Chanzyme TRPM7 Mediates the Ca2+ Influx Essential for Lipopolysaccharide-Induced Toll-Like Receptor 4 Endocytosis and Macrophage Activation.

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 Ca2+-conducting ion channel, mediates the cytosolic Ca2+ elevations essential for LPS-induced macrophage activation. LPS triggered TRPM7-dependent Ca2+ elevations essential for TLR4 endocytosis and the subsequent activation of the transcription factor IRF3. In a parallel pathway, the Ca2+ 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-1ß 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 Ca2+ signaling in macrophage activation and identifies the ion channel TRPM7 as a central component of TLR4 signaling.

Targeting TRPM2 Channels Impairs Radiation-Induced Cell Cycle Arrest and Fosters Cell Death of T Cell Leukemia Cells in a Bcl-2-Dependent Manner.

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, Ca(2+) 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 Ca(2+)-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 Ca(2+)-overload-induced mitochondrial superoxide anion formation.

The Role of Janus Kinase 3 in the Regulation of Na⁺/K⁺ ATPase under Energy Depletion.

BACKGROUND/AIMS: Janus kinase-3 (JAK3) is activated during energy depletion. Energy-consuming pumps include the Na(+)/K(+)-ATPase. The present study explored whether JAK3 regulates Na(+)/K(+)-ATPase in dendritic cells (DCs). METHODS: Ouabain (100 µM)-sensitive (Iouabain) and K(+)-induced (Ipump) outward currents were determined by utilizing whole cell patch-clamp, Na(+)/K(+)-ATPase α1-subunit mRNA levels by RT-PCR, Na(+)/K(+)-ATPase protein abundance by flow cytometry or immunofluorescence, and cellular ATP by luciferase-assay in DCs from bone marrow of JAK3-knockout (jak3(-/-)) or wild-type mice (jak3(+/+)). Ipump was further determined by voltage clamp in Xenopus oocytes expressing JAK3, active (A568V)JAK3 or inactive (K851A)JAK3. RESULTS: Na(+)/K(+)-ATPase α1-subunit mRNA and protein levels, as well as Ipump and Iouabain were significantly higher in jak3(-/-)DCs than in jak3(+/+)DCs. Energy depletion by 4h pre-treatment with 2,4-dinitro-phenol significantly decreased Ipump in jak3(+/+) DCs but not in jak3(-/-)DCs. Cellular ATP was significantly lower in jak3(-/-)DCs than in jak3(+/+)DCs and decreased in both genotypes by 2,4-dinitro-phenol, an effect significantly more pronounced in jak3(-/-)DCs than in jak3(+/+)DCs and strongly blunted by ouabain in both jak3(+/+) and jak3(-/-)DCs. Ipump and Iouabain in oocytes were decreased by expression of JAK3 and of (A568V)JAK3 but not of (K851A)JAK3. JAK3 inhibitor WHI-P154 (4-[(3'-bromo-4'-hydroxyphenyl)amino]-6,7-dimethoxyquinazoline, 22 µM) enhanced Ipump and Iouabain in JAK3 expressing oocytes. The difference between (A568V)JAK3 and (K851A)JAK3 expressing oocytes was virtually abrogated by actinomycin D (50 nM). CONCLUSIONS: JAK3 down-regulates Na(+)/K(+)-ATPase activity, an effect involving gene expression and profoundly curtailing ATP consumption.

Akt2- and ETS1-dependent IP3 receptor 2 expression in dendritic cell migration.

BACKGROUND/AIMS: The protein kinase Akt2/PKBß 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 Ca(2+) signaling. We thus explored whether Akt2 regulates DC Ca(2+) 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 Ca(2+) 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 Ca(2+) concentration, thapsigargin-induced release of Ca(2+) from intracellular stores with subsequent store-operated Ca(2+) entry (SOCE), ATP-induced inositol 1,4,5-trisphosphate (IP3)-dependent Ca(2+) release as well as Ca(2+) release-activated Ca(2+) (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 Ca(2+) 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.

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.

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 (I(pump)) as well as ouabain-inhibited current (I(ouabain)) 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, I(pump) and I(ouabain) 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 I(pump) 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.

Altered regulation of cytosolic Ca²âº concentration in dendritic cells from klotho hypomorphic mice.

The function of dendritic cells (DCs), antigen-presenting cells regulating naïve T-cells, is regulated by cytosolic Ca²âº concentration ([Ca²âº]i). [Ca²âº]i is increased by store-operated Ca²âº entry and decreased by K⁺-independent (NCX) and K⁺-dependent (NCKX) Na⁺/Ca²âº 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 the antiaging protein Klotho. Thus 1,25(OH)2D3 plasma levels are excessive in Klotho-deficient mice (klothohm). The present study explored whether Klotho deficiency modifies [Ca²âº]i regulation in DCs. DCs were isolated from the bone marrow of klothohm mice and wild-type mice (klotho+/+) and cultured for 7-9 days with granulocyte-macrophage colony-stimulating factor. According to major histocompatibility complex II (MHC II) and CD86 expression, differentiation and lipopolysaccharide (LPS)-induced maturation were similar in klothohm DCs and klotho+/+ DCs. However, NCKX1 membrane abundance and NCX/NCKX-activity were significantly enhanced in klothohm DCs. The [Ca²âº]i increase upon acute application of LPS (1 µg/ml) was significantly lower in klothohm DCs than in klotho+/+ DCs, a difference reversed by the NCKX blocker 3',4'-dichlorobenzamyl (DBZ; 10 µM). CCL21-dependent migration was significantly less in klothohm DCs 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 klothohm mice a vitamin D-deficient diet decreased NCKX activity, augmented LPS-induced increase of [Ca²âº]i, and enhanced migration of klothohm DCs, thus dissipating the differences between klothohm DCs and klotho+/+ DCs. In conclusion, Klotho deficiency upregulates NCKX1 membrane abundance and Na⁺/Ca²âº-exchange activity, thus blunting the increase of [Ca²âº]i following LPS exposure and CCL21-mediated migration. The effects are in large part due to excessive 1,25(OH)2D3 formation.

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 Ca(2+) entry, and thus influences Ca(2+)-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 Ca(2+) sensor. Orai1 and STIM1 transcript levels were decreased in mast cells from SGK1 knock-out mice and increased in HEK293 cells transfected with active (S422D)SGK1 but not with inactive (K127N)SGK1 or in (S422D)SGK1-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 Ca(2+) 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.

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

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.

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.