title: Endothelin-1 regulates hypoxia-inducible factor-1α and -2α stability through prolyl hydroxylase domain 2 inhibition in human lymphatic endothelial cells.
abstract: OBJECTIVE
  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.
  
  
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  These results reveal a PHD2-mediated mechanism through which ET-1 stabilizes HIF-1α and HIF-2α pathway thereby regulating LEC behavior and lymphangiogenesis.
authors.affiliation: Molecular Pathology, Regina Elena National Cancer Institute, Rome, Italy.
authors.forename: Valentina
authors.initials: V
authors.lastname: Caprara
authors.affiliation: Molecular Pathology, Regina Elena National Cancer Institute, Rome, Italy.
authors.forename: Silvia
authors.initials: S
authors.lastname: Scappa
authors.affiliation: Microbiology Section, Department of Experimental Medicine, University of Brescia, Italy.
authors.forename: Emirena
authors.initials: E
authors.lastname: Garrafa
authors.affiliation: Molecular Pathology, Regina Elena National Cancer Institute, Rome, Italy.
authors.forename: Valeriana
authors.initials: V
authors.lastname: Di Castro
authors.affiliation: Molecular Pathology, Regina Elena National Cancer Institute, Rome, Italy.
authors.forename: Laura
authors.initials: L
authors.lastname: Rosanò
authors.affiliation: Molecular Pathology, Regina Elena National Cancer Institute, Rome, Italy. Electronic address: bagnato@ifo.it.
authors.forename: Anna
authors.initials: A
authors.lastname: Bagnato
authors.affiliation: Molecular Pathology, Regina Elena National Cancer Institute, Rome, Italy. Electronic address: spinella@ifo.it.
authors.forename: Francesca
authors.initials: F
authors.lastname: Spinella
mesh_terms: D051792:Basic Helix-Loop-Helix Proteins / Q000737:chemistry* / Q000378:metabolism*; D002465:Cell Movement / Q000187:drug effects; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D019332:Endothelin-1 / Q000494:pharmacology*; D006801:Humans; D006900:Hydroxylation / Q000187:drug effects; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000737:chemistry* / Q000378:metabolism*; D018919:Neovascularization, Physiologic / Q000187:drug effects; D055550:Protein Stability / Q000187:drug effects; D017434:Protein Structure, Tertiary; D034741:RNA, Small Interfering / Q000378:metabolism; D044023:Receptor, Endothelin B / Q000378:metabolism; D000099162:Endothelial PAS Domain-Containing Protein 1
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D019332:Endothelin-1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D034741:RNA, Small Interfering; D044023:Receptor, Endothelin B; D000099162:Endothelial PAS Domain-Containing Protein 1
keywords: Endothelin B receptor; Endothelin-1; Hypoxia-inducible factor 1; Lymphatic endothelial cells; Prolyl hydroxylases enzymes