Endothelin-1 regulates hypoxia-inducible factor-1α and-2α 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)-1 alpha and HIF-2 alpha. The stability of these transcriptional factors is essential for lymph/angiogenesis and tumor progression. Here we analyze the molecular mechanism through which ET-I regulates HIF-1 alpha and HIF-2 alpha 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-1 alpha oxygen-dependent degradation domain we monitored the capacity of ET-1 to increase HIF-1 alpha and HIF-2 alpha stability and nuclear accumulation. In addition, using siRNA against HIF-1 alpha or HIF-2 alpha, we investigated the implication of these transcriptional factors in ET-1-mediated tube-like structure formation. As HIF-1 alpha 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-alpha stability and nuclear accumulation by inhibiting prolyl hydroxylation and reduces PHD2 mRNA and protein levels. Transfection with HIF-1 alpha or HIF-2 alpha 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-1 alpha and HIF-2 alpha pathway thereby regulating LEC behavior and lymphangiogenesis. (C) 2014 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).