RESUMEN
The F-BAR domain containing protein CIP4 (Cdc42 interacting protein 4) interacts with Cdc42 and WASP/N-WASP and is thought to participate in the assembly of filamentous actin. CIP4(-/-) mice had normal T- and B-lymphocyte development but impaired T cell-dependent antibody production, IgG antibody affinity maturation, and germinal center (GC) formation, despite an intact CD40L-CD40 axis. CIP4(-/-) mice also had impaired contact hypersensitivity (CHS) to haptens, and their T cells failed to adoptively transfer CHS. Ovalbumin-activated CD4(+) effector T cells from CIP4(-/-)/OT-II mice migrated poorly to antigen-challenged skin. Activated CIP4(-/-) T cells exhibited impaired adhesion and polarization on immobilized VCAM-1 and ICAM-1 and defective arrest and transmigration across murine endothelial cell monolayers under shear flow conditions. These results demonstrate an important role for CIP4 in integrin-dependent T cell-dependent antibody responses and GC formation and in integrin-mediated recruitment of effector T cells to cutaneous sites of antigen-driven immune reactions.
Asunto(s)
Movimiento Celular , Integrinas/inmunología , Proteínas Asociadas a Microtúbulos/inmunología , Linfocitos T/citología , Linfocitos T/inmunología , Animales , Linfocitos B/inmunología , Adhesión Celular , Polaridad Celular , Dermatitis Alérgica por Contacto/genética , Dermatitis Alérgica por Contacto/inmunología , Molécula 1 de Adhesión Intercelular/inmunología , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/deficiencia , Antígenos de Histocompatibilidad Menor , Molécula 1 de Adhesión Celular Vascular/inmunologíaRESUMEN
ERG (Ets-related gene) is an ETS transcription factor that has recently been shown to regulate a number of endothelial cell (EC)-restricted genes including VE-cadherin, von Willebrand factor, endoglin, and intercellular adhesion molecule-2. Our preliminary data demonstrate that unlike other ETS factors, ERG exhibits a highly EC-restricted pattern of expression in cultured primary cells and several adult mouse tissues including the heart, lung, and brain. In response to inflammatory stimuli, such as tumor necrosis factor-alpha, we observed a marked reduction of ERG expression in ECs. To further define the role of ERG in the regulation of normal EC function, we used RNA interference to knock down ERG. Microarray analysis of RNA derived from ERG small interfering RNA- or tumor necrosis factor-alpha-treated human umbilical vein (HUV)ECs revealed significant overlap (P<0.01) in the genes that are up- or downregulated. Of particular interest to us was a significant change in expression of interleukin (IL)-8 at both protein and RNA levels. Exposure of ECs to tumor necrosis factor-alpha is known to be associated with increased neutrophil attachment. We observed that knockdown of ERG in HUVECs is similarly associated with increased neutrophil attachment compared to control small interfering RNA-treated cells. This enhanced adhesion could be blocked with IL-8 neutralizing or IL-8 receptor blocking antibodies. ERG can inhibit the activity of the IL-8 promoter in a dose dependent manner. Direct binding of ERG to the IL-8 promoter in ECs was confirmed by chromatin immunoprecipitation. In summary, our findings support a role for ERG in promoting antiinflammatory effects in ECs through repression of inflammatory genes such as IL-8.
Asunto(s)
Células Endoteliales/metabolismo , Inflamación/prevención & control , Interleucina-8/metabolismo , Regiones Promotoras Genéticas , Transactivadores/metabolismo , Transcripción Genética , Animales , Adhesión Celular , Células Cultivadas , Técnicas de Cocultivo , Modelos Animales de Enfermedad , Regulación hacia Abajo , Células Endoteliales/efectos de los fármacos , Endotoxemia/metabolismo , Humanos , Inflamación/metabolismo , Interleucina-8/genética , Lipopolisacáridos/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Neutrófilos/metabolismo , Proteínas Oncogénicas/metabolismo , Interferencia de ARN , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Factores de Tiempo , Transactivadores/genética , Factores de Transcripción , Regulador Transcripcional ERG , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
BACKGROUND: The molecular mechanisms that govern stem cell differentiation along the endothelial lineage remain largely unknown. Ets related gene (ERG) has recently been shown to participate in the transcriptional regulation of a number of endothelial specific genes including VE-cadherin (CD144), endoglin, and von Willebrand's Factor (vWF). The specific role of the ETS factor ERG during endothelial differentiation has not been evaluated. RESULTS: ERG expression and function were evaluated during the differentiation of embryonic stem cells into embryoid bodies (EB). The results of our study demonstrate that ERG is first expressed in a subpopulation of vascular endothelial growth factor receptor 2 (VEGF-R2) expressing cells that also express VE-cadherin. During ES cell differentiation, ERG expression remains restricted to cells of the endothelial lineage that eventually coalesce into primitive vascular structures within embryoid bodies. ERG also exhibits an endothelial cell (EC)-restricted pattern during embryogenesis. To further define the role of ERG during ES cell differentiation, we used a knockdown strategy to inhibit ERG expression. Delivery of three independent shRNA led to 70-85% reductions in ERG expression during ES cell differentiation compared to no change with control shRNA. ERG knockdown was associated with a marked reduction in the number of ECs, the expression of EC-restricted genes, and the formation of vascular structures. CONCLUSION: The ETS factor ERG appears to be a critical regulator of EC differentiation.