RESUMO
The thiazide-sensitive NaCl cotransporter (NCC) is the primary mediator of salt reabsorption in the distal convoluted tubule and is a key determinant of the blood pressure set point. Given its complex topology, NCC is inefficiently processed and prone to endoplasmic reticulum (ER)-associated degradation (ERAD), although the mechanisms governing this process remain obscure. Here, we identify factors that impact the ER quality control of NCC. Analyses of NCC immunoprecipitates revealed that the cotransporter formed complexes with the core chaperones Hsp90, Hsp70, and Hsp40. Disruption of Hsp90 function accelerated NCC degradation, suggesting that Hsp90 promotes NCC folding. In addition, two cochaperones, the C terminus of Hsp70-interacting protein (CHIP) and the Hsp70/Hsp90 organizer protein, were associated with NCC. Although CHIP, an E3 ubiquitin ligase, promoted NCC ubiquitination and ERAD, the Hsp70/Hsp90 organizer protein stabilized NCC turnover, indicating that these two proteins differentially remodel the core chaperone systems to favor cotransporter degradation and biogenesis, respectively. Adjusting the folding environment in mammalian cells via reduced temperature enhanced NCC biosynthetic trafficking, increased Hsp90-NCC interaction, and diminished binding to Hsp70. In contrast, cotransporters harboring disease-causing mutations that impair NCC biogenesis failed to escape ERAD as efficiently as the wild type protein when cells were incubated at a lower temperature. Instead, these mutants interacted more strongly with Hsp70, Hsp40, and CHIP, consistent with a role for the Hsp70/Hsp40 system in selecting misfolded NCC for ERAD. Collectively, these observations indicate that Hsp70 and Hsp90 comprise two functionally distinct ER quality control checkpoints that sequentially monitor NCC biogenesis.
Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Complexos Multiproteicos/metabolismo , Proteólise , Receptores de Droga/metabolismo , Simportadores/metabolismo , Animais , Linhagem Celular , Cães , Retículo Endoplasmático/genética , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP90/genética , Humanos , Camundongos , Complexos Multiproteicos/genética , Dobramento de Proteína , Receptores de Droga/genética , Membro 3 da Família 12 de Carreador de Soluto , Simportadores/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
The thiazide-sensitive NaCl cotransporter (NCC, SLC12A3) mediates salt reabsorption in the distal nephron of the kidney and is the target of thiazide diuretics, which are commonly prescribed to treat hypertension. Mutations in NCC also give rise to Gitelman syndrome, a hereditary salt-wasting disorder thought in most cases to arise from impaired NCC biogenesis through enhanced endoplasmic reticulum-associated degradation (ERAD). Because the machinery that mediates NCC quality control is completely undefined, we employed yeast as a model heterologous expression system to identify factors involved in NCC degradation. We confirmed that NCC was a bona fide ERAD substrate in yeast, as the majority of NCC polypeptide was integrated into ER membranes, and its turnover rate was sensitive to proteasome inhibition. NCC degradation was primarily dependent on the ER membrane-associated E3 ubiquitin ligase Hrd1. Whereas several ER luminal chaperones were dispensable for NCC ERAD, NCC ubiquitination and degradation required the activity of Ssa1, a cytoplasmic Hsp70 chaperone. Compatible findings were observed when NCC was expressed in mammalian kidney cells, as the cotransporter was polyubiquitinated and degraded by the proteasome, and mammalian cytoplasmic Hsp70 (Hsp72) coexpression stimulated the degradation of newly synthesized NCC. Hsp70 also preferentially associated with the ER-localized NCC glycosylated species, indicating that cytoplasmic Hsp70 plays a critical role in selecting immature forms of NCC for ERAD. Together, these results provide the first survey of components involved in the ERAD of a mammalian SLC12 cation chloride cotransporter and provide a framework for future studies on NCC ER quality control.
Assuntos
Retículo Endoplasmático/metabolismo , Cloreto de Sódio/química , Tiazidas/química , Animais , Transporte Biológico , Cátions , Linhagem Celular , Cloretos/química , Cicloeximida/farmacologia , Cães , Glicosilação , Células HEK293 , Proteínas de Choque Térmico HSP70/química , Humanos , Camundongos , Complexo de Endopeptidases do Proteassoma/química , Conformação Proteica , Inibidores da Síntese de Proteínas/farmacologia , Saccharomyces cerevisiae/metabolismo , Ubiquitina/químicaRESUMO
The liver X receptor (LXR) and constitutive androstane receptor (CAR) are two nuclear receptors postulated to have distinct functions. LXR is a sterol sensor that promotes lipogenesis, whereas CAR is a xenosensor that controls xenobiotic responses. Here, we show that LXRα and CAR are functionally related in vivo. Loss of CAR increased the expression of lipogenic LXR target genes, leading to increased hepatic triglyceride accumulation, whereas activation of CAR inhibited the expression of LXR target genes and LXR ligand-induced lipogenesis. On the other hand, a combined loss of LXR α and ß increased the basal expression of xenobiotic CAR target genes, whereas activation of LXR inhibited the expression of CAR target genes and sensitized mice to xenobiotic toxicants. The mutual suppression between LXRα and CAR was also observed in cell culture and reporter gene assays. LXRα, like CAR, exhibited constitutive activity in the absence of an exogenously added ligand by recruiting nuclear receptor coactivators. Interestingly, although CAR competed with LXRα for coactivators, the constitutive activity and recruitment of coactivators was not required for CAR to suppress the activity of LXRα. In vivo chromatin immunoprecipitation assay showed that cotreatment of a CAR agonist compromised the LXR agonist responsive recruitment of LXRα to Srebp-1c, whereas an LXR agonist inhibited the CAR agonist-responsive recruitment of CAR to Cyp2b10. In conclusion, our results have revealed dual functions of LXRα and CAR in lipogenesis and xenobiotic responses, establishing a unique role of these two receptors in integrating xenobiotic and endobiotic homeostasis.
Assuntos
Lipogênese/fisiologia , Receptores Nucleares Órfãos/metabolismo , Receptor Cross-Talk/fisiologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Células Cultivadas , Receptor Constitutivo de Androstano , Feminino , Lipogênese/efeitos dos fármacos , Lipogênese/genética , Receptores X do Fígado , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Receptores Nucleares Órfãos/deficiência , Receptores Nucleares Órfãos/fisiologia , Piridinas/farmacologia , Receptor Cross-Talk/efeitos dos fármacos , Receptores Citoplasmáticos e Nucleares/deficiência , Receptores Citoplasmáticos e Nucleares/fisiologia , Xenobióticos/metabolismo , Xenobióticos/farmacologiaRESUMO
Prostate cancer is the most commonly diagnosed and the second leading cause of cancer death in men. The androgens-androgen receptor signaling plays an important role in normal prostate development, as well as in prostatic diseases, such as benign hyperplasia and prostate cancer. Accordingly, androgen ablation has been the most effective endocrine therapy for hormone-dependent prostate cancer. Here, we report a novel nuclear receptor-mediated mechanism of androgen deprivation. Genetic or pharmacological activation of the liver X receptor (LXR) in vivo lowered androgenic activity by inducing the hydroxysteroid sulfotransferase 2A1, an enzyme essential for the metabolic deactivation of androgens. Activation of LXR also inhibited the expression of steroid sulfatase in the prostate, which may have helped to prevent the local conversion of sulfonated androgens back to active metabolites. Interestingly, LXR also induced the expression of selected testicular androgen synthesizing enzymes. At the physiological level, activation of LXR in mice inhibited androgen-dependent prostate regeneration in castrated mice. Treatment with LXR agonists inhibited androgen-dependent proliferation of prostate cancer cells in a LXR- and sulfotransferase 2A1-dependent manner. In summary, we have revealed a novel function of LXR in androgen homeostasis, an endocrine role distinct to the previously known sterol sensor function of this receptor. LXR may represent a novel therapeutic target for androgen deprivation, and may aid in the treatment and prevention of hormone-dependent prostate cancer.
Assuntos
Androgênios/biossíntese , Benzoatos/uso terapêutico , Benzilaminas/uso terapêutico , Proteínas de Ligação a DNA/agonistas , Hidroxicolesteróis/uso terapêutico , Neoplasias Hormônio-Dependentes/tratamento farmacológico , Neoplasias da Próstata/tratamento farmacológico , Receptores Citoplasmáticos e Nucleares/agonistas , Androgênios/farmacologia , Animais , Benzoatos/farmacologia , Benzilaminas/farmacologia , Células Cultivadas , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Hidroxicolesteróis/farmacologia , Receptores X do Fígado , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neoplasias Hormônio-Dependentes/metabolismo , Receptores Nucleares Órfãos , Próstata/efeitos dos fármacos , Próstata/fisiologia , Neoplasias da Próstata/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores Citoplasmáticos e Nucleares/fisiologia , Regeneração/efeitos dos fármacos , Sulfotransferases/genética , Testosterona/sangueRESUMO
UNLABELLED: Gallstone disease is a hepatobiliary disorder due to biochemical imbalances in the gallbladder bile. In this report, we show that activation of nuclear receptor liver X receptor (LXR) sensitized mice to lithogenic diet-induced gallbladder cholesterol crystallization, which was associated with dysregulation of several hepatic transporters that efflux cholesterol, phospholipids, and bile salts. The combined effect of increased biliary concentrations of cholesterol and phospholipids and decreased biliary concentrations of bile salts in LXR-activated mice led to an increased cholesterol saturation index and the formation of cholesterol crystals. Interestingly, the lithogenic effect of LXR was completely abolished in the low-density lipoprotein receptor (Ldlr) null background or when the mice were treated with Ezetimibe, a cholesterol-lowering drug that blocks intestinal dietary cholesterol absorption. These results suggest that LDLR-mediated hepatic cholesterol uptake and intestinal cholesterol absorption play an essential role in LXR-promoted lithogenesis. CONCLUSION: The current study has revealed a novel lithogenic role of LXR as well as a functional interplay between LXR and LDLR in gallbladder cholesterol crystallization and possibly cholesterol gallstone disease (CGD). We propose that LXR is a lithogenic factor and that the LXR transgenic mice may offer a convenient CGD model to develop therapeutic interventions for this disease.
Assuntos
Colesterol/metabolismo , Proteínas de Ligação a DNA/metabolismo , Vesícula Biliar/metabolismo , Cálculos Biliares/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Anticolesterolemiantes/uso terapêutico , Azetidinas/uso terapêutico , Ácidos e Sais Biliares/metabolismo , Colecistite/metabolismo , Cristalização , Dieta/efeitos adversos , Ezetimiba , Feminino , Cálculos Biliares/prevenção & controle , Perfilação da Expressão Gênica , Mucosa Intestinal/metabolismo , Metabolismo dos Lipídeos/fisiologia , Fígado/metabolismo , Receptores X do Fígado , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores Nucleares Órfãos , Fosfolipídeos/metabolismo , Receptores de LDL/metabolismoRESUMO
BACKGROUND & AIMS: Liver X receptor (LXR) is known to promote hepatic lipogenesis by activating the lipogenic transcriptional factor sterol regulatory element-binding protein (Srebp). Pregnane X receptor (PXR), a previously known "xenobiotic receptor," could mediate a Srebp-independent lipogenic pathway by activating the free fatty acid uptake transporter Cd36. The goal of this study is to investigate further the role of Cd36 in hepatic steatosis. METHODS: Wild-type, LXR transgenic, PXR transgenic, and Cd36 null mice were used to study the regulation of Cd36 and other hepatic lipogenic genes and the implication of this regulation in hepatic steatosis. Promoter sequences of Cd36 and peroxisome proliferator-activated receptor (PPAR) gamma were cloned, and their respective regulation by LXR and PXR was investigated by combinations of receptor-DNA binding and reporter gene assays. RESULTS: We showed that genetic (transgene) or pharmacologic (ligands) activation of LXR induced Cd36. Promoter analysis established Cd36 as a novel transcription target of LXRalpha. Moreover, the hepatic steatosis induced by LXR agonists was largely abolished in Cd36 null mice. We also showed that PPARgamma, a positive regulator of Cd36, is a transcriptional target of PXR, suggesting that PXR can regulate Cd36 directly or through its activation of PPARgamma. Interestingly, both LXR-mediated Cd36 regulation and PXR-mediated PPARgamma regulation are liver specific. CONCLUSIONS: We conclude that Cd36 is a shared target of LXR, PXR, and PPARgamma. The network of CD36 regulation by LXR, PXR, and PPARgamma establishes this free fatty acid transporter as a common target of orphan nuclear receptors in their mediation of lipid homeostasis.
Assuntos
Antígenos CD36/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fígado Gorduroso/metabolismo , PPAR gama/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Esteroides/metabolismo , Animais , Sequência de Bases , Antígenos CD36/genética , Proteínas de Ligação a DNA/genética , Fígado Gorduroso/patologia , Fígado Gorduroso/fisiopatologia , Feminino , Regulação da Expressão Gênica/fisiologia , Homeostase/fisiologia , Metabolismo dos Lipídeos/fisiologia , Fígado/metabolismo , Fígado/patologia , Receptores X do Fígado , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dados de Sequência Molecular , Receptores Nucleares Órfãos , PPAR gama/genética , Receptor de Pregnano X , Regiões Promotoras Genéticas/fisiologia , Receptores Citoplasmáticos e Nucleares/genética , Receptores de Esteroides/genéticaRESUMO
The retinoid-related orphan receptors (RORs) and liver X receptors (LXRs) were postulated to have distinct functions. RORs play a role in tissue development and circadian rhythm, whereas LXRs are sterol sensors that affect lipid homeostasis. In this study, we revealed a novel function of RORalpha (NR1F1) in regulating the oxysterol 7alpha-hydroxylase (Cyp7b1), an enzyme critical for the homeostasis of cholesterol, bile acids, and oxysterols. The expression of Cyp7b1 gene was suppressed in the RORalpha null (RORalpha(sg/sg)) mice, suggesting RORalpha as a positive regulator of Cyp7b1. Promoter analysis established Cyp7b1 as a transcriptional target of RORalpha, and transfection of RORalpha induced the expression of endogenous Cyp7b1 in the liver. Interestingly, Cyp7b1 regulation seemed to be RORalpha-specific, because RORgamma had little effect. Reporter gene analysis showed that the activation of Cyp7b1 gene promoter by RORalpha was suppressed by LXRalpha (NR1H3), whereas RORalpha inhibited both the constitutive and ligand-dependent activities of LXRalpha. The mutual suppression between RORalpha and LXR was supported by the in vivo observation that loss of RORalpha increased the expression of selected LXR target genes, leading to hepatic triglyceride accumulation. Likewise, mice deficient of LXR alpha and beta isoforms showed activation of selected RORalpha target genes. Our results have revealed a novel role for RORalpha and a functional interplay between RORalpha and LXR in regulating endo- and xenobiotic genes, which may have broad implications in metabolic homeostasis.