REGULATION OF GENE-EXPRESSION FOR TYROSINE-HYDROXYLASE IN OXYGEN SENSITIVE CELLS BY HYPOXIA

Carotid body type I cells and the O-2-sensitive pheochromocytoma (PC12 ) cells release dopamine during hypoxia. Reduced O-2 tension causes in hibition of an outward rectifying the O-2-sensitive potassium (K) chan nel in the O-2-sensitive pheochromocytoma (PC12) cell line, which lead s 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. T hese findings suggest that membrane depolarization and regulation of i ntracellular free Ca2+ are critical signal transduction events that re gulate expression of the TH gene in PC12 cells during hypoxia. Gene ex pression of tyrosine hydroxylase (TH), the rate-limiting enzyme in the biosynthesis of dopamine, is stimulated by reduced O-2 tension in bot h type I cells and PC12 cells. The increase in TH gene expression in P C12 cells during hypoxia is due to increases in both the rate of trans cription and mRNA stability. Analysis of reporter-gene constructs reve aled that increased transcription of the TH gene during hypoxia is reg ulated by a region of the proximal promoter that extends from -284 to -150 bases, relative to the transcription start site. This region of t he gene contains a number of cis-acting regulatory elements including AP1, AP2 and hypoxia-inducible factor (HIF-1). Competition assays reve aled that hypoxia-induced binding occurs at both the AP1 and HIF-1 sit es. Results from super-shift and shift Western assays showed that a he terodimer consisting of c-Fos and JunB binds to the AP1 site during hy poxia. Mutagenesis experiments revealed that the AP1 site is required for increased transcription of the TH gene during hypoxia. We also fou nd that the genes that encode the c-Fos and JunB transcription factor proteins are regulated by reduced O-2 tension.