Serum- And Glucocorticoid-regulated Kinase Research Articles

Oximes: Novel Therapeutics with Anticancer and Anti-Inflammatory Potential.

Oximes have been studied for decades because of their significant roles as acetylcholinesterase reactivators. Over the last twenty years, a large number of oximes have been reported with useful pharmaceutical properties, including compounds with antibacterial, anticancer, anti-arthritis, and anti-stroke activities. Many oximes are kinase inhibitors and have been shown to inhibit over 40 different kinases, including AMP-activated protein kinase (AMPK), phosphatidylinositol 3-kinase (PI3K), cyclin-dependent kinase (CDK), serine/threonine kinases glycogen synthase kinase 3 α/β (GSK-3α/β), Aurora A, B-Raf, Chk1, death-associated protein-kinase-related 2 (DRAK2), phosphorylase kinase (PhK), serum and glucocorticoid-regulated kinase (SGK), Janus tyrosine kinase (JAK), and multiple receptor and non-receptor tyrosine kinases. Some oximes are inhibitors of lipoxygenase 5, human neutrophil elastase, and proteinase 3. The oxime group contains two H-bond acceptors (nitrogen and oxygen atoms) and one H-bond donor (OH group), versus only one H-bond acceptor present in carbonyl groups. This feature, together with the high polarity of oxime groups, may lead to a significantly different mode of interaction with receptor binding sites compared to corresponding carbonyl compounds, despite small changes in the total size and shape of the compound. In addition, oximes can generate nitric oxide. This review is focused on oximes as kinase inhibitors with anticancer and anti-inflammatory activities. Oximes with non-kinase targets or mechanisms of anti-inflammatory activity are also discussed.

Identification and Kinetic Characterization of Serum- and Glucocorticoid-Regulated Kinase Inhibitors Using a Fluorescence Polarization-Based Assay.

The serum- and glucocorticoid-regulated kinase (SGK) family consists of three isoforms (SGK1, SGK2, and SGK3) that have been implicated in the regulation of tumor growth, metastasis, autophagy, and epithelial ion transport. SGK1 and SGK3 play essential roles in protein kinase B (AKT or PKB)-independent phosphoinositide 3-kinases (PI3K)-mediated tumorigenesis, as evidenced by the significantly elevated expression levels of SGK1 and SGK3 in many cancers, including prostate cancer, colorectal carcinoma, estrogen-dependent breast cancer, and glioblastoma. Therefore, SGK is a potential target for anticancer therapy. A small kinase-focused library comprising 160 compounds was screened against SGK1 using a fluorescence polarization-based kinase assay that yielded a Z'-factor of 0.82. Among the 39 compounds obtained as initial hits in a primary screen, 12 compounds contained the thiazolidine-2,4-dione scaffold. The inhibitory mechanisms of the most potent hit, KMU010402, were further investigated using kinetic analyses, followed by determination of the inhibition constants for SGK1, SGK2, and SGK3. Molecular modeling was used to propose a potential binding mode of KMU010402 to SGK1.

Aberrant Claudin-6-Adhesion Signaling Promotes Endometrial Cancer Progression via Estrogen Receptor α.

Cell adhesion proteins not only maintain tissue integrity, but also possess signaling abilities to organize diverse cellular events in a variety of physiologic and pathologic processes; however, the underlying mechanism remains obscure. Among cell adhesion molecules, the claudin (CLDN) family is often aberrantly expressed in various cancers, but the biological relevance and molecular basis for this observation have not yet been established. Here, we show that high CLDN6 expression accelerates cellular proliferation and migration in two distinct human endometrial cancer cell lines in vitro. Using a xenograft model, we also revealed that aberrant CLDN6 expression promotes tumor growth and invasion in endometrial cancer tissues. The second extracellular domain and Y196/200 of CLDN6 were required to recruit and activate Src-family kinases (SFK) and to stimulate malignant phenotypes. Knockout and overexpression of ESR1 in endometrial carcinoma cells showed that the CLDN6-adhesion signal links to estrogen receptor α (ERα) to advance tumor progression. In particular, aberrant CLDN6-ERα signaling contributed to collective cell behaviors in the leading front of endometrial cancer cells. Importantly, we demonstrate that CLDN6/SFK/PI3K-dependent AKT and SGK (serum- and glucocorticoid-regulated kinase) signaling in endometrial cancer cells targets Ser518 in the human ERα to activate ERα transcriptional activity in a ligand-independent manner, thereby promoting tumor progression. Furthermore, CLDN6, at least in part, also regulated gene expression in an ERα-independent manner. IMPLICATIONS: The identification of this machinery highlights regulation of the transcription factors by cell adhesion to advance tumor progression.

Activation of Serum/Glucocorticoid Regulated Kinase 1/Nuclear Factor-κB Pathway Are Correlated with Low Sensitivity to Bortezomib and Ixazomib in Resistant Multiple Myeloma Cells

Multiple myeloma (MM) is an incurable malignancy often associated with primary and acquired resistance to therapeutic agents, such as proteasome inhibitors. However, the mechanisms underlying the proteasome inhibitor resistance are poorly understood. Here, we elucidate the mechanism of primary resistance to bortezomib and ixazomib in the MM cell lines, KMS-20, KMS-26, and KMS-28BM. We find that low bortezomib and ixazomib concentrations induce cell death in KMS-26 and KMS-28BM cells. However, high bortezomib and ixazomib concentrations induce cell death only in KMS-20 cells. During Gene Expression Omnibus analysis, KMS-20 cells exhibit high levels of expression of various genes, including anti-phospho-fibroblast growth factor receptor 1 (FGFR1), chemokine receptor type (CCR2), and serum and glucocorticoid regulated kinase (SGK)1. The SGK1 inhibitor enhances the cytotoxic effects of bortezomib and ixazomib; however, FGFR1 and CCR2 inhibitors do not show such effect in KMS-20 cells. Moreover, SGK1 activation induces the phosphorylation of NF-κB p65, and an NF-κB inhibitor enhances the sensitivity of KMS-20 cells to bortezomib and ixazomib. Additionally, high levels of expression of SGK1 and NF-κB p65 is associated with a low sensitivity to bortezomib and a poor prognosis in MM patients. These results indicate that the activation of the SGK1/NF-κB pathway correlates with a low sensitivity to bortezomib and ixazomib, and a combination of bortezomib and ixazomib with an SGK1 or NF-κB inhibitor may be involved in the treatment of MM via activation of the SGK1/NF-κB pathway.

Abstract 1432: TORC2 dependent control of cell growth and size in cancer cells

Abstract Severe defects in cell growth and size are a nearly universal feature of cancer cells and have provided a basis for cancer pathology for over 100 years; however, the underlying mechanisms remain largely unknown. Recent work in budding yeast has shown that a highly conserved signaling network surrounding Tor complex 2 (TORC2) controls cell growth and size. A key component of the network is the yeast homolog of serum and glucocorticoid-regulated kinase (SGK), which controls production of ceramide signaling lipids that control cell growth and size. Here, we present evidence that a similar TORC2 network controls cell growth and size in mammalian cells. In addition, we show that expression of oncogenic drivers in NIH3T3 cells causes defects in cell size, as well as defects in TORC2-dependent control of cell size. Cancer cells also show severe defects in TORC2-dependent control of cell size. Our working hypothesis is that the TORC2 network is downstream of multiple oncogenes, and that aberrant TORC2 signaling is a major cause of the nearly universal size defects observed in cancer cells. A better understanding of the mechanisms that control cell growth and size in normal cells, and how they go awry in cancer, could identify new targets for drugs to improve cancer therapies. Citation Format: Jerry T. DeWitt, Douglas Kellogg. TORC2 dependent control of cell growth and size in cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1432.

GR, Sgk1, and NDRG1 in esophageal squamous cell carcinoma: their correlation with therapeutic outcome of neoadjuvant chemotherapy

BackgroundEsophageal squamous cell carcinoma (ESCC) is a highly malignant neoplasm.The glucocorticoid (GC)-glucocorticoid receptor (GR) pathway plays pivotal roles in cellular response to various stresses of tumor cells, including chemotherapy. However, the status of the GC-GR pathway in ESCC, including its correlation with chemotherapeutic responses, is largely unknown.MethodsGR, serum-and glucocorticoid-regulated kinase 1 (Sgk1), and N-myc down regulation gene 1 (NDRG1) were immunolocalized in 98 patients with ESCC who had undergone esophagectomy following neoadjuvant chemotherapy (NAC) with 2 courses of 5-fluorouracil + cisplatin. We also examined biopsy specimens before NAC in 42 cases and compared the results between those before and after NAC.ResultsOverall survival (OS) of the patients treated with surgery following NAC was significantly shorter in the group with high GR than that with low GR status (P = 0.0473). Both OS and disease-free survival (DFS) were significantly shorter in both Sgk1- and NDRG1-high groups than in the low groups (OS: Sgk1, P = 0.0055; NDRG1, P = 0.0021; DFS: Sgk1, P = 0.0240; NDRG1, P = 0.0086). Biopsy specimens before NAC showed significantly shorter DFS in the high Sgk1 group (P = 0.0095), while both OS and DFS were shorter in the high NDRG1 group (OS, P = 0.0233; DFS, P = 0.0006) than in the respective low groups. In the high NDRG1 group of biopsy specimens before NAC, the tumor reduction rate by NAC was significantly attenuated (P = 0.021).ConclusionsHigh GR, Sgk1, and NDRG1 statuses in ESCC after NAC was significantly associated with an overall worse prognosis, with no significant changes in their expression levels before and after NAC. Therefore, increased activity of the GC-GR pathway with enhanced induction of Sgk1 and NDRG1 in carcinoma cells play pivotal roles in tumor progression and development of chemo-resistance in patients with ESCC undergoing NAC.

Oocyte Maturation in Starfish.

Oocyte maturation is a process that occurs in the ovaries, where an immature oocyte resumes meiosis to attain competence for normal fertilization after ovulation/spawning. In starfish, the hormone 1-methyladenine binds to an unidentified receptor on the plasma membrane of oocytes, inducing a conformational change in the heterotrimeric GTP-binding protein α-subunit (Gα), so that the α-subunit binds GTP in exchange of GDP on the plasma membrane. The GTP-binding protein βγ-subunit (Gβγ) is released from Gα, and the released Gβγ activates phosphatidylinositol-3 kinase (PI3K), followed by the target of rapamycin kinase complex2 (TORC2) and 3-phosphoinositide-dependent protein kinase 1 (PDK1)-dependent phosphorylation of serum- and glucocorticoid-regulated kinase (SGK) of ovarian oocytes. Thereafter, SGK activates Na+/H+ exchanger (NHE) to increase the intracellular pH (pHi) from ~6.7 to ~6.9. Moreover, SGK phosphorylates Cdc25 and Myt1, thereby inducing the de-phosphorylation and activation of cyclin B–Cdk1, causing germinal vesicle breakdown (GVBD). Both pHi increase and GVBD are required for spindle assembly at metaphase I, followed by MI arrest at pHi 6.9 until spawning. Due to MI arrest or SGK-dependent pHi control, spawned oocytes can be fertilized normally

Phosphoproteomics reveals that the hVPS34 regulated SGK3 kinase specifically phosphorylates endosomal proteins including Syntaxin-7, Syntaxin-12, RFIP4 and WDR44.

The serum- and glucocorticoid-regulated kinase (SGK) isoforms contribute resistance to cancer therapies targeting the PI3K pathway. SGKs are homologous to Akt and these kinases display overlapping specificity and phosphorylate several substrates at the same residues, such as TSC2 to promote tumor growth by switching on the mTORC1 pathway. The SGK3 isoform is up-regulated in breast cancer cells treated with PI3K or Akt inhibitors and recruited and activated at endosomes, through its phox homology domain binding to PtdIns(3)P. We undertook genetic and pharmacological phosphoproteomic screens to uncover novel SGK3 substrates. We identified 40 potential novel SGK3 substrates, including four endosomal proteins STX7 (Ser126) and STX12 (Ser139), RFIP4 (Ser527) and WDR44 (Ser346) that were efficiently phosphorylated in vitro by SGK3 at the sites identified in vivo, but poorly by Akt. We demonstrate that these substrates are inefficiently phosphorylated by Akt as they possess an n + 1 residue from the phosphorylation site that is unfavorable for Akt phosphorylation. Phos-tag analysis revealed that stimulation of HEK293 cells with IGF1 to activate SGK3, promoted phosphorylation of a significant fraction of endogenous STX7 and STX12, in a manner that was blocked by knock-out of SGK3 or treatment with a pan SGK inhibitor (14H). SGK3 phosphorylation of STX12 enhanced interaction with the VAMP4/VTI1A/STX6 containing the SNARE complex and promoted plasma membrane localization. Our data reveal novel substrates for SGK3 and suggest a mechanism by which STX7 and STX12 SNARE complexes are regulated by SGK3. They reveal new biomarkers for monitoring SGK3 pathway activity.

SGK regulates pH increase and cyclin B-Cdk1 activation to resume meiosis in starfish ovarian oocytes.

Tight regulation of intracellular pH (pHi) is essential for biological processes. Fully grown oocytes, having a large nucleus called the germinal vesicle, arrest at meiotic prophase I. Upon hormonal stimulus, oocytes resume meiosis to become fertilizable. At this time, the pHi increases via Na+/H+ exchanger activity, although the regulation and function of this change remain obscure. Here, we show that in starfish oocytes, serum- and glucocorticoid-regulated kinase (SGK) is activated via PI3K/TORC2/PDK1 signaling after hormonal stimulus and that SGK is required for this pHi increase and cyclin B-Cdk1 activation. When we clamped the pHi at 6.7, corresponding to the pHi of unstimulated ovarian oocytes, hormonal stimulation induced cyclin B-Cdk1 activation; thereafter, oocytes failed in actin-dependent chromosome transport and spindle assembly after germinal vesicle breakdown. Thus, this SGK-dependent pHi increase is likely a prerequisite for these events in ovarian oocytes. We propose a model that SGK drives meiotic resumption via concomitant regulation of the pHi and cell cycle machinery.

Identification, structure modification, and characterization of potential small-molecule SGK3 inhibitors with novel scaffolds.

The serum and glucocorticoid-regulated kinase (SGK) family has been implicated in the regulation of many cellular processes downstream of the PI3K pathway. It plays a crucial role in PI3K-mediated tumorigenesis, making it a potential therapeutic target for cancer. SGK family consists of three isoforms (SGK1, SGK2, and SGK3), which have high sequence homology in the kinase domain and similar substrate specificity with the AKT family. In order to identify novel compounds capable of inhibiting SGK3 activity, a high-throughput screening campaign against 50,400 small molecules was conducted using a fluorescence-based kinase assay that has a Z' factor above 0.5. It identified 15 hits (including nitrogen-containing aromatic, flavone, hydrazone, and naphthalene derivatives) with IC50 values in the low micromolar to sub-micromolar range. Four compounds with a similar scaffold (i.e., a hydrazone core) were selected for structural modification and 18 derivatives were synthesized. Molecular modeling was then used to investigate the structure-activity relationship (SAR) and potential protein-ligand interactions. As a result, a series of SGK inhibitors that are active against both SGK1 and SGK3 were developed and important functional groups that control their inhibitory activity identified.

Mechanistic target of rapamycin: integrating growth factor and nutrient signaling in the collecting duct.

The renal collecting duct and other postmacula densa sites are the primary tubular regions for fine-tuning of electrolyte homeostasis in the body. A role for the mechanistic target of rapamycin (mTOR), a serine-threonine kinase, has recently been appreciated in this regulation. mTOR exists in two distinct multiprotein functional complexes, i.e., mTORC1 and mTORC2. Upregulation of mTORC1, by growth factors and amino acids, is associated with cell cycle regulation and hypertrophic changes. In contrast, mTORC2 has been demonstrated to have a role in regulating Na+ and K+ reabsorptive processes, including those downstream of insulin and serum- and glucocorticoid-regulated kinase (SGK). In addition, mTORC2 can upregulate mTORC1. A number of elegant in vitro and in vivo studies using cell systems and genetically modified mice have revealed mechanisms underlying activation of the epithelial Na+ channel (ENaC) and the renal outer medullary K+ channel (ROMK) by mTORC2. Overall, mTOR in its systematic integration of phosphorylative signaling facilitates the delicate balance of whole body electrolyte homeostasis in the face of changes in metabolic status. Thus, inappropriate regulation of renal mTOR has the potential to result in electrolyte disturbances, such as acidosis/alkalosis, hyponatremia, and hypertension. The goal of this minireview is to highlight the physiological role of mTOR in its complexes in regulating electrolyte homeostasis in the aldosterone-sensitive distal nephron.

SGK Kinase Activity in Multiple Myeloma Cells Protects against ER Stress Apoptosis via a SEK-Dependent Mechanism.

To assess the role of the serum and glucocorticoid-regulated kinase (SGK) kinase in multiple myeloma, we ectopically expressed wild type or a phosphomimetic version of SGK into multiple myeloma cell lines. These cells were specifically resistant to the ER stress inducers tunicamycin, thapsigargin, and bortezomib. In contrast, there was no alteration of sensitivity to dexamethasone, serum starvation, or mTORC inhibitors. Mining of genomic data from a public database indicated that low baseline SGK expression in multiple myeloma patients correlated with enhanced ability to undergo a complete response to subsequent bortezomib treatment and a longer time to progression and overall survival following treatment. SGK overexpressing multiple myeloma cells were also relatively resistant to bortezomib in a murine xenograft model. Parental/control multiple myeloma cells demonstrated a rapid upregulation of SGK expression and activity (phosphorylation of NDRG-1) during exposure to bortezomib and an SGK inhibitor significantly enhanced bortezomib-induced apoptosis in cell lines and primary multiple myeloma cells. In addition, a multiple myeloma cell line selected for bortezomib resistance demonstrated enhanced SGK expression and SGK activity. Mechanistically, SGK overexpression constrained an ER stress-induced JNK proapoptotic pathway and experiments with a SEK mutant supported the notion that SGK's protection against bortezomib was mediated via its phosphorylation of SEK (MAP2K4) which abated SEK/JNK signaling. These data support a role for SGK inhibitors in the clinical setting for myeloma patients receiving treatment with ER stress inducers like bortezomib. Enhanced SGK expression and activity in multiple myeloma cells contributes to resistance to ER stress, including bortezomib challenge.

Highlights of This Issue

Inhibitors targeting Bromodomain and Extra-Terminal (BET) proteins are currently in clinical trials for a number of cancer types. In pre-clinical models, BET inhibitors have shown great promise as epigenetic therapies that alter a number of BET protein interactions, including interactions with transcription factors. In the Rapid Impact article, Asangani and colleagues develop clonal cell lines that are enzalutamide-resistant. In addition to evaluating androgen receptor (AR) signaling and transcriptional consequences in these enzalutamide-resistant model systems, BET inhibitor efficacy is also assessed. The data offer a strong rationale for combining BET inhibitors with AR antagonists for the treatment of advanced prostate cancer.Here a PCR-based miRNA (miR) profiling analysis identified miRNA-137 (miR-137) as downregulated in colon cancer stem cells (CSCs) compared to normal colon stem cells (NCSCs) and regulated doublecortin-like kinase 1 (DCLK1) gene expression. Moreover, experimental data demonstrated that miR-137 has the capacity to suppress the tumorigenicity of colon CSCs and that maintained expression of miR-137 in NCSCs contributes to suppressing uncontrolled cell proliferation through the inhibition of DCLK1 expression. The insight provided by this study concerning the miR-137/DCLK1 axis lends support for the development of potential gene therapeutic strategies targeting colon CSCs.miR-155 is an oncogenic microRNA (miR) associated with poor prognosis in a variety of cancer types including lymphoma, lung, breast, and colon cancer. Czochor and colleagues report that miR-155 over-expression drives an increase in mutation frequency in cell culture and in vivo in a transgenic mouse model. Mechanistic evidence suggests that miR-155 impacts DNA repair and genome stability by reducing expression of the high-fidelity DNA polymerase delta and by promoting the error-prone non-homologous end-joining (NHEJ) double-strand break (DSB) repair pathway. Elucidating the role of miR-155 in promoting tumorigenesis is an important step in developing therapies to target miR-155 over-expressing tumors.Multiple myeloma (MM) tumor cells are vulnerable to endoplasmic reticulum (ER) stress because of the huge amount of Ig protein they produce. One of the most successful anti-myeloma therapeutics, bortezomib, actually enhances ER stress and that may be the mechanism of its effect. This study shows that expression and activity of the serum and glucocorticoid-regulated kinase (SGK) is a resistance mechanism for sensitivity of MM cells to cytotoxicity of ER stress inducers including bortezomib. This was found in vitro in cell lines and primary cells as well as in vivo in xenografted mice. Mechanistically, resistance was mediated by restraint of a Jun kinase pro-apoptotic pathway in which SGK-induced phosphorylation of SEK down-regulated the pathway. These results support the development of SGK inhibitors in MM to be combined with bortezomib.

Abstract 11005: Activation of Hypothalamic Mineralocorticoid Receptor-Independent Epithelial Sodium Channels Leads to Salt-Sensitive Hypertension in Mouse Model of Metabolic Syndrome

Introduction and Hypothesis: Metabolic syndrome (MetS) is often associated with salt-sensitive hypertension. In genetic salt-sensitive hypertensive models, activation of the hypothalamic mineralocorticoid receptor (MR)-epithelial sodium channel (ENaC) pathway has been established as a major worsening factor. However, in MetS, contribution of the hypothalamic MR-ENaC pathway to salt-sensitive hypertension remains unknown. Therefore, we determined whether the hypothalamic MR-ENaC pathway activation leads to salt-sensitive hypertension in MetS. Methods and Results: Diet-induced obesity (DIO) was achieved in male C57BL/6 mice by feeding them a high-fat diet from 4 weeks of age. Mice fed a normal diet served as controls (CON). Mice at 12 weeks of age were given tap water with regular salt (RS) or 1% NaCl (high salt; HS) for 8 weeks. HS led to increased systolic blood pressure only in DIO-mice, and not in CON-mice (DIO-HS: 122±3 mmHg vs DIO-RS: 107±2 mmHg, CON-HS: 109±2 mmHg vs CON-RS: 105±1 mmHg, n=6/group, p<0.05). In DIO-mice, 24-h urinary norepinephrine excretion after salt loading was significantly higher than that before salt loading (244±18 ng/day vs 371±29 ng/day, n=6 in each, p<0.05). HS-induced hypertension was significantly prevented by intracerebroventricular infusion of the ENaC blocker benzamil (Ben), but not the MR blocker spironolactone (Spl; DIO-HS-Vehicle 122±1 mmHg, DIO-HS-Ben 108±1 mmHg, DIO-HS-Spl 119±1 mmHg, n=6 in each, p<0.05). Expression of hypothalamic serum and glucocorticoid-regulated kinase (SGK) 1, a marker of MR activity, did not differ between in CON-mice and DIO-mice. However, the SGK1 phosphorylation level was greater in DIO-mice than in CON-mice, suggesting MR-independent SGK1 activation. Water intake and urinary sodium excretion did not differ between in DIO-HS-Vehicle and DIO-HS-Ben. Interestingly, salt loading-induced plasma arginine vasopressin (AVP) secretion was significantly prevented in DIO-HS-Ben. Conclusions: These findings suggest that salt-induced hypertension in DIO-mice results from MR-independent hypothalamic SGK1-ENaC activation with systemic AVP secretion. Hypothalamic ENaC could be a novel therapeutic target in salt-induced hypertension of MetS.

Inhibition of serum- and glucocorticoid-inducible kinase 1 enhances TLR-mediated inflammation and promotes endotoxin-driven organ failure.

Serum- and glucocorticoid-regulated kinase (SGK)1 is associated with several important pathologic conditions and plays a modulatory role in adaptive immune responses. However, the involvement and functional role of SGK1 in innate immune responses remain entirely unknown. In this study, we establish that SGK1 is a novel and potent negative regulator of TLR-induced inflammation. Pharmacologic inhibition of SGK1 or suppression by small interfering RNA enhances proinflammatory cytokine (TNF, IL-12, and IL-6) production in TLR-engaged monocytes, a result confirmed in Cre-loxP-mediated SGK1-deficient cells. SGK1 inhibition or gene deficiency results in increased phosphorylation of IKK, IκBα, and NF-κB p65 in LPS-stimulated cells. Enhanced NF-κB p65 DNA binding also occurs upon SGK1 inhibition. The subsequent enhancement of proinflammatory cytokines is dependent on the phosphorylation of TGF-β-activated kinase 1 (TAK1), as confirmed by TAK1 gene silencing. In vivo relevance was established in a murine endotoxin model, in which we found that SGK1 inhibition aggravates the severity of multiple organ damage and enhances the inflammatory response by heightening both proinflammatory cytokine levels and neutrophil infiltration. These findings have identified an anti-inflammatory function of SGK1, elucidated the underlying intracellular mechanisms, and establish, for the first time, that SGK1 holds potential as a novel target for intervention in the control of inflammatory diseases.

Renal NCC is unchanged in the midpregnant rat and decreased in the late pregnant rat despite avid renal Na+ retention.

Pregnancy is characterized by plasma volume expansion due to Na(+) retention, driven by aldosterone. The aldosterone-responsive epithelial Na(+) channel is activated in the kidney in pregnancy. In the present study, we investigated the aldosterone-responsive Na(+)-Cl(-) cotransporter (NCC) in mid- and late pregnant rats compared with virgin rats. We determined the abundance of total NCC, phosphorylated NCC (pNCC; pT53, pS71 and pS89), phosphorylated STE20/SPS-1-related proline-alanine-rich protein kinase (pSPAK; pS373), and phosphorylated oxidative stress-related kinase (pOSR1; pS325) in the kidney cortex. We also measured mRNA expression of NCC and members of the SPAK/NCC regulatory kinase network, serum and glucocorticoid-regulated kinase (SGK)1, total with no lysine kinase (WNK)1, WNK3, and WNK4. Additionally, we performed immunohistochemistry for NCC kidneys from virgin and pregnant rats. Total NCC, pNCC, and pSPAK/OSR1 abundance were unchanged in midpregnant versus virgin rats. In late pregnant versus virgin rats, total NCC and pNCC were decreased; however, pSPAK/OSR1 was unchanged. We detected no differences in mRNA expression of NCC, SGK1, total WNK1, WNK3, and WNK4. By immunohistochemistry, NCC was mainly localized to the apical region in virgin rats, and density in the apical region was reduced in late pregnancy. Therefore, despite high circulating aldosterone levels in pregnancy, the aldosterone-responsive transporter NCC is not increased in total or activated (phosphorylated) abundance or in apical localization in midpregnant rats, and all are reduced in late pregnancy. This contrasts to the mineralocorticoid-mediated activation of the epithelial Na(+) channel, which we have previously reported. Why and how NCC escapes aldosterone activation in pregnancy is not clear but may relate to regional differences in aldosterone sensitivity the increased K(+) intake or other undefined mechanisms.

Abstract MP15: Single And Joint Associations Of Genetic Variants In The Serum/glucocorticoid Regulated Kinase (sgk) Genes With Blood Pressure Responses To Sodium Intake: The Gensalt Study

Background: Serum and glucocorticoid regulated kinase (SGK) plays a critical role in the regulation of renal sodium transport. We examined the association between SGK genes and salt sensitivity of blood pressure (BP) using single-marker and gene-based association analyses. Methods: A 7-day low-sodium (51.3 mmol sodium/day) followed by a 7-day high-sodium intervention (307.8 mmol sodium/day) was conducted among 1,906 Chinese participants. BP measurements were obtained at baseline and each intervention using a random-zero sphygmomanometer. Additive associations between each SNP and salt-sensitivity phenotypes were assessed using a mixed linear regression model to account for family dependencies. Gene-based analyses were conducted using the truncated p-value method. The Bonferroni-method was used to adjust for multiple testing in all analyses. Results: In single-marker association analyses, SGK1 marker rs2758151 was significantly associated with diastolic BP (DBP) response to high-sodium intervention (P=0.0010). DBP responses (95% confidence interval) to high-sodium intervention for genotypes C/C, C/T, and T/T were 2.04 (1.57 to 2.52), 1.79 (1.42 to 2.16), and 0.85 (0.30 to 1.41) mmHg, respectively. Similar non-significant trends were observed for SBP and MAP responses (P=0.15 and 0.0026, respectively). In addition, gene-based analyses demonstrated significant associations between SGK1 and SBP, DBP and MAP responses to high sodium intervention (P=0.0002, 0.0076, and 0.00001, respectively). Neither SGK2 nor SGK3 were associated with the salt-sensitivity phenotypes in single-maker and gene-based analyses. Conclusions: The current study identified single-marker and gene-based association of the SGK1 gene and BP salt-sensitivity in the Han Chinese population. Further studies are warranted to identify causal SGK1 gene variants.

Elevated SGK1 predicts resistance of breast cancer cells to Akt inhibitors

The majority of human cancers harbour mutations promoting activation of the Akt protein kinase, and Akt inhibitors are being evaluated in clinical trials. An important question concerns the understanding of the innate mechanisms that confer resistance of tumour cells to Akt inhibitors. SGK (serum- and glucocorticoid-regulated kinase) is closely related to Akt and controlled by identical upstream regulators {PI3K (phosphoinositide 3-kinase), PDK1 (phosphoinositide-dependent kinase 1) and mTORC2 [mTOR (mammalian target of rapamycin) complex 2]}. Mutations that trigger activation of Akt would also stimulate SGK. Moreover, Akt and SGK possess analogous substrate specificities and are likely to phosphorylate overlapping substrates to promote proliferation. To investigate whether cancers possessing high SGK activity could possess innate resistance to Akt-specific inhibitors (that do not target SGK), we analysed SGK levels and sensitivity of a panel of breast cancer cells towards two distinct Akt inhibitors currently in clinical trials (AZD5363 and MK-2206). This revealed a number of Akt-inhibitor-resistant lines displaying markedly elevated SGK1 that also exhibited significant phosphorylation of the SGK1 substrate NDRG1 [N-Myc (neuroblastoma-derived Myc) downstream-regulated gene 1]. In contrast, most Akt-inhibitor-sensitive cell lines displayed low/undetectable levels of SGK1. Intriguingly, despite low SGK1 levels, several Akt-inhibitor-sensitive cells showed marked NDRG1 phosphorylation that was, unlike in the resistant cells, suppressed by Akt inhibitors. SGK1 knockdown markedly reduced proliferation of Akt-inhibitor-resistant, but not -sensitive, cells. Furthermore, treatment of Akt-inhibitor-resistant cells with an mTOR inhibitor suppressed proliferation and led to inhibition of SGK1. The results of the present study suggest that monitoring SGK1 levels as well as responses of NDRG1 phosphorylation to Akt inhibitor administration could have a use in predicting the sensitivity of tumours to compounds that target Akt. Our findings highlight the therapeutic potential that SGK inhibitors or dual Akt/SGK inhibitors might have for treatment of cancers displaying elevated SGK activity.

SGK regulation of renal sodium transport.

The serum and glucocorticoid regulated kinase (SGK) family of protein kinases shares similar biochemical and hormonal signaling properties; however, the SGK kinases also exhibit distinct differences in regulating renal sodium (Na(+)) transport. This review will highlight recent advances in our understanding of the specificity of SGK kinase signaling and regulation of renal Na(+) transport. Differential expression of SGK kinases at the cellular and subcellular levels contributes to signaling specificity. New evidence indicates that SGK1 associates with the apical cell membrane of cortical collecting duct cells to regulate open probability of the epithelial Na(+) channel (ENaC). Scaffold proteins can also recruit SGK1 to multiprotein complexes for regulation of ENaC expression in the apical membrane. Recent SGK1 knockout models have implicated the NaCl co-transporter (NCC) as another target of SGK1 regulation. Less is known about the function of SGK2 or SGK3, but both kinases can regulate Na(+)/H(+) exchanger 3 (NHE3) activity. The SGK kinases assume distinct roles in regulating Na transport in both proximal and distal elements of the kidney tubule. Future examination of the molecular mechanisms by which the SGK kinases regulate specific substrates will inform our understanding of how these kinases contribute to the physiology of renal Na(+) transport.

P.4.017 Cortisol decreases hippocampal neurogenesis by regulating the enzyme serum- and glucocorticoid-regulated kinase 1

Chronic stress increases circulating glucocorticoid hormones, which impair hippocampal neurogenesis by activating the glucocorticoid receptor (GR) [1], and thereby potentially contribute to the development of depressive symptoms [2]. However, the molecular mechanisms underlying this effect are unknown. The enzyme, serum-and glucocorticoid-regulated kinase 1 (SGK1) has been shown to mediate some glucocorticoid effects on the brain [3]. We therefore hypothesized that SGK1 may also mediate the effects of glucocorticoids on hippocampal neurogenesis in humans. We treated human hippocampal progenitor cells (HPC03A/07, ReNeuron, UK) with high concentrations of the glucocorticoid, cortisol (100mM), and with the SGK1-inhibitor, GSK650394 (100nM) (Tocris Biosciences). We analysed cell proliferation by 5′-bromodeoxyuridine (BrdU, 10mM) incorporation, and neuronal differentiation by immunocytochemistry for the neuronal marker, microtubule-associated protein-2 (MAP2). GR activation was examined by Western blot using specific antibodies for the GR phosphorylation-sites, S203, S211 and S226. Cortisol decreased BrdU-positive, proliferating cells (by −13±2%; p = 0.03, n = 3), and neuronal differentiation into MAP2positive neurons (by −16±2%; p = 0.04, n = 3). The SGK1inhibitor, GSK650394 (100nM), counteracted the effects of cortisol on proliferation (p = 0.0027, n = 4) and on neuronal differentiation (p = 0.03, n = 3). Moreover, cortisol induced GR phosphorylation at S203 (1.5±0.1 fold; p = 0.01, n = 3), S211 (3.0±0.5 fold; p = 0.004, n = 3) and S226 (1.6±0.2 fold; p = 0.04, n = 3). Cotreatment with GSK650394 (100nM) counteracted the cortisolinduced GR phosphorylation at S203 (p = 0.01, n = 3) and S211 (p = 0.03, n = 3), but not at the S226 phospho-site (p = 0.8, n = 3). In summary, our data identify the kinase, SGK1, as a novel mechanism mediating the effects of cortisol on human hippocampal neurogenesis and GR function.