RESUMO
Neuropilin-1 (NRP-1) is involved in angiogenesis, but the role of NRP-1 in megakaryocytopoiesis is not yet fully understood. In this study, we investigated whether thrombopoietin (TPO) regulates the expression of platelet-derived growth factor (PDGF) and its receptors (PDGFRs) on TPO-dependent UT-7/TPO cells and whether PDGFRs and NRP-1 on UT-7/TPO cells form complexes during megakaryocytic differentiation. When UT-7/TPO cells were starved of TPO for 24 h and then stimulated with 5 ng/ml TPO, the expression of PDGF-B, PDGFRα, and PDGFRß were significantly up-regulated after the addition of TPO. TPO also induced tyrosine phosphorylation of PDGFRα but not PDGFRß, and promoted the formation of PDGFRαß heterodimer complexes. Furthermore, megakaryocytic differentiation of UT-7/TPO cells on treatment with phorbol myristate acetate (PMA) was accompanied by a marked up-regulation of PDGFRß and NRP-1 protein expression, complex formation between PDGFRs and NRP-1, PDGFRαß heterodimer complexes, and an increase in PDGF-BB-binding activity. Immunocytochemistry confirmed complex formation between PDGFRs and NRP-1 and PDGFRαß heterodimer complexes in PMA-differentiated UT-7/TPO cells. Our observations suggest that NRP-1 is involved in megakaryocytopoiesis through complex formation with PDGFRs, and that NRP-1-PDGFR-complexes may contribute to effective cellular functions mediated by TPO and PDGF in megakaryocytic cells.
Assuntos
Megacariócitos/citologia , Megacariócitos/metabolismo , Neuropilina-1/química , Neuropilina-1/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/química , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/química , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Trombopoese/fisiologia , Trombopoetina/metabolismo , Becaplermina , Linhagem Celular , Humanos , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-sis/química , Proteínas Proto-Oncogênicas c-sis/genética , Proteínas Proto-Oncogênicas c-sis/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Proteínas Recombinantes/metabolismo , Trombopoese/genéticaRESUMO
We investigated whether the gene expression of vascular endothelial growth factor (VEGF) and its receptors (VEGFR and neuropilin-1 [NRP-1]) could be specifically regulated during the megakaryocytic differentiation of human thrombopoietin (TPO)-dependent UT-7/TPO cells. Undifferentiated UT-7/TPO cells expressed a functional VEGFR-2, leading to VEGF binding and VEGF(165)-induced tyrosine phosphorylation, cell proliferation, and apoptosis inhibition. The megakaryocytic differentiation of UT-7/TPO cells on treatment with phorbol myristate acetate (PMA) was accompanied by a marked up-regulation of NRP-1 mRNA and protein expression and by an increase in VEGF-binding activity, which was mainly mediated by VEGFR-2. VEGF(165) promoted the formation of complexes containing NRP-1 and VEGFR-2 in undifferentiated UT-7/TPO cells in a dose-dependent manner. Unlike human umbilical vein endothelial cells, PMA-differentiated UT-7/TPO cells exhibited complex formation between NRP-1 and VEGFR-2 even in the absence of VEGF(165). These findings suggest that NRP-1-VEGFR-2-complex formation may contribute to effective cellular functions mediated by VEGF(165) in megakaryocytic cells.
Assuntos
Diferenciação Celular , Megacariócitos/citologia , Neuropilina-1/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Linhagem Celular , Humanos , Megacariócitos/metabolismoRESUMO
We investigated the gene expression profiles of vascular endothelial growth factor (VEGF) and its receptors in HL-60 leukemia cells. In the VEGF family, both mRNA and protein expression of VEGF-C were up-regulated in phorbol myristate acetate (PMA)-differentiated HL-60 cells. We detected two bands of approximately 31 and approximately 60kDa in cell lysates, and the higher expression of approximately 31kDa band was further increased after stimulation with tumor necrosis factor (TNF)-alpha and lipopolysaccharide (LPS). A approximately 31kDa VEGF-C protein was also detected in conditioned media from PMA-differentiated HL-60 cells after LPS stimulation. The mRNA expression of VEGFR-1, VEGFR-2, and neuropilin-1 (NRP-1) was markedly up-regulated in PMA-differentiated HL-60 cells, corresponding to the results from VEGF binding studies, in which VEGF binding activity was increased in PMA-differentiated HL-60 cells. These did not occur in dimethylsulfoxide (DMSO)-differentiated HL-60 cells. The expression of VEGF-C and VEGF receptors is regulated specifically in HL-60 cells during macrophage differentiation.