Takahashi Y, Takahashi S, Shiga Y, Yoshimi T, Miura T
Laboratory of Environmental Molecular Physiology, School of Life Science, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.
J Biol Chem. 2000 May 12;275(19):14139-46. doi: 10.1074/jbc.275.19.14139.
Accumulated evidence indicates that hypoxia activates collagen synthesis in tissues. To explore the molecular mechanism of activation, we screened genes that are up-regulated or down-regulated by hypoxia. Fibroblasts isolated from fetal rat lung were cultured under hypoxia. Differential display technique showed that the mRNA level of prolyl 4-hydroxylase (PH) alpha(I), an active subunit that catalyzes the oxygen-dependent hydroxylation of proline residue in procollagen, increased 2-3-fold after an 8-h exposure to hypoxia. This elevated level was maintained over 40 h and returned to the basal level after reoxygenation. The transcription rate, protein level, and hydroxyproline content (an indicator of the prolyl hydroxylation) were all elevated by hypoxic culture. Analysis of the promotor region of PHalpha(I) gene indicated that a motif similar to hypoxia-responsive element (HRE) of hypoxia-inducible genes such as erythropoietin, was identified within a 120-base pair sequence upstream of the transcription start site. Luciferase reporter assay and mutational analysis showed that a site similar to the HRE in this motif is functionally essential to hypoxic response. Electrophoretic mobility shift assay revealed that hypoxia-inducible factor-1 was stimulated and bound to the PHalpha(I) HRE upon hypoxic challenge. Our results indicate that PHalpha(I), an essential enzyme for collagen synthesis, is a target gene for hypoxia-inducible factor-1.
越来越多的证据表明，缺氧可激活组织中的胶原蛋白合成。为了探究激活的分子机制，我们筛选了受缺氧上调或下调的基因。从胎鼠肺中分离出的成纤维细胞在缺氧条件下培养。差异显示技术表明，脯氨酰4-羟化酶（PH）α（I）的mRNA水平在缺氧8小时后增加了2至3倍，PHα（I）是一种活性亚基，可催化前胶原中脯氨酸残基的氧依赖性羟化。这种升高的水平持续了40多个小时，并在复氧后恢复到基础水平。缺氧培养可提高转录速率、蛋白质水平和羟脯氨酸含量（脯氨酰羟化的指标）。对PHα（I）基因启动子区域的分析表明，在转录起始位点上游120个碱基对的序列中，鉴定出了一个与促红细胞生成素等缺氧诱导基因的缺氧反应元件（HRE）相似的基序。荧光素酶报告基因检测和突变分析表明，该基序中与HRE相似的位点对缺氧反应在功能上至关重要。电泳迁移率变动分析显示，缺氧诱导因子-1在缺氧刺激下被激活并与PHα（I）HRE结合。我们的结果表明，PHα（I）是胶原蛋白合成的关键酶，是缺氧诱导因子-1的靶基因。
