Identification of Oxygen Sensor Expressed in Newborn Rats

分子氧(O2)对好氧生物是必不可少的。组织或细胞暴露在低氧环境中会引起各种适应性或致病反应。越来越多的证据表明，低氧激活了组织中胶原的合成。为了探索其分子机制，我们筛选了受低氧诱导表达上调或下调的基因。从胎鼠肺分离出成纤维细胞，在低氧条件下培养。差异显示技术显示，缺氧8h后，前胶原蛋白中的一个活性亚基--脯氨基4-羟基酶(PH)a(I)的mRNA水平增加了2~3倍。这一升高的水平维持在40h以上，并在复氧后恢复到基础水平。低氧培养后，细胞的转录速率、蛋白质水平和羟基脯氨酸含量均升高。对PHA(I)基因启动子区域的分析表明，在转录起始点上游120个碱基对的序列中，发现了一个类似于促红细胞生成素等低氧诱导基因的低氧反应元件(HRE)的基序。荧光素酶报告基因分析和突变分析表明，与该基序中的HRE类似的位点在功能上对低氧反应是必需的。凝胶迁移率改变分析显示，缺氧诱导因子-1(HIF-1)在低氧刺激下被激活并与PHA(I)HRE结合。我们的结果表明，胶原合成的关键酶PHA(I)是HIF-1的靶基因。

Molecular oxygen (O2) is essential for aerobic organisms. Exposure of tissues or cells to hypoxia induces a variety of adaptive or pathogenic responses. Accumulated evidence indicates that hypoxia activities collagen synthesis in tissues. To explore the molecular mechanism we screened those genes which 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) a(I), an active subunit which catalyzes the oxygen-dependent hydroxylation of proline residue in procollagen, was increased by 2 to 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, the protein level and the hydroxy proline content, an indicator of the prolyl hydroxylation, were all elevated by hypoxic culture. Analysis of the promotor region of PHa(I) gene indicated that a motif similar to hypoxia responsive element (HRE) of hypoxia-inducible genes such as erythropoietin was identified within 120-base pair sequence upstream 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 (HIF-1) was stimulated and bound to the PHa(I) HRE upon hypoxic challenge. Our results indicate that PHa(I), an essential enzyme for collagen synthesis, is a target gene for HIF-1.