RESUMO
Vascular endothelial growth factor (VEGF)-A has essential roles in vasculogenesis and angiogenesis, but the downstream steps and mechanisms by which human VEGF-A acts are incompletely understood. We report here that human VEGF-A exerts much of its angiogenic activity by up-regulating the expression of TR3 (mouse homologue Nur77), an immediate-early response gene and orphan nuclear receptor transcription factor previously implicated in tumor cell, lymphocyte, and neuronal growth and apoptosis. Overexpression of TR3 in human umbilical vein endothelial cells (HUVECs) resulted in VEGF-A-independent proliferation, survival, and induction of several cell cycle genes, whereas expression of antisense TR3 abrogated the response to VEGF-A in these assays and also inhibited tube formation. Nur77 was highly expressed in several types of VEGF-A-dependent pathological angiogenesis in vivo. Also, using a novel endothelial cell-selective retroviral targeting system, overexpression of Nur77 DNA potently induced angiogenesis in the absence of exogenous VEGF-A, whereas Nur77 antisense strongly inhibited VEGF-A-induced angiogenesis. B16F1 melanoma growth and angiogenesis were greatly inhibited in Nur77-/- mice. Mechanistic studies with TR3/Nur77 mutants revealed that TR3/Nur77 exerted most of its effects on cultured HUVECs and its pro-angiogenic effects in vivo, through its transactivation and DNA binding domains (i.e., through transcriptional activity).
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica/fisiologia , Neovascularização Fisiológica/fisiologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Esteroides/metabolismo , Fatores de Transcrição/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Feminino , Sangue Fetal/citologia , Sangue Fetal/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Linfócitos/metabolismo , Camundongos , Camundongos Knockout , Camundongos Nus , Neovascularização Fisiológica/efeitos dos fármacos , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares , Estrutura Terciária de Proteína/genética , Receptores Citoplasmáticos e Nucleares/genética , Receptores de Esteroides/genética , Retroviridae , Fatores de Transcrição/genética , Transdução Genética/métodos , Fator A de Crescimento do Endotélio Vascular/administração & dosagemRESUMO
Tumor necrosis factor (TNF) alpha-converting enzyme (TACE/ADAM-17) has diverse roles in the proteolytic processing of cell surface molecules and, due to its ability to process TNFalpha, is a validated therapeutic target for anti-inflammatory therapies. Unlike a number of other ADAM proteins, which interact with integrin receptors via their disintegrin domains, there is currently no evidence for an ADAM-17-integrin association. By analyzing the adhesion of a series of cell lines with recombinant fragments of the extracellular domain of ADAM-17, we now demonstrate a functional interaction between ADAM-17 and alpha(5)beta(1) integrin in a trans orientation. Because ADAM-17-mediated adhesion was sensitive to RGD peptides and EDTA, and the integrin-binding site within ADAM-17 was narrowed down to the disintegrin/cysteine-rich region, the two molecules appear to have a ligand-receptor relationship mediated by the alpha(5)beta(1) ligand binding pocket. Intriguingly, ADAM-17 and alpha(5)beta(1) were found to co-localize in both membrane ruffles and focal adhesions in HeLa cells. When confluent HeLa cell monolayers were wounded, ADAM-17 and alpha(5)beta(1) redistributed to the leading edge and co-localized, which is suggestive of a cis orientation. We postulate that the interaction of ADAM-17 with alpha(5)beta(1) may target or modulate its metalloproteolytic activity.
