Wenting Yang

Transcription Factor NF-κB Regulates Expression of Pore-forming Ca2+ Channel Unit, Orai1, and Its Activator, STIM1, to Control Ca2+ Entry and Affect Cellular Functions

The serum and glucocorticoid-inducible kinase SGK1 increases the activity of Orai1, the pore forming unit of store-operated Ca2+ entry, and thus influences Ca2+-dependent cellular functions such as migration. SGK1 further regulates transcription factor nuclear factor κB (NF-κB). This study explored whether SGK1 influences transcription of Orai1 and/or STIM1, the Orai1-activating Ca2+ sensor. Orai1 and STIM1 transcript levels were decreased in mast cells from SGK1 knock-out mice and increased in HEK293 cells transfected with active S422DSGK1 but not with inactive K127NSGK1 or in S422DSGK1-transfected cells treated with the NF-κB inhibitor Wogonin (100 μm). Treatment with the stem cell factor enhanced transcript levels of STIM1 and Orai1 in sgk1+/+ but not in sgk1−/− mast cells and not in sgk1+/+ cells treated with Wogonin. Orai1 and STIM1 transcript levels were further increased in sgk1+/+ and sgk1−/− mast cells by transfection with active NF-κB subunit p65 as well as in HEK293 cells by transfection with NF-κB subunits p65/p50 or p65/p52. They were decreased by silencing of NF-κB subunits p65, p50, or p52 or by NF-κB inhibitor Wogonin (100 μm). Luciferase assay and chromatin immunoprecipitation defined NF-κB-binding sites in promoter regions accounting for NF-κB sensitive genomic regulation of STIM1 and Orai1. Store-operated Ca2+ entry was similarly increased by overexpression of p65/p50 or p65/p52 and decreased by treatment with Wogonin. Transfection of HEK293 cells with p65/p50 or p65/p52 further augmented migration. The present observations reveal powerful genomic regulation of Orai1/STIM1 by SGK1-dependent NF-κB signaling.

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.

Expression and functional significance of the Ca(2+)-activated Cl(-) channel ANO6 in dendritic cells.

Background/Aims: Migration of dendritic cells (DCs), antigen presenting cells that link innate and adaptive immunity, is critical for initiation of immune responses. DC migration is controlled by the activity of different ion channels, which mediate Ca2+ flux or set the membrane potential. Moreover, cell migration requires local volume changes at the leading and rear end of travelling cells, which might be mediated by the fluxes of osmotically active solutes, including Cl-. The present study explored the functional expression, regulation and role of Cl- channels in mouse bone marrow-derived DCs. Methods/Results: In whole-cell patch clamp experiments we detected outwardly rectifying Cl- currents which were activated by elevation of cytosolic Ca2+, triggered either by ionomycin in the presence of extracellular Ca2+ or mobilization of Ca2+ by IP3 Most importantly, Ca2+-activated Cl- channels (CaCCs) were activated by CCL21 (75 ng/ml), an agonist of the chemokine receptor CCR7. The currents showed sensitivity to Cl- channel blockers such as tannic acid (10 µM), digallic acid (100 µM) and more specific CaCC blockers niflumic acid (300 µM) and AO1 (20 µM). According to RT-PCR and Western blot data, Anoctamin 6 (ANO6) is expressed in DCs. Knock-down of ANO6 with siRNA led to inhibition of CaCC currents in DCs. Moreover, chemokine-induced migration of both immature and LPS-matured DCs was reduced upon ANO6 knock-down. Conclusion: Our data identify ANO6 as a Ca2+-activated Cl- channel in mouse DCs, show its activation upon chemokine receptor ligation and establish an important role of ANO6 in chemokine-induced DC migration.

Enhanced Ca2+ entry and Na+/Ca2+ exchanger activity in dendritic cells from AMP-activated protein kinase-deficient mice

In dendritic cells (DCs), chemotactic chemokines, such as CXCL12, rapidly increase cytosolic Ca2+ concentrations ([Ca2+]i) by triggering Ca2+ release from intracellular stores followed by store‐operated Ca2+ (SOC) entry. Increase of [Ca2+]i is blunted and terminated by Ca2+ extrusion, accomplished by K+independent Na+/Ca2+ exchangers (NCXs) and K+‐dependent Na+/Ca2+ exchangers (NCKXs). Increased [Ca2+]i activates energy‐sensing AMP‐activated protein kinase (AMPK), which suppresses proinflammatory responses of DCs and macrophages. The present study explored whether AMPK participates in the regulation of DC [Ca2+]i and migration. DCs were isolated from AMPKα1‐deficient (ampk–/–) mice and, as control, from their wild‐type (ampk+/+) littermates. AMPKα1, Orai1‐2, STIM1‐2, and mitochondrial calcium uniporter protein expression was determined by Western blotting, [Ca2+]i by Fura‐2 fluorescence, SOC entry by inhibition of endosomal Ca2+ ATPase with thapsigargin (1 μM), Na+/Ca2+ exchanger activity from increase of [Ca2+]i, and respective whole‐cell current in patch clamp following removal of extracellular Na+. Migration was quantified utilizing transwell chambers. AMPKα1 protein is expressed in ampk+/+ DCs but not in ampk–/– DCs. CXCL12 (300 ng/ml)‐induced increase of [Ca2+]i, SOC entry, Orai 1 protein abundance, NCX, and NCKX were all significantly higher in ampk–/– DCs than in ampk+/+ DCs. NCX and NCKX currents were similarly increased in ampk–/– DCs. Moreover, CXCL12 (50 ng/ml)‐induced DC migration was enhanced in ampk–/– DCs. AMPK thus inhibits SOC entry, Na+/Ca2+ exchangers, and migration of DCs.—Nurbaeva, M. K., Schmid, E., Szteyn, K., Yang, W., Viollet, B., Shumilina, E., Lang, F. Enhanced Ca2+ entry and Na+/Ca2+ exchanger activity in dendritic cells from AMP‐activated protein kinase‐deficient mice. FASEB J. 26, 3049–3058 (2012). www.fasebj.org

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.

1,25( OH)(2) vitamin D-3-dependent inhibition of platelet Ca2+ signaling and thrombus formation in klotho-deficient mice

Platelets are activated by increased cytosolic Ca2+ concentration ([Ca2+]i) following store‐operated calcium entry (SOCE) accomplished by calcium‐release‐activated calcium (CRAC) channel moiety Orai1 and its regulator STIM1. In other cells, Ca2+ transport is regulated by 1,25(OH)2 vitamin D3 [1,25(OH)2D3]. 1,25(OH)2D3 formation is inhibited by klotho and excessive in klotho‐deficient mice (kl/kl). The present study explored the effect of klotho deficiency on platelet Ca2+ signaling and activation. Platelets and megakaryocytes isolated from WT and kl/kl‐mice were analyzed by RT‐PCR, Western blotting, confocal microscopy, Fura‐2‐fluorescence, patch clamp, flow cytometry, aggregometry, and flow chamber. STIM1/Orai1 transcript and protein levels, SOCE, agonist‐induced [Ca2+]i increase, activation‐dependent degranulation, integrin αIIbβ3 activation and aggregation, and thrombus formation were significantly blunted in kl/kl platelets (by 27–90%). STIM1/Orai1 transcript and protein levels, as well as CRAC currents, were significantly reduced in kl/kl megakaryocytes (by 38–73%) and 1,25(OH)2D3‐treated WT megakaryocytes. Nuclear NF‐κB subunit p50/p65 abundance was significantly reduced in kl/kl‐megakaryocytes (by 51–76%). Transfection with p50/p65 significantly increased STIM1/Orai1 transcript and protein levels in megakaryocytic MEG‐01 cells (by 46–97%). Low‐vitamin D diet (LVD) of kl/kl mice normalized plasma 1,25(OH)2D3 concentration and function of platelets and megakaryocytes. Klotho deficiency inhibits platelet Ca2+ signaling and activation, an effect at least partially due to 1,25(OH)2D3‐dependent down‐regulation of NF‐κB activity and STIM1/Orai1 expression in megakaryocytes.—Borst, O., Münzer, P., Schmid, E., Schmidt, E.‐M., Russo, A., Walker, B., Yang, W., Leibrock, C., Szteyn, K., Schmidt, S., Elvers, M., Faggio, C., Shumilina, E., Kuro‐o, M., Gawaz, M., Lang, F. 1,25(OH)2 vitamin D3‐dependent inhibition of platelet Ca2+ signaling and thrombus formation in klotho‐deficient mice. FASEB J. 28, 2108–2119 (2014). www.fasebj.org

Akt2-and ETS1-Dependent IP3 Receptor 2 Expression in Dendritic Cell Migration

Background/Aims: The protein kinase Akt2/PKBβ is a known regulator of macrophage and dendritic cell (DC) migration. The mechanisms linking Akt2 activity to migration remained, however, elusive. DC migration is governed by Ca²⁺ signaling. We thus explored whether Akt2 regulates DC Ca²⁺ signaling. Methods: DCs were derived from bone marrow of Akt2-deficient mice (akt2^-/-) and their wild type littermates (akt2^+/+). DC maturation was induced by lipopolysaccharides (LPS) and evaluated by flow cytometry. Cytosolic Ca²⁺ concentration was determined by Fura-2 fluorescence, channel activity by whole cell recording, transcript levels by RT-PCR, migration utilizing transwells. Results: Upon maturation, chemokine CCL21 stimulated migration of akt2^+/+ but not akt2^-/- DCs. CCL21-induced increase in cytosolic Ca²⁺ concentration, thapsigargin-induced release of Ca²⁺ from intracellular stores with subsequent store-operated Ca²⁺ entry (SOCE), ATP-induced inositol 1,4,5-trisphosphate (IP₃)-dependent Ca²⁺ release as well as Ca²⁺ release-activated Ca²⁺ (CRAC) channel activity were all significantly lower in mature akt2^-/- than in mature akt2^+/+ DCs. Transcript levels of IP₃ receptor IP₃R2 and of IP₃R2 regulating transcription factor ETS1 were significantly higher in akt2^+/+ than in akt2^-/- DCs prior to maturation and were upregulated by LPS stimulation (1h) in akt2^+/+ and to a lower extent in akt2^-/- DCs. Following maturation, protein abundance of IP₃R2 and ETS1 were similarly higher in akt2^+/+ than in akt2^-/- DCs. The IP₃R inhibitor Xestospongin C significantly decreased CCL21-induced migration of akt2^+/+DCs and abrogated the differences between genotypes. Finally, knock-down of ETS1 with siRNA decreased IP₃R2 mRNA abundance, thapsigargin- and ATP-induced Ca²⁺ release, SOCE and CRAC channel activation, as well as DC migration. Conclusion: Akt2 upregulates DC migration at least in part by ETS1-dependent stimulation of IP₃R2 transcription.

Lipopolysaccharide-sensitive H+ current in dendritic cells

Dendritic cells (DCs) are the most potent antigen-presenting cells equipped to transport antigens from the periphery to lymphoid tissues and to present them to T cells. Ligation of Toll-like receptor 4 (TLR4), expressed on the DC surface, by lipopolysaccharides (LPS), elements of the Gram-negative bacteria outer wall, induces DC maturation. Initial steps of maturation include stimulation of antigen endocytosis and enhanced reactive oxygen species (ROS) production with eventual downregulation of endocytic capacity in fully matured DCs. ROS production depends on NADPH oxidase (NOX2), the activity of which requires continuous pH and charge compensation. The present study demonstrates, for the first time, the functional expression of voltage-gated proton (Hv1) channels in mouse bone marrow-derived DCs. In whole cell patch-clamp experiments, we recorded Zn(2+) (50 μM)-sensitive outwardly rectifying currents activated upon depolarization, which were highly selective for H(+), with the reversal potential shift of 38 mV per pH unit. The threshold voltage of activation (V(threshold)) was dependent on the pH gradient and was close to the empirically predicted V(threshold) for the Hv1 currents. LPS (1 μg/ml) had bimodal effects on Hv1 channels: acute LPS treatment increased Hv1 channel activity, whereas 24 h of LPS incubation significantly inhibited Hv1 currents and decreased ROS production. Activation of H(+) currents by acute application of LPS was abolished by PKC inhibitor GFX (10 nM). According to electron current measurements, acute LPS application was associated with increased NOX2 activity.

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.

Effect of dexamethasone on Na+/Ca2+ exchanger in dendritic cells.

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.