Raju V S, McCoubrey W K, Maines M D
Department of Biophysics, University of Rochester, School of Medicine, NY 14642, USA.
Biochim Biophys Acta. 1997 Mar 20;1351(1-2):89-104. doi: 10.1016/s0167-4781(96)00183-2.
Heme oxygenase-2 (HO-2) is constitutively expressed in mammalian tissues; together with HO-1 (HSP32) it catalyzes the cleavage of heme to produce biliverdin IX alpha, CO and Fe. Detection of a consensus sequence of the glucocorticoid response element (GRE) in the promoter region of the HO-2 gene prompted the present study which has investigated the role of glucocorticoids (Gcs) in the regulation of HO-2 protein and transcript development in the newborn rat brain and has examined the promoter activity of the GRE in HeLa cells. Using in situ hybridization histochemistry, we noted a pronounced increase in signal for HO-2 mRNA in the brain of 14-day-old rats postnatally treated with corticosterone (5 microg/g, 4 x, starting 24-36 h after birth). And, using immunohistochemistry, a striking increase in neuronal HO-2 immunostaining in treated brains was detected. The HO-2 GRE was tested for responsiveness to dexamethasone (DX) using both a promoterless CAT expression vector, and a heterologous promoter containing luciferase expression vector in HeLa cells. The HO-2 promoter containing the GRE and transcription start site induced CAT reporter gene activity in response to DX, whereas mutation or deletion in the GRE abolished hormone responsiveness. Similarly, constructs containing the GRE conferred responsiveness to DX in an orientation-independent manner and increased relative luciferase activity. Further, specific binding of glucocorticoid receptor protein to the GRE was observed; binding could be competed out only by excess cold GRE and not by mutated HO-2 GRE, or AP1. HO-2 mRNAs (approximately 1.3 and approximately 1.9 kb) increased in HeLa cells treated with DX (5 microM), the level reached a maximum at 24 h. DX did not effect HO-1 mRNA level. The increase in the HO-2 transcript was accompanied by an increase in HO-2 protein, as assessed by Western blot analysis, and an increase in HO activity, as measured by bilirubin formation. Also, an increase in intensity of immunostaining was noted in DX-treated HeLa cells. We conclude that the GRE present in the HO-2 gene promoter region is functional, and propose the direct involvement of the adrenal glucocorticoids in modulation of HO-2 gene expression. In the context of biological functions of heme degradation products, we suggest that this regulation may be of significance, particularly to the neurons.
血红素加氧酶-2（HO-2）在哺乳动物组织中组成性表达；它与HO-1（热休克蛋白32）一起催化血红素裂解，生成胆绿素IXα、一氧化碳和铁。在HO-2基因启动子区域检测到糖皮质激素反应元件（GRE）的共有序列，促使本研究调查糖皮质激素（Gcs）在新生大鼠脑HO-2蛋白和转录物发育调控中的作用，并检测HeLa细胞中GRE的启动子活性。使用原位杂交组织化学，我们发现出生后用皮质酮（5微克/克，4次，出生后24 - 36小时开始）处理的14日龄大鼠脑中HO-2 mRNA信号显著增加。并且，使用免疫组织化学，在处理过的大脑中检测到神经元HO-2免疫染色显著增加。使用无启动子的CAT表达载体和含有荧光素酶表达载体的异源启动子，在HeLa细胞中测试HO-2 GRE对地塞米松（DX）的反应性。含有GRE和转录起始位点的HO-2启动子响应DX诱导CAT报告基因活性，而GRE中的突变或缺失消除了激素反应性。同样，含有GRE的构建体以方向独立的方式赋予对DX的反应性并增加相对荧光素酶活性。此外，观察到糖皮质激素受体蛋白与GRE的特异性结合；只有过量的冷GRE能竞争结合，而突变的HO-2 GRE或AP1则不能。用DX（5微摩尔）处理的HeLa细胞中HO-2 mRNA（约1.3和约1.9 kb）增加，在24小时达到最大值。DX不影响HO-1 mRNA水平。通过蛋白质印迹分析评估，HO-2转录物的增加伴随着HO-2蛋白的增加，通过胆红素形成测量，HO活性也增加。并且，在DX处理的HeLa细胞中观察到免疫染色强度增加。我们得出结论，HO-2基因启动子区域存在的GRE具有功能，并提出肾上腺糖皮质激素直接参与HO-2基因表达的调节。鉴于血红素降解产物的生物学功能，我们认为这种调节可能具有重要意义，特别是对神经元而言。
