RESUMO
Glucagon-like peptide-1 (GLP-1) induces several immediate early response genes such as c-fos, c-jun, and early growth response-1 (Egr-1), which are involved in cell proliferation and differentiation. We recently reported that exendin-4 (EX-4), a potent GLP-1 agonist, upregulated Egr-1 expression via phosphorylation of CREB, a transcription factor in INS-1 beta-cells. This study was designed to investigate the role of another transcription factors, serum response factor (SRF) and Yin Yang-1 (YY1), in EX-4-induced Egr-1 expression. EX-4 significantly increased Egr-1 mRNA and subsequently its protein level. EX-4-induced Egr-1 expression was inhibited by pretreatment with a PKA inhibitor, H-89, and an MEK inhibitor, PD 98059. The siRNA-mediated inhibition of PKA and ERK1 resulted in significant reduction of EX-4-induced Egr-1 expression. Promoter analyses showed that SRE clusters were essential for Egr-1 transcription, and YY1 overexpression did not affect Egr-1 promoter activity. EMSA results demonstrated that EX-4-induced transient increase in DNA-protein complex on SRE site, and that both SRF and phospho-SRF were bound to this site. Treatment of either YY1 consensus oligonucleotide or YY1 antibody did not effect the change of density or migration of the DNA-protein complex. Collectively, EX-4-induced Egr-1 expression is largely dependent on cAMP-mediated extracellular signal-regulated kinase activation, and EX-4 induces Egr-1 transcription via the interaction of SRF and phospho-SRF to SRE sites.
Assuntos
Proteína 1 de Resposta de Crescimento Precoce/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Peptídeos/farmacologia , Elementos de Resposta/genética , Fator de Resposta Sérica/metabolismo , Peçonhas/farmacologia , Fator de Transcrição YY1/metabolismo , Animais , Linhagem Celular , AMP Cíclico/metabolismo , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Exenatida , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/enzimologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Transcrição Gênica/efeitos dos fármacosRESUMO
Trichostatin A and helixor A increased thrombospondin-1 expression by ECV304 cells at both mRNA and protein levels by transcriptional activation through the enhancement of tsp-1 promoter activity. The induction of thrombospondin-1 by these agents potently reduced ECV 304 cell migration and capillary-like tube formation on Matrigel; these findings were confirmed by the neutralization of thrombospondin-1 using a specific antibody. In the presence of exogenous vascular endothelial growth factor, however, these agents had a different effect on the vascular endothelial growth factor-induced tube formation; trichostatin A remarkably inhibited tube formation regardless of the presence of exogenous vascular endothelial growth factor, whereas helixor A reduced it to 70-80% of the control level. Interestingly, when the helixor A-generated conditioned media were concentrated three-fold and the endogenous vascular endothelial growth factor was removed, tube formation was remarkably inhibited compared with the effect of three-fold concentrated conditioned media that had endogenous vascular endothelial growth factor. Additionally, in media with endogenous vascular endothelial growth factor that were concentrated five-fold, tube formation was markedly blocked regardless of the presence of exogenous or endogenous vascular endothelial growth factor. Thus, our results indicate that trichostatin A-induced or helixor A-induced antiangiogenesis is mediated by both agents; increased, absolute and relative levels of thrombospondin-1 to the vascular endothelial growth factor level are critical in angiogenesis.
Assuntos
Inibidores da Angiogênese/farmacologia , Células Endoteliais/efeitos dos fármacos , Ácidos Hidroxâmicos/farmacologia , Extratos Vegetais/farmacologia , Trombospondina 1/biossíntese , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Meios de Cultivo Condicionados , Relação Dose-Resposta a Droga , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Humanos , Neovascularização Patológica/metabolismo , Neovascularização Patológica/prevenção & controle , Trombospondina 1/genética , Regulação para Cima , Fatores de Crescimento do Endotélio Vascular/farmacologiaRESUMO
Thrombospondin-1 (TSP-1) level is tightly regulated at the transcriptional level. To determine the detailed molecular mechanisms of TSP-1 expression, nine serial 5'-deletion constructs of the human genomic tsp-1 promoter (nucleotides -2,220 to +756) were prepared, inserted into luciferase reporter plasmids, and transiently transfected into the Hep3B human hepatocarcinoma cell. Among the nine 5'-deletion constructs, pTSP-Luc-4 (-767 approximately +756) had consistently decreased luciferase activity with or without PMA stimulation, whereas a further truncated construct [pTSP-Luc-4' (-407 approximately +756)] had increased levels of expression. By searching the nucleotides from -767 to -407, a consensus binding sequence (5'-CCATTTT-3') for the repressor Yin Yang-1 (YY-1) at nucleotide -440 was identified. The suppression induced by this site was weakened in the presence of the region upstream of nucleotide -767 (pTSP-Luc-1 and -2). Nuclear protein directly bound to an oligonucleotide containing the repressive YY-1 sequence but the binding capacity of the sequence was decreased by the increased c-Jun levels. Moreover, proteins immunoprecipitated with anti-YY-1 revealed an interaction between c-Jun and YY-1 factor. These data suggest that the repressive YY-1 site of the tsp-1 promoter could not be functional via activating positive cis-elements on the upstream from this site and weakened via c-Jun/YY-1 interactions.