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Ekaterina Shumilina,
Meerim K Nurbaeva,
Wenting Yang,
Evi Schmid,
Kalina Szteyn,
Antonella Russo, Nicole Heise,
Christina B Leibrock,
Nguyen Thi Xuan,
Caterina Faggio,
Makoto Kuro-O,
Florian Lang
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ABSTRACT: Function of dendritic cells (DCs), antigen-presenting cells regulating naïve T-cells, is regulated by cytosolic Ca(2+) concentration ([Ca(2+)]i). [Ca(2+)]i is increased by store operated Ca(2+) entry and decreased by K(+)-independent (NCX) and K(+)-dependent (NCKX) Na(+)/Ca(2+)-exchangers. NCKX-exchangers are stimulated by immunosuppressive 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), the biologically active form of vitamin D. Formation of 1,25(OH)2D3 is inhibited by anti-aging protein Klotho. Thus, 1,25(OH)2D3 plasma levels are excessive in Klotho-deficient mice (klothohm). The present study explored whether Klotho-deficiency modifies [Ca(2+)]i regulation in DCs. DCs were isolated from bone marrow of klothohm mice and wild type mice (klotho(+/+)) and cultured for 7-9 days with GM-CSF. According to MHCII and CD86 expression, differentiation and lipopolysaccharide (LPS)-induced maturation was similar in klothohmDCs and klotho(+/+)DCs. However, NCKX1 membrane abundance and NCX/NCKX-activity were significantly enhanced in klothohmDCs. The [Ca(2+)]i increase upon acute application of LPS (1 µg/ml) was significantly lower in klothohmDCs than in klotho(+/+)DCs, a difference reversed by NCKX-blocker 3',4'-dichlorobenzamyl (DBZ, 10 µM). CCL21-dependent migration was significantly less in klothohmDCs than in klotho(+/+)DCs but could be restored by DBZ. NCKX-activity was enhanced by pretreatment of klotho(+/+)DC precursors with 1,25(OH)2D3 the first 2 days after isolation from bone marrow. Feeding klothohmmice vitamin D deficient diet decreased NCKX activity, augmented LPS-induced increase of [Ca(2+)]i, and enhanced migration of klothohmDCs, thus dissipating the differences between klothohmDCs and klotho(+/+)DCs. In conclusion, Klotho deficiency up-regulates NCKX1 membrane abundance and Na(+)/Ca(2+)-exchange activity thus blunting the increase of [Ca(2+)]i following LPS-exposure and CCL21-mediated migration. The effects are in large part due to excessive 1,25(OH)2D3 formation.
AJP Cell Physiology 04/2013; · 3.54 Impact Factor
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Anja Eylenstein,
Eva-Maria Gehring, Nicole Heise,
Ekaterina Shumilina,
Sebastian Schmidt,
Kalina Szteyn,
Patrick Münzer,
Meerim K Nurbaeva,
Melanie Eichenmüller,
Leonid Tyan,
Ivonne Regel,
Michael Föller,
Dietmar Kuhl,
Jonathan Soboloff,
Reinhold Penner,
Florian Lang
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ABSTRACT: Ca(2+) signaling includes store-operated Ca(2+) entry (SOCE) following depletion of endoplasmic reticulum (ER) Ca(2+) stores. On store depletion, the ER Ca(2+) sensor STIM1 activates Orai1, the pore-forming unit of Ca(2+)-release-activated Ca(2+) (CRAC) channels. Here, we show that Orai1 is regulated by serum- and glucocorticoid-inducible kinase 1 (SGK1), a growth factor-regulated kinase. Membrane Orai1 protein abundance, I(CRAC), and SOCE in human embryonic kidney (HEK293) cells stably expressing Orai1 and transfected with STIM1 were each significantly enhanced by coexpression of constitutively active (S422D)SGK1 (by+81, +378, and+136%, respectively) but not by inactive (K127N)SGK1. Coexpression of the ubiquitin ligase Nedd4-2, an established negatively regulated SGK1 target, down-regulated SOCE (by -48%) and I(CRAC) (by -60%), an effect reversed by expression of (S422D)SGK1 (by +175 and +173%, respectively). Orai1 protein abundance and SOCE were significantly lower in mast cells from SGK1-knockout (sgk1(-/-)) mice (by -37% and -52%, respectively) than in mast cells from wild-type (sgk1(+/+)) littermates. Activation of SOCE by sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase-inhibitor thapsigargin (2 μM) stimulated migration, an effect significantly higher (by +306%) in (S422D)SGK1-expressing than in (K127N)SGK1-expressing HEK293 cells, and also significantly higher (by +108%) in sgk1(+/+) than in sgk1(-/-) mast cells. SGK1 is thus a novel key player in the regulation of SOCE.
The FASEB Journal 03/2011; 25(6):2012-21. · 5.71 Impact Factor
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Nicole Heise,
Ekaterina Shumilina,
Meerim K Nurbaeva,
Evi Schmid,
Kalina Szteyn,
Wenting Yang,
Nguyen Thi Xuan,
Kan Wang,
Irina M Zemtsova,
Michael Duszenko,
Florian Lang
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ABSTRACT: Ca(+)-dependent signaling regulates the function of dendritic cells (DCs), antigen-presenting cells linking innate and adaptive immunity. The activity of DCs is suppressed by glucocorticoids, potent immunosuppressive hormones. The present study explored whether the glucocorticoid dexamethasone influences the cytosolic Ca(2+) concentration ([Ca(2+)](i)) in DCs. To this end, DCs were isolated from mouse bone marrow. According to fura-2 fluorescence, exposure of DCs to lipopolysaccharide (LPS, 100 ng/ml) increased [Ca(2+)](i), an effect significantly blunted by overnight incubation with 10 nM dexamethasone before LPS treatment. Dexamethasone did not affect the Ca(2+) content of intracellular stores, sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)2 and SERCA3 expression, ryanodine receptor (RyR)1 expression, or Ca(2+) entry through store-operated Ca(2+) channels. In contrast, dexamethasone increased the transcript level and the membrane protein abundance of the Na(+)/Ca(2+) exchanger NCX3. The activity of Na(+)/Ca(2+) exchangers was assessed by removal of extracellular Na(+) in the presence of external Ca(2+), a maneuver triggering the Ca(2+) influx mode. Indeed, Na(+) removal resulted in a rapid transient increase of [Ca(2+)](i) and induced an outwardly directed current as measured in whole cell patch-clamp experiments. Dexamethasone significantly augmented the increase of [Ca(2+)](i) and the outward current following removal of extracellular Na(+). The NCX blocker KB-R7943 reversed the inhibitory effect of dexamethasone on LPS-induced increase in [Ca(2+)](i). Dexamethasone blunted LPS-induced stimulation of CD86 expression and TNF-α production, an effect significantly less pronounced in the presence of NCX blocker KB-R7943. In conclusion, our results show that glucocorticoid treatment blunts LPS-induced increase in [Ca(2+)](i) in DCs by increasing expression and activity of Na(+)/Ca(2+) exchanger NCX3. The effect contributes to the inhibitory effect of the glucocorticoid on DC maturation.
AJP Cell Physiology 02/2011; 300(6):C1306-13. · 3.54 Impact Factor
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ABSTRACT: Previous studies have shown that pharmacological inhibition of the phosphoinositol-3 (PI3) kinase disrupts the activation of mast cells. Through phosphoinositide-dependent kinase PDK1, PI3 kinase activates the serum- and glucocorticoid-inducible kinase 3 (SGK3). The present study explored the role of SGK3 in mast cell function. Mast cells were isolated and cultured from bone marrow (BMMCs) of gene-targeted mice lacking SGK3 (sgk3(-/-)) and their wild-type littermates (sgk3(+/+)). BMMC numbers in the ear conch were similar in both genotypes. Stimulation with IgE and cognate antigen triggered the release of intracellular Ca(2+) and entry of extracellular Ca(2+). Influx of extracellular Ca(2+) but not Ca(2+) release from intracellular stores was significantly blunted in sgk3(-/-) BMMCs compared with sgk3(+/+) BMMCs. Antigen stimulation further led to a rapid increase of a K(+)-selective conductance in sgk3(+/+) BMMCs, an effect again blunted in sgk3(-/-) BMMCs. In contrast, the Ca(2+) ionophore ionomycin activated K(+) currents to a similar extent in sgk3(-/-) and in sgk3(+/+) BMMCs. β-Hexosaminidase release, triggered by antigen stimulation, was also significantly decreased in sgk3(-/-) BMMCs. IgE-dependent anaphylaxis measured as a sharp decrease in body temperature upon injection of DNP-HSA antigen was again significantly blunted in sgk3(-/-) compared with sgk3(+/+) mice. Serum histamine levels measured 30 min after induction of an anaphylactic reaction were significantly lower in sgk3(-/-) than in sgk3(+/+) mice. In conclusion, both in vitro and in vivo function of BMMCs are impaired in gene targeted mice lacking SGK3. Thus SGK3 is critical for proper mast cell function.
AJP Cell Physiology 11/2010; 299(5):C1007-14. · 3.54 Impact Factor
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ABSTRACT: The function of mast cells is modified by the phosphoinositol-3 (PI3)-kinase pathway. The kinase signals partially through the phosphoinositide-dependent kinase PDK1, which on the one hand activates the serum- and glucocorticoid- inducible kinase SGK1 and on the other hand activates protein kinase PKCδ. SGK1 participates in the stimulation of Ca(2+) entry and degranulation, PKCδ inhibits degranulation. The present experiments explored the role of PDK1 in mast cell function. As mice completely lacking PDK1 are not viable, experiments have been performed in mast cells isolated from bone marrow (BMMCs) of PDK1 hypomorphic mice (pdk1(hm)) and their wild-type littermates (pdk1(wt)). Antigen stimulation via the FceRI receptor was followed by Ca(2+) entry leading to increase of cytosolic Ca(2+) activity in pdk1(wt) BMMCs, an effect significantly blunted in pdk1(hm) BMMCs. In contrast, Ca(2+) release from intracellular stores was not different between BMMCs of the two genotypes. The currents through Ca(2+)-activated K(+) channels following antigen exposure were again significantly larger in pdk1(wt) than in pdk1(hm) cells. The Ca(2+) ionophore ionomycin (1 μM) increased the K(+) channel conductance to similar values in both genotypes. β-hexosaminidase and histamine release were similar in pdk1(wt) BMMCs and pdk1(hm) BMMCs. PKCδ inhibitor rottlerin increased β-hexosaminidase release in pdk1(wt) BMMCs but not in pdk1(hm) BMMCs. Phosphorylation of PKCδ and of the SGK1 target NDRG1, was stimulated by the antigen in pdk1(wt) but not in pdk1(hm) cells. The observations reveal a role for PDK1 in the regulation of Ca(2+) entry into and degranulation of murine mast cells.
Cellular Physiology and Biochemistry 01/2010; 26(4-5):699-706. · 2.86 Impact Factor
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ABSTRACT: Genotoxic stress induces cell cycle arrest and DNA repair which may enable tumor cells to survive radiation therapy. Here, we defined the role of Ca(2+) signaling in the cell cycle control and survival of chronic myeloid leukemia (CML) cells subjected to ionizing radiation (IR). To this end, K562 erythroid leukemia cells were irradiated (0-10 Gy). Tumor survival was analyzed by clonogenic survival assay and cell cycle progression via flow cytometry. Plasma membrane cation conductance was assessed by patch-clamp whole-cell recording and the cytosolic free Ca(2+) concentration ([Ca(2+)](i)) was measured by fura-2 Ca(2+) imaging. Nuclear activity of Ca(2+)/calmodulin-dependent kinase II (CaMKII) was defined by Western blotting. In addition, the effect of IR (5 Gy) on the cation conductance of primary CML cells was determined. The results indicated that IR (10 Gy) induced a G(2)/M cell cycle arrest of K562 cells within 24 h post-irradiation (p.i.) and decreased the clonogenic survival to 0.5 % of that of the control cells. In K562 cells, G(2)/M cell cycle arrest was preceded by activation of TRPV5/6-like nonselective cation channels in the plasma membrane 1-5 h p.i., resulting in an elevated Ca(2+) entry as evident from fura-2 Ca(2+) imaging. Similarly, IR stimulated a Ca(2+)-permeable nonselective cation conductance in primary CML cells within 2-4 h p.i.. Ca(2+) entry, into K562 cells was paralleled by an IR-induced activation of nuclear CaMKII. The IR-stimulated accumulation in G(2) phase was delayed upon buffering [Ca(2+)](i) with the Ca(2+) chelator BAPTA-AM or inhibiting CaMKII with KN93 (1 nM). In addition, KN93 decreased the clonogenic survival of irradiated cells but not of control cells. In conclusion, the data suggest that IR-stimulated cation channel activation, Ca(2+) entry and CaMKII activity participate in control of cell cycle progression and survival of irradiated CML cells.
Cellular Physiology and Biochemistry 01/2010; 26(4-5):597-608. · 2.86 Impact Factor
