Hypoxia induces complex pattern or gene expression in the O2-sensitive type Icells of the carotid body, and in the PC12 cells which are used as an experimental model for type I cells. One of the genes regulated by hypoxia in type I cells and in PC 12 cells is tyrosine hydroxylase (TH), the regulatory enzyme in catecholamine synthesis. Our laboratory has used the TH gene as a model to identify the molecular mechanisms that regulate hypoxia-responsive genes in carotid body. Hypoxia stimulates TH gene transcription by promoting interactions of c-fos, junB and hypoxia-inducible factors (HIFs) with cis-acting elements within the TH promoter. We discovered that the von Hippel-Lindau protein (pVhl), a component of a multiprotein complex that has E3 ubiquity ligase activity, and ubiquitinates and targets the HIFs for degradation, is a potent modulator of TH gene expression during normoxia and hypoxia. Very little is known about the molecular mechanisms involved in regulation of hypoxia-responsive genes by pVhl, the overall goal of this proposal is to study the role of the pVhl complex in the physiological regulation of gene expression by hypoxia. Specifically, we will identify the molecular mechanism by which pVhl regulates transcription of the TH gene during hypoxia in PC 12 cells, and determine if similar regulation occurs in the carotid body and other catecholaminergic tissues such as adrenal glands and superior cervical ganglia. The main hypothesis is that pVhl inhibits the activity of the TH promoter by targeting hypoxia-regulated transcription factors for ubiquitination and degradation. Regulation of accumulation of transcription factors by ubiquitination and degradation is a novel, and as yet little understood mechanism. We will: (1) Identify the mechanism by which pVhl modulates hypoxic activation of the TH gene expression. We hypothesize that this modulation occurs at the level of transcription and activity of the TH promoter. (2) Identify the transcription factors that are involved in hypoxic activation of the TH promoter and are regulated by pVhl in PC12 cells, carotid bodies and other catecholaminergic tissues. (3) Determine if the E3 ubiquity ligase activity of the pVhl-associated complex towards the HIFs is regulated by hypoxia, and in a tissue specific manner in extracts from PC12 cells and catecholaminergic tissues from rats exposed to normoxia or hypoxia. The major biological significance of this proposals is that it will provide normal insights to a novel mechanism for regulation of transcription of the hypoxia-responsive genes. An understanding of the physiological role of pVhl in regulation of gene expression during hypoxia will provide much needed information on how O2-sensitive cells respond and adapt to hypoxia.
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