Madhuri Bhandaru

SGK3 regulates Ca(2+) entry and migration of dendritic cells.

Background/Aims: Dendritic cells (DCs) are antigen-presenting cells linking innate and adaptive immunity. DC maturation and migration are governed by alterations of cytosolic Ca²⁺ concentrations ([Ca²⁺]_i). Ca²⁺ entry is in part accomplished by store-operated Ca²⁺ (SOC) channels consisting of the membrane pore-forming subunit Orai and the ER Ca²⁺ sensing subunit STIM. Moreover, DC functions are under powerful regulation of the phosphatidylinositol-3-kinase (PI3K) pathway, which suppresses proinflammatory cytokine production but supports DC migration. Downstream targets of PI3K include serum- and glucocorticoid-inducible kinase isoform SGK3. The present study explored, whether SGK3 participates in the regulation of [Ca²⁺]_i and Ca²⁺-dependent functions of DCs, such as maturation and migration. Methods/ Results: Experiments were performed with bone marrow derived DCs from gene targeted mice lacking SGK3 (sgk3^-/-) and DCs from their wild type littermates (sgk3^+/+). Maturation, phagocytosis and cytokine production were similar in sgk3^-/- and sgk3^+/+ DCs. However, SOC entry triggered by intracellular Ca²⁺ store depletion with the endosomal Ca²⁺ ATPase inhibitor thapsigargin (1 µM) was significantly reduced in sgk3^-/- compared to sgk3^+/+ DCs. Similarly, bacterial lipopolysaccharide (LPS, 1 µg/ml)- and chemokine CXCL12 (300 ng/ml)- induced increase in [Ca²⁺]_i was impaired in sgk3^-/- DCs. Moreover, currents through SOC channels were reduced in sgk3^-/- DCs. STIM2 transcript levels and protein abundance were significantly lower in sgk3^-/- DCs than in sgk3^+/+ DCs, whereas Orai1, Orai2, STIM1 and TRPC1 transcript levels and/or protein abundance were similar in sgk3-/- and sgk3^+/+ DCs. Migration of both, immature DCs towards CXCL12 and LPS-matured DCs towards CCL21 was reduced in sgk3^-/- as compared to sgk3^+/+ DCs. Migration of sgk3^+/+ DCs was further sensitive to SOC channel inhibitor 2-APB (50 µM) and to STIM1/STIM2 knock-down. Conclusion: SGK3 contributes to the regulation of store-operated Ca²⁺ entry into and migration of dendritic cells, effects at least partially mediated through SGK3-dependent upregulation of STIM2 expression.

Relative resistance of SGK1 knockout mice against chemical carcinogenesis

The serum and glucocorticoid inducible kinase SGK1 was originally cloned from mammary tumor cells. SGK1 was found to be up‐regulated in a variety of tumors, but down‐regulated in several distinct tumors. Thus, evidence for a role of SGK1 in tumor growth remained conflicting. According to in vitro observations, SGK1 is up‐regulated by the oncogene β‐catenin and negatively regulates the proapoptotic transcription factor FOXO3a, which in turn stimulates transcription of the Bcl2‐interacting mediator BIM. This study aimed to define the role of SGK1 in colon carcinoma in vivo. SGK1 knockout mice (sgk1−/−) and their wild type littermates (sgk1+/+) were subjected to chemical cancerogenesis (intraperitoneal injection of 20 mg/kg 1,2‐dimethylhydrazine followed by three cycles of 30 g/L synthetic dextran sulfate sodium for 7 days). Moreover, SGK1 was silenced in HEK293 cells. FOXO3a and BIM protein abundance was determined by Western blotting and immunohistochemistry. Following chemical cancerogenesis, sgk1−/−mice developed significantly less colonic tumors than sgk1+/+mice. According to Western blotting and immunohistochemistry, SGK1 deficiency enhanced the expression of FOXO3a and BIM both, in vitro and in vivo. SGK1 deficiency counteracts the development of colonic tumors, an effect at least in part due to up‐regulation of FOXO3a and BIM.

Regulation of calcium signaling in dendritic cells by 1,25-dihydroxyvitamin D3

Dendritic cells (DCs) are antigen‐presenting cells that provide a link between innate and adaptive immunity. Ca2+‐dependent signaling plays a central regulatory role in DC responses to diverse antigens. DCs are a primary target of 1,25‐dihydroxyvitamin D3 [1,25(OH)2D3], a secosteroid hormone, that, in addition to its wellestablished action on Ca2+ homeostasis, possesses immunomodulatory properties. Surprisingly, nothing is known about its effects on DC cytosolic Ca2+ activity. The present study explored whether 1,25(OH)2D3 modifies the intracellular Ca2+ concentration ([Ca2+]i) in DCs. Here we show that mouse DCs expressed K+‐independent (NCX1–3) and K+‐dependent (NCKX1, 3, 4, and 5) Na+/Ca2+ exchangers. Acute application of LPS (100 ng/ml) to DCs increased [Ca2+]i, an effect significantly blunted by prior incubation with 1,25(OH)2D3. 1,25(OH)2D3 increased the membrane abundance of the NCKX1 protein, up‐regulated the K+‐ and Na+‐dependent Ca2+ entry and enhanced the K+‐dependent Na+/Ca2+ exchanger currents. The NCKX blocker 3′,4′‐dichlorobenzamyl (DBZ) reversed the inhibitory effect of 1,25(OH)2D3 on the LPS‐induced increase of [Ca2+]i. Expression of the costimulatory molecule CD86 was down‐regulated by 1,25(OH)2D3, an effect reversed by DBZ. In summary, 1,25(OH)2D3 blunts the LPS‐induced increase in [Ca2+]i by stimulation of Na+/Ca2+ exchangerdependent Ca2+ extrusion, an effect that contributes to 1,25(OH)2D3‐mediated immunosuppression. The results disclose completely novel mechanisms in the regulation of DC maturation and function.—Shumilina, E., Xuan, N. T., Matzner, N., Bhandaru, M., Zemtsova, I. M., Lang, F. Regulation of calcium signaling in dendritic cells by 1,25‐dihydroxyvitamin D3. FASEB J. 24, 1989–1996 (2010). www.fasebj.org

APC sensitive gastric acid secretion.

Adenomatous polyposis coli (APC) is a tumor suppressor gene inactivated in familial adenomatous polyposis and sporadic colorectal cancer. Mice carrying a loss-of-function mutation in the apc gene (apc^Min/+) spontaneously develop gastrointestinal tumors. APC fosters degradation of β-catenin, which in turn upregulates the serum- and glucocorticoid-inducible kinase SGK1. SGK1 stimulates KCNQ1, which is required for luminal K⁺ recycling and thus for gastric acid secretion. BCECF-fluorescence was utilized to determine gastric acid secretion in isolated gastric glands from apc^Min/+ mice and their wild type littermates (apc^+/+). Western blotting was employed to analyse β-catenin and SGK1 expression and immunohistochemistry to determine KCNQ1 protein abundance. β-catenin and SGK1 expression were enhanced in apc^Min/+ mice. Cytosolic pH was similar in apc^Min/+ mice and apc^+/+ mice. Na⁺-independent pH recovery following an ammonium pulse (ΔpH/min), which reflects H⁺/K⁺ ATPase activity, was, however, significantly faster in apc^Min/+ mice than in apc^+/+mice. In both genotypes ΔpH/min was abolished in the presence of H⁺/K⁺ ATPase inhibitor omeprazole (100 μM). Treatment of apc^Min/+ and apc^+/+mice with 5 μM forskolin 15 minutes prior to the experiment or increase in local K⁺-concentrations to 35 mM (replacing Na⁺/NMDG) significantly increased ΔpH/min and abrogated the differences between genotypes. The increase of ΔpH/min in apc^Min/+mice required SGK1, as it was abolished by additional knockout of SGK1 (apc^Min/+/sgk1^-/-). In conclusion, basal gastric acid secretion is significantly enhanced in apc^Min/+mice, pointing to a role of APC in the regulation of gastric acid secretion. The effect of APC requires H⁺/K⁺ ATPase activity and is at least partially due to SGK1-dependent upregulation of KCNQ1.

Upregulation of Na+/H+ exchanger by the AMP-activated protein kinase

AMP-activated protein kinase (AMPK) is activated upon energy depletion and serves to restore energy balance by stimulating energy production and limiting energy utilization. Specifically, it enhances cellular glucose uptake by stimulating GLUT and SGLT1 and glucose utilization by stimulating glycolysis. During O(2) deficiency glycolytic degradation of glucose leads to formation of lactate and H(+), thus imposing an acid load to the energy-deficient cell. Cellular acidification inhibits glycolysis and thus impedes glucose utilization. Maintenance of glycolysis thus requires cellular H(+) export. The present study explored whether AMPK influences Na(+)/H(+) exchanger (NHE) activity and/or Na(+)-independent acid extrusion. NHE1 expression was determined by RT-PCR and Western blotting. Cytosolic pH (pH(i)) was estimated utilizing BCECF fluorescence and Na(+)/H(+) exchanger activity from the Na(+)-dependent re-alkalinization (DeltapH(i)) after an ammonium pulse. As a result, human embryonic kidney (HEK) cells express NHE1. The pH(i) and DeltapH(i) in those cells were significantly increased by treatment with AMPK stimulator AICAR (1mM) and significantly decreased by AMPK inhibitor compound C (10 microM). The effect of AICAR on pH(i) and DeltapH(i) was blunted in the presence of the Na(+)/H(+) exchanger inhibitor cariporide (10microM), but not by the H(+) ATPase inhibitor bafilomycin (10nM). AICAR significantly enhanced lactate formation, an effect significantly blunted in the presence of cariporide. These observations disclose a novel function of AMPK, i.e. regulation of cytosolic pH.

Hyperaldosteronism, hypervolemia, and increased blood pressure in mice expressing defective APC

Adenomatous polyposis coli (APC) fosters degradation of beta-catenin, a multifunctional protein upregulating the serum- and glucocorticoid-inducible kinase (SGK1). SGK1 regulates a wide variety of renal transport processes. The present study explored the possibility that APC influences renal function. To this end, metabolic cage experiments were performed in mice carrying a loss-of-function mutation in the APC gene (apc(Min/+)), their wild-type littermates (apc(+/+)), and apc(Min/+) mice lacking functional SGK1 (apc(Min/+)/sgk1(-/-)). As a result, mean body weight, food intake, fluid intake, salt appetite, urinary flow, as well as plasma Na(+) and K(+) concentrations were similar in apc(Min/+) mice, apc(+/+) mice, and apc(Min/+)/sgk1(-/-) mice. Glomerular filtration rate and absolute renal Na(+) excretion were decreased, and fractional urinary K(+) excretion was enhanced in apc(Min/+) mice. The antinatriuresis, but not the hypofiltration and kaliuresis was partially reversed by additional lack of SGK1. Plasma corticosterone and aldosterone concentrations were significantly enhanced in apc(Min/+) mice. While the plasma corticosterone concentration was similar in apc(+/+) mice and apc(Min/+)/sgk1(-/-) mice, plasma aldosterone was even higher in apc(Min/+)/sgk1(-/-) mice than in apc(Min/+) mice. The hyperaldosteronism of apc(Min/+) mice was paralleled by significantly elevated plasma volume and blood pressure. The experiments reveal an influence of defective APC on adrenal hormone release and renal function, effects partially but not completely explained by APC dependence of SGK1 expression.

Influence of dexamethasone on na+/h+ exchanger activity in dendritic cells.

Glucocorticoids regulate the function of dendritic cells (DCs), antigen-presenting cells linking innate and adaptive immunity. Glucocorticoids influence the function of other cell types by modulating the activity of the Na⁺/H⁺exchanger (NHE), a carrier involved in the regulation of cytosolic pH and cell volume. The present study explored whether dexamethasone influences Na⁺/H⁺ exchanger activity in DCs. The DCs were isolated from mouse bone marrow, cell volume was estimated from forward scatter in FACS analysis, cytosolic pH (pH_i) utilizing BCECF fluorescence and Na⁺/H⁺ exchanger activity from the Na⁺ dependent realkalinization after an ammonium pulse. Treatment with the glucocorticoid dexamethasone (100 nM; 1, 4, 16 and 24h) significantly decreased pH_i (≧4 h) and gradually increased Na⁺/H⁺ exchanger activity (=16 h). The stimulation of Na⁺/H⁺ exchanger activity by dexamethasone was virtually abrogated by glucocorticoid receptor blocker mefiprestone (1 µM) and NHE3 inhibitor dimethyl amiloride (5 µM), but not prevented by NHE1 inhibitor cariporide (10 µM). Dexamethasone treatment significantly increased SGK1 mRNA levels. Stimulation of Na⁺/H⁺ exchanger activity by dexamethasone was blunted in DCs lacking SGK1. Dexamethasone treatment did not significantly alter ROS formation but significantly decreased the forward scatter. Exposure of DCs to lipopolysacharide (LPS, 1 µg/ml) led to a transient increase followed by a decline of Na⁺/H⁺ exchanger activity and to enhanced forward scatter as well as ROS formation, all effects significantly blunted in the presence of dexamethasone (100 nM). In conclusion, glucocorticoid treatment decreased pH_i and cell volume, effects paralleled by upregulation of Na⁺/H⁺ exchanger activity in DCs. Moreover, glucocorticoids blunted the stimulation of Na⁺/H⁺ exchanger activity, cell swelling and ROS formation following LPS treatment.

Decreased bone density and increased phosphaturia in gene-targeted mice lacking functional serum- and glucocorticoid-inducible kinase 3

Insulin and growth factors activate the phosphatidylinositide-3-kinase pathway, leading to stimulation of several kinases including serum- and glucocorticoid-inducible kinase isoform SGK3, a transport regulating kinase. Here, we explored the contribution of SGK3 to the regulation of renal tubular phosphate transport. Coexpression of SGK3 and sodium-phosphate cotransporter IIa significantly enhanced the phosphate-induced current in Xenopus oocytes. In sgk3 knockout and wild-type mice on a standard diet, fluid intake, glomerular filtration and urine flow rates, and urinary calcium ion excretion were similar. However, fractional urinary phosphate excretion was slightly but significantly larger in the knockout than in wild-type mice. Plasma calcium ion, phosphate concentration, and plasma parathyroid hormone levels were not significantly different between the two genotypes, but plasma calcitriol and fibroblast growth factor 23 concentrations were significantly lower in the knockout than in wild-type mice. Moreover, bone density was significantly lower in the knockouts than in wild-type mice. Histological analysis of the femur did not show any differences in cortical bone but there was slightly less prominent trabecular bone in sgk3 knockout mice. Thus, SGK3 has a subtle but significant role in the regulation of renal tubular phosphate transport and bone density.

SGK1-sensitive renal tubular glucose reabsorption in diabetes.

The hyperglycemia of diabetes mellitus increases the filtered glucose load beyond the maximal tubular transport rate and thus leads to glucosuria. Sustained hyperglycemia, however, may gradually increase the maximal renal tubular transport rate and thereby blunt the increase of urinary glucose excretion. The mechanisms accounting for the increase of renal tubular glucose transport have remained ill-defined. A candidate is the serum- and glucocorticoid-inducible kinase SGK1. The kinase has been shown to stimulate Na(+)-coupled glucose transport in vitro and mediate the stimulation of electrogenic intestinal glucose transport by glucocorticoids in vivo. SGK1 expression is confined to glomerula and distal nephron in intact kidneys but may extend to the proximal tubule in diabetic nephropathy. To explore whether SGK1 modifies glucose transport in diabetic kidneys, Akita mice (akita(+/-)), which develop spontaneous diabetes, have been crossbred with gene-targeted mice lacking SGK1 on one allele (sgk1(+/-)) to eventually generate either akita(+/-)/sgk1(-/-) or akita(+/-)/sgk1(+/+) mice. Both akita(+/-)/sgk1(-/-) and akita(+/-)/sgk1(+/+) mice developed profound hyperglycemia (>20 mM) within approximately 6 wk. Body weight and plasma glucose concentrations were not significantly different between these two genotypes. However, urinary excretion of glucose and urinary excretion of fluid, Na(+), and K(+), as well as plasma aldosterone concentrations, were significantly higher in akita(+/-)/sgk1(-/-) than in akita(+/-)/sgk1(+/+) mice. Studies in isolated perfused proximal tubules revealed that the electrogenic glucose transport was significantly lower in akita(+/-)/sgk1(-/-) than in akita(+/-)/sgk1(+/+) mice. The data provide the first evidence that SGK1 participates in the stimulation of renal tubular glucose transport in diabetic kidneys.

Rapamycin Sensitive ROS Formation and Na + /H + Exchanger Activity in Dendritic Cells

Rapamycin, a widely used immunosuppressive drug, has been shown to interfere with the function of dendritic cells (DCs), antigen-presenting cells contributing to the initiation of primary immune responses and the establishment of immunological memory. DC function is governed by the Na+/H+ exchanger (NHE), which is activated by bacterial lipopolysaccharides (LPS) and is required for LPS-induced cell swelling, reactive oxygen species (ROS) production and TNF-α release. The present study explored, whether rapamycin influences NHE activity and/or ROS formation in DCs. Mouse DCs were treated with LPS in the absence and presence of rapamycin (100 nM). ROS production was determined from 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence, cytosolic pH (pHi) from 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) fluorescence, NHE activity from the Na+-dependent realkalinization following an ammonium pulse, cell volume from forward scatter in FACS analysis, and TNF-α production utilizing ELISA. In the absence of LPS, rapamycin did not significantly modify cytosolic pH, NHE activity or cell volume but significantly decreased ROS formation. LPS stimulated NHE activity, enhanced forward scatter, increased ROS formation, and triggered TNF-α release, effects all blunted in the presence of rapamycin. NADPH oxidase inhibitor Vas-2870 (10 µM) mimicked the effect of rapamycin on LPS induced stimulation of NHE activity and TNF-α release. The effect of rapamycin on TNF-α release was also mimicked by the antioxidant ROS scavenger Tempol (30 µM) and partially reversed by additional application of tert-butylhydroperoxide (10 µM). In conclusion, in DCs rapamycin disrupts LPS induced ROS formation with subsequent inhibition of NHE activity, cell swelling and TNF-α release.