The regulation of hypoxic genes by calcium involves c-Jun/AP-1, which cooperates with hypoxia-inducible factor 1 in response to hypoxia

Hypoxia causes the accumulation of the transcription factor hypoxia-inducible factor 1 (HIF-1), culminating in the expression of hypoxia-inducible genes such as those for vascular endothelial growth factor (VEGF) and NDRG-1/Cap43. Previously, we have demonstrated that intracellular calcium (Ca2+) is required for the expression of hypoxia-inducible genes. Here we found that, unlike with hypoxia or hypoxia-mimicking conditions, the elevation of intracellular Ca2+ neither induced the HIF-1alpha protein nor stimulated HIF-1-dependent transcription. Furthermore, the elevation of intracellular Ca2+ induced NDRG-1/Cap43 mRNA in HIF-1alpha-deficient cells. It also increased levels of c-Jun protein, causing its phosphorylation. The protein kinase inhibitor K252a abolished c-Jun induction and activator protein 1 (AP-1)-dependent reporter expression caused by Ca2+ ionophore or hypoxia. K252a also significantly decreased hypoxia-induced VEGF and NDRG-1/Cap43 gene expression in both human and mouse cells. Using a set of deletion VEGF-Luc promoter constructs, we found that both HIF-1 and two AP-1 sites contribute to hypoxia-mediated induction of transcription. In contrast, only AP-1 sites contributed to Ca2+-mediated VEGF-Luc induction. A dominant-negative AP-1 prevented Ca2+-dependent transcription and partially impaired hypoxia-mediated transcription. In addition, dominant-negative AP-1 diminished the expression of the NDRG-1/Cap43 gene following hypoxia. We conclude that during hypoxia, an increase in intracellular Ca2+ activates a HIF-1-independent signaling pathway that involves AP-1-dependent transcription. Cooperation between the HIF-1 and AP-1 pathways allows fine regulation of gene expression during hypoxia.