Higashi K, Kouba D J, Song Y J, Uitto J, Mauviel A
Department of Dermatology and Cutaneous Biology, Jefferson Medical College, Philadelphia, Pennsylvania 19107, USA.
Matrix Biol. 1998 Mar;16(8):447-56. doi: 10.1016/s0945-053x(98)90016-6.
Previous studies have shown that interferon-gamma (IFN-gamma) inhibits type I collagen gene expression through both transcriptional and post-transcriptional mechanisms (Kahäri et al., 1990). In the present study, using transient cell transfections of human dermal fibroblast cultures with a series of 5' deletion promoter/CAT reporter gene constructs, we have identified the IFN-gamma-response element of the human alpha 2(I) collagen gene (COL1A2) promoter. Specifically, we have identified a segment of the proximal promoter region, located between nucleotides -161 and -125 relative to the transcription start site, as critical for down-regulation of COL1A2 promoter activity by IFN-gamma. This IFN-gamma response element (IgRE) is clearly distinct from the previously described tumor necrosis factor-alpha response element (TaRE) located between nucleotides -265 and -241 of the COL1A2 promoter, a difference which is likely to explain the additive inhibitory effect of these two cytokines. The inhibitory effect of IFN-gamma was dose-dependent and rapidly induced, requiring less than 5 min exposure of fibroblast cultures. Gel mobility shift assays indicated that a highly specific nuclear protein complex bound to this 37-base pair region of promoter. Competition experiments with oligonucleotides spanning discrete segments of this promoter region mapped the binding element within a distinctive pyrimidine-rich sequence. Point mutations within the latter revealed that this element plays a crucial role not only in the IFN-gamma response, but also in the basal activity of the proximal promoter. Substitution mutations within the IgRE of the -161/CAT construct attenuated the promoter response to IFN-gamma, as measured in transient cell transfections, and eliminated specific DNA/protein complex formation, as measured by gel mobility shift assay. UV-crosslinking experiments indicated that two DNA/protein complexes were formed with the IgRE, with molecular weights around 55 kDa and 30 kDa, corresponding to proteins of approximately 30 kDa and approximately 6 kDa, respectively. Our results further clarify the molecular mechanisms involved in the regulation of type I collagen gene expression by IFN-gamma.
先前的研究表明，干扰素-γ（IFN-γ）通过转录和转录后机制抑制I型胶原基因的表达（卡哈里等人，1990年）。在本研究中，我们利用一系列5'缺失启动子/CAT报告基因构建体对人皮肤成纤维细胞培养物进行瞬时细胞转染，确定了人α2（I）胶原基因（COL1A2）启动子的IFN-γ反应元件。具体而言，我们确定了近端启动子区域的一个片段，相对于转录起始位点位于核苷酸-161至-125之间，该片段对于IFN-γ下调COL1A2启动子活性至关重要。这个IFN-γ反应元件（IgRE）与先前描述的位于COL1A2启动子核苷酸-265至-241之间的肿瘤坏死因子-α反应元件（TaRE）明显不同，这种差异可能解释了这两种细胞因子的累加抑制作用。IFN-γ的抑制作用呈剂量依赖性且诱导迅速，成纤维细胞培养物暴露不到5分钟即可出现。凝胶迁移率变动分析表明，一种高度特异性的核蛋白复合物与启动子的这个37个碱基对区域结合。用跨越该启动子区域离散片段的寡核苷酸进行的竞争实验将结合元件定位在一个独特的富含嘧啶的序列内。后者中的点突变表明，该元件不仅在IFN-γ反应中起关键作用，而且在近端启动子的基础活性中也起关键作用。在瞬时细胞转染中测量，-161/CAT构建体的IgRE内的取代突变减弱了启动子对IFN-γ的反应，并如凝胶迁移率变动分析所测消除了特异性DNA/蛋白质复合物的形成。紫外线交联实验表明，与IgRE形成了两种DNA/蛋白质复合物，分子量分别约为55 kDa和30 kDa，分别对应于约30 kDa和约6 kDa的蛋白质。我们的结果进一步阐明了IFN-γ调节I型胶原基因表达所涉及的分子机制。
