The Hypoxic Regulation and Function of Hypoxiainducible Factor 2α (HIF-2α) In an Adrenomedullary Chromaffin Cell Line

Abstract

<p> Exposure to chronic low oxygen (hypoxia) leads to a series of adaptive responses involving changes in gene expression that are critical for cell, tissue, and organismal survival. These changes are mediated by an important set of regulators belonging to the hypoxia inducible factor (HIF) family of transcription factors (e.g. HIF-lα, HIF-2α, HIF3α) which undergo rapid degradation during normal oxygen (normoxia) but are rapidly stabilized during hypoxia. While the role of HIF-1α has been extensively studied in many cell types, there have been relatively few studies on the role of HIF-2α, though recent evidence suggests its function maybe tissue specific. This thesis examined the hypothesis that HIF-2α plays a central role in the development and function of catecholaminergic cells of the sympathoadrenal (SA) lineage. The study was aided by use of an immortalized line of rat adrenomedullary chromaffin cells (i.e. MAH cells), derived from fetal SA progenitors, which express several hypoxia-sensitive properties characteristic of native cells in the adrenal gland. In Chapter 2, I investigated the potential contributions of mitochondrial reactive oxygen species (ROS) and 0 2 consumption to HIF-2α induction in MAH cells exposed to chronic hypoxia (2% O(2); 24 hr). In MAH cells, chronic hypoxia caused an increase in HIF-2α induction which was blocked by inhibition of any of the mitochondrial complexes using pharmacological agents, or by specific inhibition of complexes III and IV using RNAi techniques. It was found that in this 0 2-sensitive chromaffin cell line mitochondrial O(2) consumption, rather than changes in ROS, regulated HIF-2α induction during hypoxia. In Chapter 3, I investigated the hypothesized role of HIF-2α in the development of the catecholaminergic phenotype in cells of the SA lineage using the MAH cell line as a model. Mutant MAH cells, with depleted HIF-2α due to siRNA knock-down, showed dramatically lower levels of dopamine and noradrenaline compared to untransfected and scrambled control cells, regardless of whether the cells were cultured under normoxia or chronic hypoxia. This was correlated with a marked reduction in the expression of DOPA decarboxylase (DDC) and dopamine B hydroxylase (DBH), though the expression of tyrosine hydroxylase (TH) was unaffected. Moreover, HIF-2α was able to bind to a region of the DDC gene promoter which contains two putative hypoxia response elements (HREs). These data suggest that a basal level of HIF-2α function is required for the normal developmental expression of DDC and DBH in SA progenitor cells, and that loss of this function leads to impaired catecholamine (CA) biosynthesis. In Chapter 4, I investigated genes regulated by chronic hypoxia in MAH cells, with a focus on those involved in CA metabolism, storage, and secretion. Using microarray analysis combined with QPCR and RNAi knock-down methodology I uncovered several genes, involved in amine vesicular packaging, trafficking and secretion, which were upregulated during chronic hypoxia. One gene specifically, the adenosine A(2A) receptor (A(2A)R) gene, which appears to modulate CA secretion via autocrine or paracrine actions of extracellular adenosine, was dramatically upregulated in chronic hypoxia. Interestingly, this effect was completely abolished in HIF-2α knockdown MAH cells, suggesting a critical involvement of HIF-2α. Chromatin immunoprecipitation (ChIP) assays revealed that HIF-2α bound to the promoter region of the A(2A)R gene which contains a putative hypoxia response element (HRE) immediately upstream of exon 1. Ratiometric fluorescence measurements of intracellular Ca(2+) revealed that adenosine (50 μM) potentiated the high K(+)-evoked rise in [Ca(2+)]i in MAH cells. This effect of adenosine was further enhanced after chronic hypoxia, but was abolished in HIF-2α knock-down cells. In conclusion, these data suggest that HIF-2α is a key regulator of several genes involved in CA biosynthesis, and of others that mediate the facilitatory effects of chronic hypoxia on CA secretion in sympathoadrenal derivatives.