RESUMEN
NADPH oxidase (NOX)-derived reactive oxygen species (ROS) and copper (Cu), an essential micronutrient, have been implicated in vascular inflammatory diseases. We reported that in proinflammatory cytokine TNF-α-stimulated endothelial cells (ECs), cytosolic Cu chaperone antioxidant-1 (Atox1) functions as a Cu-dependent transcription factor for the NOX organizer p47phox, thereby increasing ROS-dependent inflammatory gene expression. However, the role and mechanism of Atox1 nuclear translocation in inflamed ECs remain unclear. Using enface staining and nuclear fractionation, here we show that Atox1 was localized in the nucleus in inflamed aortas from ApoE-/- mice with angiotensin II infusion on a high-fat diet, while it was found in cytosol in those from control mice. In cultured human ECs, TNF-α stimulation promoted Atox1 nuclear translocation within 15 min, which was associated with Atox1 binding to TNF-α receptor-associated factor 4 (TRAF4) in a Cu-dependent manner. TRAF4 depletion by siRNA significantly inhibited Atox1 nuclear translocation, p47phox expression, and ROS production as well as its downstream VCAM1/ICAM1 expression and monocyte adhesion to inflamed ECs, which were rescued by overexpression of nuclear targeted Atox1. Furthermore, Atox1 colocalized with TRAF4 at the nucleus in TNF-α-stimulated inflamed ECs and vessels. In summary, Cu-dependent Atox1 binding to TRAF4 plays an important role in Atox1 nuclear translocation and ROS-dependent inflammatory responses in TNF-α-stimulated ECs. Thus the Atox1-TRAF4 axis is a novel therapeutic target for vascular inflammatory disease such as atherosclerosis.
Asunto(s)
Aterosclerosis/genética , Proteínas Transportadoras de Cobre/genética , Chaperonas Moleculares/genética , NADPH Oxidasas/genética , Especies Reactivas de Oxígeno/metabolismo , Factor 4 Asociado a Receptor de TNF/genética , Angiotensina II/administración & dosificación , Animales , Aorta/metabolismo , Aorta/patología , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Aterosclerosis/etiología , Aterosclerosis/metabolismo , Aterosclerosis/patología , Cobre/metabolismo , Proteínas Transportadoras de Cobre/metabolismo , Dieta Alta en Grasa/efectos adversos , Regulación de la Expresión Génica , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Inflamación , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados para ApoE , Chaperonas Moleculares/metabolismo , NADPH Oxidasas/metabolismo , Unión Proteica , Transporte de Proteínas/efectos de los fármacos , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Factor 4 Asociado a Receptor de TNF/antagonistas & inhibidores , Factor 4 Asociado a Receptor de TNF/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Molécula 1 de Adhesión Celular Vascular/genética , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
Receptor tyrosine kinases (RTKs) are important drivers of cancers. In addition to genomic alterations, aberrant activation of WT RTKs plays an important role in driving cancer progression. However, the mechanisms underlying how RTKs drive prostate cancer remain incompletely characterized. Here we show that non-proteolytic ubiquitination of RTK regulates its kinase activity and contributes to RTK-mediated prostate cancer metastasis. TRAF4, an E3 ubiquitin ligase, is highly expressed in metastatic prostate cancer. We demonstrated here that it is a key player in regulating RTK-mediated prostate cancer metastasis. We further identified TrkA, a neurotrophin RTK, as a TRAF4-targeted ubiquitination substrate that promotes cancer cell invasion and found that inhibition of TrkA activity abolished TRAF4-dependent cell invasion. TRAF4 promoted K27- and K29-linked ubiquitination at the TrkA kinase domain and increased its kinase activity. Mutation of TRAF4-targeted ubiquitination sites abolished TrkA tyrosine autophosphorylation and its interaction with downstream proteins. TRAF4 knockdown also suppressed nerve growth factor (NGF) stimulated TrkA downstream p38 MAPK activation and invasion-associated gene expression. Furthermore, elevated TRAF4 levels significantly correlated with increased NGF-stimulated invasion-associated gene expression in prostate cancer patients, indicating that this signaling axis is significantly activated during oncogenesis. Our results revealed a posttranslational modification mechanism contributing to aberrant non-mutated RTK activation in cancer cells.
Asunto(s)
Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Receptor trkA/metabolismo , Factor 4 Asociado a Receptor de TNF/metabolismo , Animales , Línea Celular Tumoral , Movimiento Celular , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Xenoinjertos , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundario , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Invasividad Neoplásica , Trasplante de Neoplasias , Células PC-3 , Neoplasias de la Próstata/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , Receptor trkA/química , Receptor trkA/genética , Factor 4 Asociado a Receptor de TNF/antagonistas & inhibidores , Factor 4 Asociado a Receptor de TNF/genética , Ubiquitinación , Regulación hacia ArribaRESUMEN
TRAF4 is an adapter protein overexpressed in certain cancers, but its contributions to tumorigenesis are unclear. In lung cancer cells and primary lung tumors, we found that TRAF4 is overexpressed. RNA interference-mediated attenuation of TRAF4 expression blunted the malignant phenotype in this setting, exerting inhibitory effects on cell proliferation, anchorage-independent growth, and tumor development in a xenograft mouse model. Unexpectedly, we discovered that TRAF4, but not Skp2, was required for activation of the pivotal cell survival kinase Akt through ubiquitination. Furthermore, TRAF4 attenuation impaired glucose metabolism by inhibiting expression of Glut1 and HK2 mediated by the Akt pathway. Overall, our work suggests that TRAF4 offers a candidate molecular target for lung cancer prevention and therapy.