Millhorn D E, Raymond R, Conforti L, Zhu W, Beitner-Johnson D, Filisko T, Genter M B, Kobayashi S, Peng M
Department of Molecular and Cellular Physiology, College of Medicine, University of Cincinnati, Ohio, USA.
Kidney Int. 1997 Feb;51(2):527-35. doi: 10.1038/ki.1997.73.
Carotid body type I cells and the O2 sensitive pheochromocytoma (PC12) cells release dopamine during hypoxia. Reduced O2 tension causes inhibition of an outward rectifying the O2-sensitive potassium (K) channel in the O2-sensitive pheochromocytoma (PC12) cell line, which leads to membrane depolarization and increased intracellular free Ca2+. We found that removal of Ca2+ from the extracellular milieu, inhibition of voltage-dependent Ca2+ channels, and chelation of intracellular Ca2+ prevents full activation of the TH gene expression during hypoxia. These findings suggest that membrane depolarization and regulation of intracellular free Ca2+ are critical signal transduction events that regulate expression of the TH gene in PC12 cells during hypoxia. Gene expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in the biosynthesis of dopamine, is stimulated by reduced O2 tension in both type I cells and PC12 cells. The increase in TH gene expression in PC12 cells during hypoxia is due to increases in both the rate of transcription and mRNA stability. Analysis of reporter-gene constructs revealed that increased transcription of the TH gene during hypoxia is regulated by a region of the proximal promoter that extends from -284 to -150 bases, relative to the transcription start site. This region of the gene contains a number of cis-acting regulatory elements including AP1, AP2 and hypoxia-inducible factor (HIF-1). Competition assays revealed that hypoxia-induced binding occurs at both the AP1 and HIF-1 sites. Results from super-shift and shift Western assays showed that a heterodimer consisting of c-Fos and JunB binds to the AP1 site during hypoxia. Mutagenesis experiments revealed that the AP1 site is required for increased transcription of the TH gene during hypoxia. We also found that the genes that encode the c-Fos and JunB transcription factor proteins are regulated by reduced O2 tension.
颈动脉体I型细胞和对氧敏感的嗜铬细胞瘤（PC12）细胞在缺氧时释放多巴胺。氧张力降低会抑制对氧敏感的嗜铬细胞瘤（PC12）细胞系中一种外向整流的对氧敏感钾（K）通道，这会导致膜去极化并增加细胞内游离钙离子浓度。我们发现，从细胞外环境中去除钙离子、抑制电压依赖性钙离子通道以及螯合细胞内钙离子可防止缺氧期间TH基因表达的完全激活。这些发现表明，膜去极化和细胞内游离钙离子的调节是缺氧期间调节PC12细胞中TH基因表达的关键信号转导事件。酪氨酸羟化酶（TH）是多巴胺生物合成中的限速酶，其基因表达在I型细胞和PC12细胞中均受氧张力降低的刺激。缺氧期间PC12细胞中TH基因表达的增加是由于转录速率和mRNA稳定性的增加。报告基因构建体分析表明，缺氧期间TH基因转录的增加受近端启动子中一个区域的调节，该区域相对于转录起始位点从-284到-150个碱基。该基因区域包含许多顺式作用调节元件，包括AP1、AP2和缺氧诱导因子（HIF-1）。竞争分析表明，缺氧诱导的结合发生在AP1和HIF-1位点。超迁移和迁移Western分析结果表明，缺氧期间由c-Fos和JunB组成的异二聚体与AP1位点结合。诱变实验表明，AP1位点是缺氧期间TH基因转录增加所必需的。我们还发现，编码c-Fos和JunB转录因子蛋白的基因受氧张力降低的调节。
