Toll-like receptors (TLRs) sense pathogen-associated molecular patterns to activate the production of inflammatory mediators. TLR4 recognizes lipopolysaccharide (LPS) and drives the secretion of inflammatory cytokines, often contributing to sepsis. We report that transient receptor potential melastatin-like 7 (TRPM7), a non-selective but Ca-conducting ion channel, mediates the cytosolic Ca elevations essential for LPS-induced macrophage activation. LPS triggered TRPM7-dependent Ca elevations essential for TLR4 endocytosis and the subsequent activation of the transcription factor IRF3. In a parallel pathway, the Ca signaling initiated by TRPM7 was also essential for the nuclear translocation of NFκB. Consequently, TRPM7-deficient macrophages exhibited major deficits in the LPS-induced transcriptional programs in that they failed to produce IL-1β and other key pro-inflammatory cytokines. In accord with these defects, mice with myeloid-specific deletion of Trpm7 are protected from LPS-induced peritonitis. Our study highlights the importance of Ca signaling in macrophage activation and identifies the ion channel TRPM7 as a central component of TLR4 signaling.

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.

Background/Aims: The serum- and glucocorticoid-inducible kinase Sgk1 contributes to cardiac remodeling and development of heart failure, which is paralelled by Sgk1-dependent stimulation of the cardiac Na⁺/H⁺ exchanger Nhe1. Glucocorticoids are powerful stimulators of Sgk1 expression and influence cardiac remodeling. The present study thus explored whether the glucocorticoid receptor agonist dexamethasone influenced cardiac Sgk1 expression, as well as activity, expression and phosphorylation at Ser⁷⁰³ of the cardiac Na⁺/H⁺ exchanger Nhe1. Methods: Experiments were performed in HL-1 cardiomyocytes and gene targeted mice lacking functional Sgk1 (sgk1^-/-) and respective wild type mice (sgk1^+/+). Gene expression was determined by quantitative RT-PCR and Nhe1 phosphorylation was determined utilizing a specific antibody against a 14-3-3 binding motif at P-Ser⁷⁰³, which represents a putative phosphorylation site recognition motif for Sgk1 and is involved in Nhe1 activation. Cytosolic pH (pH_i) was determined utilizing 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) fluorescence and Nhe activity by the Na⁺-dependent realkalinization after an ammonium pulse. Results: Treatment of HL-1 cardiomyocytes with dexamethasone was followed by a significant increase in Sgk1 mRNA expression, parallelled by increased Na⁺/H⁺ exchanger activity. Furthermore, dexamethasone significantly increased Nhe1 and Spp1 mRNA expression. The effects of dexamethasone were blunted by cotreatment of HL-1 cardiomyocytes with the Sgk1 inhibitor EMD638683. Cotreatment with Nhe1 inhibitor cariporide similarly prevented dexamethasone-stimulated Spp1 mRNA expression. In sgk1^+/+ mice, dexamethasone significantly increased cardiac Sgk1 mRNA levels. In sgk1^+/+ mice, but not in sgk1^-/- mice, dexamethasone significantly increased cardiac Nhe1 mRNA expression and Nhe1 phosphorylation at Ser⁷⁰³. Furthermore, cardiac Spp1, Ctgf, Nppa and Nppb mRNA levels were significantly increased in dexamethasone treated sgk1^+/+ mice, effects significantly blunted in sgk1^-/- mice. Conclusions: Sgk1 is critically involved in the phosphorylation and activation of the cardiac Na⁺/H⁺ exchanger Nhe1.

Alterations of cytosolic Ca 2ϩ activity participate in the regulation of a wide variety of cellular functions including excitation-contraction coupling, exocytosis, migration, cell proliferation, and cell death (1-4). Cytosolic Ca 2ϩ is increased by release of Ca 2ϩ from intracellular stores and/or Ca 2ϩ entry across the cell membrane (5). Ca 2ϩ release from intracellular stores results in the stimulation of Ca 2ϩ release-activated Ca 2ϩ channel (CRAC) 2 (6, 7), which consists of the pore forming units Orai1, -2, and/or -3 (8 -10) and the endoplasmic reticulum-located regulatory subunit STIM1 or -2 (11-13). The stimulation of the channel leads to the inward current I CRAC and the store-operated Ca 2ϩ entry (SOCE). Recent observations uncovered the powerful stimulation of I CRAC and SOCE by the serum and glucocorticoid-inducible kinase SGK1 (14), a kinase stimulated by growth factors and involved in stress response (15) and regulation of cell survival (16). SGK1 is partially effective through phosphorylation of the ubiquitin ligase Nedd4-2 (neuronal precursor cells expressed developmentally down-regulated). Nedd4-2 ubiquitinates Orai1, thus preparing the channel protein for degradation (14). The effect of Nedd4-2 on Orai1 parallels that of Nedd4-2 on the epithelial Na ϩ channel ENaC (16, 17). The phosphorylation of Nedd4-2 leads to binding of the ubiquitin ligase to the protein 14-3-3, which prevents the interaction with the channel protein (18). Accordingly, SGK1 enhances Orai1 protein abundance in the cell membrane (14). STIM is similarly regulated by ubiquitination (19). However, the effect of SGK1 on Orai1 protein abundance is only in part explained by Nedd4-2-dependent protein degradation. Therefore, further experiments were performed to explore whether SGK1, in addition, stimulates Orai1 and/or STIM1 expression. As a matter of fact, RT-PCR revealed an increase of Orai1 and STIM1 transcript levels after expression of constitutively active SGK1. Thus, further experiments were performed to uncover the transcription factor involved. Previously, SGK1 has been shown to foster nuclear translocation and activation of nuclear factor B (NF-B) (20 -22). Accordingly, this study explored the putative involvement of NF-B subunits p65 (RELA), p50 (NFKB1), and p52 (NFKB2) in the regulation of Orai1 and STIM1 expression. EXPERIMENTAL PROCEDURES

Messenger RNA data of lymphohematopoietic cancer lines suggest a correlation between expression of the cation channel TRPM2 and the antiapoptotic protein Bcl-2. The latter is overexpressed in various tumor entities and mediates therapy resistance. Here, we analyzed the crosstalk between Bcl-2 and TRPM2 channels in T cell leukemia cells during oxidative stress as conferred by ionizing radiation (IR). To this end, the effects of TRPM2 inhibition or knock-down on plasma membrane currents, Ca2+ signaling, mitochondrial superoxide anion formation, and cell cycle progression were compared between irradiated (0–10 Gy) Bcl-2-overexpressing and empty vector-transfected Jurkat cells. As a result, IR stimulated a TRPM2-mediated Ca2+-entry, which was higher in Bcl-2-overexpressing than in control cells and which contributed to IR-induced G2/M cell cycle arrest. TRPM2 inhibition induced a release from G2/M arrest resulting in cell death. Collectively, this data suggests a pivotal function of TRPM2 in the DNA damage response of T cell leukemia cells. Apoptosis-resistant Bcl-2-overexpressing cells even can afford higher TRPM2 activity without risking a hazardous Ca2+-overload-induced mitochondrial superoxide anion formation.

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.

The large-conductance calcium- and voltage-activated K+ channel (BKCa) are encoded by the Kcnma1 gene. They are ubiquitously expressed in neuronal, smooth muscle, astrocytes, and neuroendocrine cells where they are known to play an important role in physiological and pathological processes. They are usually localized to the plasma membrane of the majority of the cells with an exception of adult cardiomyocytes, where BKCa is known to localize to mitochondria. BKCa channels couple calcium and voltage responses in the cell, which places them as unique targets for a rapid physiological response. The expression and activity of BKCa have been linked to several cardiovascular, muscular, and neurological defects, making them a key therapeutic target. Specifically in the heart muscle, pharmacological and genetic activation of BKCa channels protect the heart from ischemia-reperfusion injury and also facilitate cardioprotection rendered by ischemic preconditioning. The mechanism involved in cardioprotection is assigned to the modulation of mitochondrial functions, such as regulation of mitochondrial calcium, reactive oxygen species, and membrane potential. Here, we review the progress made on BKCa channels and cardioprotection and explore their potential roles as therapeutic targets for preventing acute myocardial infarction.

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.