Kalina Szteyn

Stimulation of Ca2+-channel Orai1/STIM1 by serum- and glucocorticoid-inducible kinase 1 (SGK1)

Ca2+ signaling includes store‐operated Ca2+ entry (SOCE) following depletion of endoplas‐mic reticulum (ER) Ca2+ stores. On store depletion, the ER Ca2+ sensor STIM1 activates Orai1, the pore‐forming unit of Ca2+‐release‐activated Ca2+ (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, /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 SGK1 (by+81, +378, and+136%, respectively) but not by inactive K12 NSGK1. Coexpression of the ubiquitin ligase Nedd4‐2, an established negatively regulated SGK1 target, down‐regulated SOCE (by –48%) and /CRAc (by –60%), an effect reversed by expression of 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 Ca2+‐ATPase‐inhibitor thapsi‐gargin (2 μΜ) stimulated migration, an effect significantly higher (by +306%) in SGKl‐expressing than in SGKl‐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.—Eylenstein, A., Geh‐ring, E.‐M., Heise, N., Shumilina, E., Schmidt, S., Szteyn, K., Münzer, P., Nurbaeva, M. K., Eichenmüller, M., Tyan, L., Regel, I., Föller, M., Kuhl, D., Soboloff, J., Penner, R., Lang, R. Stimulation of Ca2+‐channel Orai1/STIM1 by serum‐ and glucocorticoid‐inducible kinase 1 (SGK1). FASEB J. 25, 2012‐2021 (2011). www.fasebj.org

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.

Down-Regulation of Na+/K+ ATPase Activity by Human Parvovirus B19 Capsid Protein VP1

Background/Aims: Human parvovirus B19 (B19V) may cause inflammatory cardiomyopathy (iCMP) which is accompanied by endothelial dysfunction. The B19V capsid protein VP1 contains a lysophosphatidylcholine producing phospholipase A2 (PLA) sequence. Lysophosphatidylcholine has in turn been shown to inhibit Na+/K+ ATPase. The present study explored whether VP1 modifies Na+/K+ ATPase activity. Methods: Xenopus oocytes were injected with cRNA encoding VP1 isolated from a patient suffering from fatal B19V-iCMP or cRNA encoding PLA2-negative VP1 mutant (H153A) and K+ induced pump current (Ipump) as well as ouabain-inhibited current (Iouabain) both reflecting Na+/K+-ATPase activity were determined by dual electrode voltage clamp. Results: Injection of cRNA encoding VP1, but not of VP1(H153A) or water, was followed by a significant decrease of both, Ipump and Iouabain in Xenopus oocytes. The effect was not modified by inhibition of transcription with actinomycin (10 µM for 36 hours) but was abrogated in the presence of PLA2 specific blocker 4-bromophenacylbromide (50 µM) and was mimicked by lysophosphatidylcholine (0.5 - 1 µg/ml). According to whole cell patch clamp, lysophosphatidylcholine (1 µg /ml) similarly decreased Ipump in human microvascular endothelial cells (HMEC). Conclusion: The B19V capsid protein VP1 is a powerful inhibitor of host cell Na+/K+ ATPase, an effect at least partially due to phospholipase A2 (PLA2) dependent formation of lysophosphatidylcholine.

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

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.

Energy-sensitive regulation of Na+/K+-ATPase by Janus kinase 2

Janus kinase 2 (JAK2) contributes to intracellular signaling of leptin and erythropoietin, hormones protecting cells during energy depletion. The present study explores whether JAK2 is activated by energy depletion and regulates Na(+)/K(+)-ATPase, the major energy-consuming pump. In Jurkat cells, JAK2 activity was determined by radioactive kinase assay, phosphorylated JAK2 detected by Western blotting, ATP levels measured by luciferase assay, as well as Na(+)/K(+)-ATPase α1-subunit transcript and protein abundance determined by real-time PCR and Western blotting, respectively. Ouabain-sensitive K(+)-induced currents (Ipump) were measured by whole cell patch clamp. Ipump was further determined by dual-electrode voltage clamp in Xenopus oocytes injected with cRNA-encoding JAK2, active (V617F)JAK2, or inactive (K882E)JAK2. As a result, in Jurkat T cells, JAK2 activity significantly increased following energy depletion by sodium azide (NaN3) or 2,4- dinitro phenol (DNP). DNP- and NaN3-induced decrease of cellular ATP was significantly augmented by JAK2 inhibitor AG490 and blunted by Na(+)/K(+)-ATPase inhibitor ouabain. DNP decreased and AG490 enhanced Ipump as well as Na(+)/K(+)-ATPase α1-subunit transcript and protein abundance. The α1-subunit transcript levels were also enhanced by signal transducer and activator of transcription-5 inhibitor CAS 285986-31-4. In Xenopus oocytes, Ipump was significantly decreased by expression of JAK2 and (V617F)JAK2 but not of (K882E)JAK2, effects again reversed by AG490. In (V617F)JAK2-expressing Xenopus oocytes, neither DNP nor NaN3 resulted in further decline of Ipump. In Xenopus oocytes, the effect of (V617F)JAK2 on Ipump was not prevented by inhibition of transcription with actinomycin. In conclusion, JAK2 is a novel energy-sensing kinase that curtails energy consumption by downregulating Na(+)/K(+)-ATPase expression and activity.