RESUMEN
We recently cloned a novel human nuclear factor (designated THAP1) from postcapillary venule endothelial cells (ECs) that contains a DNA-binding THAP domain, shared with zebrafish E2F6 and several Caenorhabditis elegans proteins interacting genetically with retinoblastoma gene product (pRB). Here, we show that THAP1 is a physiologic regulator of EC proliferation and cell-cycle progression, 2 essential processes for angiogenesis. Retroviral-mediated gene transfer of THAP1 into primary human ECs inhibited proliferation, and large-scale expression profiling with microarrays revealed that THAP1-mediated growth inhibition is due to coordinated repression of pRB/E2F cell-cycle target genes. Silencing of endogenous THAP1 through RNA interference similarly inhibited EC proliferation and G1/S cell-cycle progression, and resulted in down-regulation of several pRB/E2F cell-cycle target genes, including RRM1, a gene required for S-phase DNA synthesis. Chromatin immunoprecipitation assays in proliferating ECs showed that endogenous THAP1 associates in vivo with a consensus THAP1-binding site found in the RRM1 promoter, indicating that RRM1 is a direct transcriptional target of THAP1. The similar phenotypes observed after THAP1 overexpression and silencing suggest that an optimal range of THAP1 expression is essential for EC proliferation. Together, these data provide the first links in mammals among THAP proteins, cell proliferation, and pRB/E2F cell-cycle pathways.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción E2F/metabolismo , Células Endoteliales/metabolismo , Regulación de la Expresión Génica/fisiología , Genes cdc/fisiología , Proteínas Nucleares/metabolismo , Proteína de Retinoblastoma/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/farmacología , Secuencia de Bases , Ciclo Celular/efectos de los fármacos , Ciclo Celular/fisiología , Línea Celular , Proliferación Celular/efectos de los fármacos , ADN/biosíntesis , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/farmacología , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Factores de Transcripción E2F/antagonistas & inhibidores , Factores de Transcripción E2F/genética , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Fase G1/fisiología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/genética , Genes cdc/efectos de los fármacos , Humanos , Ratones , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/farmacología , Regiones Promotoras Genéticas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteína de Retinoblastoma/antagonistas & inhibidores , Proteína de Retinoblastoma/genética , Ribonucleósido Difosfato Reductasa , Fase S/efectos de los fármacos , Fase S/fisiología , Proteínas Supresoras de Tumor/genéticaRESUMEN
OBJECTIVE: To identify and characterize which endothelial heparan sulfate proteoglycans (HSPGs) bind the chemokine CXCL8 (interleukin-8) in human rheumatoid arthritis (RA) and nonrheumatoid synovia. METHOD: CXCL8 binding to endothelial HSPGs in RA and nonrheumatoid synovia was determined by heparinase treatment followed by an in situ binding assay and autoradiography. Endothelial HSPGs were characterized by immunohistochemical analysis and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Phosphatidyinositol-specific phospholipase C (PI-PLC) and antibodies to HSPGs were used in in situ binding experiments to identify which HSPGs bound CXCL8. RESULTS: The expression of heparan sulfate on microvascular endothelial cells was demonstrated in RA and nonrheumatoid synovia. Using antibodies to syndecan-1-4 and glypican-1, -3, and -4, the selective expression of syndecan-3 by endothelial cells was detected in RA and nonrheumatoid synovia. In addition, RT-PCR showed the presence of syndecan-3 messenger RNA in endothelial cells extracted from RA and nonrheumatoid synovia. (125)I-CXCL8 bound to venular endothelial cells; treatment with heparinases I and III significantly reduced this binding in RA but not nonrheumatoid synovia. (125)I-CXCL8 binding was not reduced after treatment with PI-PLC, which cleaves glycosyl phosphatidylinositol linkages, suggesting that CXCL8 did not bind to glypicans. Treatment of synovia with a syndecan-3 antibody reduced CXCL8 binding to RA but not nonrheumatoid endothelial cells; however, no reduction in binding was observed with syndecan-2 or glypican-4 antibodies. CONCLUSION: Our results show the selective induction of a CXCL8 binding site on endothelial syndecan-3 in RA synovium. This site may be involved in leukocyte trafficking into RA synovial tissue.