Serum- and glucocorticoid-induced protein kinase 1 (SGK1) is regulated by store-operated Ca2+ entry and mediates cytoprotection against necrotic cell death.

Serum and glucocorticoid-regulated kinase 1 (SGK1) encodes a phosphatidylinositol 3-kinase-dependent serine/threonine kinase that is rapidly induced in response to cellular stressors and is an important cell survival signal. Previous studies have suggested that an increase in cytoplasmic Ca(2+) concentration ([Ca(2+)]c) is required for increased SGK1 expression, but the subcellular source of Ca(2+) regulating SGK1 transcription remains uncertain. Activation of endoplasmic reticulum stress (ERS) with thapsigargin (TG) increased SGK1 mRNA and protein expression in MDA-MB-231 cells. Intracellular Ca(2+) imaging revealed that store-operated Ca(2+) entry played a prominent role in SGK1 induction by TG. Neither ERS nor release of Ca(2+) from the ER was sufficient to activate SGK1. Prolonged elevation of intracellular Ca(2+) levels, however, triggered cell death with a much greater proportion of the cells undergoing necrosis rather than apoptosis. A relative increase in the percentage of cells undergoing necrosis was observed in cells expressing a short hairpin RNA targeted to the SGK1 gene. Necrotic cell death evoked by cytoplasmic Ca(2+) overloading was associated with persistent hyperpolarization of the inner mitochondrial membrane and a modest increase in calpain activation, but did not involve detectable caspase 3 or caspase 7 activation. The effects of cytoplasmic Ca(2+) overloading on mitochondrial membrane potential were significantly reduced in cells expressing SGK1 compared with SGK1-depleted cells. Our findings indicate that store-operated Ca(2+) entry regulates SGK1 expression in epithelial cells and suggest that SGK1-dependent cytoprotective signaling involves effects on maintaining mitochondrial function.

Administration of glucocorticoids to ovarian cancer patients is associated with expression of the anti-apoptotic genes SGK1 and MKP1/DUSP1 in ovarian tissues.

PURPOSE: To prevent chemotherapy-related side effects, synthetic glucocorticoids, for example, dexamethasone, are routinely administered to patients with ovarian cancer. However, preclinical data implicate glucocorticoids in suppressing chemotherapy-mediated apoptosis in epithelial tumors. The anti-apoptotic mechanisms underlying this increased survival have been shown to require up-regulation of prosurvival genes, including serum and glucocorticoid-regulated kinase 1 (SGK1) and map kinase phosphatase 1 (MKP1)/dual specificity phosphatase 1 (DUSP1). Despite abundant preclinical data, there are no correlative studies in patients. We therefore evaluated anti-apoptotic gene expression in tumor samples from patients randomized to dexamethasone or normal saline. EXPERIMENTAL DESIGN: Eighteen patients were randomized before exploratory laparotomy for suspected ovarian cancer. Dexamethasone or normal saline was administered i.v. following anesthesia. Ovarian and omental tumor samples were collected intra-operatively before and after infusion. Samples were analyzed for histology and glucocorticoid receptor expression by immunohistochemistry. SGK1 and MKP1/DUSP1 mRNA levels were determined using quantitative real-time PCR. RESULTS: Ten patients were evaluable. At 30 min postinfusion, tumor samples from five patients receiving dexamethasone revealed an average SGK1 mRNA induction of 6.1-fold (SEM, +/-2.6) compared with only 1.5-fold (SEM, +/-0.4) in tumor samples from five patients receiving normal saline (P = 0.028). Average MKP1/DUSP1 mRNA expression was increased by 8.2-fold (SEM, +/-2.9) following dexamethasone versus 1.1-fold (SEM, +/-0.4) following normal saline (P = 0.009). All samples expressed glucocorticoid receptor. CONCLUSION: Glucocorticoid administration to patients is associated with rapid up-regulation of SGK1 and MKP1 expression in ovarian tumors. This finding supports the hypothesis that pharmacologic doses of glucocorticoids may decrease chemotherapy effectiveness in ovarian cancer patients through increased anti-apoptotic gene expression.

Hsp90 regulates the phosphorylation and activity of serum- and glucocorticoid-regulated kinase-1.

SGK-1 (serum- and glucocorticoid-regulated kinase-1), a member of the AGC protein kinase family, plays an important role in regulating ion channel expression and contributes to malignant epithelial cell proliferation and survival. SGK-1 activity is regulated on three levels: transcriptional induction following a variety of environmental and intracellular stresses, proteasomal degradation, and phosphorylation. Here we report that phosphoinositide 3-kinase (PI3K)-dependent phosphorylation of SGK-1 requires formation of a complex between SGK-1 and heat-shock protein 90 (Hsp90). Inactivation of Hsp90 by geldanamycin led to decreased SGK-1 phosphorylation independently of increased proteasomal protein degradation, and inhibition of PI3K activity by LY294002 appeared to eliminate SGK-1 phosphorylation at the same residues as those affected by geldanamycin treatment. Interestingly, geldanamycin-targeted phosphorylation sites were not limited to the known conserved PI3K-dependent sites Thr-256 and Ser-422 in SGK-1 but included additional unknown PI3K-dependent residues. Inhibition of Hsp90 also resulted in a complete loss of SGK-1 kinase activity, suggesting that Hsp90 activity is essential for regulating the PI3K/SGK-1 pathway.

Glucocorticoid (GC)-mediated down-regulation of urokinase plasminogen activator expression via the serum and GC regulated kinase-1/forkhead box O3a pathway.

The glucocorticoid receptor (GR) and its ligand, cortisol, play a central role in human physiology. The exact mechanisms by which GR activation regulates these processes are the subject of intensive investigation. We and others have shown that GR activation can indirectly down-regulate specific genes via serum and glucocorticoid (GC) regulated kinase-1-mediated inhibition of forkhead box O3a (FOXO3a) transcriptional activity. We previously used gene expression microarrays, together with bioinformatic analyses, to identify putative FOXO3a target genes in breast epithelial cells. In this paper we refine our analysis through the use of FOXO3a chromatin immunoprecipitation (ChIP) microarrays. ChIP microarray results reveal urokinase plasminogen activator (uPA) as a putative novel target of FOXO3a in breast epithelial and breast cancer cell lines. We further show that uPA down-regulation after GC treatment requires serum and GC regulated kinase-1-mediated inactivation of FOXO3a activity. ChIP and luciferase assays confirm that FOXO3a can both occupy and transactivate the uPA promoter. Our data suggest that inactivation of FOXO3a after GR activation is an important mechanism contributing to GC-mediated repression of uPA gene expression in breast epithelial and cancer cells.

Ubiquitin-proteasome degradation of serum- and glucocorticoid-regulated kinase-1 (SGK-1) is mediated by the chaperone-dependent E3 ligase CHIP.

SGK-1 (serum- and glucocorticoid-regulated kinase-1) is a stress-induced serine/threonine kinase that is phosphorylated and activated downstream of PI3K (phosphoinositide 3-kinase). SGK-1 plays a critical role in insulin signalling, cation transport and cell survival. SGK-1 mRNA expression is transiently induced following cellular stress, and SGK-1 protein levels are tightly regulated by rapid proteasomal degradation. In the present study we report that SGK-1 forms a complex with the stress-associated E3 ligase CHIP [C-terminus of Hsc (heat-shock cognate protein) 70-interacting protein]; CHIP is required for both the ubiquitin modification and rapid proteasomal degradation of SGK-1. We also show that CHIP co-localizes with SGK-1 at or near the endoplasmic reticulum. CHIP-mediated regulation of SGK-1 steady-state levels alters SGK-1 kinase activity. These data suggest a model that integrates CHIP function with regulation of the PI3K/SGK-1 pathway in the stress response.

A novel N-terminal hydrophobic motif mediates constitutive degradation of serum- and glucocorticoid-induced kinase-1 by the ubiquitin-proteasome pathway.

Serum- and glucocorticoid-induced protein kinase-1 (SGK-1) plays a critical role in regulation of the epithelial sodium channel, ENaC. SGK-1 also shares significant catalytic domain homology with protein kinase B (PKB/AKT-1) and is a downstream effector of antiapoptotic phosphoinositide 3-kinase signaling. Steady-state levels of an active SGK-1 are tightly regulated by rapid transcriptional activation and post-translational modification including phosphorylation. We show here that endogenous SGK-1 protein is polyubiquitinated and rapidly degraded by the 26S proteasome. In contrast to other rapidly degraded kinases, neither the catalytic activity of SGK-1 nor activation site phosphorylation was required for its ubiquitin modification and degradation. Instead, SGK-1 degradation required a lysine-less six-amino-acid (amino acids 19-24) hydrophobic motif (GMVAIL) within the N-terminal domain. Deletion of amino acids 19-24 significantly increased the half-life of SGK1 and prevented its ubiquitin modification. Interestingly, this minimal region was also required for the association of SGK-1 with the endoplasmic reticulum. Ubiquitin modification and degradation of SGK-1 were increasingly inhibited by the progressive mutation of six N-terminal lysine residues surrounding the GMVAIL motif. Mutation of all six lysines to arginine did not disrupt the subcellular localization of SGK-1 despite a significant decrease in ubiquitination, implying that this modification per se was not required for targeting to the endoplasmic reticulum. These results suggest that constitutive ubiquitin-mediated degradation of SGK-1 is an important mechanism regulating its biological activity.

Glucocorticoid receptor activation signals through forkhead transcription factor 3a in breast cancer cells.

Activation of the glucocorticoid receptor (GR) plays a critical role in the stress response of virtually all cell types. Despite recent advances in large-scale genomic and proteomic data acquisition, identification of physiologically relevant molecular events downstream of nuclear hormone receptor activation remains challenging. By analyzing gene expression changes 30 min after dexamethasone (Dex) treatment, we previously found that immediate induction of serum and glucocorticoid-regulated kinase-1 (SGK-1) expression is required for GR-mediated mammary epithelial cell survival signaling. We now report that activation of the GR mediates Forkhead transcription factor 3a (FOXO3a) phosphorylation and inactivation in mammary epithelial cells. GR-mediated induction of SGK-1 expression is required for FOXO3a inactivation; additional growth factor stimulation is not required. To further explore the gene expression changes that occur downstream of GR-mediated FOXO3a inactivation, we analyzed temporal gene expression data and selected GR-down-regulated genes containing core FOXO3a binding motifs in their proximal promoters. This approach revealed several previously unrecognized transcriptional target genes of FOXO3a, including IGF binding protein-3 (IGFBP-3). Endogenous IGFBP-3 expression was confirmed to be dependent on the GR-SGK-1-FOXO3a signaling pathway. Moreover, GR activation decreased FOXO3a-induced apoptosis in SK-BR-3 breast cancer cells. Collectively, our data suggest that GR-mediated FOXO3a inactivation is an important mechanism contributing to glucocorticoid-mediated mammary epithelial cell survival.

Coordinate expression of the PI3-kinase downstream effectors serum and glucocorticoid-induced kinase (SGK-1) and Akt-1 in human breast cancer.

The phosphatidylinositol 3-kinase (PI3-K) signalling pathway has been implicated in breast cancer development and resistance to therapy. Akt-1 and serum and glucocorticoid-induced kinase-1 (SGK-1) are homologous kinases which are important downstream effectors of PI3-K signalling. We sought to determine the individual expression patterns of these two kinases in order to better understand their respective roles in PI3-K signalling in breast cancer. To this end, we examined the expression of both p-Akt-1 and SGK-1 in 40 breast cancers. p-Akt-1 expression was seen in 58% of tumour samples, while SGK-1 overexpression was detected in 48%. Interestingly, a highly significant association was found between the expression of p-Akt-1 and SGK-1 (P=0.002), suggesting complementary physiological functions in PI3-K signalling. This finding is consistent with recent genetic data from Caenorhabditis elegans suggesting that both SGK-1 and Akt-1 are required for signalling downstream of insulin receptor activation.

Anti-insulin receptor autoantibodies are not required for type 2 diabetes pathogenesis in NZL/Lt mice, a New Zealand obese (NZO)-derived mouse strain.

The New Zealand obese (NZO) mouse strain shares with the related New Zealand black (NZB) strain a number of immunophenotypic traits. Among these is a high proportion of B-1 B lymphocytes, a subset associated with autoantibody production. Approximately 50% of NZO/HlLt males develop a chronic insulin-resistant type 2 diabetes syndrome associated with 2 unusual features: the presence of B lymphocyte-enriched peri-insular infiltrates and the development of anti-insulin receptor autoantibodies (AIRAs). To establish the potential pathogenic contributions of B lymphocytes and AIRAs in this model, a disrupted immunoglobulin heavy chain gene (Igh-6) congenic on the NZB/BlJ background was backcrossed 4 generations into the NZO/HlLt background and was then intercrossed to produce mice that initially segregated for wild-type versus the mutant Igh-6 allele and thus permitted comparison of syndrome development. A new flow cytometric assay (AIRA binding to transfected Chinese hamster ovary cells stably expressing mouse insulin receptor) showed IgM and IgG subclass AIRAs in serum from Igh-6 intact males, but not in Igh-6null male serum. However, the absence of B lymphocytes and antibodies distinguishing mutant from wild-type males failed to significantly affect diabetes-free survival. The Igh-6null males gained weight less rapidly than wild-type males, probably accounting for a retardation, but not prevention, of hyperglycemia. Thus, AIRA and the B-lymphocyte component of the peri-insulitis in chronic diabetics were not essential either to development of insulin resistance or to eventual pancreatic beta cell failure and loss. A new substrain, designated NZL, was generated by inbreeding Igh-6 wild-type segregants. Currently at the F10 generation, NZL mice exhibit the same juvenile-onset obesity as NZO/HlLt males, but develop type 2 diabetes at a higher frequency (> 80%). Also, unlike NZO/HlLt mice that are difficult to breed, the NZL/Lt strain breeds well and thus offers clear advantages to obesity/diabetes researchers.

Microarray analysis reveals glucocorticoid-regulated survival genes that are associated with inhibition of apoptosis in breast epithelial cells.

Activation of the glucocorticoid receptor (GR) results in diverse physiological effects depending on cell type. For example, glucocorticoids (GC) cause apoptosis in lymphocytes but can rescue mammary epithelial cells from growth factor withdrawal-induced death. However, the molecular mechanisms of GR-mediated survival remain poorly understood. In this study, a large-scale oligonucleotide screen of GR-regulated genes was performed. Several of the genes that were found to be induced 30 min after GR activation encode proteins that function in cell survival signaling pathways. We also demonstrate that dexamethasone pretreatment of breast cancer cell lines inhibits chemotherapy-induced apoptosis in a GR-dependent manner and is associated with the transcriptional induction of at least two genes identified in our screen, serum and GC-inducible protein kinase-1 (SGK-1) and mitogen-activated protein kinase phosphatase-1 (MKP-1). Furthermore, GC treatment alone or GC treatment followed by chemotherapy increases both SGK-1 and MKP-1 steady-state protein levels. In the absence of GC treatment, ectopic expression of SGK-1 or MKP-1 inhibits chemotherapy-induced apoptosis, suggesting a possible role for these proteins in GR-mediated survival. Moreover, specific inhibition of SGK-1 or MKP-1 induction by the introduction of SGK-1- or MKP-1-small interfering RNA reversed the anti-apoptotic effects of GC treatment. Taken together, these data suggest that GR activation in breast cancer cells regulates survival signaling through direct transactivation of genes that encode proteins that decrease susceptibility to apoptosis. Given the widespread clinical administration of dexamethasone before chemotherapy, understanding GR-induced survival mechanisms is essential for achieving optimal therapeutic responses.

Ubiquitin modification of serum and glucocorticoid-induced protein kinase-1 (SGK-1).

The serum and glucocorticoid-induced protein kinase gene (sgk-1) encodes a multifunctional kinase that can be phosphorylated and activated through a phosphatidylinositol 3-kinase-dependent signaling pathway. In many cell types, endogenous SGK-1 steady-state protein levels are very low but can be acutely up-regulated after glucocorticoid receptor-mediated transcriptional activation; in breast epithelial and cancer cell lines, this up-regulation is associated with promotion of cell survival. We and others have noted that ectopically introduced full-length SGK-1 is poorly expressed, although SGK-1 lacking the first 60 amino acids (delta60SGK-1) is expressed at much higher-fold protein levels than wild-type SGK-1 in both human embryonic kidney 293T and MCF10A mammary epithelial cells. In this report, we demonstrate for the first time that the low steady-state expression level of SGK-1 is due to polyubiquitination and subsequent degradation by the 26S proteasome. Deletion of the amino-terminal 60 amino acids of SGK-1 results in a mutant SGK-1 protein that is neither efficiently polyubiquitinated nor degraded by the 26S proteasome, accounting for the higher steady-state levels of the truncated protein. We also demonstrate that a subset of SGK-1 localizes to the plasma membrane and that the polyubiquitin-modified SGK-1 localizes to a membrane-associated fraction of the cell. Taken together, these data suggest that a significant fraction of SGK-1 is membrane-associated and ubiquitinated. These findings are consistent with the recently described role of SGK-1 in phosphorylating the membrane-associated protein Nedd4-2 and the integral membrane Na+/H+ exchanger isoform 3 (NHE3) and suggest a novel mechanism of regulation of SGK-1.