Anand Rotte

Impact of Cyclin-Dependent Kinase CDK4 Inhibition on Eryptosis

Background/Aims: The cyclin-dependent kinase 4 (CDK4) participates in the regulation of apoptosis of nucleated cells by altering transcriptional regulation of genes governing cell proliferation and cell death. Similar to apoptosis of nucleated cells, erythrocytes may enter eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine (PS) exposure at the cell surface. As mature erythrocytes lack nuclei, acute stimulation of eryptosis cannot result from altered gene expression. Eryptosis is triggered by isotonic cell shrinkage following Cl- removal (replacement with the impermeant organic anion gluconate) or by oxidative stress (exposure to 0.3 mM tertbutyl-hydroperoxide [tBOOH]). The present study explored whether CDK4 is expressed in erythrocytes and whether the CDK4 inhibitors II (NSC625987) and III (ryuvidine) influence eryptosis. Methods: Western blotting was utilized for determination of the presence of CDK4 protein in human erythrocytes, and FACS analysis to determine Fluo3 fluorescence (reflecting cytosolic Ca2+), annexin-V-binding (reflecting PS-exposure) and forward scatter (reflecting cell volume). Results: CDK4 protein was present in human erythrocytes. Cl- removal was followed by decrease of forward scatter and increase of both annexin-V-binding and Fluo3 fluorescence, an effect significantly curtailed by CDK4 inhibitors II and III. Furthermore, CDK4 inhibition blunted enhanced PS-exposure elicited by tBOOH treatment. Conclusions: The present observations disclose the presence of CDK4 protein in human erythrocytes and the suppression of suicidal erythrocyte death by pharmacological inhibition of CDK4.

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.

Effect of bacterial lipopolysaccharide on Na(+)/H(+) exchanger activity in dendritic cells.

The function of dendritic cells (DCs), antigen-presenting cells linking innate and adaptive immunity, is stimulated by bacterial lipopolysaccharides (LPS), which trigger the formation of reactive oxygen species (ROS). In macrophages, ROS formation is paralleled by activation of the Na+/H+ exchanger, a carrier involved in the regulation of cytosolic pH and cell volume. The present study explored whether LPS influence Na+/H+ exchanger activity in DCs. The DCs were isolated from murine bone marrow, cell volume was estimated from forward scatter in FACS analysis, ROS production from 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence, apoptosis from annexin V binding, cytosolic pH (pHi) from 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) fluorescence and Na+/H+ exchanger activity from the Na+ dependent realkalinization following an ammonium pulse. Exposure of DCs to LPS (1 µg/ml) led to a transient increase of Na+/H+ exchanger activity. Moreover, LPS increased forward scatter and ROS formation and decreased apoptosis. The NHE1 inhibitor cariporide (10 µM) virtually abrogated Na+/H+ exchanger activity, inhibited LPS-induced cell swelling, blunted LPS-induced ROS formation and reversed the antiapoptotic effect of LPS. Na+/H+ exchanger activity was stimulated by oxidative stress and LPS induced stimulation of NHE activity was abolished in presence of ROS chelators (Tempol, Tiron and Vitamin C). In conclusion, LPS treatment transiently upregulates the Na+/H+ exchanger in DCs, an effect required for the effects of LPS on DC survival, cell volume and ROS formation.

PI3 kinase and PDK1 in the regulation of the electrogenic intestinal dipeptide transport.

The phosphoinositol 3 kinase (PI3K) and the phosphoinositide dependent kinase (PDK1) stimulate the serum and glucocorticoid inducible kinase (SGK) and protein kinase B (PKB/Akt) isoforms, kinases stimulating a variety of transporters. Most recently, SGK1 was shown to stimulate the peptide transporters PepT1 and PepT2, and to mediate the glucocorticoid stimulation of PepT1. Basal electrogenic intestinal peptide transport was, however, not dependent on the presence of SGK1. The present study explored whether basal electrogenic intestinal peptide transport is dependent on PI3K or PDK1. To this end, peptide transport in intestinal segments was determined utilizing Ussing chamber analysis. Cytosolic pH (pHi) was determined by BCECF fluorescence. The luminal addition of 5 mM dipeptide gly-gly induced a current (Ip) across intestinal segments. Ip was significantly decreased in the presence of PI3 kinase inhibitors Wortmannin (1 µM) or LY294002 (50 µM). Exposure of isolated intestinal cells to 5 mM gly-gly was followed by cytosolic acidification (ΔpHi), which was significantly blunted by Wortmannin and by LY294002. Both, Ip and ΔpHi were significantly smaller in PDK1 hypomorphic mice (pdk1flfl) than in their wild type littermates (pdk1wt). In conclusion, PI3K and PDK1 participate in the regulation of basal peptide transport.

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.

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.

Effect of thymoquinone on cytosolic pH and Na+/H+ exchanger activity in mouse dendritic cells.

The anti-inflammatory Nigella sativa component thymoquinone compromises the function of dendritic cells (DCs), key players in the regulation of innate and adaptive immunity. DC function is regulated by the Na+/H+ exchanger (NHE), which is stimulated by lipopolysaccharides (LPS) and required for LPS-induced cell swelling, reactive oxygen species (ROS) production, TNF-α release and migration. Here we explored, whether thymoquinone influences NHE activity in DCs. To this end, bone marrow derived mouse DCs were treated with LPS in the absence and presence of thymoquinone (10 µM). Cytosolic pH (pHi) was determined 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, ROS production from 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence, TNF-α production utilizing ELISA and DC migration with transwell migration assays. As a result, exposure of DCs to LPS (1 µg/ml) led within 4 hours to transient increase of NHE activity. Thymoquinone did not significantly modify cytosolic pH or cellular NHE activity in the absence of LPS, but abrogated the effect of LPS on NHE activity. Accordingly, in the presence of thymoquinone LPS-treatment resulted in cytosolic acidification. LPS further increased forward scatter and ROS formation, effects similarly abrogated by thymoquinone. Again, in the absence of LPS, thymoquinone did not significantly modify ROS formation and cell volume. LPS further triggered TNF-α release and migration, effects again blunted in the presence of thymoquinone. NHE1 inhibitor cariporide (10 µM) blunted LPS induced TNF-α release and migration. The effects of thymoquinone on NHE activity and migration were reversed upon treatment of the cells with t-butyl hydroperoxide (TBOOH, 5 µM). In conclusion, thymoquinone blunts LPS induced NHE activity, cell swelling, oxidative burst, cytokine release and migration of bone marrow derived murine dendritic cells. NHE inhibition may thus contribute to the antiinflammatory action of thymoquinone.