Chung K Y, Agarwal A, Uitto J, Mauviel A
Department of Dermatology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
J Biol Chem. 1996 Feb 9;271(6):3272-8. doi: 10.1074/jbc.271.6.3272.
Previous studies have shown that transforming growth factor-beta (TGF-be ta) and tumor necrosis factor-alpha (TNF-alpha) modulate type I collagen gene expression in fibroblasts. To fine-map the corresponding response elements in the human alpha2(I) collagen (COL1A2) promoter, we have generated a series of 5' deletion promoter/chloramphenicol acetyltransferase (CAT) reporter gene constructs. Transient cell transfection assays using human dermal fibroblasts and stable transfection experiments using NIH 3T3 fibroblasts identified the region located between residues -265 and -241, as critical for TGF-beta response. Specifically, we demonstrate that this 25-base pair region mediates the up-regulatory effect of TGF-beta on COL1A2 promoter activity and allows antagonistic activity of TNF-alpha on the TGF-beta effect. Gel mobility shift assays indicate that nuclear factor binding to this 25-base pair region of COL1A2 promoter is competed by AP-1, but not NF-1 or NF-kappaB, oligonucleotides. Transient cell transfection experiments with plasmid constructs in which the potential AP-1-binding site located within this short region of promoter was modified by site-directed mutagenesis indicated that this element plays a significant role in the basal activity of the promoter. Furthermore, this sequence is essential for TGF-beta response and does not require the presence of the three Sp-1-binding sites located further upstream, between nucleotides -273 and -304. In addition, overexpression of c-jun in co-transfection experiments with COL1A2 promoter/CAT constructs blocks the TGF- beta response, further implicating AP-1 in the regulation of COL1A2 gene expression. Our results clarify the molecular mechanisms involved in the regulation of type I collagen gene expression and further emphasize the importance of AP-1 in mediating some of the TGF-beta effects on gene transcription.
先前的研究表明，转化生长因子-β（TGF-β）和肿瘤坏死因子-α（TNF-α）可调节成纤维细胞中I型胶原基因的表达。为了精细定位人α2（I）胶原（COL1A2）启动子中的相应反应元件，我们构建了一系列5'缺失启动子/氯霉素乙酰转移酶（CAT）报告基因构建体。使用人皮肤成纤维细胞的瞬时细胞转染试验和使用NIH 3T3成纤维细胞的稳定转染实验确定，位于-265至-241位残基之间的区域对TGF-β反应至关重要。具体而言，我们证明了这个25个碱基对的区域介导了TGF-β对COL1A2启动子活性的上调作用，并允许TNF-α对TGF-β作用产生拮抗活性。凝胶迁移率变动分析表明，与COL1A2启动子的这个25个碱基对区域结合的核因子可被AP-1竞争，但不能被NF-1或NF-κB寡核苷酸竞争。用质粒构建体进行的瞬时细胞转染实验，其中通过定点诱变修饰了启动子这个短区域内潜在的AP-1结合位点，表明该元件在启动子的基础活性中起重要作用。此外，该序列对于TGF-β反应必不可少，并且不需要位于更上游、核苷酸-273至-304之间的三个Sp-1结合位点的存在。此外，在与COL1A2启动子/CAT构建体共转染实验中c-jun的过表达阻断了TGF-β反应，进一步表明AP-1参与了COL1A2基因表达的调控。我们的结果阐明了I型胶原基因表达调控中涉及的分子机制，并进一步强调了AP-1在介导TGF-β对基因转录的某些作用中的重要性。
