RESUMEN
OBJECTIVE: The sterol-responsive nuclear receptors, liver X receptors α (LXRα, NR1H3) and ß (LXRß, NR1H2), are key determinants of cellular cholesterol homeostasis. LXRs are activated under conditions of high cellular sterol load and induce expression of the cholesterol efflux transporters ABCA1 and ABCG1 to promote efflux of excess cellular cholesterol. However, the full set of genes that contribute to LXR-stimulated cholesterol efflux is unknown, and their identification is the objective of this study. APPROACH AND RESULTS: We systematically compared the global transcriptional response of macrophages to distinct classes of LXR ligands. This allowed us to identify both common and ligand-specific transcriptional responses in macrophages. Among these, we identified endonuclease-exonuclease-phosphatase family domain containing 1 (EEPD1/KIAA1706) as a direct transcriptional target of LXRs in human and murine macrophages. EEPD1 specifically localizes to the plasma membrane owing to the presence of a myristoylation site in its N terminus. Accordingly, the first 10 amino acids of EEPD1 are sufficient to confer plasma membrane localization in the context of a chimeric protein with GFP. Functionally, we report that silencing expression of EEPD1 blunts maximal LXR-stimulated Apo AI-dependent efflux and demonstrate that this is the result of reduced abundance of ABCA1 protein in human and murine macrophages. CONCLUSIONS: In this study, we identify EEPD1 as a novel LXR-regulated gene in macrophages and propose that it promotes cellular cholesterol efflux by controlling cellular levels and activity of ABCA1.
Asunto(s)
Transportador 1 de Casete de Unión a ATP/metabolismo , Membrana Celular/enzimología , Colesterol/metabolismo , Endodesoxirribonucleasas/metabolismo , Receptores X del Hígado/metabolismo , Macrófagos/enzimología , Transportador 1 de Casete de Unión a ATP/genética , Animales , Apolipoproteína A-I/metabolismo , Transporte Biológico , Células COS , Membrana Celular/efectos de los fármacos , Chlorocebus aethiops , Endodesoxirribonucleasas/genética , Perfilación de la Expresión Génica/métodos , Regulación Enzimológica de la Expresión Génica , Células HeLa , Células Hep G2 , Humanos , Ligandos , Receptores X del Hígado/agonistas , Receptores X del Hígado/deficiencia , Receptores X del Hígado/genética , Macrófagos/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células RAW 264.7 , Interferencia de ARN , Transcriptoma , TransfecciónRESUMEN
Cholesterol metabolism is subject to complex transcriptional and nontranscriptional regulation. Herein, the role of ubiquitylation is emerging as an important post-translational modification that regulates cholesterol synthesis and uptake. Similar to other post-translational modifications, ubiquitylation is reversible in a process dependent on activity of deubiquitylating enzymes (DUBs). Yet whether these play a role in cholesterol metabolism is largely unknown. As a first step to test this possibility, we used pharmacological inhibition of cellular DUB activity. Short term (2 h) inhibition of DUBs resulted in accumulation of high molecular weight ubiquitylated proteins. This was accompanied by a dramatic decrease in abundance of the LDLR and attenuated LDL uptake into hepatic cells. Importantly, this occurred in the absence of changes in the mRNA levels of the LDLR or other SREBP2-regulated genes, in line with this phenotype being a post-transcriptional event. Mechanistically, we identify transcriptional induction of the E3 ubiquitin ligase IDOL in human and rodent cells as the underlying cause for ubiquitylation-dependent lysosomal degradation of the LDLR following DUB inhibition. In contrast to the established transcriptional regulation of IDOL by the sterol-responsive liver X receptor (LXR) transcription factors, induction of IDOL by DUB inhibition is LXR-independent and occurs in Lxrαß(-/-) MEFs. Consistent with the role of DUBs in transcriptional regulation, we identified a 70-bp region in the proximal promoter of IDOL, distinct from that containing the LXR-responsive element, which mediates the response to DUB inhibition. In conclusion, we identify a sterol-independent mechanism to regulate IDOL expression and IDOL-mediated lipoprotein receptor degradation.
Asunto(s)
Lipoproteínas LDL/metabolismo , Regiones Promotoras Genéticas , Receptores de LDL/metabolismo , Transcripción Genética , Ubiquitina-Proteína Ligasas/metabolismo , Proteasas Ubiquitina-Específicas/metabolismo , Absorción Fisiológica/efectos de los fármacos , Sustitución de Aminoácidos , Animales , Línea Celular , Células Cultivadas , Inhibidores Enzimáticos/farmacología , Genes Reporteros , Células Endoteliales de la Vena Umbilical Humana/citología , Humanos , Receptores X del Hígado , Lisosomas/efectos de los fármacos , Lisosomas/enzimología , Lisosomas/metabolismo , Ratones , Mutación , Receptores Nucleares Huérfanos/genética , Receptores Nucleares Huérfanos/metabolismo , Regiones Promotoras Genéticas/efectos de los fármacos , Proteolisis/efectos de los fármacos , Receptores de LDL/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Transcripción Genética/efectos de los fármacos , Ubiquitina-Proteína Ligasas/química , Proteasas Ubiquitina-Específicas/antagonistas & inhibidores , Proteasas Ubiquitina-Específicas/genética , Ubiquitinación/efectos de los fármacosRESUMEN
Besides their well-known role in initiating adaptive immune responses, several groups have studied the role of dendritic cells (DCs) in the context of chronic metabolic inflammation, such as in diet-induced obesity (DIO) or metabolic-associated fatty liver disease. DCs also have an important function in maintaining metabolic tissue homeostasis in steady-state conditions. In this review, we will briefly describe the different DC subsets, the murine models available to assess their function, and discuss the role of DCs in regulating energy balance and maintaining tissue homeostasis.