ABSTRACT
Autophagy is required in diverse paradigms of lifespan extension, leading to the prevailing notion that autophagy is beneficial for longevity. However, why autophagy is harmful in certain contexts remains unexplained. Here, we show that mitochondrial permeability defines the impact of autophagy on aging. Elevated autophagy unexpectedly shortens lifespan in C. elegans lacking serum/glucocorticoid regulated kinase-1 (sgk-1) because of increased mitochondrial permeability. In sgk-1 mutants, reducing levels of autophagy or mitochondrial permeability transition pore (mPTP) opening restores normal lifespan. Remarkably, low mitochondrial permeability is required across all paradigms examined of autophagy-dependent lifespan extension. Genetically induced mPTP opening blocks autophagy-dependent lifespan extension resulting from caloric restriction or loss of germline stem cells. Mitochondrial permeability similarly transforms autophagy into a destructive force in mammals, as liver-specific Sgk knockout mice demonstrate marked enhancement of hepatocyte autophagy, mPTP opening, and death with ischemia/reperfusion injury. Targeting mitochondrial permeability may maximize benefits of autophagy in aging.
Subject(s)
Aging/metabolism , Mitochondrial Membrane Transport Proteins/physiology , Mitochondrial Membranes/physiology , Animals , Autophagy/physiology , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans Proteins/physiology , Caloric Restriction , HEK293 Cells , Humans , Longevity/physiology , Male , Mice , Mice, Knockout , Mitochondria , Mitochondrial Membrane Transport Proteins/metabolism , Mitochondrial Permeability Transition Pore , Permeability , Primary Cell Culture , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Protein Serine-Threonine Kinases/physiology , Reperfusion Injury/metabolism , Signal TransductionABSTRACT
SGK1 is an AGC kinase that regulates the expression of membrane sodium channels in renal tubular cells in a manner dependent on the metabolic checkpoint kinase complex mTORC2. We hypothesized that SGK1 might represent an additional mTORC2-dependent regulator of the differentiation and function of T cells. Here we found that after activation by mTORC2, SGK1 promoted T helper type 2 (TH2) differentiation by negatively regulating degradation of the transcription factor JunB mediated by the E3 ligase Nedd4-2. Simultaneously, SGK1 repressed the production of interferon-γ (IFN-γ) by controlling expression of the long isoform of the transcription factor TCF-1. Consistent with those findings, mice with selective deletion of SGK1 in T cells were resistant to experimentally induced asthma, generated substantial IFN-γ in response to viral infection and more readily rejected tumors.
Subject(s)
Asthma/immunology , Immediate-Early Proteins/metabolism , Melanoma, Experimental/immunology , Multiprotein Complexes/immunology , Poxviridae Infections/immunology , Protein Serine-Threonine Kinases/metabolism , TOR Serine-Threonine Kinases/immunology , Th1 Cells/immunology , Th2 Cells/immunology , Vaccinia virus/immunology , Adaptive Immunity/genetics , Animals , Cell Differentiation/genetics , Cells, Cultured , Endosomal Sorting Complexes Required for Transport/metabolism , Gene Expression Regulation/genetics , Hepatocyte Nuclear Factor 1-alpha , Immediate-Early Proteins/genetics , Interferon-gamma/genetics , Interferon-gamma/metabolism , Mechanistic Target of Rapamycin Complex 2 , Mice , Mice, Inbred C57BL , Mice, Knockout , Nedd4 Ubiquitin Protein Ligases , Protein Serine-Threonine Kinases/genetics , T Cell Transcription Factor 1/genetics , T Cell Transcription Factor 1/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Tumor Burden/genetics , Ubiquitin-Protein Ligases/metabolismABSTRACT
OBJECTIVE: Restenosis after percutaneous coronary intervention remains to be a serious medical problem. Although mineralocorticoid receptor (MR) has been implicated as a potential target for treating restenosis, the cellular and molecular mechanisms are largely unknown. This study aims to explore the functions of macrophage MR in neointimal hyperplasia and to delineate the molecular mechanisms. APPROACH AND RESULTS: Myeloid MR knockout (MMRKO) mice and controls were subjected to femoral artery injury. MMRKO reduced intima area and intima/media ratio, Ki67- and BrdU-positive vascular smooth muscle cells, expression of proinflammatory molecules, and macrophage accumulation in injured arteries. MMRKO macrophages migrated less in culture. MMRKO decreased Ki67- and BrdU-positive macrophages in injured arteries. MMRKO macrophages were less Ki67-positive in culture. Conditioned media from MMRKO macrophages induced less migration, Ki67 positivity, and proinflammatory gene expression of vascular smooth muscle cells. After lipopolysaccharide treatment, MMRKO macrophages had decreased p-cFos and p-cJun compared with control macrophages, suggesting suppressed activation of activator protein-1 (AP1). Nuclear factor-κB (NF-κB) pathway was also inhibited by MMRKO, manifested by decreased p-IκB kinase-ß and p-IκBα, increased IκBα expression, decreased nuclear translocation of p65 and p50, as welll as decreased phosphorylation and expression of p65. Finally, overexpression of serum-and-glucocorticoid-inducible-kinase-1 (SGK1) attenuated the effects of MR deficiency in macrophages. CONCLUSIONS: Selective deletion of MR in myeloid cells limits macrophage accumulation and vascular inflammation and, therefore, inhibits neointimal hyperplasia and vascular remodeling. Mechanistically, MR deficiency suppresses migration and proliferation of macrophages and leads to less vascular smooth muscle cell activation. At the molecular level, MR deficiency suppresses macrophage inflammatory response via SGK1-AP1/NF-κB pathways.
Subject(s)
Immediate-Early Proteins/metabolism , Inflammation/enzymology , Macrophages/enzymology , Muscle, Smooth, Vascular/enzymology , Myocytes, Smooth Muscle/enzymology , NF-kappa B/metabolism , Neointima , Protein Serine-Threonine Kinases/metabolism , Receptors, Mineralocorticoid/deficiency , Transcription Factor AP-1/metabolism , Vascular System Injuries/enzymology , Animals , Cell Movement , Cell Proliferation , Coculture Techniques , Disease Models, Animal , Femoral Artery/enzymology , Femoral Artery/injuries , Femoral Artery/metabolism , Genetic Predisposition to Disease , Hyperplasia , Immediate-Early Proteins/genetics , Inflammation/genetics , Inflammation/pathology , Inflammation/prevention & control , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Muscle, Smooth, Vascular/injuries , Muscle, Smooth, Vascular/pathology , Myocytes, Smooth Muscle/pathology , Paracrine Communication , Phenotype , Protein Serine-Threonine Kinases/genetics , RAW 264.7 Cells , RNA Interference , Receptors, Mineralocorticoid/genetics , Signal Transduction , Time Factors , Transfection , Vascular Remodeling , Vascular System Injuries/genetics , Vascular System Injuries/pathology , Vascular System Injuries/prevention & controlABSTRACT
Insulin resistance is a major hallmark of metabolic syndromes, including Type 2 diabetes. Although numerous functions of SGK1 (serum- and glucocorticoid-regulated kinase 1) have been identified, a direct effect of SGK1 on insulin sensitivity has not been previously reported. In the present study, we generated liver-specific SGK1-knockout mice and found that these mice developed glucose intolerance and insulin resistance. We also found that insulin signalling is enhanced or impaired in Hep1-6 cells infected with adenoviruses expressing SGK1 (Ad-SGK1) or shRNA directed against the coding region of SGK1 (Ad-shSGK1) respectively. In addition, we determined that SGK1 inhibits ERK1/2 (extracellular-signal-regulated kinase 1/2) activity in liver and Ad-shERK1/2-mediated inhibition of ERK1/2 reverses the attenuated insulin sensitivity in Ad-shSGK1 mice. Finally, we found that SGK1 functions are compromised under insulin-resistant conditions and overexpression of SGK1 by Ad-SGK1 significantly ameliorates insulin resistance in both glucosamine-treated HepG2 cells and livers of db/db mice, a genetic model of insulin resistance.
Subject(s)
Diabetes Mellitus, Type 2/metabolism , Immediate-Early Proteins/metabolism , Insulin Resistance , Insulin/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Protein Serine-Threonine Kinases/metabolism , Animals , Diabetes Mellitus, Type 2/etiology , Diabetes Mellitus, Type 2/pathology , Glucose Intolerance , Hep G2 Cells , Humans , Immediate-Early Proteins/chemistry , Insulin/genetics , Liver/metabolism , Liver/pathology , Mice , Mice, Knockout , Mitogen-Activated Protein Kinase 3/antagonists & inhibitors , Protein Serine-Threonine Kinases/chemistry , Signal Transduction/geneticsABSTRACT
The mineralocorticoid receptor (MR) is a transcription factor for genes mediating diverse, cell-specific functions, including trophic effects as well as promoting fluid/electrolyte homeostasis. It was reported that in intercalated cells, phosphorylation of the MR at serine 843 (S843) by Unc-51-like kinase (ULK1) inhibits MR activation and that phosphorylation of ULK1 by mechanistic target of rapamycin (mTOR) inactivates ULK1, and thereby prevents MR inactivation. We extended these findings with studies in M1 mouse cortical collecting duct cells stably expressing the rat MR and a reporter gene. Pharmacological inhibition of ULK1 dose-dependently increased ligand-induced MR transactivation, while ULK1 activation had no effect. Pharmacological inhibition of mTOR and CRISPR/gRNA gene knockdown of rapamycin-sensitive adapter protein of mTOR (Raptor) or rapamycin-insensitive companion of mTOR (Rictor) decreased phosphorylated ULK1 and ligand-induced activation of the MR reporter gene, as well as transcription of endogenous MR-target genes. As predicted, ULK1 inhibition had no effect on aldosterone-mediated transcription in M1 cells with the mutated MR-S843A (alanine cannot be phosphorylated). In contrast, mTOR inhibition dose-dependently decreased transcription in the MR-S843A cells, though not as completely as in cells with the wild-type MR-S843. mTOR, Raptor, and Rictor coprecipitated with the MR and addition of aldosterone increased their phosphorylated, active state. These results suggest that mTOR significantly regulates MR activity in at least 2 ways: by suppressing MR inactivation by ULK1, and by a yet ill-defined mechanism that involves direct association with MR. They also provide new insights into the diverse functions of ULK1 and mTOR, 2 key enzymes that monitor the cell's energy status.
Subject(s)
Aldosterone , Receptors, Mineralocorticoid , Animals , Mice , Rats , Autophagy-Related Protein-1 Homolog/genetics , Autophagy-Related Protein-1 Homolog/metabolism , Ligands , Mechanistic Target of Rapamycin Complex 1/metabolism , Multiprotein Complexes/metabolism , Phosphorylation , Rapamycin-Insensitive Companion of mTOR Protein/metabolism , Receptors, Mineralocorticoid/genetics , Receptors, Mineralocorticoid/metabolism , Regulatory-Associated Protein of mTOR , RNA, Guide, CRISPR-Cas Systems , Sirolimus/pharmacology , TOR Serine-Threonine Kinases/metabolism , Transcription Factors/metabolismABSTRACT
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.
Subject(s)
Immediate-Early Proteins/physiology , Kidney/physiology , Protein Serine-Threonine Kinases/physiology , Sodium/urine , Aldosterone/blood , Animals , Blood Pressure/physiology , Diet, Sodium-Restricted , Endosomal Sorting Complexes Required for Transport/metabolism , Epithelial Sodium Channels/biosynthesis , Immediate-Early Proteins/genetics , Immediate-Early Proteins/metabolism , Kidney/metabolism , Mice , Mice, Knockout , Nedd4 Ubiquitin Protein Ligases , Phosphorylation , Potassium/blood , Potassium/urine , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Sodium/blood , Sodium Chloride Symporters/biosynthesis , Sodium Chloride, Dietary/metabolism , Ubiquitin-Protein Ligases/metabolismABSTRACT
A hallmark of type 2 diabetes (T2D) is hepatic resistance to insulin's glucose-lowering effects. The serum- and glucocorticoid-regulated family of protein kinases (SGK) is activated downstream of mechanistic target of rapamycin complex 2 (mTORC2) in response to insulin in parallel to AKT. Surprisingly, despite an identical substrate recognition motif to AKT, which drives insulin sensitivity, pathological accumulation of SGK1 drives insulin resistance. Liver-specific Sgk1-knockout (Sgk1Lko) mice display improved glucose tolerance and insulin sensitivity and are protected from hepatic steatosis when fed a high-fat diet. Sgk1 promotes insulin resistance by inactivating AMP-activated protein kinase (AMPK) via phosphorylation on inhibitory site AMPKαSer485/491. We demonstrate that SGK1 is dominant among SGK family kinases in regulation of insulin sensitivity, as Sgk1, Sgk2, and Sgk3 triple-knockout mice have similar increases in hepatic insulin sensitivity. In aggregate, these data suggest that targeting hepatic SGK1 may have therapeutic potential in T2D.
Subject(s)
AMP-Activated Protein Kinases/metabolism , Immediate-Early Proteins/metabolism , Liver/metabolism , Protein Serine-Threonine Kinases/metabolism , AMP-Activated Protein Kinases/antagonists & inhibitors , Animals , Diabetes Mellitus, Type 2/pathology , Diet, High-Fat , Forkhead Box Protein O1/metabolism , Glucose/metabolism , Immediate-Early Proteins/deficiency , Immediate-Early Proteins/genetics , Insulin/metabolism , Insulin Resistance , Mechanistic Target of Rapamycin Complex 2/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Phosphorylation , Protein Serine-Threonine Kinases/deficiency , Protein Serine-Threonine Kinases/genetics , Proto-Oncogene Proteins c-akt/metabolism , RNA, Guide, Kinetoplastida/metabolism , Signal TransductionABSTRACT
Drugs of abuse cause significant neuroadaptations within the ventral tegmental area (VTA), with alterations in gene expression tied to changes in reward behavior. Serum- and glucocorticoid-inducible kinase 1 (SGK1) transcription, catalytic activity, and phosphorylation are upregulated in the VTA by chronic cocaine or morphine treatment, positioning SGK1 as a critical mediator of reward behavior. Using transgenic mouse models, we investigated the effect of SGK1 knockout in the VTA and in dopamine (DA) neurons to evaluate the necessity of protein expression for natural and drug reward behaviors. SGK1 knockdown in the VTA did not impact reward behaviors. Given VTA cellular heterogeneity, we also investigated a DA neuron-specific SGK1 knockout (KO). DA SGK1 KO significantly decreased body weight of adult mice as well as increased general locomotor activity; however, reward behaviors were similarly unaltered. Given that SGK1 mutants virally overexpressed in the VTA are capable of altering drug-associated behavior, our current results suggest that changes in SGK1 protein signaling may be distinct from expression. This work yields novel information on the impact of SGK1 deletion, critical for understanding the role of SGK1 signaling in the central nervous system and evaluating SGK1 as a potential therapeutic target for treatment of substance use disorders.
Subject(s)
Behavior, Animal/drug effects , Cocaine/pharmacology , Dopaminergic Neurons/drug effects , Immediate-Early Proteins/physiology , Protein Serine-Threonine Kinases/physiology , Reward , Ventral Tegmental Area/drug effects , Animals , Dopamine Uptake Inhibitors/pharmacology , Dopaminergic Neurons/pathology , Female , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Signal Transduction , Ventral Tegmental Area/pathologyABSTRACT
Nitrones (e.g. α-phenyl-N-tert-butyl nitrone; PBN) are cerebroprotective in experimental stroke. Free radical trapping is their proposed mechanism. As PBN has low radical trapping potency, we tested Sgk1 induction as another possible mechanism. PBN was injected (100 mg/kg, i.p.) into adult male rats and mice. Sgk1 was quantified in cerebral tissue by microarray, quantitative RT-PCR and western analyses. Sgk1+/+ and Sgk1-/- mice were randomized to receive PBN or saline immediately following transient (60 min) occlusion of the middle cerebral artery. Neurological deficit was measured at 24 h and 48 h and infarct volume at 48 h post-occlusion. Following systemic PBN administration, rapid induction of Sgk1 was detected by microarray (at 4 h) and confirmed by RT-PCR and phosphorylation of the Sgk1-specific substrate NDRG1 (at 6 h). PBN-treated Sgk1+/+ mice had lower neurological deficit ( p < 0.01) and infarct volume ( p < 0.01) than saline-treated Sgk1+/+ mice. PBN-treated Sgk1-/- mice did not differ from saline-treated Sgk1-/- mice. Saline-treated Sgk1-/- and Sgk1+/+ mice did not differ. Brain Sgk3:Sgk1 mRNA ratio was 1.0:10.6 in Sgk1+/+ mice. Sgk3 was not augmented in Sgk1-/- mice. We conclude that acute systemic treatment with PBN induces Sgk1 in brain tissue. Sgk1 may play a part in PBN-dependent actions in acute brain ischemia.
Subject(s)
Cyclic N-Oxides/therapeutic use , Immediate-Early Proteins/drug effects , Protein Serine-Threonine Kinases/drug effects , Animals , Brain/metabolism , Brain Ischemia/drug therapy , Cyclic N-Oxides/pharmacology , Immediate-Early Proteins/genetics , Immediate-Early Proteins/pharmacology , Infarction, Middle Cerebral Artery/drug therapy , Male , Mice , Mice, Knockout , Nitrogen Oxides/pharmacology , Nitrogen Oxides/therapeutic use , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Protein Serine-Threonine Kinases/pharmacology , Rats , Stroke/drug therapy , Transcriptional Activation/drug effectsABSTRACT
Although the central nervous system has been implicated in glucocorticoid-induced gain of fat mass, the underlying mechanisms are poorly understood. The aim of this study was to investigate the possible involvement of hypothalamic serum- and glucocorticoid-regulated kinase 1 (SGK1) in glucocorticoid-increased adiposity. It is well known that SGK1 expression is induced by acute glucocorticoid treatment, but it is interesting that we found its expression to be decreased in the arcuate nucleus of the hypothalamus, including proopiomelanocortin (POMC) neurons, following chronic dexamethasone (Dex) treatment. To study the role of SGK1 in POMC neurons, we produced mice that developed or experienced adult-onset SGK1 deletion in POMC neurons (PSKO). As observed in Dex-treated mice, PSKO mice exhibited increased adiposity and decreased energy expenditure. Mice overexpressing constitutively active SGK1 in POMC neurons consistently had the opposite phenotype and did not experience Dex-increased adiposity. Finally, Dex decreased hypothalamic α-melanocyte-stimulating hormone (α-MSH) content and its precursor Pomc expression via SGK1/FOXO3 signaling, and intracerebroventricular injection of α-MSH or adenovirus-mediated FOXO3 knockdown in the arcuate nucleus largely reversed the metabolic alterations in PSKO mice. These results demonstrate that POMC SGK1/FOXO3 signaling mediates glucocorticoid-increased adiposity, providing new insights into the mechanistic link between glucocorticoids and fat accumulation and important hints for possible treatment targets for obesity.
Subject(s)
Adiposity/drug effects , Dexamethasone/pharmacology , Forkhead Box Protein O3/genetics , Glucocorticoids/pharmacology , Immediate-Early Proteins/genetics , Neurons/drug effects , Pro-Opiomelanocortin/metabolism , Protein Serine-Threonine Kinases/genetics , Adiposity/genetics , Animals , Arcuate Nucleus of Hypothalamus/drug effects , Arcuate Nucleus of Hypothalamus/metabolism , Energy Metabolism/drug effects , Forkhead Box Protein O3/metabolism , Hypothalamus/cytology , Hypothalamus/drug effects , Hypothalamus/metabolism , Immediate-Early Proteins/metabolism , Male , Mice , Neurons/metabolism , Protein Serine-Threonine Kinases/metabolism , alpha-MSH/drug effects , alpha-MSH/metabolismABSTRACT
Recent clinical studies demonstrated beneficial effects of mineralocorticoid receptor (MR) antagonists in patients with heart failure and other cardiovascular diseases. However, the underlying molecular mechanisms are poorly understood, and the genes that mediate direct effects of aldosterone in the cardiovascular system are yet to be identified. The goal of this study was to identify genes that are directly regulated by aldosterone in cardiomyocytes and thus potentially play a role in initiating MR-mediated effects in the heart. We generated clonal cell lines of cardiomyocytes (H9C2 cells) stably expressing the MR. Using these cell lines and Affymetrix microarrays, we determined the effects of physiological concentrations of aldosterone on the gene expression profile. In two independent microarrays we identified 48 genes that were induced more than 1.5-fold (27 known genes and 21 expressed sequence tags) and five (three known genes and two expressed sequence tags) that were suppressed by a 2-h aldosterone treatment. We focused on eight genes that have a potential function in cardiovascular regulation and verified their aldosterone regulation using quantitative RT-PCR. These include genes related to extracellular matrix regulation (tenascin-X, ADAMTS1, PAI-1, UPAR, and hyaluronic acid synthase-2), signaling, and regulation of vascular tone (RGS2, adrenomedullin) and inflammation (orosomucoid). Protein synthesis inhibitors did not prevent aldosterone induction of these genes. We conclude that in cardiomyocytes aldosterone rapidly and directly regulates the expression of several genes that are involved in cardiac remodeling and regulation of blood pressure and thus might be mediators of the physiological and pathophysiological effects of aldosterone on the cardiovascular system.
Subject(s)
Aldosterone/pharmacology , Gene Expression Regulation/drug effects , Genes, Immediate-Early/drug effects , Myocytes, Cardiac/drug effects , Ventricular Remodeling , Animals , Cell Line , Extracellular Matrix Proteins/genetics , Extracellular Matrix Proteins/metabolism , Myocytes, Cardiac/metabolism , Oligonucleotide Array Sequence Analysis , Orosomucoid/genetics , Orosomucoid/metabolism , Protein Biosynthesis/drug effects , Rats , Receptors, Mineralocorticoid/genetics , Receptors, Mineralocorticoid/metabolism , Transfection , Vasodilation/genetics , Ventricular Remodeling/drug effectsABSTRACT
The mineralocorticoid receptor (MR) is a member of the steroid-thyroid hormone receptor superfamily of ligand-dependent transcription factors with diverse functions including the biological actions of aldosterone. Identification of the various transcriptional coregulators of MR is essential for understanding the complexity of MR signaling pathways under physiological and pathological conditions. We used a yeast two-hybrid system to find proteins that interact with a full-length MR and found, among other proteins, that MR interacted specifically with receptor for activated C kinase 1 (RACK1), a scaffolding protein. Overexpression of RACK1 using a tetracycline-inducible lentivirus in mouse cortical collecting duct M1 cells stably expressing the rat MR and a Gaussia luciferase gene reporter under a hormone-response element promoter resulted in enhanced agonist-dependent MR transactivation. Knockdown of RACK1 protein expression by short hairpin RNAs led to a significant reduction in MR activation of the reporter gene and the endogenous genes Ctla2α and Psca. We also demonstrated that RACK1 regulation of MR action is mediated through phosphorylation by the PKC-ß signaling pathway. MR and RACK1 were coimmunoprecipitated using an MR antibody in male Sprague-Dawley brain tissue and M1-rMR cells, and colocalization in M1-rMR cells and male rat brains was confirmed by immunofluorescence and immunohistochemistry. The scaffolding protein RACK1 is associated with MR under basal and agonist-stimulated conditions and facilitates agonist-stimulated MR actions through PKC-ß. These findings indicate that RACK1 is a newly described coactivator of MR.
Subject(s)
Aldosterone/metabolism , GTP-Binding Proteins/metabolism , Receptors, Mineralocorticoid/metabolism , Animals , Cells, Cultured , GTP-Binding Proteins/genetics , Male , Promoter Regions, Genetic , Protein Binding , Rats , Rats, Sprague-Dawley , Receptors for Activated C Kinase , Receptors, Mineralocorticoid/genetics , Signal Transduction/genetics , Transcriptional ActivationABSTRACT
Sodium deficiency increases angiotensin II (ATII) and aldosterone, which synergistically stimulate sodium retention and consumption. Recently, ATII-responsive neurons in the subfornical organ (SFO) and aldosterone-sensitive neurons in the nucleus of the solitary tract (NTSHSD2 neurons) were shown to drive sodium appetite. Here we investigate the basis for NTSHSD2 neuron activation, identify the circuit by which NTSHSD2 neurons drive appetite, and uncover an interaction between the NTSHSD2 circuit and ATII signaling. NTSHSD2 neurons respond to sodium deficiency with spontaneous pacemaker-like activity-the consequence of "cardiac" HCN and Nav1.5 channels. Remarkably, NTSHSD2 neurons are necessary for sodium appetite, and with concurrent ATII signaling their activity is sufficient to produce rapid consumption. Importantly, NTSHSD2 neurons stimulate appetite via projections to the vlBNST, which is also the effector site for ATII-responsive SFO neurons. The interaction between angiotensin signaling and NTSHSD2 neurons provides a neuronal context for the long-standing "synergy hypothesis" of sodium appetite regulation.
Subject(s)
Aldosterone/physiology , Angiotensin II/physiology , Biological Clocks/physiology , Neurons/physiology , Signal Transduction , Sodium/physiology , Solitary Nucleus/physiology , Animals , Eating/physiology , Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels/physiology , Male , Mice , Mice, Transgenic , NAV1.5 Voltage-Gated Sodium Channel/physiology , Neural Pathways/physiology , Septal Nuclei/physiology , Sodium/deficiencyABSTRACT
CONTEXT: Pseudohypoaldosteronism type 1 (PHA1) is a rare salt-wasting syndrome. Mutations in the NR3C2 gene coding for the mineralocorticoid receptor (MR) cause autosomal dominant PHA1. OBJECTIVE: Our objective was to reveal the cause of renal salt loss in six PHA1 patients and analyze the mutants' functional impact on MR function. DESIGN: Our study included the following: clinical and hormonal characterization of the patients' phenotype, analysis of the NR3C2 gene, determination of receptor affinities to aldosterone and the transcriptional activation abilities of the MR mutants, investigation of subcellular translocation using fluorescence-labeled MR, and studying changes in mutant receptor conformation with proteolysis experiments and three-dimensional modeling. RESULTS: Six heterozygous NR3C2 mutations were detected. One frameshift mutation (c.1131dupT) has been reported previously. The second frameshift mutation (c.2871dupC), which has only recently been reported by our group, showed no aldosterone binding and no transactivation because of a major change in receptor conformation. Two novel nonsense mutations generate a truncated receptor protein. Two missense mutations differently affect MR function. S818L was reported recently without complete in vitro data. S818L does not bind aldosterone or activate transcription or translocate into the nucleus. A major displacement of several residues involved in aldosterone binding was PHA1 causing. The novel E972G mutation showed a significantly lower ligand-binding affinity and only 9% of wild-type transcriptional activity caused by major changes in receptor conformation. CONCLUSIONS: Our data on six mutations extend the spectrum of PHA1-causing NR3C2 gene mutations. Studying naturally occurring mutants helps to clarify their pathogenicity and to identify crucial residues for MR structure and function.
Subject(s)
Mutation , Pseudohypoaldosteronism/etiology , Pseudohypoaldosteronism/genetics , Receptors, Mineralocorticoid/genetics , Amino Acid Sequence , Animals , Cells, Cultured , Child , Child, Preschool , DNA Mutational Analysis , Female , Gene Expression , Humans , Imaging, Three-Dimensional , Male , Mice , Models, Molecular , Molecular Sequence Data , Mutant Proteins/analysis , Mutant Proteins/metabolism , Pedigree , Protein Structure, Secondary , Protein Transport , Pseudohypoaldosteronism/metabolism , Rabbits , Receptors, Mineralocorticoid/chemistry , Receptors, Mineralocorticoid/metabolism , Sequence Homology, Amino Acid , Tissue Distribution , Transcriptional Activation , TransfectionABSTRACT
Serum- and glucocorticoid-induced kinases (SGK) are members of the serine-threonine kinase family. SGK1, the isoform identified first, is rapidly induced by aldosterone. In this study, we determined that the two recently described isoforms, SGK2 and SGK3 are also expressed in renal cortical collecting duct (CCD) cells; however, their expression is not induced by aldosterone or glucocorticoids. SGK1 increases the activity of the epithelial sodium channel (ENaC) in oocytes but its cellular targets in native mineralocorticoid target cells and its mechanism of action are still unknown. We studied the role of SGK1 in corticosteroid-regulated Na transport in M-1 mouse CCD cell lines that stably over-express or down-regulate SGK1. Basal rates of transepithelial Na transport were significantly lower in CCD cells in which SGK1 expression or activity was down-regulated than in SGK1 overexpressing cells. Importantly, corticosteroid treatment failed to stimulate Na transport in cells with down-regulated SGK1 while it significantly increased Na transport in parent and SGK1 overexpressing M-1 cells. To determine if C-terminal PDZ interactions are important for SGK's effect on ENaC activity or trafficking, we examined the effects of mutant SGK1 in which the conserved PDZ binding domain has been eliminated. However, such mutations did not decrease its stimulatory effect on ENaC current in Xenopus oocytes. Fluorescence confocal microscopy revealed that the intracellular localization of full-length and PDZ binding mutated SGK1 was identical: they both localize to intracellular vesicular structures. On the other hand, N-terminally truncated (delta 60)-SGK1 did not increase ENaC activity. We conclude that SGK1 is a critical component in corticosteroid-regulated Na transport in mammalian CCD cells. Our data also indicate that the N-terminal of SGK1 is necessary for its stimulatory effect on Na transport while elimination of the C-terminal PDZ binding domain did not change its function.
Subject(s)
Aldosterone/physiology , Kidney Tubules, Collecting/physiology , Nuclear Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Sodium Channels/metabolism , Sodium/metabolism , Animals , Biological Transport, Active/physiology , Cell Line , Gene Expression Regulation/genetics , Gene Expression Regulation/physiology , Humans , Immediate-Early Proteins , Ion Transport/physiology , Kidney Tubules, Collecting/cytology , Kidney Tubules, Collecting/metabolism , Mice , Nuclear Proteins/genetics , Protein Serine-Threonine Kinases/genetics , Protein Structure, Tertiary/genetics , Protein Structure, Tertiary/physiology , Structure-Activity Relationship , XenopusABSTRACT
Na(+)/H(+) exchanger 3 (NHE3) is the major Na(+) transporter in the intestine. Serum- and glucocorticoid-induced kinase (SGK) 1 interacts with NHE regulatory factor 2 (NHERF2) and mediates activation of NHE3 by dexamethasone (Dex) in cultured epithelial cells. In this study, we compared short-term regulation of NHE3 by Dex in SGK1-null and NHERF2-null mice. In comparison to wild-type mice, loss of SGK1 or NHERF2 significantly attenuated regulation of NHE3 by Dex but did not completely obliterate the effect. We show that transfection of SGK2 or SGK3 in PS120 cells resulted in robust activation of NHE3 by Dex. However, unlike SGK1 or SGK2, SGK3 rapidly activated NHE3 within 15 min of Dex treatment in both PS120 and Caco-2bbe cells. Immunofluorescence analysis showed that SGK3 colocalized with NHE3 in recycling endosomes, whereas SGK1 and SGK2 were diffusely distributed. Mutation of Arg-90 of SGK3 disrupted the endosomal localization of SGK3 and delayed NHE3 activation. Activation of SGK3 and NHE3 by Dex was dependent on phosphoinositide 3-kinase (PI3K) and phosphoinositide-dependent kinase 1 (PDK1), and Dex induced translocation of PDK1 to endosomes. Our study identifies SGK3 as a novel endosomal kinase that acutely regulates NHE3 in a PI3K-dependent mechanism.
Subject(s)
Dexamethasone/pharmacology , Endosomes/drug effects , Epithelial Cells/drug effects , Gene Expression Regulation , Protein Serine-Threonine Kinases/metabolism , Signal Transduction/genetics , Sodium-Hydrogen Exchangers/metabolism , Sodium/metabolism , Animals , Cell Line , Endosomes/metabolism , Epithelial Cells/cytology , Epithelial Cells/metabolism , Glucocorticoids/pharmacology , Humans , Immediate-Early Proteins/genetics , Immediate-Early Proteins/metabolism , Mice , Mice, Knockout , Phosphatidylinositol 3-Kinases , Phosphoproteins/genetics , Phosphoproteins/metabolism , Plasmids , Protein Serine-Threonine Kinases/genetics , Pyruvate Dehydrogenase Acetyl-Transferring Kinase , Sodium-Hydrogen Exchanger 3 , Sodium-Hydrogen Exchangers/genetics , TransfectionABSTRACT
The serum and glucocorticoid-inducible kinase 1 (SGK1) is an inducible kinase the physiological function of which has been characterized primarily in the kidney. Here we show that SGK1 is expressed in white adipose tissue and that its levels are induced in the conversion of preadipocytes into fat cells. Adipocyte differentiation is significantly diminished via small interfering RNA inhibition of endogenous SGK1 expression, whereas ectopic expression of SGK1 in mesenchymal precursor cells promotes adipogenesis. The SGK1-mediated phenotypic effects on differentiation parallel changes in the mRNA levels for critical regulators and markers of adipogenesis, such as peroxisome proliferator-activated receptor gamma, CCAAT enhancer binding protein alpha, and fatty acid binding protein aP2. We demonstrate that SGK1 affects differentiation by direct phosphorylation of Foxo1, thereby changing its cellular localization from the nucleus to the cytosol. In addition we show that SGK1-/- cells are unable to relocalize Foxo1 to the cytosol in response to dexamethasone. Together these results show that SGK1 influences adipocyte differentiation by regulating Foxo1 phosphorylation and reveal a potentially important function for this kinase in the control of fat mass and function.
Subject(s)
Adipocytes, White/metabolism , Adipogenesis , Forkhead Transcription Factors/metabolism , Glucocorticoids/pharmacology , Immediate-Early Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , 3T3-L1 Cells , Adipocytes, White/cytology , Adipocytes, White/drug effects , Adipogenesis/drug effects , Adipogenesis/genetics , Animals , Animals, Genetically Modified , Biomarkers/metabolism , Cell Line , Cells, Cultured , Embryo, Mammalian , Female , Fibroblasts , Forkhead Box Protein O1 , Forkhead Transcription Factors/genetics , Gene Expression Regulation, Enzymologic/drug effects , Humans , Immediate-Early Proteins/antagonists & inhibitors , Immediate-Early Proteins/genetics , Male , Mice , Mice, Inbred C57BL , Organ Specificity , Phosphorylation/drug effects , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/genetics , Protein Transport/drug effects , RNA, Messenger/metabolism , RNA, Small InterferingABSTRACT
Excess mineralocorticoid signaling is deleterious for cardiovascular functions, as demonstrated by the beneficial effects of mineralocorticoid receptor (MR) antagonism on morbidity and mortality in patients with heart failure. However, the understanding of signaling pathways after MR activation in the heart remains limited. We performed transcriptomic analyses in the heart of double-transgenic mice with conditional, cardiomyocyte-specific, overexpression of the MR (MRcardio mice) or the glucocorticoid receptor (GR; GRcardio mice). Some of the genes induced in MRcardio mice were selected for comparative evaluation (real time PCR) in vivo in the heart of mice and ex vivo in the MR-expressing cardiomyocyte H9C2 cell line after aldosterone or corticosterone treatment. We demonstrate that chronic MR overexpression in the heart results in a limited number of induced (n = 24) and repressed (n = 22) genes compared with their control littermates. These genes are specifically modulated by MR because there is limited overlap (three induced, four repressed) with the genes that are regulated in the heart of GRcardio mice (compared with control mice: 70 induced, 73 repressed). Interestingly, some MR-induced genes that are up-regulated in vivo in mice are also induced by 24-h aldosterone treatment in H9C2 cells, such as plasminogen activator inhibitor 1 and Serpina-3 (alpha1-antichymotrypsin). The signaling pathways that are affected by long-term activation of MR may be of particular interest to design novel therapeutic targets in cardiac diseases.
Subject(s)
Gene Expression Profiling , Myocytes, Cardiac/metabolism , Receptors, Mineralocorticoid/physiology , Signal Transduction/physiology , Aldosterone/pharmacology , Animals , Blotting, Western , Cell Line , Corticosterone/pharmacology , Doxycycline/pharmacology , Female , Gene Expression/drug effects , Male , Mice , Mice, Transgenic , Myocardium/metabolism , Myocytes, Cardiac/cytology , Oligonucleotide Array Sequence Analysis , Pregnancy , Rats , Receptors, Mineralocorticoid/genetics , Reverse Transcriptase Polymerase Chain Reaction , Serpins/genetics , Serpins/metabolism , Signal Transduction/geneticsABSTRACT
Aldosterone is the principal regulator of Na homeostasis, and thereby blood pressure. One of the main targets of aldosterone is the epithelial Na channel (ENaC) located in the apical membrane of target cells. Previous studies identified several genes involved in the regulation of ENaC such as SGK1; however, SGK1 knockout mice have only a mild salt-losing phenotype, indicating that further genes must be involved in the action of aldosterone. In our search for further aldosterone-regulated genes, we discovered that aldosterone, at physiological concentrations, induces the expression of the promyelocytic leukemia zinc finger protein (PLZF) in renal cortical collecting duct (CCD) cell lines that stably express mineralocorticoid receptors (MRs). This effect is rapid and does not require de novo protein synthesis, suggesting a direct action. Surprisingly, stable overexpression of human or mouse PLZF isoforms significantly decreased transepithelial Na transport in CCD cells while having no effect on the integrity of the monolayers. In parallel with the decline in Na transport, PLZF suppressed the mRNA levels of beta- and gamma-ENaC subunits. These observations suggest that PLZF is a negative regulator of ENaC in renal epithelial cells and might be part of a negative feedback loop that limits aldosterone's stimulatory effects on sodium reabsorption.
Subject(s)
Aldosterone/physiology , Epithelial Sodium Channels/physiology , Kruppel-Like Transcription Factors/physiology , Animals , Doxycycline/pharmacology , Epithelial Sodium Channels/drug effects , Histone Deacetylase Inhibitors , Humans , Mice , Promyelocytic Leukemia Zinc Finger Protein , RNA, Messenger/metabolism , Rats , Zinc Fingers/physiology , p300-CBP Transcription Factors/antagonists & inhibitorsABSTRACT
Amiloride-sensitive Na(+) channel activity was examined in the cortical collecting ducts of a mouse line (SGK1(-/-)) deficient in the serum- and glucocorticoid-dependent protein kinase SGK1. This activity was correlated with changes in renal Na handling and in the maturation of epithelial Na(+) channel (ENaC) protein. Neither SGK1(-/-) mice nor paired SGK1(+/+) animals expressed detectable channel activity, measured as amiloride-sensitive whole-cell current (I(Na)), under control conditions with standard chow. Administration of aldosterone (0.5 microg/h via osmotic minipump for 7 days) increased I(Na) to a similar extent in SGK1(+/+) (378 +/- 61 pA/cell at -100 mV) and in SGK1(-/-) (350 +/- 57 pA/cell) animals. However, the maturation of ENaC, assessed as the ratio of cleaved to full-length forms of gamma-ENaC, was more pronounced in SGK(+/+) mice. The SGK1(-/-) animals exhibited a salt-wasting phenotype when kept on a low-Na diet for up to 2 days, losing significantly more Na in the urine than wild-type mice. Under these conditions, I(Na) was enhanced more in SGK1(-/-) (94 +/- 14 pA/cell) than in SGK(+/+) (23 +/- 5 pA/cell) genotypes. Despite the larger currents, the ratio of cleaved to full-length gamma-ENaC was lower in the knockout animals. The mice also expressed a smaller amount of Na(+)-Cl(-) cotransporter protein under Na-depleted conditions. These results indicated that SGK1 is essential for optimal processing of ENaC but is not required for activation of the channel by aldosterone.