RESUMEN
White adipose tissue (WAT) is not only the main tissue for energy storage but also an endocrine organ that secretes adipokines. Obesity is the most common metabolic disorder and is related to alterations in WAT characteristics, such as chronic inflammation and increasing oxidative stress. WW domain containing E3 ubiquitin protein ligase 1 (WWP1) is a HECT-type ubiquitin E3 ligase that has been implicated in various pathologies. In the present study, we found that WWP1 was upregulated in obese WAT in a p53-dependent manner. To investigate the functions of WWP1 in adipocytes, a proteome analysis of WWP1 overexpression (OE) and knockdown (KD) 3T3-L1 cells was performed. This analysis showed a positive correlation between WWP1 expression and the abundance of several antioxidative proteins. Thus, we measured reactive oxygen species (ROS) in WWP1 OE and KD cells. Consistent with the proteome results, WWP1 OE reduced ROS levels, whereas KD increased them. These findings indicate that WWP1 is an obesity-inducible E3 ubiquitin ligase that can protect against obesity-associated oxidative stress in WAT.
Asunto(s)
Adipocitos/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Células 3T3-L1 , Tejido Adiposo Blanco/metabolismo , Animales , Dieta Alta en Grasa/efectos adversos , Modelos Animales de Enfermedad , Técnicas de Silenciamiento del Gen , Genes p53 , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Obesidad/etiología , Obesidad/genética , Obesidad/metabolismo , Estrés Oxidativo , Proteoma/genética , Proteoma/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Ubiquitina-Proteína Ligasas/genética , Regulación hacia ArribaRESUMEN
The p53 protein is known as a guardian of the genome and is involved in energy metabolism. Since the metabolic system is uniquely regulated in each tissue, we have anticipated that p53 also would play differential roles in each tissue. In this study, we focused on the functions of p53 in white adipose tissue (adipocytes) and skeletal muscle (myotubes), which are important peripheral tissues involved in energy metabolism. We found that in 3T3-L1 preadipocytes, but not in C2C12 myoblasts, p53 stabilization or overexpression downregulates the expression of Ppargc1a, a master regulator of mitochondrial biogenesis. Next, by using p53-knockdown C2C12 myotubes or 3T3-L1 preadipocytes, we further examined the relationship between p53 and mitochondrial regulation. In C2C12 myoblasts, p53 knockdown did not significantly affect Ppargc1a expression and mtDNA, but did suppress differentiation to myotubes, as previously reported. However, in 3T3-L1 preadipocytes and mouse embryonic fibroblasts, p53 downregulation enhanced both differentiation into adipocytes and mitochondrial DNA content. Furthermore, p53-depleted 3T3-L1 cells showed increase in mitochondrial proteins and enhancement of both Citrate Synthase and Complex IV activities during adipogenesis. These results show that p53 differentially regulates cell differentiation and mitochondrial biogenesis between adipocytes and myotubes, and provide evidence that p53 is an inhibitory factor of mitochondrial regulation in adipocyte lineage.
Asunto(s)
Adipogénesis/fisiología , Mitocondrias/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Células 3T3-L1 , Adipocitos/citología , Adipocitos/metabolismo , Adipogénesis/genética , Animales , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Línea Celular , Linaje de la Célula/genética , Linaje de la Célula/fisiología , Células Cultivadas , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Expresión Génica , Técnicas de Silenciamiento del Gen , Genes p53 , Ratones , Ratones Noqueados , Mitocondrias/genética , Mioblastos Esqueléticos/citología , Mioblastos Esqueléticos/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Factores de Transcripción/genética , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/genéticaRESUMEN
PARP1 is an important enzyme involved in various patho-physiological phenomena such as ischemia/reperfusion (I/R) injury, which occurs when blood flow is restored after cerebral infarction, myocardial infarction and transplantation of various organs. I/R-induced PARP1 over-activation is mediated by production of reactive oxygen species and is involved in NF-κB transactivation. For these reasons, PARP1 is an attractive target for strategies to protect against I/R injury. We previously reported that an MDM2 inhibitor Nutlin3a, a cis-imidazoline compound, induces PARP1 degradation in a p53 and proteasome-dependent manner. In this study, we evaluated the effect of Nutlin3a analogs, Nutlin3b and Caylin2, on PARP1 degradation. Like Nutlin3a, Caylin2, but not Nutlin3b, induced PARP1 degradation in both 3T3-L1 and 3T3-F442A. This result occurred almost in parallel with p53 accumulation. Furthermore Caylin2-induced PARP1 degradation was not observed in p53 deficient mouse embryonic fibroblasts or in the presence of the proteasome inhibitor MG132. These results suggest that Caylin2 induces PARP1 degradation by the same mechanism as Nutlin3a. Finally, we showed that Nutlin3a or Caylin2 treatment induces reversible PARP1 down-regulation without an inflammatory response. For protection against I/R injury, our results support the usability of the p53 inducible cis-imidazoline compounds, Nutlin3a and its analogs, as PARP1 inhibitors.