Anikó Náray-Fejes-Tóth

sgk Is an Aldosterone-induced Kinase in the Renal Collecting Duct EFFECTS ON EPITHELIAL Na+ CHANNELS

The early phase of the stimulatory effect of aldosterone on sodium reabsorption in renal epithelia is thought to involve activation of apical sodium channels. However, the genes initiating this effect are unknown. We used a combination of polymerase chain reaction-based subtractive hybridization and differential display techniques to identify aldosterone-regulated immediate early genes in renal mineralocorticoid target cells. We report here that aldosterone rapidly increases mRNA levels of a putative Ser/Thr kinase,sgk (or serum- andglucocorticoid-regulated kinase), in its native target cells, i.e. in cortical collecting duct cells. The effect occurs within 30 min of the addition of aldosterone, is mediated through mineralocorticoid receptors, and does not require de novo protein synthesis. The full-length sequences of rabbit and mouse sgk cDNAs were determined. Both cDNAs show significant homology to rat and human sgk (88–94% at the nucleotide level, and 96–99% at the amino acid level). Coexpression of the mouse sgk in Xenopus oocytes with the three subunits of the epithelial Na+ channel results in a significantly enhanced Na+ current. These results suggest that sgk is an immediate early aldosterone-induced gene, and this protein kinase plays an important role in the early phase of aldosterone-stimulated Na+ transport.

Glucocorticoids and Stress: Permissive and Suppressive Actions

Protection against stress by glucocorticoids is discussed in relation to their permissive and suppressive actions. Evidence from the last decade is summarized regarding the physiological nature of the suppressive actions, and the hypothesis that they prevent stress-activated defense mechanisms from overshooting and damaging the organism. Support for this hypothesis has come from observations on how endogenous or administered glucocorticoids control inflammatory and immune responses, protect in endotoxic and hemorrhagic shock, regulate central nervous system responses to stimuli, and moderate many defense reactions through suppression of cytokines and other mediators. Studies showing that glucocorticoids permissively induce receptors for several mediators that they suppress have led to a model in which stimulated activity of a mediator system is increased permissively through induction of mediator receptors and decreased through suppression of mediator production.

Epithelial Na channel activation and processing in mice lacking SGK1

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.

The AGC kinase SGK1 regulates TH1 and TH2 differentiation downstream of the mTORC2 complex

Serum- and glucocorticoid-regulated kinase 1 (SGK1) is an AGC kinase that regulates membrane sodium channel expression in renal tubular cells in an mTORC2-dependent manner. We hypothesized that SGK1 might represent a novel mTORC2-dependent regulator of T cell differentiation and function. Here we demonstrate that upon activation by mTORC2, SGK1 promoted TH2 differentiation by negatively regulating the NEDD4-2 E3 ligase-mediated destruction of transcription factor JunB. Simultaneously, SGK1 repressed the production of interferon-γ (IFN-γ) by controlling the expression of the long isoform of transcription factor TCF-1. Consistent with these findings, mice with a selective deletion of SGK1 in T cells were resistant to experimentally induced asthma, generated robust amounts of IFN-γ in response to viral infections and more readily rejected tumors.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.

Subcellular localization of the type 2 11beta-hydroxysteroid dehydrogenase. A green fluorescent protein study.

11β-Hydroxysteroid dehydrogenase (11β-HSD) is thought to confer aldosterone specificity to mineralocorticoid target cells by protecting the inherently non-selective mineralocorticoid receptor (MR) from occupancy by endogenous glucocorticoids. Recently, we characterized a novel isoform of 11β-HSD in aldosterone target cells, which has high affinity for its substrate, is unidirectional, and prefers NAD as cofactor. In this study we utilized a green fluorescent protein (GFP) technique to determine the subcellular localization of this isoform, 11β-HSD2. We generated a chimeric gene encoding the full-length rabbit 11β-HSD2 and, fused to its C terminus, the coding sequence of GFP. This construct was stably transfected into CHO cells. The enzymatic characteristics of the expressed 11β-HSD2/GFP fusion protein were undistinguishable from those of the native enzyme: high affinity for corticosterone (KM 8–10 nM), NAD dependence, and lack of reductase activity. The intracellular location of the recombinant protein was determined by fluorescence microscopy. 11β-HSD2-associated fluorescence was observed as a reticular network over the cytoplasm and nuclear envelope, whereas the plasma membrane and the nucleus were negative, suggesting endoplasmic reticulum (ER) localization. Staining of CHO cells expressing 11β-HSD2/GFP with established subcellular organelle markers revealed a colocalization of 11β-HSD2/GFP only with ER markers and tubulin. To examine the orientation of 11β-HSD2 within the ER, we selectively permeabilized CHO cells and stained them with an anti-GFP antibody. Fluorescence microscopy indicated that the C-terminal region of 11β-HSD2 is on the cytoplasmic surface of the ER membrane, since it was accessible to the GFP antibody. This conclusion was confirmed by trypsin treatment of permeabilized cells followed by Western blotting. The C-terminal region of 11β-HSD2 was accessible to trypsin, indicating that it is on the cytoplasmic side of the ER membrane. These results indicate that 11β-HSD2 is localized exclusively to the ER. Since 11β-HSD2 does not contain any known ER retrieval signal, experiments are currently under way to determine what structural motifs are responsible for its ER localization.

Inducible kidney-specific Sgk1 knockout mice show a salt-losing phenotype

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.

SGK is a primary glucocorticoid-induced gene in the human.

Serum- and glucocorticoid-induced kinase (sgk) is transcriptionally regulated by corticosteroids in several cell types. Recent findings suggest that sgk is an important gene in the early action of corticosteroids on epithelial sodium reabsorption. Surprisingly, the human sgk was reported not to be transcriptionally regulated by corticosteroids in a hepatoma cell line, and thus far no glucocorticoid response element has been identified in the human SGK gene. Since humans clearly respond to both aldosterone and glucocorticoids in cells where sgk action seems to be important, in this study we determined sgk mRNA levels following dexamethasone treatment for various duration in five human cell lines. These cell lines included epithelial cells (H441, T84 and HT29) and lymphoid/monocyte (U937 and THP-1) lines. Using quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), we found that sgk mRNA levels are markedly induced by glucocorticoids in all of the five cell lines studied. Time course analyses revealed that sgk mRNA levels are elevated as early as 30 min after addition of the glucocorticoid, and remain elevated for several hours. Northern analysis in H441 cells confirmed that sgk is an early induced gene. The induction of sgk by dexamethasone was unaffected by cycloheximide, indicating that it does not require de novo protein synthesis. These results indicate that the human sgk, just like its counterparts in other species, is a primary glucocorticoid-induced gene.

Regulation of sodium transport in mammalian collecting duct cells by aldosterone-induced kinase, SGK1: structure/function studies.

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 SGKs 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.

Serum- and glucocorticoid-inducible kinase 1 (SGK1) regulates adipocyte differentiation via forkhead box O1.

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.

The role of 11β-hydroxysteroid dehydrogenase in steroid hormone specificity

11Beta-hydroxysteroid dehydrogenase (11beta-HSD) is thought to confer aldosterone specificity to mineralocorticoid target cells by protecting the mineralocorticoid receptor (MR) from occupancy by endogenous glucocorticoids. In aldosterone target cells the type 2 11beta-HSD is present, which, in contrast to the type 1 11beta-HSD, has very high affinity for its substrate, is unidirectional and prefers NAD as cofactor. cDNAs encoding 11beta-HSD2 have been recently cloned from different species, and the cell-specific expression of its mRNA and protein were determined. 11Beta-HSD2 is expressed in every aldosterone target tissue. Northern analysis revealed that the rabbit 11beta-HSD2 is expressed at high levels in the renal collecting duct and at much lower levels in the colon. RT-PCR experiments demonstrated that 11beta-HSD2 mRNA is present only in aldosterone target cells within the kidney. We determined the subcellular localization of the rabbit 11beta-HSD2 using a chimera encoding 11beta-HSD2 and the green fluorescent protein (GFP). This construct was stably transfected into CHO and MDCK cells. The expressed 11beta-HSD2/GFP protein retained high enzymatic activity, and its characteristics were undistinguishable from those of the native enzyme. The intracellular localization of this protein was determined by fluorescence microscopy. 11Beta-HSD2-associated fluorescence was observed as a reticular network over the cytoplasm whereas the plasma membrane and the nucleus were negative, suggesting endoplasmic reticulum (ER) localization. Co-staining with markers for ER proteins, the Golgi membrane, mitochondria and nucleus confirmed that 11beta-HSD2 is localized exclusively to the ER. To determine what structural motifs are responsible for the ER localization, we generated deletion mutants missing the C-terminal 42 and 118 amino acids, and fused them to GFP. Similarly as with the intact 11beta-HSD2, these mutants localized exclusively to the ER. Both C-terminal deletion mutants completely lost dehydrogenase activity, independently whether activity was determined in intact cells or homogenates. These results indicate that 11beta-HSD2 has a novel ER retrieval signal which is not localized to the C-terminal region. In addition, the C-terminal 118 amino acids are essential for NAD-dependent 11beta-HSD activity.