Chen S J, Yuan W, Lo S, Trojanowska M, Varga J
Section of Rheumatology, University of Illinois College of Medicine at Chicago, Chicago, Illinois 60607-7171, USA.
J Cell Physiol. 2000 Jun;183(3):381-92. doi: 10.1002/(SICI)1097-4652(200006)183:3<381::AID-JCP11>3.0.CO;2-O.
Transcription of the alpha2(I) collagen gene (COL1A2) in fibroblasts is potently induced by transforming growth factor-beta (TGF-beta). Smad family proteins function as intracellular signal transducers for TGF-beta that convey information from the cell membrane to the nucleus. In the present study, we establish the functional requirement for endogenous Smad3 and Smad4 in TGF-beta-stimulated COL1A2 transcription in human skin fibroblasts in vitro. Furthermore, using transfections with a series of 5' deletions of the human COL1A2 promoter, we identify a proximal region between -353 and -148 bp, which is required for full stimulation of transcription by a constitutively active TGF-beta type I receptor. This region of the COL1A2 promoter contains a CAGA motif also found in the promoter of the plasminogen activator inhibitor-1. Substitutions disrupting this sequence decreased the binding of nuclear extracts or recombinant Smad3 to the CAGACA oligonucleotide, and markedly reduced the transcriptional response to TGF-beta or overexpressed Smad3 in transient transfection assays. The insertion of tandem repeats of CAGACA conferred TGF-beta stimulation to a heterologous minimal promoter-reporter construct. Inhibition of endogenous Smad expression in fibroblasts by antisense oligonucleotides or cDNA against Smad3 or Smad4, and transfection of COL1A2 promoter constructs into Smad4-deficient breast adenocarcinoma cells, indicated the critical role of Smads for the full TGF-beta response. The importance of Smad binding to the CAGACA box of COL1A2 was further established by transcriptional decoy oligonucleotide competition. Taken together, the results identify a functional Smad-binding element of the COL1A2 promoter harboring a CAGACA consensus sequence that is both necessary and sufficient for stimulation by TGF-beta, and demonstrate that interaction of this Smad-binding element with endogenous Smads is required for the full TGF-beta response in fibroblasts.
成纤维细胞中α2（I）型胶原蛋白基因（COL1A2）的转录可被转化生长因子-β（TGF-β）有效诱导。Smad家族蛋白作为TGF-β的细胞内信号转导分子，将信息从细胞膜传递至细胞核。在本研究中，我们确定了内源性Smad3和Smad4在体外人皮肤成纤维细胞中TGF-β刺激的COL1A2转录中的功能需求。此外，通过用一系列人COL1A2启动子的5'缺失进行转染，我们确定了-353至-148 bp之间的近端区域，该区域是组成型活性TGF-β I型受体完全刺激转录所必需的。COL1A2启动子的该区域含有纤溶酶原激活物抑制剂-1启动子中也发现的CAGA基序。破坏该序列的取代减少了核提取物或重组Smad3与CAGACA寡核苷酸的结合，并在瞬时转染实验中显著降低了对TGF-β或过表达Smad3的转录反应。CAGACA串联重复序列的插入赋予异源最小启动子-报告基因构建体TGF-β刺激。通过反义寡核苷酸或针对Smad3或Smad4的cDNA抑制成纤维细胞中的内源性Smad表达，以及将COL1A2启动子构建体转染到Smad4缺陷的乳腺腺癌细胞中，表明Smad对完整的TGF-β反应起关键作用。转录诱饵寡核苷酸竞争进一步证实了Smad与COL1A2的CAGACA框结合的重要性。综上所述，结果确定了COL1A2启动子的一个功能性Smad结合元件，其具有CAGACA共有序列，该序列对于TGF-β刺激既是必需的也是充分的，并证明该Smad结合元件与内源性Smads的相互作用是成纤维细胞中完整TGF-β反应所必需的。
