HYPOXIA INCREASES RATE OF TRANSCRIPTION AND STABILITY OF TYROSINE-HYDROXYLASE MESSENGER-RNA IN PHEOCHROMOCYTOMA (PC12) CELLS

Reduced arterial oxygen tension (i.e. hypoxia) is a powerful physiolog ical stimulus that induces synthesis and release of dopamine from O2-s ensitive (type I) cells in the mammalian carotid bodies. We reported r ecently that hypoxia stimulates gene expression for tyrosine hydroxyla se (TH), the rate-limiting enzyme in catecholamine synthesis in type I cells of the carotid body. Efforts to identify the mechanisms regulat ing TH gene expression in O2-sensitive cells during hypoxia have been hampered by the lack of an appropriate model cell culture system. Here we report that TH gene expression in the rat pheochromocytoma cell li ne (PC12) is regulated during hypoxia in a manner similar to that meas ured in carotid body type I cells. PC12 cells might therefore be usefu l as an experimental model for identifying the molecular mechanisms th at regulate TH gene expression during hypoxia. Nuclear runoff assays r evealed that transcription of the wild type TH gene was enhanced durin g exposures to hypoxia lasting 12 h. Chloramphenicol acetyltransferase assays with constructs that contained different fragments of TH promo ter revealed that the regulatory sequences that mediate the hypoxia-in duced increase in transcription are located between bases -272 and +27 of the TH gene. Findings from experiments in which transcription was inhibited either with actinomycin D or 5,6-dichloro-1-D-ribofuranosylb enzimidazole, as well as pulse-chase experiments using 4-thiouridine s howed that the half-life of TH mRNA was substantially increased during hypoxia. Thus, in the present paper we show that TH gene expression i n PC12 cells during hypoxia is regulated by increases in both the rate of TH gene transcription and TH mRNA stability.