RESUMO
The metalloprotease ADAMTS9 participates in melanoblast development and is a tumor suppressor in esophageal and nasopharyngeal cancer. ADAMTS9 null mice die before gastrulation, but, ADAMTS9+/- mice were initially thought to be normal. However, when congenic with the C57Bl/6 strain, 80% of ADAMTS9+/- mice developed spontaneous corneal neovascularization. beta-Galactosidase staining enabled by a lacZ cassette targeted to the ADAMTS9 locus showed that capillary endothelial cells (ECs) in embryonic and adult tissues and in capillaries growing into heterotopic tumors expressed ADAMTS9. Heterotopic B.16-F10 melanomas elicited greater vascular induction in ADAMTS9+/- mice than in wild-type littermates, suggesting a potential inhibitory role in tumor angiogenesis. Treatment of cultured human microvascular ECs with ADAMTS9 small-interfering RNA resulted in enhanced filopodial extension, decreased cell adhesion, increased cell migration, and enhanced formation of tube-like structures on Matrigel. Conversely, overexpression of catalytically active, but not inactive, ADAMTS9 in ECs led to fewer tube-like structures, demonstrating that the proteolytic activity of ADAMTS9 was essential. However, unlike the related metalloprotease ADAMTS1, which exerts anti-angiogenic effects by cleavage of thrombospondins and sequestration of vascular endothelial growth factor165, ADAMTS9 neither cleaved thrombospondins 1 and 2, nor bound vascular endothelial growth factor165. Taken together, these data identify ADAMTS9 as a novel, constitutive, endogenous angiogenesis inhibitor that operates cell-autonomously in ECs via molecular mechanisms that are distinct from those used by ADAMTS1.
Assuntos
Proteínas ADAM/metabolismo , Células Endoteliais/enzimologia , Células Endoteliais/patologia , Microvasos/enzimologia , Microvasos/patologia , Neovascularização Patológica/enzimologia , Proteínas ADAM/genética , Proteína ADAMTS9 , Envelhecimento/metabolismo , Animais , Biocatálise , Movimento Celular , Neovascularização da Córnea/enzimologia , Neovascularização da Córnea/patologia , Embrião de Mamíferos/enzimologia , Embrião de Mamíferos/patologia , Ativação Enzimática , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , Neoplasias/irrigação sanguínea , Neoplasias/enzimologia , Neoplasias/patologia , Neovascularização Patológica/patologia , Especificidade de Órgãos , Fosforilação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Trombospondina 1/metabolismo , Trombospondinas/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
The expression of Cyp19, the key gene of estrogen biosynthesis, in granulosa cells (GC) is essential for follicular growth and coordination of the ovulatory process. The goal of this study was to examine the effect of PGE2 and PGF2alpha on Cyp19 expression in undifferentiated and luteinized GC (UGC and LGC). In UGC, PGE2 increased Cyp19 mRNA and Cyp19 protein levels whereas PGF2alpha had no effect. In LGC, PGF2alpha decreased Cyp19 expression whereas PGE2 had no effect. Gene-reporter experiments demonstrated that PGE2 increases Cyp19 transcription in UGC. A protein kinase A inhibitor blocked PGE2-induced increase in Cyp19 promoter activity. PGE2 increased GATA-4 binding to the Cyp19 promoter. Mutation of the GATA binding site resulted in the loss of PGE2 stimulation. This study demonstrates that PGE2 stimulates Cyp19 expression in rat GC and suggests that GATA-4 may mediate (at least in part) the stimulatory effect of PGE2.