RESUMEN
A disintegrin and metalloproteinase (ADAM) family are crucial enzymes for ectodomain shedding of multiple substrates and are involved in diverse biologic and pathologic processes. However, the molecular mechanism underlying substrate selectivity of ADAMs is poorly understood. In this study, we observed that disruption of actin polymerization by pharmacological inhibitors, latrunculin A (LatA) and cytochalasin D (CyD), induced ectodomain shedding of epidermal growth factor (EGF) family ligands. Induced shedding activity by LatA or CyD was suppressed by a metalloprotease inhibitor KB-R7785, indicating that ADAMs-mediated shedding is tightly controlled by actin cytoskeleton. We also investigated roles of cullin family, a component of cullin-RING based E3 ubiquitin ligases, in ectodomain shedding, since cullin family is implicated in the regulation of cytoskeletal dynamics. Knockdown of cullin 3 (Cul3) by a specific siRNA inhibited ectodomain shedding of amphiregulin (AREG), a member of EGF family, and responses were associated with activation of RhoA GTPase and induction of stress fiber formation. On the other hand, the RhoA inhibitor C3 transferase rescued AREG shedding reduced by Cul3 knockdown. These results describe a novel molecular mechanism of Cul3 to regulate AREG shedding by modulating cytoskeletal dynamics in a RhoA dependent manner.
Asunto(s)
Proteína ADAM17/genética , Citoesqueleto de Actina/metabolismo , Anfirregulina/genética , Proteínas Cullin/genética , Fibroblastos/metabolismo , Proteína ADAM17/antagonistas & inhibidores , Proteína ADAM17/metabolismo , ADP Ribosa Transferasas/farmacología , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/ultraestructura , Anfirregulina/metabolismo , Animales , Toxinas Botulínicas/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/antagonistas & inhibidores , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Línea Celular Tumoral , Proteínas Cullin/antagonistas & inhibidores , Proteínas Cullin/metabolismo , Citocalasina D/antagonistas & inhibidores , Citocalasina D/farmacología , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Regulación de la Expresión Génica , Glicina/análogos & derivados , Glicina/farmacología , Humanos , Ácidos Hidroxámicos/farmacología , Isoenzimas/antagonistas & inhibidores , Isoenzimas/genética , Isoenzimas/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Acetato de Tetradecanoilforbol/análogos & derivados , Acetato de Tetradecanoilforbol/farmacología , Tiazolidinas/antagonistas & inhibidores , Tiazolidinas/farmacología , Proteína de Unión al GTP rhoA/antagonistas & inhibidores , Proteína de Unión al GTP rhoA/genética , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Vascular endothelial (VE)-cadherin, a major endothelial adhesion molecule, regulates vascular permeability, and increased vascular permeability has been observed in several cancers. The aim of this study was to elucidate the role of the NEDD8-Cullin E3 ligase, in maintaining barrier permeability. To this end, we investigated the effects of the inhibition of Cullin E3 ligases, by using inhibitors and knockdown techniques in HUVECs. Furthermore, we analyzed the mRNA and protein levels of the ligases by quantitative RT-PCR and Western blotting, respectively. The results revealed that NEDD8-conjugated Cullin 3 is required for VE-cadherin-mediated endothelial barrier functions. Treatment of HUVECs with MLN4924, a chemical inhibitor of the NEDD8-activating enzyme, led to high vascular permeability due to impaired cell-cell contact. Similar results were obtained when HUVECs were treated with siRNA directed against Cullin 3, one of the target substrates of NEDD8. Immunocytochemical staining showed that both treatments equally depleted VE-cadherin protein localized at the cell-cell borders. However, quantitative RT-PCR showed that there was no significant difference in the VE-cadherin mRNA levels between the treatment and control groups. In addition, cycloheximide chase assay revealed that the half-life of VE-cadherin protein was dramatically reduced by Cullin 3 depletion. Together, these findings suggest that neddylated Cullin 3 plays a crucial role in endothelial cell barrier function by regulating VE-cadherin.
Asunto(s)
Antígenos CD/fisiología , Cadherinas/fisiología , Permeabilidad Capilar/fisiología , Proteínas Cullin/fisiología , Endotelio Vascular/fisiología , Ubiquitinas/fisiología , Antígenos CD/efectos de los fármacos , Antígenos CD/genética , Cadherinas/efectos de los fármacos , Cadherinas/genética , Permeabilidad Capilar/efectos de los fármacos , Comunicación Celular/efectos de los fármacos , Proteínas Cullin/análisis , Proteínas Cullin/antagonistas & inhibidores , Cicloheximida/farmacología , Ciclopentanos/farmacología , Endotelio Vascular/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana , Humanos , Proteína NEDD8 , Inhibidores de la Síntesis de la Proteína , Pirimidinas/farmacología , ARN Mensajero/metabolismo , ARN Interferente Pequeño/farmacología , Ubiquitinas/análisisRESUMEN
Vascular endothelial cell growth factor receptor 2 (VEGFR2) is an essential receptor for the homeostasis of endothelial cells. In this study, we showed that NEDD8-conjugated Cullin3 (CUL3)-based ubiquitin E3 (UbE3) ligase plays a crucial role in VEGFR2 mRNA expression. Human umbilical vein endothelial cells treated with MLN4924, an inhibitor of NEDD8-activating enzyme, or with CUL3 siRNA drastically lost their response to VEGF due to the intense decrease in VEGFR2 expression. Moreover, speckle-type POZ protein (SPOP) and death-domain associated protein (DAXX) were involved in the CUL3 UbE3 ligase complex as a substrate adaptor and a substrate, respectively. Knockdown of SPOP and CUL3 led to the upregulation of DAXX protein and downregulation of VEGFR2 levels. These levels were inversely correlated with one another. In addition, simultaneous knockdown of SPOP and DAXX completely reversed the downregulation of VEGFR2 levels. Moreover, the CUL3-SPOP-DAXX axis had the same effects on NOTCH1, DLL4 and NRP1 expression. Taken together, these findings suggest that the CUL3-SPOP-DAXX axis plays a very important role in endothelial cell function by targeting key angiogenic regulators.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Cullin/metabolismo , Células Endoteliales/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Represoras/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Co-Represoras , Proteínas Cullin/genética , Ciclopentanos/farmacología , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Células Endoteliales de la Vena Umbilical Humana , Humanos , Chaperonas Moleculares , Proteínas Nucleares/genética , Pirimidinas/farmacología , Proteínas Represoras/genética , Transducción de Señal/efectos de los fármacos , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
This corrects the article DOI: 10.1038/srep42845.