Regulation of gene expression for tyrosine hydroxylase in oxygen sensitive cells by hypoxia.
Regulation of gene expression for tyrosine hydroxylase in oxygen sensitive cells by hypoxia.
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DOI：
10.1038/ki.1997.73
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发表时间：
1997-02
影响因子：
19.6
通讯作者：
D. Millhorn;R. Raymond;L. Conforti;W. Zhu;D. Beitner‐Johnson;T. Filisko;M. Genter;S. Kobayashi;M. Peng
中科院分区：
文献类型：
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作者：
D. Millhorn;R. Raymond;L. Conforti;W. Zhu;D. Beitner‐Johnson;T. Filisko;M. Genter;S. Kobayashi;M. Peng
缺氧对氧敏感细胞中酪氨酸羟化酶基因表达的调节。I型颈动脉体细胞和O2敏感的嗜铬细胞瘤(PC12)细胞在缺氧期间释放多巴胺。氧张力降低会抑制 O2 敏感性嗜铬细胞瘤 (PC12) 细胞系中 O2 敏感性钾 (K) 通道的向外整流，从而导致膜去极化和细胞内游离 Ca2+ 增加。我们发现，从细胞外环境中去除 Ca2+、抑制电压依赖性 Ca2+ 通道以及细胞内 Ca2+ 的螯合可防止缺氧期间 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 Ca2+ 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。 I 型细胞和 PC12 细胞中氧张力降低会刺激酪氨酸羟化酶 (TH) 的基因表达，TH 是多巴胺生物合成中的限速酶。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 稳定性的增加所致。对报告基因构建体的分析表明，缺氧期间TH基因转录的增加受到近端启动子区域的调节，该区域相对于转录起始位点从-284个碱基延伸到-150个碱基。该基因区域包含许多顺式作用调控元件，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。 Super-shift 和 shift Western 测定的结果表明，由 c-Fos 和 JunB 组成的异二聚体在缺氧期间与 API 位点结合。诱变实验表明，缺氧期间，TH 基因的转录增加需要 AP1 位点。我们还发现编码 c-FosandJunB 转录因子蛋白的基因受到氧气张力降低的调节。
Regulation of gene expression for tyrosine hydroxylase in oxygen sensitive cells by hypoxia.Carotid body type I cells and the O2-sensitive pheochromocytoma (PC12) cells release dopamine during hypoxia. Reduced O2tension 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 O2tension 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 ofcis-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-FosandJunBbinds to the API 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-FosandJunBtranscription factor proteins are regulated by reduced O2tension.