Endothelin-1 inhibits prolyl hydroxylase domain 2 to activate hypoxia-inducible factor-1α in melanoma cells

Francesca Spinella, Laura Rosanó, Martina del Duca, Valeriana di Castro, Maria Rita Nicotra, Pier Giorgio Natali, Anna Bagnato

Research output: Contribution to journalArticle

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Abstract

Background: The endothelin B receptor (ETBR) promotes tumorigenesis and melanoma progression through activation by endothelin (ET)-1, thus representing a promising therapeutic target. The stability of hypoxia-inducible factor (HIF)-1a is essential for melanomagenesis and progression, and is controlled by site-specific hydroxylation carried out by HIF-prolyl hydroxylase domain (PHD) and subsequent proteosomal degradation. Principal Findings: Here we found that in melanoma cells ET-1, ET-2, and ET-3 through ETBR, enhance the expression and activity of HIF-1a and HIF-2a that in turn regulate the expression of vascular endothelial growth factor (VEGF) in response to ETs or hypoxia. Under normoxic conditions, ET-1 controls HIF-a stability by inhibiting its degradation, as determined by impaired degradation of a reporter gene containing the HIF-1a oxygen-dependent degradation domain encompassing the PHD-targeted prolines. In particular, ETs through ETBR markedly decrease PHD2 mRNA and protein levels and promoter activity. In addition, activation of phosphatidylinositol 3-kinase (PI3K)-dependent integrin linked kinase (ILK)-AKTmammalian target of rapamycin (mTOR) pathway is required for ETBR-mediated PHD2 inhibition, HIF-1α HIF-2α and VEGF expression. At functional level, PHD2 knockdown does not further increase ETs-induced in vitro tube formation of endothelial cells and melanoma cell invasiveness, demonstrating that these processes are regulated in a PHD2-dependent manner. In human primary and metastatic melanoma tissues as well as in cell lines, that express high levels of HIF-1α ETBR expression is associated with low PHD2 levels. In melanoma xenografts, ETBR blockade by ETBR antagonist results in a concomitant reduction of tumor growth, angiogenesis, HIF-1α and HIF-2α expression, and an increase in PHD2 levels. Conclusions: In this study we identified the underlying mechanism by which ET-1, through the regulation of PHD2, controls HIF-1α stability and thereby regulates angiogenesis and melanoma cell invasion. These results further indicate that targeting ETBR may represent a potential therapeutic treatment of melanoma by impairing HIF-1α stability.

Original languageEnglish
Article numbere11241
JournalPLoS One
Volume5
Issue number6
DOIs
Publication statusPublished - 2010

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procollagen-proline dioxygenase
Endothelin B Receptors
Prolyl Hydroxylases
Hypoxia-Inducible Factor 1
endothelins
Endothelin-1
melanoma
Melanoma
hypoxia
Degradation
cells
receptors
Vascular Endothelial Growth Factor A
Chemical activation
Endothelin-2
Phosphatidylinositol 3-Kinase
Endothelin-3
Hydroxylation
degradation
vascular endothelial growth factors

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

Cite this

Endothelin-1 inhibits prolyl hydroxylase domain 2 to activate hypoxia-inducible factor-1α in melanoma cells. / Spinella, Francesca; Rosanó, Laura; del Duca, Martina; di Castro, Valeriana; Nicotra, Maria Rita; Natali, Pier Giorgio; Bagnato, Anna.

In: PLoS One, Vol. 5, No. 6, e11241, 2010.

Research output: Contribution to journalArticle

Spinella, Francesca ; Rosanó, Laura ; del Duca, Martina ; di Castro, Valeriana ; Nicotra, Maria Rita ; Natali, Pier Giorgio ; Bagnato, Anna. / Endothelin-1 inhibits prolyl hydroxylase domain 2 to activate hypoxia-inducible factor-1α in melanoma cells. In: PLoS One. 2010 ; Vol. 5, No. 6.
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AU - Rosanó, Laura

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AU - di Castro, Valeriana

AU - Nicotra, Maria Rita

AU - Natali, Pier Giorgio

AU - Bagnato, Anna

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N2 - Background: The endothelin B receptor (ETBR) promotes tumorigenesis and melanoma progression through activation by endothelin (ET)-1, thus representing a promising therapeutic target. The stability of hypoxia-inducible factor (HIF)-1a is essential for melanomagenesis and progression, and is controlled by site-specific hydroxylation carried out by HIF-prolyl hydroxylase domain (PHD) and subsequent proteosomal degradation. Principal Findings: Here we found that in melanoma cells ET-1, ET-2, and ET-3 through ETBR, enhance the expression and activity of HIF-1a and HIF-2a that in turn regulate the expression of vascular endothelial growth factor (VEGF) in response to ETs or hypoxia. Under normoxic conditions, ET-1 controls HIF-a stability by inhibiting its degradation, as determined by impaired degradation of a reporter gene containing the HIF-1a oxygen-dependent degradation domain encompassing the PHD-targeted prolines. In particular, ETs through ETBR markedly decrease PHD2 mRNA and protein levels and promoter activity. In addition, activation of phosphatidylinositol 3-kinase (PI3K)-dependent integrin linked kinase (ILK)-AKTmammalian target of rapamycin (mTOR) pathway is required for ETBR-mediated PHD2 inhibition, HIF-1α HIF-2α and VEGF expression. At functional level, PHD2 knockdown does not further increase ETs-induced in vitro tube formation of endothelial cells and melanoma cell invasiveness, demonstrating that these processes are regulated in a PHD2-dependent manner. In human primary and metastatic melanoma tissues as well as in cell lines, that express high levels of HIF-1α ETBR expression is associated with low PHD2 levels. In melanoma xenografts, ETBR blockade by ETBR antagonist results in a concomitant reduction of tumor growth, angiogenesis, HIF-1α and HIF-2α expression, and an increase in PHD2 levels. Conclusions: In this study we identified the underlying mechanism by which ET-1, through the regulation of PHD2, controls HIF-1α stability and thereby regulates angiogenesis and melanoma cell invasion. These results further indicate that targeting ETBR may represent a potential therapeutic treatment of melanoma by impairing HIF-1α stability.

AB - Background: The endothelin B receptor (ETBR) promotes tumorigenesis and melanoma progression through activation by endothelin (ET)-1, thus representing a promising therapeutic target. The stability of hypoxia-inducible factor (HIF)-1a is essential for melanomagenesis and progression, and is controlled by site-specific hydroxylation carried out by HIF-prolyl hydroxylase domain (PHD) and subsequent proteosomal degradation. Principal Findings: Here we found that in melanoma cells ET-1, ET-2, and ET-3 through ETBR, enhance the expression and activity of HIF-1a and HIF-2a that in turn regulate the expression of vascular endothelial growth factor (VEGF) in response to ETs or hypoxia. Under normoxic conditions, ET-1 controls HIF-a stability by inhibiting its degradation, as determined by impaired degradation of a reporter gene containing the HIF-1a oxygen-dependent degradation domain encompassing the PHD-targeted prolines. In particular, ETs through ETBR markedly decrease PHD2 mRNA and protein levels and promoter activity. In addition, activation of phosphatidylinositol 3-kinase (PI3K)-dependent integrin linked kinase (ILK)-AKTmammalian target of rapamycin (mTOR) pathway is required for ETBR-mediated PHD2 inhibition, HIF-1α HIF-2α and VEGF expression. At functional level, PHD2 knockdown does not further increase ETs-induced in vitro tube formation of endothelial cells and melanoma cell invasiveness, demonstrating that these processes are regulated in a PHD2-dependent manner. In human primary and metastatic melanoma tissues as well as in cell lines, that express high levels of HIF-1α ETBR expression is associated with low PHD2 levels. In melanoma xenografts, ETBR blockade by ETBR antagonist results in a concomitant reduction of tumor growth, angiogenesis, HIF-1α and HIF-2α expression, and an increase in PHD2 levels. Conclusions: In this study we identified the underlying mechanism by which ET-1, through the regulation of PHD2, controls HIF-1α stability and thereby regulates angiogenesis and melanoma cell invasion. These results further indicate that targeting ETBR may represent a potential therapeutic treatment of melanoma by impairing HIF-1α stability.

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