RESUMEN
Vascular endothelial growth factor A (VEGF-A) is a key molecule in angiogenesis acting through VEGF receptors (VEGFRs), ανß3 integrin, receptor protein tyrosine phosphatase beta/zeta (RPTPß/ζ) and cell surface nucleolin (NCL). Pleiotrophin (PTN) stimulates endothelial cell migration and limits the angiogenic effects of VEGF-A165 to the levels of its own effect, possibly acting as a VEGF-A165 modifier. Since PTN and VEGF-A165 share receptors and actions on endothelial cells, in the present work we studied whether and how VEGF-A165 affects PTN expression or secretion. VEGF-A165 decreased PTN mRNA and protein levels acting at the transcriptional level. Bevacizumab, a selective VEGFR2 tyrosine kinase inhibitor and down-regulation of VEGFR2 expression by siRNA did not affect this decrease, suggesting that it is VEGFR-independent. VEGF-A121 also decreased PTN mRNA and protein levels, suggesting that heparin binding of VEGF-A165 is not involved. Blockage of cell surface NCL, lack of expression or mutation of ß3 integrin and down-regulation of RPTPß/ζ abolished the inhibitory effect of VEGF-A165 on PTN expression and secretion. Down-regulation of endogenous PTN in endothelial cells enhanced VEGF-A165-induced increase in migration and tube formation on matrigel. Collectively, these data suggest that VEGF-A down-regulates PTN expression and secretion through the RPTPß/ζ-ανß3-NCL axis to enhance its own effect on cell migration and further highlight the role of RPTPß/ζ in VEGF-A actions.
Asunto(s)
Proteínas Portadoras , Movimiento Celular , Citocinas , Expresión Génica/efectos de los fármacos , Fragmentos de Péptidos/fisiología , Proteínas Tirosina Fosfatasas Clase 5 Similares a Receptores/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/fisiología , Animales , Bevacizumab/farmacología , Western Blotting , Células CHO , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Cricetulus , Citocinas/genética , Citocinas/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Ligandos , Fragmentos de Péptidos/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptor Cross-Talk/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/farmacologíaRESUMEN
BACKGROUND: Receptor protein tyrosine phosphatase beta/zeta (RPTPß/ζ) is a chondroitin sulphate (CS) transmembrane protein tyrosine phosphatase and is a receptor for pleiotrophin (PTN). RPTPß/ζ interacts with ανß3 on the cell surface and upon binding of PTN leads to c-Src dephosphorylation at Tyr530, ß3 Tyr773 phosphorylation, cell surface nucleolin (NCL) localization and stimulation of cell migration. c-Src-mediated ß3 Tyr773 phosphorylation is also observed after vascular endothelial growth factor 165 (VEGF165) stimulation of endothelial cells and is essential for VEGF receptor type 2 (VEGFR2) - ανß3 integrin association and subsequent signaling. In the present work, we studied whether RPTPß/ζ mediates angiogenic actions of VEGF. METHODS: Human umbilical vein endothelial, human glioma U87MG and stably transfected Chinese hamster ovary cells expressing different ß3 subunits were used. Protein-protein interactions were studied by a combination of immunoprecipitation/Western blot, immunofluorescence and proximity ligation assays, properly quantified as needed. RPTPß/ζ expression was down-regulated using small interference RNA technology. Migration assays were performed in 24-well microchemotaxis chambers, using uncoated polycarbonate membranes with 8 µm pores. RESULTS: RPTPß/ζ mediates VEGF165-induced c-Src-dependent ß3 Tyr773 phosphorylation, which is required for VEGFR2-ανß3 interaction and the downstream activation of phosphatidylinositol 3-kinase (PI3K) and cell surface NCL localization. RPTPß/ζ directly interacts with VEGF165, and this interaction is not affected by bevacizumab, while it is interrupted by both CS-E and PTN. Down-regulation of RPTPß/ζ by siRNA or administration of exogenous CS-E abolishes VEGF165-induced endothelial cell migration, while PTN inhibits the migratory effect of VEGF165 to the levels of its own effect. CONCLUSIONS: These data identify RPTPß/ζ as a cell membrane binding partner for VEGF that regulates angiogenic functions of endothelial cells and suggest that it warrants further validation as a potential target for development of additive or alternative anti-VEGF therapies.
Asunto(s)
Unión Proteica/genética , Proteínas Tirosina Fosfatasas Clase 5 Similares a Receptores/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Células CHO , Línea Celular , Movimiento Celular/genética , Cricetulus , Regulación hacia Abajo/genética , Glioma , Células Endoteliales de la Vena Umbilical Humana , Humanos , Integrinas/genética , Integrinas/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación/genética , Mapas de Interacción de Proteínas/genética , ARN Interferente Pequeño/genética , Proteínas Tirosina Fosfatasas Clase 5 Similares a Receptores/genética , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
In the present work, a series of conjugates of amino acids with all-trans-retinoic acid (ATRA) and shorter polyene chain analogues were rationally designed, synthesized by coupling the succinimidyl active esters of the acidic retinoids with appropriately protected amino acids or peptides followed by deprotection, and examined for their possible effect on viability of human prostate cancer LNCaP cells. In contrast to ATRA, all conjugates bearing amino acids with polar side chains showed no inhibitory effect on LNCaP cell proliferation, while conjugates with alpha-amino acids with lipophilic side chain, such as 7, or linear amino acids, such as 9, significantly decreased prostate cancer LNCaP cell number. Interestingly, while the effect of ATRA was RARalpha-dependent, the effect of its active analogues was not inhibited by a selective RARalpha antagonist. Cell cycle analysis showed no effect on cell cycle, while quantitative analysis by annexin V-propidium iodide staining revealed that neither ATRA nor its analogues affected LNCaP cell apoptosis or necrosis. These results demonstrate that compounds 7 and 9 are potentially useful agents that warrant further preclinical development for treatment of prostate cancer.
Asunto(s)
Aminoácidos/química , Antineoplásicos/química , Antineoplásicos/farmacología , Polienos/química , Neoplasias de la Próstata/patología , Tretinoina/química , Tretinoina/farmacología , Animales , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Masculino , Receptores de Ácido Retinoico/antagonistas & inhibidores , Receptor alfa de Ácido Retinoico , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Pleiotrophin (PTN) is a heparin-binding growth factor with diverse biological activities, the most studied of these being those related to the nervous system, tumor growth and angiogenesis. Although interest in the involvement of PTN in tumor growth is increasing, many questions remain unanswered, particularly concerning the receptors and the signaling pathways involved. In this review, we briefly introduce PTN, and summarize data on its involvement in tumor growth and angiogenesis, and on what is known to date concerning the receptors and pathways involved.