外和体内增强了内源性Egr-1与共有Egr-1结合位点元件以及人COL1A2启动子富含GC的DNA序列的相互作用。单独强制表达Egr-1会导致COL1A2启动子活性呈剂量依赖性上调，并进一步增强TGF-β诱导的刺激。相反，当COL1A2启动子的Egr-1结合位点发生突变或缺失时，TGF-β反应被消除。此外，Egr-1缺陷的胚胎小鼠成纤维细胞尽管Smad激活完整，但TGF-β反应减弱，在这些细胞中强制表达异位EGR-1可以剂量依赖性方式恢复COL1A2刺激。综上所述这些发现表明Egr-1是一种新的细胞内TGF-β靶点，对于成纤维细胞中胶原蛋白基因表达的最大刺激是必需的。因此，结果表明Egr-1参与TGF-β引发的促纤维化反应，并提示Egr-1可能在纤维化发病机制中发挥新的重要作用。
