All-trans-retinoic acid inhibition of Pro alpha1(I) collagen gene expression in fetal rat skin fibroblasts: identification of a retinoic acid response element in the Pro alpha1(I) collagen gene.
All-trans-retinoic acid inhibition of Pro alpha1(I) collagen gene expression in fetal rat skin fibroblasts: identification of a retinoic acid response element in the Pro alpha1(I) collagen gene.
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全反式视黄酸抑制胎鼠皮肤成纤维细胞中 Pro α1(I) 胶原基因表达：鉴定 Pro α1(I) 胶原基因中的视黄酸反应元件。
DOI：
10.1111/1523-1747.ep12289723
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Cutroneo,KR
中科院分区：
文献类型：
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作者：
Meisler,NT;Parrelli,J;Gendimenico,GJ;Mezick,JA;Cutroneo,KR
本研究旨在确定类维甲酸全反式维甲酸调节胎鼠皮肤成纤维细胞中原α1(I)胶原基因表达的机制。用ColCat3.6质粒稳定转染FRS成纤维细胞，该质粒含有大鼠原α1(I)胶原基因的5 '侧区，该基因与报告基因氯霉素乙酰转移酶相连。t-RA对CAT活性的影响是浓度和孵育时间的函数。t-RA对CAT活性的最大抑制发生在处理48 h后10−8M。转化生长因子-β1；并不能阻断t-RA对CAT活性的抑制作用。对含有大鼠原α1(I)胶原基因启动子的3.6 kb DNA片段进行计算机序列分析，发现了一个直接重复的RARE序列，该序列分别由位于- 1345和- 1335位置的一个多样化（5 ' -AG-TAGA-3 ‘）和一个理想化（5 ’ -GGGTCA-3 '）半位点组成。在凝胶迁移实验中发现，细菌中表达的两种核类视黄酸受体-γ和类视黄酸X受体-α特异性地与含有RARE的双链寡核苷酸结合。理想半位点的突变消除了受体蛋白与寡核苷酸的结合。用t- ra处理的FRS成纤维细胞制备的核蛋白提取物进行凝胶迁移试验表明，与含有RARE的寡核苷酸的结合比对照值降低。对突变的寡核苷酸进行相同的测定，结果只有轻微的结合。这些研究表明，t-RA通过减少核蛋白与基因5 '侧区RARE序列的结合，下调大鼠原α1(I)胶原基因的启动子活性。
The current study was undertaken to determine the mechanism by which the retinoid all-trans-retinoic acid regulates proα1(I) collagen gene expression in fetal rat skin fibroblasts. FRS fibroblasts were stably transfected with the ColCat3.6 plasmid, which contains a portion of the 5′ flanking region of the rat proα1(I) collagen gene linked to a reporter gene, chloramphenicol acetyltransferase. The effect of t-RA on CAT activity was determined as a function of concentration and incubation time. Maximal inhibition of CAT activity by t-RA occurred at 10−8M after 48 h of treatment. Transforming growth factor-β1; did not block the inhibitory effect of t-RA on CAT activity. Computer sequence analysis of the 3.6-kb DNA fragment that contains the promoter for the rat proα1(I) collagen gene identified a direct repeat RARE sequence composed of one diverse (5′-AG-TAGA-3′) and one idealized (5′-GGGTCA-3′) half site located at positions −1345 and −1335, respectively. Two nuclear retinoid receptors that were expressed in bacteria, retinoic acid receptor-γand retinoid X receptor-α, were found to bind specifically to a double-stranded oligonucleotide containing the RARE in gel mobility shift assays. Mutation of the idealized half-site eliminated the binding of receptor proteins to the oligonucleotide. Gel mobility shift assays using nuclear protein extracts prepared from t-RA-treated FRS fibroblasts showed that binding to the oligonucleotide containing the RARE was decreased from control values. The same assays performed with the mutated oligonucleotide resulted in only slight bindbig. These studies indicate that t-RA downregulates the promoter activity of the rat proα1(I) collagen gene by decreasing the binding of nuclear protein to the RARE sequence in the 5′ flanking region of the gene.