Hypoxia increases rate of transcription and stability of tyrosine hydroxylase mRNA in pheochromocytoma (PC12) cells

Reduced arterial oxygen tension (i.e. hypoxia) is a powerful physiological stimulus that induces synthesis and release of dopamine from O₂-sensitive (type I) cells in the mammalian carotid bodies. We reported recently that hypoxia stimulates gene expression for tyrosine hydroxylase (TH), the rate- limiting enzyme in catecholamine synthesis in type I cells of the carotid body. Efforts to identify the mechanisms regulating 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 line (PC12) is regulated during hypoxia in a manner similar to that measured in carotid body type I cells. PC12 cells might therefore be useful as an experimental model for identifying the molecular mechanisms that regulate TH gene expression during hypoxia. Nuclear runoff assays revealed that transcription of the wild type TH gene was enhanced during exposures to hypoxia lasting 12 h. Chloramphenicol acetyltransferase assays with constructs that contained different fragments of TH promoter revealed that the regulatory sequences that mediate the hypoxia-induced 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- ribofuranosylbenzimidazole, as well as pulse-chase experiments using 4- thiouridine showed that the half-life of TH mRNA was substantially increased during hypoxia. Thus, in the present paper we show that TH gene expression in PC12 cells during hypoxia is regulated by increases in both the rate of TH gene transcription and TH mRNA stability.