Abstract 2626: Oxidative Stress Stabilizes HIF-2alpha Involving NOX4: Possible Role In Pulmonary Vascular Remodeling
The hypoxia-inducible transcription factor HIF-2 consists of a constitutively expressed β-subunit and an α-subunit which is unstable under normoxia due to proteasomal degradation upon oxygen-dependent prolyl hydroxylation. HIF-2 appears to play a prominent role in the vascular response to hypoxia and in the pathogenesis of hypoxia-induced pulmonary hypertension (PH). However, it is unclear whether HIF-2 is also active under normoxic conditions. Since reactive oxygen species (ROS) have been shown to contribute to PH, we investigated whether HIF-2α is responsive to ROS in pulmonary artery smooth muscle cells (PASMC). Exposure to H2O2 or overexpression of the NADPH oxidase subunit NOX4 increased HIF-2α protein whereas depletion of NOX4 by siRNA reduced HIF-2α induction by thrombin. Treatment with ascorbate, a cofactor required for prolyl hydroxylation of HIF-2α, diminished upregulation by H2O2, NOX4 or thrombin. Since binding of the von Hippel Lindau protein (pVHL) to hydroxylated HIF-2α mediates degradation of HIF-2α, we determined the interaction of HIF-2α and pVHL by a pVHL-GST pull-down assay. H2O2 and thrombin reduced interaction between pVHL and HIF-2α whereas ascorbate reversed this effect. Since HIF-2α is hydroxylated at prolyl residues in the N-terminal transactivation domain (N-TAD), a mammalian yeast two hybrid assay was used to determine activation of the HIF-2α N-TAD. Whereas H2O2 and thrombin activated the wildtype N-TAD dependent on ascorbate or NOX4, no effect was observed when the prolyl residues were mutated. In addition, depletion of HIF-2α by siRNA decreased the expression of its target gene plasminogen activator inhibitor-1 and PASMC proliferation. HIF-2α was also found upregulated in remodeled pulmonary vessels in tissue samples from patients with PH. These findings show that HIF-2α is a novel redox-sensitive transcription factor which is upregulated by oxidative stress involving NOX4 due to inhibition of prolyl hydroxylation and subsequent HIF-2α stabilisation. Since HIF-2α contributed to PASMC proliferation by ROS and was upregulated in remodeled pulmonary vessels, our data suggest that HIF-2α may be critically involved in pulmonary vascular remodeling also independently of hypoxia.