Endothelin-1 regulates hypoxia-inducible factor-1 alpha and-2 alpha stability through prolyl hydroxylase domain 2 inhibition in human lymphatic endothelial cells

AIMS: Lymphangiogenesis, the formation of new lymphatic vessels, is thought to constitute a route for the tumor cells to metastasize. We previously demonstrated that endothelin-1 (ET-1) induces the expression of lymphangiogenic factors through hypoxia-inducible factor (HIF)-1α and HIF-2α. The stability of these transcriptional factors is essential for lymph/angiogenesis and tumor progression. Here we analyze the molecular mechanism through which ET-1 regulates HIF-1α and HIF-2α protein levels and how these transcriptional factors are implicated in controlling lymphatic endothelial cell (LEC) behavior. MAIN METHODS: Using Western blotting assay and a reporter gene containing the HIF-1α oxygen-dependent degradation domain we monitored the capacity of ET-1 to increase HIF-1α and HIF-2α stability and nuclear accumulation. In addition, using siRNA against HIF-1α or HIF-2α, we investigated the implication of these transcriptional factors in ET-1-mediated tube-like structure formation. As HIF-1α proteosomal degradation is controlled by site-specific hydroxylation carried out by HIF-prolyl hydroxylase domain (PHD) enzymes, we analyzed the expression of PHD-2 isoform. KEY FINDINGS: We show that ET-1 through its receptor, ETBR, controls HIF-α stability and nuclear accumulation by inhibiting prolyl hydroxylation and reduces PHD2 mRNA and protein levels. Transfection with HIF-1α or HIF-2α siRNA abrogated the capacity of ET-1 to induce tube-like structure formation. SIGNIFICANCE: These results reveal a PHD2-mediated mechanism through which ET-1 stabilizes HIF-1α and HIF-2α pathway thereby regulating LEC behavior and lymphangiogenesis. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.