Carbon Monoxide Promotes VEGF Expression by Increasing HIF-1α Protein Level via Two Distinct Mechanisms, Translational Activation and Stabilization of HIF-1α Protein

Carbon monoxide (CO) plays a significant role in vascular functions. We here examined the molecular mechanism by which CO regulates HIF-1 (hypoxia-inducible transcription factor-1)-dependent expression of vascular endothelial growth factor (VEGF), which is an important angiogenic factor. We found that astrocytes stimulated with CORM-2 (CO-releasing molecule) promoted angiogenesis by increasing VEGF expression and secretion. CORM-2 also induced HO-1 (hemeoxygenase-1) expression and increased nuclear HIF-1 alpha protein level, without altering its promoter activity and mRNA level. VEGF expression was inhibited by treatment with HIF-1 alpha siRNA and a hemeoxygenase inhibitor, indicating that CO stimulates VEGF expression via up-regulation of HIF-1 alpha protein level, which is partially associated with HO-1 induction. CORM-2 activated the translational regulatory proteins p70(S6k) and eIF-4E as well as phosphorylating their upstream signal mediators Akt and ERK. These translational signal events and HIF-1 alpha protein level were suppressed by inhibitors of phosphatidylinositol 3-kinase (PI3K), MEK, and mTOR, suggesting that the PI3K/Akt/mTOR and MEK/ERK pathways are involved in a translational increase in HIF-1 alpha. In addition, CORM-2 also increased stability of the HIF-1 alpha protein by suppressing its ubiquitination, without altering the proline hydroxylase-dependent HIF-1 alpha degradation pathway. CORM-2 increased HIF-1 alpha/HSP90 alpha interaction, which is responsible for HIF-1 alpha stabilization, and HSP90-specific inhibitors decreased this interaction, HIF-1 alpha protein level, and VEGF expression. Furthermore, HSP90 alpha knockdown suppressed CORM-2-induced increases in HIF-1 alpha and VEGF protein levels. These results suggest that CO stimulates VEGF production by increasing HIF-1 alpha protein level via two distinct mechanisms, translational stimulation and protein stabilization of HIF-1 alpha.