Ca2+ Mediates HIF-dependent Upregulation of Aquaporin 1 in Pulmonary Arterial Smooth Muscle Cells

Abstract

Prolonged exposure to hypoxia causes structural remodeling and sustained contraction of the pulmonary vasculature, resulting in the development of pulmonary hypertension. Both pulmonary arterial smooth muscle cell (PASMC) proliferation and migration contribute to the vascular remodeling. We previously showed that the protein expression of aquaporin 1 (AQP1), a membrane water channel protein, is elevated in PASMCs during following in vivo or in vitro exposure to hypoxia. Studies in other cell types suggest that AQP1 is a direct transcriptional target of hypoxia inducible factor (HIF)-1. Moreover, we and others have shown that an increase in intracellular calcium concentration ([Ca2+]i) is a hallmark of hypoxic exposure in PASMCs. Thus, we wanted to determine whether HIF regulates AQP1 in PASMCs and, if so, whether the process occurred via transcriptional regulation or was Ca2+-dependent. PASMCs were exposed to hypoxia, incubated with DMOG, which inhibits HIF protein degradation or infected with constitutively active forms of HIF-1 or HIF-2. Hypoxia, DMOG and HIF1/2 produced a time-dependent increase in AQP1 protein, but not mRNA. Interestingly, incubation with increasing HIF1/2a levels and DMOG increased [Ca2+]i in PASMCs, and this elevation was prevented by the voltage-gated Ca2+ channel inhibitor, verapamil (VER) and nonselective cation channel inhibitor SKF96365 (SKF). VER and SKF also blocked upregulation of AQP1 protein by DMOG or HIF1/2, but had no effect on expression of GLUT1, a canonical HIF transcriptional target. Silencing of AQP1 abrogated increases in PASMC migration and proliferation induced by HIF1/2, suggesting induction of AQP1 protein by HIF1/2 has a functional outcome in these cells. Thus, our results show that contrary to reports in other cell types, in PASMCs, AQP1 does not appear to be a direct target for HIF transcriptional regulation. Instead, AQP1 protein may be upregulated by a mechanism involving HIF-dependent increases in [Ca2+]i.