Activation of hypoxia-inducible factor-1 in pulmonary arterial smooth muscle cells by endothelin-1

Pisarcik S, Maylor J, Lu W, Yun X, Undem C, Sylvester JT, Semenza GL, Shimoda LA. Activation of hypoxia-inducible factor-1 in pulmonary arterial smooth muscle cells by endothelin-1. Am J Physiol Lung Cell Mol Physiol 304: L549-L561, 2013. First published February 15, 2013; doi: 10.1152/ajplung.00081.2012.-Numerous cellular responses to hypoxia are mediated by the transcription factor hypoxia-inducible factor-1 (HIF-1). HIF-1 plays a central role in the pathogenesis of hypoxic pulmonary hypertension. Under certain conditions, HIF-1 may utilize feedforward mechanisms to amplify its activity. Since hypoxia increases endothelin-1 (ET-1) levels in the lung, we hypothesized that during moderate, prolonged hypoxia ET-1 might contribute to HIF-1 signaling in pulmonary arterial smooth muscle cells (PASMCs). Primary cultures of rat PASMCs were treated with ET-1 or exposed to moderate, prolonged hypoxia (4% O-2 for 60 h). Levels of the oxygen-sensitive HIF-1 alpha subunit and expression of HIF target genes were increased in both hypoxic cells and cells treated with ET-1. Both hypoxia and ET-1 also increased HIF-1 alpha mRNA expression and decreased mRNA and protein expression of prolyl hydroxylase 2 (PHD2), which is the protein responsible for targeting HIF-1 alpha for O-2-dependent degradation. The induction of HIF-1 alpha by moderate, prolonged hypoxia was blocked by BQ-123, an antagonist of ET-1 receptor subtype A. The effects of ET-1 were mediated by increased intracellular calcium, generation of reactive oxygen species, and ERK1/2 activation. Neither ET-1 nor moderate hypoxia induced the expression of HIF-1 alpha or HIF target genes in aortic smooth muscle cells. These results suggest that ET-1 induces a PASMC-specific increase in HIF-1 alpha levels by upregulation of HIF-1 alpha synthesis and downregulation of PHD2-mediated degradation, thereby amplifying the induction of HIF-1 alpha in PASMCs during moderate, prolonged hypoxia.