Regulation of tyrosine hydroxylase gene expression during hypoxia: Role of Ca2+ and PKC

Gene expression for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, is regulated by reductions in oxygen tension (hypoxia). Hypoxia-induced regulation of the TH gene is due to the binding of specific transcription factors to specific sites on the 5' flanking region of the gene. The purpose of this study was to identify the second messenger system(s) responsible for regulation of the TH gene during hypoxia. Fura-2 fluorescence imaging of rat pheochromocytoma (PC12) cells, an O-2-sensitive cell line, revealed that there is an increase in cytosolic calcium (Ca2+) associated with exposure to hypoxia. Based on the evidence that the transcription factors that bind to the TH promoter during hypoxia can also be induced by elevations in cytosolic Ca2+, the role of Ca2+ in the hypoxic regulation of the TH gene was explored. To assay the effect of hypoxia on TH gene expression, Northern blot analyses of total RNA were performed on PC12 cells exposed to hypoxia in the presence or absence of specific inhibitors. The addition of the L-type calcium channel blockers nifedipine or verapamil caused partial inhibition of the hypoxia-induced increase in TH mRNA. The increase in cytosolic Ca2+ during hypoxia was also only partially inhibited by addition of nifedipine. Importantly, chelation of extracellular Ca2+ completely inhibited the increase in TH mRNA by hypoxia. Pre-treatment of PC12 cells with BAPTA/AM, an intracellular Ca2+ chelator, inhibited the hypoxic induction of TH gene expression in a dose-dependent manner. Addition of chelerythrine chloride (CHL), a protein kinase C inhibitor, to the media before exposure to hypoxia also resulted in an inhibition of TH induction by hypoxia. These results suggest that hypoxia regulates TH gene expression by a mechanism that is dependent on influx of calcium from the extracellular stores, partially but not exclusively through the L-type calcium channels. These results further suggest that a member of the PKC family is essential for this regulation.