The influence of protein kinase inhibition on hypoxia-inducible factor-1alpha during hypoxia in adult canine ventricular tissue

Evidence of an oxygen-sensitive transcriptional regulatory pathway in the heart and its role in an early cellular response to ischemia and/or hypoxia has been progressively mounting. Hypoxia-Inducible Factor-1 (HIF-1) has been shown to play a critical role in the increased transcriptional expression of vascular endothelial growth factor (VEGF), glycolytic enzymes, and glucose transporters during hypoxia. To date, the mechanism responsible for transduction of the hypoxic signal that leads to activation of HIF-1α is unknown. However, phosphorylation is suspected to be involved.; The purpose of the present study was to determine canine myocardial expression of HIF-1α following hypoxic challenge in vivo and in vitro, and to characterize an upstream activator responsible for initiation of a hypoxic signal. In order to determine the role of phosphatidylinositol-3 OH Kinase (P13-K), protein kinase B (AKT), tyrosine kinases, and protein kinase C (PKC) in the stabilization of HIF-1α, transmural ventricular biopsies from adult mongrel dogs of either sex were incubated in 12 well plates containing 2mls of Kreb's Hensleit Buffer at 37°C for four hours under hypoxic (3% O₂ 5% CO₂ balance N₂) conditions. Wortmanin (P13-K inhibitor), Genistein (broad-spectrum tyrosine kinase inhibitor), and Chelerythrine (PKC inhibitor) were administered to the wells and protein was extracted, immunoprecipitated, and analyzed by immunoblot analyses. The results from these studies indicate that P13-K attenuates hypoxia-induced stabilization of HIF-1α in the cytosol and that PKC and tyrosine kinase modulate HIF-1α activity by regulating translocation and dimerization respectively. Phosphorylation of HIF-1α was limited to serine residues. Further studies revealed that protein kinase B (AKT) responded to hypoxia in an isoform specific manner. Measurement of two down stream targets of AKT, glycogen synthase kinase-3 (GSK-3), a direct phosphorylator of HIF-1α and 4E binding protein1 (4E-BP1), repressor of translational induction, suggested isoform specific activation of GSK-3 and phosphorylative inhibition of 4E-BP1 repressor activity.; These findings suggest that hypoxic induction of the PI-3K/AKT/GSK-3 pathway may play a major role in transcriptional regulation through cytosolic stabilization of HIF-1α as well as translational induction through removal of 4E-BP1 repressor activity. Furthermore, these results show conclusively that multiple kinase interaction plays a critical role in regulation of the oxygen-sensitive transcriptional activity of hypoxia-inducible factor-1α.