Stimulation of type I collagen transcription in human skin fibroblasts by TGF-β:: Involvement of Smad 3
Stimulation of type I collagen transcription in human skin fibroblasts by TGF-β:: Involvement of Smad 3
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DOI：
10.1046/j.1523-1747.1999.00477.x
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发表时间：
1999-01-01
影响因子：
6.5
通讯作者：
Varga, J
中科院分区：
文献类型：
--
作者：
Chen, SJ;Yuan, WH;Varga, J
转化生长因子 -β（TGF -β）刺激α2（I）型前胶原基因（COL1A2）的转录，而参与这一反应的细胞内介质仍知之甚少。在本研究中，我们证明原代人皮肤成纤维细胞在体外无TGF -β存在的情况下表达Smads，这是一个新的信号分子家族。TGF -β可使Smad 7 mRNA的水平迅速且短暂地升高。Smad 3和Smad 4的瞬时过表达（而非Smad 1或Smad 2）可导致由含假定的TGF -β反应元件的人COL1A2启动子772bp片段驱动的氯霉素乙酰转移酶（CAT）报告基因的反式激活。Smad对启动子活性的刺激不依赖配体，但可被TGF -β进一步增强。一个磷酸化缺陷型Smad 3突变体或缺乏羧基末端磷酸化基序的野生型Smad 7的过表达，可特异性抑制TGF -β诱导的COL1A2启动子激活。在近端COL1A2启动子的TGF -β反应区域内发现了一个CAGACA序列，该序列在纤溶酶原激活物抑制剂 -1基因启动子中是一个功能性的Smad结合元件。凝胶迁移变动分析显示，成纤维细胞核提取物中对该序列具有蛋白质磷酸化依赖性结合活性；TGF -β处理强烈刺激了这种DNA -蛋白质复合物的形成。通过抗体超迁移鉴定，在TGF -β处理的成纤维细胞中Smad是CAGACA结合转录复合物的一个组分。这些结果表明：（i）Smad 3在人成纤维细胞中将TGF -β信号从受体传递到COL1A2启动子，并且可能在TGF -β引发的COL1A2启动子活性刺激中起重要作用；（ii）在成纤维细胞中，Smads似乎作为可诱导的DNA结合转录因子发挥作用；（iii）Smad 7可能参与TGF -β对COL1A2启动子活性调节的自分泌负反馈。
Transforming growth factor-beta (TGF-beta) stimulates the transcription of the alpha 2(I) procollagen gene (COL1A2), The intracellular mediators involved in this response remain poorly understood. In this study, we demonstrate that primary human skin fibroblasts express Smads, a novel family of signaling molecules, in vitro in the absence of TGF-beta. The levels of Smad 7 mRNA was rapidly and transiently increased by TGF-beta. Transient overexpression of Smad 3 and Smad 4, but not Smad 1 or Smad 2, caused trans-activation of a CAT reporter gene driven by a 772 bp segment of the human COL1A2 promoter containing putative TGF-beta response elements. Smad stimulation of promoter activity was ligand independent, but was further enhanced by TGF-beta, Overexpression of a phosphorylation-deficient Smad 3 mutant or wild-type Smad 7, which lacks the carboxy-terminal phosphorylation motif, specifically inhibited TGF-beta-induced activation of COL1A2 promoter. A CAGACA sequence shown to be a functional Smad-binding element in the plasminogen activator inhibitor-1 gene promoter was found within the TGF-beta-response region of the proximal COL1A2 promoter. Gel mobility shift assays showed protein phosphorylation-dependent binding activity in fibroblast nuclear extracts specific for this sequence; TGF-beta treatment strongly stimulated the formation of this DNA-protein complex. Smad was identified as a component of the CAGACA-binding transcription complex in TGF-beta-treated fibroblasts by antibody supershifting, These results demonstrate that (i) Smad 3 transmits TGF-beta signals from the receptor to the COL1A2 promoter in human fibroblasts, and is Likely to play an important role in stimulation of COL1A2 promoter activity elicited by TGF-beta; (ii) in fibroblasts, Smads appear to function as inducible DNA-binding transcription factors; and (iii) Smad 7 may be involved in autocrine negative feedback in the regulation of COL1A2 promoter activity by TGF-beta.