Regulation of calcium signaling in dendritic cells by 1,25-dihydroxyvitamin D3.

Dendritic cells (DCs) are antigen-presenting cells that provide a link between innate and adaptive immunity. Ca(2+)-dependent signaling plays a central regulatory role in DC responses to diverse antigens. DCs are a primary target of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], a secosteroid hormone, that, in addition to its well-established action on Ca(2+) homeostasis, possesses immunomodulatory properties. Surprisingly, nothing is known about its effects on DC cytosolic Ca(2+) activity. The present study explored whether 1,25(OH)(2)D(3) modifies the intracellular Ca(2+) concentration ([Ca(2+)](i)) in DCs. Here we show that mouse DCs expressed K(+)-independent (NCX1-3) and K(+)-dependent (NCKX1, 3, 4, and 5) Na(+)/Ca(2+) exchangers. Acute application of LPS (100 ng/ml) to DCs increased [Ca(2+)](i), an effect significantly blunted by prior incubation with 1,25(OH)(2)D(3). 1,25(OH)(2)D(3) increased the membrane abundance of the NCKX1 protein, up-regulated the K(+)- and Na(+)-dependent Ca(2+) entry and enhanced the K(+)-dependent Na(+)/Ca(2+) exchanger currents. The NCKX blocker 3',4'-dichlorobenzamyl (DBZ) reversed the inhibitory effect of 1,25(OH)(2)D(3) on the LPS-induced increase of [Ca(2+)](i). Expression of the costimulatory molecule CD86 was down-regulated by 1,25(OH)(2)D(3), an effect reversed by DBZ. In summary, 1,25(OH)(2)D(3) blunts the LPS-induced increase in [Ca(2+)](i) by stimulation of Na(+)/Ca(2+) exchanger-dependent Ca(2+) extrusion, an effect that contributes to 1,25(OH)(2)D(3)-mediated immunosuppression. The results disclose completely novel mechanisms in the regulation of DC maturation and function.

Impaired mast cell activation in gene-targeted mice lacking the serum- and glucocorticoid-inducible kinase SGK1.

The PI3K pathway plays a pivotal role in the stimulation of mast cells. PI3K-dependent kinases include the serum- and glucocorticoid-inducible kinase 1 (SGK1). The present study explored the role of SGK1 in mast cell function. Mast cells were isolated from bone marrow (BMMC) of SGK1 knockout mice (sgk1(-/-)) and their wild-type littermates (sgk1(+/+)). The BMMC number as well as CD117, CD34, and FcepsilonRI expression in BMCCs were similar in both genotypes. Upon Ag stimulation of the FcepsilonRI receptor, Ca(2+) entry but not Ca(2+) release from intracellular stores was markedly impaired in sgk1(-/-) BMMCs. The currents through Ca(2+)-activated K+ channels induced by Ag were significantly higher in sgk1(+/+) BMMCs than in sgk1(-/-) BMMCs. Treatment with the Ca(2+) ionophore ionomycin (1 microM) led to activation of the K+ channels in both genotypes, indicating that the Ca(2+)-activated K+ channels are similarly expressed and sensitive to activation by Ca(2+) in sgk1(+/+) and sgk1(-/-) BMMCs, and that blunted stimulation of Ca(2+)-activated K+ channels was secondary to decreased Ca(2+) entry. Ag-IgE-induced degranulation and early IL-6 secretion were also significantly blunted in sgk1(-/-) BMMCs. The decrease in body temperature following Ag treatment, which reflects an anaphylactic reaction, was substantially reduced in sgk1(-/-) mice, pointing to impaired mast cell function in vivo. Serum histamine levels measured 30 min after induction of an anaphylactic reaction were significantly lower in sgk1(-/-) than in sgk1(+/+)mice. The observations reveal a critical role for SGK1 in ion channel regulation and the function of mast cells, and thus disclose a completely novel player in the regulation of allergic reaction.

Ca2+-dependent functions in peptidoglycan-stimulated mouse dendritic cells.

Peptidoglycans (PGN) from bacterial cell walls may modify the course of an infection with bacterial pathogens. The present study explored the effect of PGN on cytosolic Ca2+ activity, cytokine production and phagocytosis of mouse dendritic cells (DCs), essential cells in the initiation and direction of antigen-specific T cell responses. Exposure of DCs to PGN was followed by a rapid increase in cytosolic Ca2+ activity ([Ca2+]i), which was due to Ca2+ release from intracellular stores and influx of extracellular Ca2+ across the cell membrane. In DCs isolated from Toll-like receptor 2 (TLR2) deficient mice the effect of PGN on [Ca2+]i was dramatically impaired. The PGN-induced increase of [Ca2+]i was dependent on voltage-gated K+ (Kv) channel activity. PGN-induced increase of [Ca2+]i was significantly blunted by margatoxin (MgTx) and perhexiline maleate (PM), inhibitors of Kv1.3 and Kv1.5, respectively. PGN further stimulated the release of tumour necrosis factor alpha (TNFalpha), interleukin-12 (IL-12) and interleukin-10 (IL-10), an effect significantly blunted by PM and the specific blocker of store-operated Ca2+ channels SKF-96365. Moreover, phagocytic capacity was dramatically increased in PGN-stimulated DCs in the presence of either Kv channel inhibitors or SKF-96365. The observations disclose Ca2+ and Kv channel-dependent cytokine production and phagocytosis in PGN-stimulated DCs.

Ion channels modulating mouse dendritic cell functions.

Ca(2+)-mediated signal transduction pathways play a central regulatory role in dendritic cell (DC) responses to diverse Ags. However, the mechanisms leading to increased [Ca(2+)](i) upon DC activation remained ill-defined. In the present study, LPS treatment (100 ng/ml) of mouse DCs resulted in a rapid increase in [Ca(2+)](i), which was due to Ca(2+) release from intracellular stores and influx of extracellular Ca(2+) across the cell membrane. In whole-cell voltage-clamp experiments, LPS-induced currents exhibited properties similar to the currents through the Ca(2+) release-activated Ca(2+) channels (CRAC). These currents were highly selective for Ca(2+), exhibited a prominent inward rectification of the current-voltage relationship, and showed an anomalous mole fraction and a fast Ca(2+)-dependent inactivation. In addition, the LPS-induced increase of [Ca(2+)](i) was sensitive to margatoxin and ICAGEN-4, both inhibitors of voltage-gated K(+) (Kv) channels Kv1.3 and Kv1.5, respectively. MHC class II expression, CCL21-dependent migration, and TNF-alpha and IL-6 production decreased, whereas phagocytic capacity increased in LPS-stimulated DCs in the presence of both Kv channel inhibitors as well as the I(CRAC) inhibitor SKF-96365. Taken together, our results demonstrate that Ca(2+) influx in LPS-stimulated DCs occurs via Ca(2+) release-activated Ca(2+) channels, is sensitive to Kv channel activity, and is in turn critically important for DC maturation and functions.

Phosphatidylinositol-3-kinase regulates mast cell ion channel activity.

Stimulation of the mast cell IgE-receptor (FcepsilonRI) by antigen leads to stimulation of Ca(2+) entry with subsequent mast cell degranulation and release of inflammatory mediators. Ca(2+) further activates Ca(2+)-activated K(+) channels, which in turn provide the electrical driving force for Ca(2+) entry. Since phosphatidylinositol (PI)-3-kinase has previously been shown to be required for mast cell activation and degranulation, we explored, whether mast cell Ca(2+) and Ca(2+)-activated K(+) channels may be sensitive to PI3-kinase activity. Whole-cell patch clamp experiments and Fura-2 fluorescence measurements for determination of cytosolic Ca(2+) concentration were performed in mouse bone marrow-derived mast cells either treated or untreated with the PI3-kinase inhibitors LY-294002 (10 muM) and wortmannin (100 nM). Antigen-stimulated Ca(2+) entry but not Ca(2+) release from the intracellular stores was dramatically reduced upon PI3-kinase inhibition. Ca(2+) entry was further inhibited by TRPV blocker ruthenium red (10 muM). Ca(2+) entry following readdition after Ca(+)-store depletion with thapsigargin was again decreased by LY-294002, pointing to inhibition of store-operated channels (SOCs). Moreover, inhibition of PI3-kinase abrogated IgE-stimulated, but not ionomycin-induced stimulation of Ca(2+)-activated K(+) channels. These observations disclose PI3-kinase-dependent regulation of Ca(2+) entry and Ca(2+)-activated K(+)-channels, which in turn participate in triggering mast cell degranulation.

Blunted IgE-mediated activation of mast cells in mice lacking the Ca2+-activated K+ channel KCa3.1.

Mast cell stimulation by Ag is followed by the opening of Ca(2+)-activated K(+) channels, which participate in the orchestration of mast cell degranulation. The present study has been performed to explore the involvement of the Ca(2+)-activated K(+) channel K(Ca)3.1 in mast cell function. To this end mast cells have been isolated and cultured from the bone marrow (bone marrow-derived mast cells (BMMCs)) of K(Ca)3.1 knockout mice (K(Ca)3.1(-/-)) and their wild-type littermates (K(Ca)3.1(+/+)). Mast cell number as well as in vitro BMMC growth and CD117, CD34, and FcepsilonRI expression were similar in both genotypes, but regulatory cell volume decrease was impaired in K(Ca)3.1(-/-) BMMCs. Treatment of the cells with Ag, endothelin-1, or the Ca(2+) ionophore ionomycin was followed by stimulation of Ca(2+)-activated K(+) channels and cell membrane hyperpolarization in K(Ca)3.1(+/+), but not in K(Ca)3.1(-/-) BMMCs. Upon Ag stimulation, Ca(2+) entry but not Ca(2+) release from intracellular stores was markedly impaired in K(Ca)3.1(-/-) BMMCs. Similarly, Ca(2+) entry upon endothelin-1 stimulation was significantly reduced in K(Ca)3.1(-/-) cells. Ag-induced release of beta-hexosaminidase, an indicator of mast cell degranulation, was significantly smaller in K(Ca)3.1(-/-) BMMCs compared with K(Ca)3.1(+/+) BMMCs. Moreover, histamine release upon stimulation of BMMCs with endothelin-1 was reduced in K(Ca)3.1(-/-) cells. The in vivo Ag-induced decline in body temperature revealed that IgE-dependent anaphylaxis was again significantly (by approximately 50%) blunted in K(Ca)3.1(-/-) mice. In conclusion, K(Ca)3.1 is required for Ca(2+)-activated K(+) channel activity and Ca(2+)-dependent processes such as endothelin-1- or Ag-induced degranulation of mast cells, and may thus play a critical role in anaphylactic reactions.

Ginkgo biloba extract and its flavonol and terpenelactone fractions do not affect beta-secretase mRNA and enzyme activity levels in cultured neurons and in mice.

Numerous clinical trials have reported beneficial effects of the Ginkgo biloba extract EGb761 in the prevention and therapy of cognitive disorders including Alzheimer's disease (AD). Although neuroprotective properties of EGb761 have been consistently reported, the molecular mechanisms of EGb761 and the specific role of its major constituents, the flavonols and terpenlactones, are largely unknown. One major hallmark of AD is the deposition of amyloid-beta (A beta) as amyloid plaques in the brain. A beta is a cleavage product of amyloid precursor protein (APP). Certain proteases, called beta-secretases (BACE), are crucial in the formation of A beta. The purpose of the present study was to investigate the efficacy of EGb761 and its flavonol and terpenelactone fraction to modulate BACE-1 enzyme activity and mRNA levels in vitro and in vivo. Neither EGb761 nor its fractions affected BACE-1 activity in vitro. Furthermore, also in Neuro-2a cells and wild-type as well as transgenic (Tg2576) laboratory mice, no significant effect of EGb761 on BACE-1 enzyme activity and mRNA levels were observed. Current findings suggest that BACE-1 may not be a major molecular target of EGb761 and its flavonol and terpenelactone fraction.

Ochratoxin a lowers mRNA levels of genes encoding for key proteins of liver cell metabolism.

Ochratoxin A (OTA) is a nephro- and hepatotoxic mycotoxin that frequently contaminates food and feedstuffs. Although recent studies have indicated that OTA modulates renal gene expression, little is known regarding its impact on differential gene expression in the liver. Therefore a microarray study of the HepG2 liver cell transcriptome in response to OTA exposure (0, 0.25, 2.5 micromol/l for 24 h) was performed using Affymetrix GeneChip technology. Selected microarray results were verified by real-time PCR and Western blotting as independent methods. Out of 14,500 genes present on the microarray, 13 and 250 genes were down-regulated by 0.25 and 2.5 micromol/l OTA, respectively. Reduced mRNA levels of calcineurin A beta (PPP3CB), which regulates inflammatory signalling pathways in immune cells, and of the uncoupling protein 2 (UCP2), which has been suggested to control the production of reactive oxygen species (ROS), were observed in response to 0.25 micromol/l OTA. A particularly strong down-regulation due to 2.5 micromol/l OTA was evident for the mRNA levels of insulin-like growth factor binding protein 1 (IGFBP1) and tubulin beta 1 (TUBB1) which have been demonstrated to function as a pro-survival factor in hepatocytes and as an important cytoskeletal component, respectively. In addition, many genes involved in energy and xenobiotic metabolism, including phosphoglycerate kinase 1 (PGK1), stearoyl-Coenzyme A desaturase 1 (SCD), and glutathione S-transferase omega 1 (GSTO1), were down-regulated by OTA. Furthermore, OTA significantly inhibited the capacitative calcium entry into the HepG2 cells, indicating an alteration of calcium homeostasis. Overall, OTA dose-dependently affects multiple genes encoding for key proteins of liver cell metabolism.