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1.
Eur J Nutr ; 61(4): 2201-2215, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35092460

RESUMO

PURPOSE: Excessive fat mass accumulation in obesity leads to diverse metabolic disorders, increased risks of cardiovascular diseases and in some cases, mortality. The aim of this study was to screen the actions of botanical extracts intended for oral use on human adipose tissue, using an in vitro screening model combining human intestinal cells with human adipose cells. This was to find the most effective extracts on lipid accumulation, UCP1 expression and ATP production in pre-adipocytes and on adipocyte lipolysis. METHODS: In this study, 25 individual plant extracts were screened for their effects on human adipose cells. Consequently, an original in vitro model was set up using the Caco-2 cell line, to mimic the intestinal passage of the extracts and then exposing human adipose cells to them. The biological actions of extracts were thus characterized, and compared with a coffee extract standard. The most effective extracts, and their combinations, were retained for their actions on lipid accumulation, the expression of the thermogenic effector UCP1 and ATP production in pre-adipocytes as well as on lipolysis activity of mature adipocytes. RESULTS: The biphasic culture system combining human Caco-2 cells with human adipose cells was verified as functional using the green coffee extract standard. Out of the 25 plant extracts studied, only 7 and their combinations were retained due to their potent effects on adipose cells biology. The data showed that compared to the coffee extract standard, Immortelle, Catechu, Carrot and Rose hip extracts were the most effective in reducing lipid accumulation and increased UCP1 expression in human pre-adipocytes. CONCLUSION: This study reveals the potential inhibitory effects on lipid accumulation and thermogenic activity of Immortelle, Catechu, Carrot and Rose hip extracts, and for the first time synergies in their combinations, using an in vitro model mimicking as closely as possible, human intestinal passage linked to adipose cells. These findings need to be confirmed by in vivo trials.


Assuntos
Café , Lipólise , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Adipócitos , Tecido Adiposo/metabolismo , Tecido Adiposo Marrom , Células CACO-2 , Café/metabolismo , Humanos , Lipídeos , Extratos Vegetais/metabolismo , Extratos Vegetais/farmacologia
2.
Cell Rep ; 37(6): 109958, 2021 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-34758301

RESUMO

Impaired hepatic glucose and lipid metabolism are hallmarks of type 2 diabetes. Increased sulfide production or sulfide donor compounds may beneficially regulate hepatic metabolism. Disposal of sulfide through the sulfide oxidation pathway (SOP) is critical for maintaining sulfide within a safe physiological range. We show that mice lacking the liver- enriched mitochondrial SOP enzyme thiosulfate sulfurtransferase (Tst-/- mice) exhibit high circulating sulfide, increased gluconeogenesis, hypertriglyceridemia, and fatty liver. Unexpectedly, hepatic sulfide levels are normal in Tst-/- mice because of exaggerated induction of sulfide disposal, with associated suppression of global protein persulfidation and nuclear respiratory factor 2 target protein levels. Hepatic proteomic and persulfidomic profiles converge on gluconeogenesis and lipid metabolism, revealing a selective deficit in medium-chain fatty acid oxidation in Tst-/- mice. We reveal a critical role of TST in hepatic metabolism that has implications for sulfide donor strategies in the context of metabolic disease.


Assuntos
Diabetes Mellitus/patologia , Dislipidemias/patologia , Gluconeogênese , Fígado/patologia , Sulfetos/metabolismo , Tiossulfato Sulfurtransferase/fisiologia , Animais , Diabetes Mellitus/etiologia , Diabetes Mellitus/metabolismo , Dislipidemias/etiologia , Dislipidemias/metabolismo , Glucose/metabolismo , Metabolismo dos Lipídeos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 2 Relacionado a NF-E2/metabolismo , Proteoma/metabolismo
3.
Mol Cell Endocrinol ; 518: 110873, 2020 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-32585168

RESUMO

By acting as a ligand-dependent transcription factor the glucocorticoid receptor (GR) mediates the actions of glucocorticoids and regulates many physiological processes. An impaired regulation of glucocorticoid action has been associated with numerous disorders. Thus, the elucidation of underlying signaling pathways is essential to understand mechanisms of disrupted glucocorticoid function and contribution to diseases. This study found increased GR transcriptional activity upon overexpression of protein phosphatase 1 alpha (PP1α) in HEK-293 cells and decreased expression levels of GR-responsive genes following PP1α knockdown in the endogenous A549 cell model. Mechanistic investigations revealed reduced phosphorylation of GR-Ser211 following PP1α silencing and provided a first indication for an involvement of glycogen synthase kinase 3 (GSK-3). Thus, the present study identified PP1α as a novel post-translational activator of GR signaling, suggesting that disruption of PP1α function could lead to impaired glucocorticoid action and thereby contribute to diseases.


Assuntos
Proteína Fosfatase 1/fisiologia , Receptores de Glucocorticoides/metabolismo , Células A549 , Sítios de Ligação , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Fosforilação/genética , Domínios e Motivos de Interação entre Proteínas/genética , Proteína Fosfatase 1/genética , Processamento de Proteína Pós-Traducional/genética , Receptores de Glucocorticoides/química , Serina/genética , Serina/metabolismo
4.
J Endocrinol ; 244(1): 149-162, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31600722

RESUMO

The serum- and glucocorticoid-induced kinase 1 (SGK1) is a transcriptional target of steroid hormones including glucocorticoids or aldosterone in addition to other stimuli such as glucose. SGK1 is activated via phosphoinositide 3-kinase, placing it downstream of insulin signaling. SGK1 participates in the upregulation of kidney Na+ reabsorption by aldosterone and has been linked to obesity-related hypertension in humans. We hypothesized that a systemic increase in SGK1 activity may trigger a multiplicity of mechanisms leading to simultaneous development of the main conditions that characterize the metabolic syndrome (MetS), including hypertension. We used a transgenic mouse model made with a bacterial artificial chromosome containing the whole mouse Sgk1 gene modified to introduce an activating point mutation. Wild type or transgenic 14-week-old male mice were fed with standard chow diet or high-fat diet for up to 18 weeks. Development of the main features of MetS and hepatic steatosis were monitored, and in vitro adipocyte differentiation was studied. Our results show that transgenic animals under high-fat diet rapidly and markedly develop MetS characterized by obesity, glucose intolerance, insulin resistance, dyslipidemia and hypertension. In addition, SGK1 gain-of-function accelerates the development of hepatic steatosis. Our study suggests that inappropriate SGK1 activity represents a risk factor in developing MetS with hypertension and related end-organ damage. Our data support SGK1 as a possible therapeutic target in MetS and related complications and provides a useful gain-of-function model for pre-clinical drug testing.


Assuntos
Dieta Hiperlipídica/efeitos adversos , Hipertensão/genética , Proteínas Imediatamente Precoces/metabolismo , Síndrome Metabólica/genética , Obesidade/genética , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Modelos Animais de Doenças , Hipertensão/etiologia , Síndrome Metabólica/etiologia , Camundongos , Camundongos Transgênicos , Obesidade/etiologia , Mutação Puntual , Fatores de Risco , Transdução de Sinais/genética
5.
J Am Soc Nephrol ; 30(5): 737-750, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30902838

RESUMO

BACKGROUND: A number of cAMP-elevating hormones stimulate phosphorylation (and hence activity) of the NaCl cotransporter (NCC) in the distal convoluted tubule (DCT). Evidence suggests that protein phosphatase 1 (PP1) and other protein phosphatases modulate NCC phosphorylation, but little is known about PP1's role and the mechanism regulating its function in the DCT. METHODS: We used ex vivo mouse kidney preparations to test whether a DCT-enriched inhibitor of PP1, protein phosphatase 1 inhibitor-1 (I1), mediates cAMP's effects on NCC, and conducted yeast two-hybrid and coimmunoprecipitation experiments in NCC-expressing MDCK cells to explore protein interactions. RESULTS: Treating isolated DCTs with forskolin and IBMX increased NCC phosphorylation via a protein kinase A (PKA)-dependent pathway. Ex vivo incubation of mouse kidney slices with isoproterenol, norepinephrine, and parathyroid hormone similarly increased NCC phosphorylation. The cAMP-induced stimulation of NCC phosphorylation strongly correlated with the phosphorylation of I1 at its PKA consensus phosphorylation site (a threonine residue in position 35). We also found an interaction between NCC and the I1-target PP1. Moreover, PP1 dephosphorylated NCC in vitro, and the PP1 inhibitor calyculin A increased NCC phosphorylation. Studies in kidney slices and isolated perfused kidneys of control and I1-KO mice demonstrated that I1 participates in the cAMP-induced stimulation of NCC. CONCLUSIONS: Our data suggest a complete signal transduction pathway by which cAMP increases NCC phosphorylation via a PKA-dependent phosphorylation of I1 and subsequent inhibition of PP1. This pathway might be relevant for the physiologic regulation of renal sodium handling by cAMP-elevating hormones, and may contribute to salt-sensitive hypertension in patients with endocrine disorders or sympathetic hyperactivity.


Assuntos
Transporte Biológico/efeitos dos fármacos , Colforsina/farmacologia , Túbulos Renais Distais/metabolismo , Proteína Fosfatase 1/antagonistas & inibidores , Proteínas/farmacologia , Análise de Variância , Animais , Transporte Biológico/genética , Humanos , Immunoblotting , Técnicas In Vitro , Camundongos , Camundongos Knockout , Fosforilação/efeitos dos fármacos , Transdução de Sinais/genética , Cloreto de Sódio/metabolismo , Membro 3 da Família 12 de Carreador de Soluto/metabolismo
6.
JCI Insight ; 3(14)2018 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-30046010

RESUMO

Aldosterone synthase inhibitors (ASIs) should alleviate obesity-related cardiovascular and renal problems resulting partly from aldosterone excess, but their clinical use may have limitations. To improve knowledge for the use of ASIs, we investigated physiology in aldosterone synthase-knockout (ASKO) mice. On regular chow diet (CD), ASKO mice ate more and weighed less than WT mice, largely because they hyperventilated to eliminate acid as CO2. Replacing CD with high-fat diet (HFD) lessened the respiratory burden in ASKO mice, as did 12- to 15-hour fasting. The latter eliminated the genotype differences in respiratory workload and energy expenditure (EE). Thus, aldosterone deficiency burdened the organism more when the animals ate carbohydrate-rich chow than when they ate a HFD. Chronic HFD exposure further promoted hyperinsulinemia in ASKO mice that contributed to visceral fat accumulation accompanied by reduced lipolysis, thermogenic reprogramming, and the absence of weight-gain-related EE increases. Intracerebroventricular aldosterone supplementation in ASKO mice attenuated the HFD-induced hyperinsulinemia, but did not affect EE, suggesting that the presence of aldosterone increased the body's energetic efficiency, thus counteracting the EE-increasing effect of low insulin. ASIs may therefore cause acid-overload-induced respiratory burden and promote obesity. Their use in patients with preexisting renal and cardiopulmonary diseases might be contraindicated.


Assuntos
Aldosterona/metabolismo , Citocromo P-450 CYP11B2/genética , Hiperinsulinismo/genética , Obesidade/genética , Adipócitos/citologia , Animais , Citocromo P-450 CYP11B2/efeitos dos fármacos , Dieta Hiperlipídica , Modelos Animais de Doenças , Metabolismo Energético , Fezes , Feminino , Hipoaldosteronismo/genética , Insulina/sangue , Resistência à Insulina/genética , Lipólise , Locomoção , Masculino , Camundongos , Camundongos Knockout , Pletismografia , Termogênese
7.
Mol Cell Endocrinol ; 450: 74-82, 2017 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-28454724

RESUMO

Stimulation of the mineralocorticoid receptor (MR) by aldosterone controls several physiological parameters including blood pressure, inflammation or metabolism. We previously showed that MR turnover constitutes a crucial regulatory step in the responses of renal epithelial cells to aldosterone. Here, we identified Protein Phosphatase 1 alpha (PP1α), as a novel cytoplasmic binding partner of MR that promotes the receptor activity. The RT-PCR expression mapping of PP1α reveals a high expression in the kidney, particularly in the distal part of the nephron. At the molecular level, we demonstrate that PP1α inhibits the ubiquitin ligase Mdm2 by dephosphorylation, preventing its interaction with MR. This results in the accumulation of the receptor due to reduction of its proteasomal degradation and consequently a greater aldosterone-induced Na+ uptake by renal cells. Thus, our findings describe an original mechanism involving a phosphatase in the regulation of aldosterone signaling and provide new and important insights into the molecular mechanism underlying the MR turnover.


Assuntos
Aldosterona/metabolismo , Rim/metabolismo , Proteína Fosfatase 1/metabolismo , Receptores de Mineralocorticoides/metabolismo , Transdução de Sinais , Animais , Linhagem Celular , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células HEK293 , Humanos , Camundongos Endogâmicos C57BL , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos , Estabilidade Proteica/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Receptores de Mineralocorticoides/química , Transdução de Sinais/efeitos dos fármacos , Sódio/metabolismo , Transcrição Gênica/efeitos dos fármacos
8.
J Endocrinol ; 230(1): 1-11, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27106108

RESUMO

The glucocorticoids bind and activate both the glucocorticoid receptor (GR) as well as the mineralocorticoid receptor in adipocytes. Despite several studies to determine the function of these two receptors in mediating glucocorticoids effects, their relative contribution in adipose tissue expansion and obesity is unclear. To investigate the effect of GR in adipose tissue function, we generated an adipocyte-specific Gr-knockout mouse model (Gr(ad-ko)). These mice were submitted either to a standard diet or a high-fat high sucrose diet. We found that adipocyte-specific deletion of Gr did not affect body weight gain or adipose tissue formation and distribution. However, the lack of Gr in adipocyte promotes a diet-induced inflammation determined by higher pro-inflammatory genes expression and macrophage infiltration in the fat pads. Surprisingly, the adipose tissue inflammation in Gr(ad-ko) mice was not correlated with insulin resistance or dyslipidemia, but with disturbed glucose tolerance. Our data demonstrate that adipocyte-specific ablation of Gr in vivo may affect the adipose tissue function but not its expansion during a high calorie diet.


Assuntos
Adipócitos/metabolismo , Tecido Adiposo/metabolismo , Inflamação/metabolismo , Receptores de Glucocorticoides/metabolismo , Animais , Dieta Hiperlipídica , Dislipidemias/genética , Dislipidemias/metabolismo , Inflamação/genética , Resistência à Insulina/genética , Metabolismo dos Lipídeos/genética , Masculino , Camundongos , Camundongos Knockout , Receptores de Glucocorticoides/genética
9.
Am J Physiol Renal Physiol ; 309(9): F779-90, 2015 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-26336159

RESUMO

The Rab GTPase-activating protein TBC1D4 (AS160) controls trafficking of the glucose transporter GLUT4 in adipocytes and skeletal muscle cells. TBC1D4 is also highly abundant in the renal distal tubule, although its role in this tubule is so far unknown. In vitro studies suggest that it is involved in the regulation of renal transporters and channels such as the epithelial sodium channel (ENaC), aquaporin-2 (AQP2), and the Na+-K+-ATPase. To assess the physiological role of TBC1D4 in the kidney, wild-type (TBC1D4+/+) and TBC1D4-deficient (TBC1D4-/-) mice were studied. Unexpectedly, neither under standard nor under challenging conditions (low Na+/high K+, water restriction) did TBC1D4-/- mice show any difference in urinary Na+ and K+ excretion, urine osmolarity, plasma ion and aldosterone levels, and blood pressure compared with TBC1D4+/+ mice. Also, immunoblotting did not reveal any change in the abundance of major renal sodium- and water-transporting proteins [Na-K-2Cl cotransporter (NKCC2) NKCC2, NaCl cotransporter (NCC), ENaC, AQP2, and the Na+-K+-ATPase]. However, the abundance of GLUT4, which colocalizes with TBC1D4 along the distal nephron of TBC1D4+/+ mice, was lower in whole kidney lysates of TBC1D4-/- mice than in TBC1D4+/+ mice. Likewise, primary thick ascending limb (TAL) cells isolated from TBC1D4-/- mice showed an increased basal glucose uptake and an abrogated insulin response compared with TAL cells from TBC1D4+/+ mice. Thus, TBC1D4 is dispensable for the regulation of renal Na+ and water transport, but may play a role for GLUT4-mediated basolateral glucose uptake in distal tubules. The latter may contribute to the known anaerobic glycolytic capacity of distal tubules during renal ischemia.


Assuntos
Proteínas Ativadoras de GTPase/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Rim/metabolismo , Sódio/metabolismo , Equilíbrio Hidroeletrolítico , Animais , Células Cultivadas , Proteínas Ativadoras de GTPase/deficiência , Proteínas Ativadoras de GTPase/genética , Regulação da Expressão Gênica , Genótipo , Glucose/metabolismo , Transportador de Glucose Tipo 4/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Insulina/farmacologia , Rim/efeitos dos fármacos , Túbulos Renais Distais/metabolismo , Alça do Néfron/metabolismo , Masculino , Camundongos Knockout , Parvalbuminas/genética , Fenótipo , Regiões Promotoras Genéticas , Sistema Renina-Angiotensina
10.
J Steroid Biochem Mol Biol ; 143: 334-42, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24820770

RESUMO

Aldosterone or glucocorticoid stimulation of the mineralocorticoid receptor (MR) is involved in numerous physiological responses, including ions and water homeostasis, blood pressure control and metabolism. The understanding of MR signaling regulation in the patho/physiological context took a new direction the last few years with a focus on the post-translational modifications of MR. Depending on its environment, cellular expression, activity or its binding partners, the MR is submitted to several post-translational modifications such as phosphorylation, ubiquitylation, sumoylation and acetylation that regulate its localization, activity and/or stability. A complex interplay between all these modifications allows a fine tuning of MR signaling depending on the physiological context. This review reports recent knowledge about post-translational modifications of MR and describes the enzymes and the molecular mechanisms involved.


Assuntos
Processamento de Proteína Pós-Traducional , Receptores de Mineralocorticoides/metabolismo , Sequência de Aminoácidos , Animais , Homeostase , Humanos , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Transdução de Sinais
11.
PLoS One ; 9(4): e94127, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24705830

RESUMO

Geldanamycin derivatives are benzoquinone ansamycin antibiotics that bind to Hsp90 and alter its function. The alteration of Hsp90 activity limits some cellular hormonal responses by inhibiting nuclear receptors activation. The nuclear receptors activity, such as PPARγ, the mineralocorticoid and glucocorticoid receptors (MR and GR) play a critical role in the conversion of preadipocytes to mature adipocytes. Given the importance of these nuclear receptors for adipogenesis, we investigated the effects of geldanamycin analogues (GA) on adipocyte differentiation and function. We found that early exposure of preadipocyte cells to GA inhibited their conversion into mature adipocytes by inhibiting the adipogenic transcriptional program and lipid droplets accumulation. Furthermore, GA altered the adipokines secretion profile of mature adipocyte. The anti-adipogenic effect of GA was also confirmed in mice fed a high fat diet. Biochemical analysis revealed that anti-adipogenic effects of geldanamycin analogues may result from the simultaneous inhibition of MR, GR and PPARγ activity. Taken together, our observations lead us to propose Hsp90 as a potent target for drug development in the control of obesity and its related metabolic complications.


Assuntos
Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Células 3T3-L1 , Adipócitos/metabolismo , Adipogenia/efeitos dos fármacos , Adipogenia/fisiologia , Adipocinas/biossíntese , Animais , Benzoquinonas/farmacologia , Dieta Hiperlipídica , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico HSP90/metabolismo , Lactamas Macrocíclicas/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Masculino , Camundongos , PPAR gama/genética , PPAR gama/metabolismo , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Receptores de Mineralocorticoides/genética , Receptores de Mineralocorticoides/metabolismo , Transcrição Gênica
12.
Cell Physiol Biochem ; 31(2-3): 462-72, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23548632

RESUMO

BACKGROUND/AIMS: Ligand activation of the mineralocorticoid receptor (MR) induces several post-translational modifications (PTMs). Among the different PTMs, MR is known to be dynamically ubiquitylated with impact on its stability and transcriptional activity. Previously, we have shown that MR is monoubiquitylated at the basal state and that aldosterone stimulation induces monoubiquitylation removal prompting polyubiquitin-dependent destabilization of the receptor and proteasomal degradation. This study investigated the role of the aldosterone induced ubiquitin-specific protease USP2-45 on the ubiquitylation state of MR. METHODS: Renal epithelial cells M1 were co-transfected with MR with or without wild-type or inactive USP2-45. The association of MR with USP2-45 or TSG101 as well as MR ubiquitylation state were determined by immunoprecipitation and immunoblotting. MR transcriptional activity was assessed via a luciferase reporter gene. RESULTS: We show that USP2-45 is able to bind MR and, similarly to aldosterone, induce MR monoubiquitylation removal, disruption of MR/TSG101 association and destabilization of MR at protein level. CONCLUSION: This study provides a novel role for USP2-45 by playing a pivotal role in the regulation of the ubiquitylation state of MR and reveals the existence of a negative feedback loop for limiting the aldosterone induced response.


Assuntos
Aldosterona/farmacologia , Endopeptidases/metabolismo , Antagonistas de Receptores de Mineralocorticoides/farmacologia , Receptores de Mineralocorticoides/metabolismo , Animais , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Endopeptidases/deficiência , Endopeptidases/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Imunoprecipitação , Camundongos , Camundongos Knockout , Ligação Proteica , Receptores de Mineralocorticoides/química , Fatores de Transcrição/metabolismo , Transfecção , Ubiquitina Tiolesterase , Proteases Específicas de Ubiquitina , Ubiquitinação
13.
FASEB J ; 26(10): 4373-82, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22798426

RESUMO

Aldosterone stimulation of the mineralocorticoid receptor (MR) is involved in numerous physiological responses, including Na+ homeostasis, blood pressure control, and heart failure. Aldosterone binding to MR promotes different post-translational modifications that regulate MR nuclear translocation, gene expression, and finally receptor degradation. Here, we show that aldosterone stimulates rapid phosphorylation of MR via ERK1/2 in a dose-dependent manner (from 0.1 to 10 nM) in renal epithelial cells. This phosphorylation induces an increase of MR apparent molecular weight, with a maximal upward shift of 30 kDa. Strikingly, these modifications are critical for the regulation of the MR ubiquitylation state. Indeed, we find that MR is monoubiquitylated in its basal state, and this status is sustained by the tumor suppressor gene 101 (Tsg101). Phosphorylation leads to disruption of MR/Tsg101 association and monoubiquitin removal. These events prompt polyubiquitin-dependent destabilization of MR and degradation. Preventing MR phosphorylation by ERK1/2 inhibition or mutation of target serines affects the sequential mechanisms of MR ubiquitylation and inhibits the aldosterone-mediated degradation. Our data provide a novel model of negative feedback of aldosterone signaling, involving sequential phosphorylation, monoubiquitin removal and subsequent polyubiquitylation/degradation of MR.


Assuntos
Aldosterona/farmacologia , Receptores de Mineralocorticoides/metabolismo , Ubiquitinação/efeitos dos fármacos , Linhagem Celular , Humanos , Immunoblotting , Imunoprecipitação , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional , Transdução de Sinais/efeitos dos fármacos
14.
Am J Physiol Renal Physiol ; 302(8): F977-85, 2012 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-22301619

RESUMO

The expression of the serum- and glucocorticoid-regulated kinase 1 (Sgk1) is induced by mineralocorticoids and, in turn, upregulates the renal epithelial Na(+) channel (ENaC). Total inactivation of Sgk1 has been associated with transient urinary Na(+) wasting with a low-Na(+) diet, while the aldosterone-mediated ENaC channel activation was unchanged in the collecting duct. Since Sgk1 is ubiquitously expressed, we aimed to study the role of renal Sgk1 and generated an inducible kidney-specific knockout (KO) mouse. We took advantage of the previously described TetOn/CreLoxP system, in which rtTA is under the control of the Pax8 promotor, allowing inducible inactivation of the floxed Sgk1 allele in the renal tubules (Sgk1fl/fl/Pax8/LC1 mice). We found that under a standard Na(+) diet, renal water and Na(+)/K(+) excretion had a tendency to be higher in doxycycline-treated Sgk1 KO mice compared with control mice. The impaired ability of Sgk1 KO mice to retain Na(+) increased significantly with a low-salt diet despite higher plasma aldosterone levels. On a low-Na(+) diet, the Sgk1 KO mice were also hyperkaliuric and lost body weight. This phenotype was accompanied by a decrease in systolic and diastolic blood pressure. At the protein level, we observed a reduction in phosphorylation of the ubiquitin protein-ligase Nedd4-2 and a decrease in the expression of the Na(+)-Cl(-)-cotransporter (NCC) and to a lesser extent of ENaC.


Assuntos
Proteínas Imediatamente Precoces/fisiologia , Rim/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Sódio/urina , Aldosterona/sangue , Animais , Pressão Sanguínea/fisiologia , Dieta Hipossódica , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Canais Epiteliais de Sódio/biossíntese , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Rim/metabolismo , Camundongos , Camundongos Knockout , Ubiquitina-Proteína Ligases Nedd4 , Fosforilação , Potássio/sangue , Potássio/urina , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Sódio/sangue , Simportadores de Cloreto de Sódio/biossíntese , Cloreto de Sódio na Dieta/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
15.
Am J Physiol Renal Physiol ; 299(6): F1462-72, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20861078

RESUMO

The mineralocorticoid receptor (MR) plays a crucial role in the regulation of Na(+) balance and blood pressure, as evidenced by gain of function mutations in the MR of hypertensive families. In the kidney, aldosterone binds to the MR, induces its nuclear translocation, and promotes a transcriptional program leading to increased transepithelial Na(+) transport via the epithelial Na(+) channel. In the unliganded state, MR is localized in the cytosol and part of a multiprotein complex, including heat shock protein 90 (Hsp90), which keeps it ligand-binding competent. 17-Allylamino-17-demethoxygeldanamycin (17-AAG) is a benzoquinone ansamycin antibiotic that binds to Hsp90 and alters its function. We investigated whether 17-AAG affects the stability and transcriptional activity of MR and consequently Na(+) reabsorption by renal cells. 17-AAG treatment lead to reduction of MR protein level in epithelial cells in vitro and in vivo, thereby interfering with aldosterone-dependent transcription. Moreover, 17-AAG inhibited aldosterone-induced Na(+) transport, possibly by interfering with MR availability for the ligand. Finally, we identified the ubiquitin-protein ligase, COOH terminus of Hsp70-interacting protein, as a novel partner of the cytosolic MR, which is responsible for its polyubiquitylation and proteasomal degradation in presence of 17-AAG. In conclusion, 17-AAG may represent a novel pharmacological tool to interfere with Na(+) reabsorption and hypertension.


Assuntos
Benzoquinonas/farmacologia , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Lactamas Macrocíclicas/farmacologia , Receptores de Mineralocorticoides/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Aldosterona/farmacologia , Animais , Feminino , Proteínas de Choque Térmico HSP70/metabolismo , Túbulos Renais Coletores/metabolismo , Leupeptinas/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Mineralocorticoides/efeitos dos fármacos , Sódio/metabolismo , Ativação Transcricional/efeitos dos fármacos , Ubiquitina-Proteína Ligases/antagonistas & inibidores
16.
EMBO J ; 27(13): 1804-15, 2008 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-18511908

RESUMO

The TGIF homoeodomain protein functions as an important negative regulator in the TGF-beta signalling pathway. The inhibitory function of TGIF is executed in part through its ability to sequester the tumour suppressor cytoplasmic promyelocytic leukaemia (cPML) in the nucleus, thereby preventing the phosphorylation of Smad2 by the activated TGF-beta type I receptor. Here, we report on the identification of PCTA (PML competitor for TGIF association), a TGIF antagonist that promotes TGF-beta-induced transcriptional and cytostatic responses. We provide evidence that PCTA functions in TGF-beta signalling by relieving the suppression of Smad2 phosphorylation by TGIF. Furthermore, we demonstrate that PCTA selectively competes with cPML for TGIF association, resulting in the accumulation of cPML in the cytoplasm, where it associates with SARA and coordinates the access of Smad2 for phosphorylation by the activated TGF-beta type I receptor. Thus, our findings on the mode of action of PCTA provide new and important insights into the molecular mechanism underlying the antagonistic interplay between TGIF and cPML in the TGF-beta signalling network.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Homeodomínio/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Proteínas Repressoras/antagonistas & inibidores , Proteína Smad2/metabolismo , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Linhagem Celular , DNA Complementar , Cães , Feminino , Biblioteca Gênica , Humanos , Fosforilação , Placenta/metabolismo , Proteína da Leucemia Promielocítica , Técnicas do Sistema de Duplo-Híbrido , Ubiquitina-Proteína Ligases
17.
Mol Cell ; 23(4): 547-59, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16916642

RESUMO

The homeodomain protein TGIF has been implicated in the negative regulation of TGF-beta signaling. In this study, we report an unexpected role of TGIF in the inhibition of Smad2 phosphorylation, which occurs by a mechanism independent of its association with Smad2. This inhibitory function of TGIF is executed in concert with c-Jun, which facilitates the interaction of TGIF with cPML, resulting in the nuclear sequestration of cPML and the disruption of the cPML-SARA complex. Notably, knockdown of TGIF by siRNA caused increased association of cPML with SARA and cytoplasmic accumulation of cPML. Furthermore, c-Jun(-/-) fibroblasts exhibit enhanced association of cPML with SARA. c-Jun(-/-) fibroblasts also lose their sensitivity to TGIF-mediated disruption of the cPML-SARA complex and of nuclear sequestration of cPML. We suggest that the interaction of TGIF with cPML through c-Jun may negatively regulate TGF-beta signaling through controlling the localization of cPML and, consequently, the assembly of the cPML-SARA complex.


Assuntos
Núcleo Celular/metabolismo , Proteínas de Homeodomínio/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Proteínas Supressoras de Tumor/metabolismo , Animais , Células COS , Células Cultivadas , Chlorocebus aethiops , Cães , Regulação para Baixo/efeitos dos fármacos , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Humanos , Camundongos , Fosforilação/efeitos dos fármacos , Proteína da Leucemia Promielocítica , Ligação Proteica , Proteínas Proto-Oncogênicas c-jun/deficiência , Proteínas Proto-Oncogênicas c-jun/metabolismo , Proteína Smad2/metabolismo , Transcrição Gênica/efeitos dos fármacos
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