Calcium signaling stimulates translation of HIF-alpha during hypoxia.

Hypoxia-inducible factors (HIFs) are ubiquitous transcription factors that mediate adaptation to hypoxia by inducing specific sets of target genes. It is well accepted that hypoxia induces accumulation and activity of HIFs by causing stabilization of their alpha subunits. We have demonstrated that hypoxia stimulates translation of HIF-1alpha and -2alpha proteins by distributing HIF-alpha mRNAs to larger polysome fractions. This requires influx of extracellular calcium, stimulation of classical protein kinase C-alpha (cPKC-alpha), and the activity of mammalian target of rapamycin, mTOR. The translational component contributes to approximately 40-50% of HIF-alpha proteins accumulation after 3 h of 1% O2. Hypoxia also inhibits general protein synthesis and mTOR activity; however, cPKC-alpha inhibitors or rapamycin reduce mTOR activity and total protein synthesis beyond the effects of hypoxia alone. These data show that during general inhibition of protein synthesis by hypoxia, cap-mediated translation of selected mRNAs is induced through the mTOR pathway. We propose that calcium-induced activation of cPKC-alpha hypoxia partially protects an activity of mTOR from hypoxic inhibition. These results provide an important physiologic insight into the mechanism by which hypoxia-stimulated influx of calcium selectively induces the translation of mRNAs necessary for adaptation to hypoxia under conditions repressing general protein synthesis.

Endogenous von Hippel-Lindau tumor suppressor protein regulates catecholaminergic phenotype in PC12 cells.

Loss of von Hippel-Lindau (VHL) gene function leads to VHL disease, which is characterized by vascular tumors of the central nervous system, renal clear cell carcinomas, and pheochromocytomas. Pheochromocytomas express high levels of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis. PC12 cells that express VHL antisense RNA had 5-10-fold reduced levels of endogenous pVHL and 2-3-fold increased levels of TH protein and mRNA. Nuclear run-on analysis revealed an augmentation of TH gene transcription with enhanced efficiency of transcript elongation in the 3' region of the gene. Transient coexpression of the VHL antisense RNA with a TH promoter reporter construct increased TH promoter activity by 2-3-fold. A decrease in pVHL accumulation also resulted in an increase in TH mRNA accumulation and transcription of the TH gene during hypoxia. This is the first evidence that endogenous pVHL is a physiological regulator of the catecholaminergic phenotype. Thus, loss of pVHL function may be causative in pheochromocytoma-associated hypercatecholaminemia and arterial hypertension.

Regulation of gene expression for neurotransmitters during adaptation to hypoxia in oxygen-sensitive neuroendocrine cells.

Reduced oxygen tension (hypoxia) in the environment stimulates oxygen-sensitive cells in the carotid body (CB). Upon exposure to hypoxia, the CB immediately triggers a reflexive physiological response, thereby increasing respiration. Adaptation to hypoxia involves changes in the expression of various CB genes, whose products are involved in the transduction and modulation of the hypoxic signal to the central nervous system (CNS). Genes encoding neurotransmitter-synthesizing enzymes and receptors are particularly important in this regard. The cellular response to hypoxia correlates closely with the release and biosynthesis of catecholamines. The gene expression of tyrosine hydroxylase (TH), the rate-limiting enzyme for catecholamine biosynthesis, is regulated by hypoxia in the CB and in the oxygen-sensitive cultured PC12 cell line. Recently, genomic microarray studies have identified additional genes regulated by hypoxia. Patterns of gene expression vary, depending on the type of applied hypoxia, e.g., intermittent vs. chronic. Construction of a hypoxia-regulated, CB-specific, subtractive cDNA library will enable us to further characterize regulation of gene expression in the CB.

Hypoxia-activated metabolic pathway stimulates phosphorylation of p300 and CBP in oxygen-sensitive cells.

Transcription co-activators and histone acetyltransferases, p300 and cyclic AMP responsive element-binding protein-binding protein (CBP), participate in hypoxic activation of hypoxia-inducible genes. Here, we show that exposure of PC12 and cells to 1-10% oxygen results in hyperphosphorylation of p300/CBP. This response is fast, long lasting and specific for hypoxia, but not for hypoxia-mimicking agents such as desferioxamine or Co2+ ions. It is also cell-type specific and occurs in pheochromocytoma PC12 cells and the carotid body of rats but not in hepatoblastoma cells. The p300 hyperphosphorylation specifically depends on the release of intracellular calcium from inositol 1,4,5-triphosphate (IP3)-sensitive stores. However, it is not inhibited by pharmacological inhibitors of any of the kinases traditionally known to be directly or indirectly calcium regulated. On the other hand, p300 hyperphosphorylation is inhibited by several different inhibitors of the glucose metabolic pathway from generation of NADH by glyceraldehyde 3-phosphate dehydrogenase, through the transfer of NADH through the glycerol phosphate shuttle to ubiquinone and complex III of the mitochondrial respiratory chain. Inhibition of IP3-sensitive calcium stores decreases generation of ATP, and this inhibition is significantly stronger in hypoxia than in normoxia. We propose that the NADH glycerol phosphate shuttle participates in generating a pool of ATP that serves either as a co-factor or a modulator of the kinases involved in the phosphorylation of p300/CBP during hypoxia.

Regulation of tyrosine hydroxylase promoter activity by the von Hippel-Lindau tumor suppressor protein and hypoxia-inducible transcription factors.

Tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, is induced by hypoxia in oxygen-sensitive cells of the carotid body and pheochromocytoma-derived PC12 cells. TH is also regulated by the von Hippel-Lindau tumor suppressor protein (pVHL). Here, we report that induction of TH gene expression involves activation of the hypoxia-inducible transcription factors (HIFs) that interact with a specific hypoxia-responsive element (HRE) in the proximal region of the TH promoter. We also show that some of the effects of pVHL on activity of the TH promoter are mediated through HIFs. Low levels of pVHL are associated with decreased HIFalpha ubiquitination, increased accumulation of HIFalpha proteins, increased binding of HIFs to the HRE within the TH promoter, and increased activity of a TH promoter-reporter construct. In contrast, high levels of pVHL repress HIF accumulation and inhibit its activity in hypoxic cells. These results indicate that HIFs may play an important role in regulation of TH gene expression in oxygen-sensitive cells and also in the development of hypercatecholaminemia in pheochromocytoma tumors.

Regulation of catecholamines by sustained and intermittent hypoxia in neuroendocrine cells and sympathetic neurons.

Chronic intermittent hypoxia, a characteristic feature of sleep-disordered breathing, induces hypertension through augmented sympathetic nerve activity and requires the presence of functional carotid body arterial chemoreceptors. In contrast, chronic sustained hypoxia does not alter blood pressure. We therefore analyzed the biosynthetic pathways of catecholamines in peripheral nervous system structures involved in the pathogenesis of intermittent hypoxia-induced hypertension, namely, carotid bodies, superior cervical ganglia, and adrenal glands. Rats were exposed to either intermittent hypoxia (90 seconds of room air alternating with 90 seconds of 10% O2) or to sustained hypoxia (10% O2) for 1 to 30 days. Dopamine, norepinephrine, epinephrine, dihydroxyphenylacetic acid, and 5-hydroxytyptamine contents were measured by high-performance liquid chromatography. Expression of tyrosine hydroxylase and its phosphorylated forms, dopamine beta-hydroxylase, phenylethanolamine N-methyltransferase, and GTP cyclohydrolase-1 were determined by Western blot analyses. Both sustained and intermittent hypoxia significantly increased dopamine and norepinephrine content in carotid bodies but not in sympathetic ganglia or adrenal glands. In carotid bodies, both types of hypoxia augmented total levels of tyrosine hydroxylase protein and its phosphorylation on serines 19, 31, 40, as well as levels of GTP cyclohydrolase-1. However, the effects of intermittent hypoxia on catecholaminergic pathways were significantly smaller and delayed than those induced by sustained hypoxia. Thus, attenuated induction of catecholaminergic phenotype by intermittent hypoxia in carotid body may play a role in development of hypertension associated with sleep-disordered breathing. The effects of both types of hypoxia on expression of catecholaminergic enzymes in superior cervical neurons and adrenal glands were transient and small.