Decreased store operated Ca2+ entry in dendritic cells isolated from mice expressing PKB/SGK-resistant GSK3.

Dendritic cells (DCs), key players of immunity, are regulated by glycogen synthase kinase GSK3. GSK3 activity is suppressed by PKB/Akt and SGK isoforms, which are in turn stimulated by the PI3K pathway. Exposure to bacterial lipopolysaccharides increases cytosolic Ca(2+)-concentration ([Ca(2+)]i), an effect augmented in DCs isolated from mutant mice expressing PKB/SGK-resistant GSK3α,β (gsk3(KI) ). Factors affecting [Ca(2+)]i include Ca(2+)-release from intracellular stores (CRIS), store-operated Ca(2+)-entry (SOCE) through STIM1/STIM2-regulated Orai1, K(+)-dependent Na(+)/Ca(2+)-exchangers (NCKX), K(+)-independent Na(+)/Ca(2+)-exchangers (NCX) and calbindin-D28k. The present study explored whether PKB/SGK-dependent GSK3α, β-activity impacts on CRIS, SOCE, NCKX, NCX or calbindin. DCs were isolated from gsk3(KI) mice and respective wild-type mice (gsk3(WT) ), [Ca(2+)]i estimated from Fura2 fluorescence, Orai1, STIM1, STIM2 as well as calbindin-D28k protein abundance determined by Western blotting and mRNA levels quantified by real time PCR. As a result, thapsigargin-induced CRIS and SOCE were significantly blunted by GSK3-inhibitors SB216763 (1-10 µM, 30 min) or GSK-XIII (10 µM, 30 min) but were significantly lower in gsk3(WT) than in gsk3(KI) DCs. Orai1, STIM1 and STIM2 protein abundance was significantly lower and calbindin-D28k abundance significantly higher in gsk3(KI) than in gsk3(WT) DCs. Activity of NCKX and NCX was significantly higher in gsk3(KI) than in gsk3(WT) DCs and was significantly increased by SB216763 (1 µM, 30 min) or GSK-XIII (10 µM, 30 min). Treatment of gsk3(WT) DCs with SB216763 (1 µM, 4-24 h) or GSK-XIII (10 µM, 4-24 h) did not significantly modify the protein abundance of Orai1, STIM1 and STIM2. The present observations point to a dual role of GSK3 in the regulation of Ca(2+) in DCs. Acute inhibition of GSK3 blunted the increase of [Ca(2+)]i following CRIS and SOCE and stimulated NCKX/NCX activity. However, expression of PKB/SGK-resistant GSK3α, β downregulated the increase of [Ca(2+)]i following CRIS and SOCE, an effect at least partially due to downregulation of Orai1, STIM1 and STIM2 expression as well as upregulation of Na(+)/Ca(2+)-exchanger activity and calbindin D28k expression

AMPKα1-Sensitivity of Orai1 and Ca2+ Entry in T - Lymphocytes

Background/Aims: T-lymphocyte activation and function critically depends on Ca2+ signaling, which is regulated by store operated Ca2+ entry (SOCE). Human and mouse T lymphocytes express AMP activated kinase AMPKα1, which is rapidly activated following elevation of cytosolic Ca2+ concentration ([Ca2+]i) by treatment of the cells with Ca2+ ionophore or following inhibition of endosomal Ca2+ ATPase with thapsigargin. AMPK is further activated by triggering of the T cell antigen receptor (TCR). The present study explored whether AMPK influences Ca2+ entry and Ca2+-sensitive regulation of T-lymphocyte function. Methods: T-lymphocytes were isolated and cultured from AMPKα1-deficient (ampk-/-) mice and from their wildtype (ampk+/+) littermates. The phenotype of the cells was analysed by flow cytometry, [Ca2+]i estimated from Fura-2 fluorescence, SOCE from increase of [Ca2+]i following thapsigargin treatment (1 µM), and cell function analysed by measuring cytokine secretion and western blotting. Results: Expression of surface markers in CD4+ and CD8+ T-cells were similar in ampk-/- and ampk+/+ T-lymphocyte blasts. Moreover, total STIM1 protein abundance was similar in ampk-/- and ampk+/+ T-lymphocyte blasts. However, Orai1 cell membrane protein abundance was significantly higher in ampk-/- than in ampk+/+ T-lymphocyte blasts. SOCE and increase of [Ca2+]i following TCR activation by triggering TCR with anti-CD3 and cross-linking secondary antibody were both significantly more pronounced in ampk-/- than in ampk+/+ T-lymphocyte blasts. The difference of Ca2+ entry between ampk-/- and ampk+/+ T-lymphocytes was abrogated by Orai1 inhibitor 2-aminoethoxydiphenyl borate (2-APB, 50 µM). Proliferation of unstimulated ampk-/- lymphocytes was higher than proliferation of ampk+/+ T-lymphocytes, a difference reversed by Orai1 silencing. Conclusions: AMPK downregulates Orai1 and thus SOCE in T-lymphocytes and thus participates in negative feed-back regulation of cytosolic Ca2+ activity

Evidence That Calcium Entry Into Calcium-Transporting Dental Enamel Cells Is Regulated by Cholecystokinin, Acetylcholine and ATP

Dental enamel is formed by specialized epithelial cells which handle large quantities of Ca2+ while producing the most highly mineralized tissue. However, the mechanisms used by enamel cells to handle bulk Ca2+ safely remain unclear. Our previous work contradicted the dogma that Ca2+ is ferried through the cytosol of Ca2+-transporting cells and instead suggested an organelle-based route across enamel cells. This new paradigm involves endoplasmic reticulum (ER)-associated Ca2+ stores and their concomitant refilling by store-operated Ca2+ entry (SOCE) mediated by Ca2+ release activated Ca2+ (CRAC) channels. Given that Ca2+ handling is maximal during the enamel-mineralization stage (maturation), we anticipated that SOCE would also be elevated then. Confirmation was obtained here using single-cell recordings of cytosolic Ca2+ concentration ([Ca2+]cyt) in rat ameloblasts. A candidate SOCE agonist, cholecystokinin (CCK), was found to be upregulated during maturation, with Cck transcript abundance reaching 30% of that in brain. CCK-receptor transcripts were also detected and Ca2+ imaging showed that CCK stimulation increased [Ca2+]cyt in a dose-responsive manner that was sensitive to CRAC-channel inhibitors. Similar effects were observed with two other SOCE activators, acetylcholine and ATP, whose receptors were also found in enamel cells. These results provide the first evidence of a potential regulatory system for SOCE in enamel cells and so strengthen the Ca2+ transcytosis paradigm for ER-based transport of bulk Ca2+. Our findings also implicate enamel cells as a new physiological target of CCK and raise the possibility of an auto/paracrine system for regulating Ca2+ transport

Store-operated Ca2+ entry controls ameloblast cell function and enamel development

Loss-of-function mutations in stromal interaction molecule 1 (STIM1) impair the activation of Ca(2+) release–activated Ca(2+) (CRAC) channels and store-operated Ca(2+) entry (SOCE), resulting in a disease syndrome called CRAC channelopathy that is characterized by severe dental enamel defects. The cause of these enamel defects has remained unclear given a lack of animal models. We generated Stim1/2(K14cre) mice to delete STIM1 and its homolog STIM2 in enamel cells. These mice showed impaired SOCE in enamel cells. Enamel in Stim1/2(K14cre) mice was hypomineralized with decreased Ca content, mechanically weak, and thinner. The morphology of SOCE-deficient ameloblasts was altered, showing loss of the typical ruffled border, resulting in mislocalized mitochondria. Global gene expression analysis of SOCE-deficient ameloblasts revealed strong dysregulation of several pathways. ER stress genes associated with the unfolded protein response were increased in Stim1/2-deficient cells, whereas the expression of components of the glutathione system were decreased. Consistent with increased oxidative stress, we found increased ROS production, decreased mitochondrial function, and abnormal mitochondrial morphology in ameloblasts of Stim1/2(K14cre) mice. Collectively, these data show that loss of SOCE in enamel cells has substantial detrimental effects on gene expression, cell function, and the mineralization of dental enamel

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

Thapsigargin-induced intacellular Ca²⁺ release and subsequent SOCE in DCs from <i>gsk3^KI</i> and <i>gsk3^WT</i> mice.

<p><b>A.</b> Representative original tracings showing [Ca²⁺]_i in Fura-2/AM loaded <i>gsk3^WT</i> (open circles) and <i>gsk3^KI</i> (closed triangels) DCs prior to and following removal of extracellular Ca²⁺, addition of SERCA inhibitor thapsigargin (1 µM) and readdition of extracellular Ca²⁺. <b>B.</b> Arithmetic means ± SEM (n = 44–59) of the peak (left) and slope (right) values of [Ca²⁺]_i increase upon Ca²⁺ release from intracellular stores (upper bars) and upon SOCE (lower bars) in <i>gsk3^WT</i> DCs (white bars) and <i>gsk3^KI</i> DCs (black bars). ***(p<0.001), unpaired <i>t</i>-test.</p

Dental enamel cells express functional SOCE channels

Dental enamel formation requires large quantities of Ca(2+) yet the mechanisms mediating Ca(2+) dynamics in enamel cells are unclear. Store-operated Ca(2+) entry (SOCE) channels are important Ca(2+) influx mechanisms in many cells. SOCE involves release of Ca(2+) from intracellular pools followed by Ca(2+) entry. The best-characterized SOCE channels are the Ca(2+) release-activated Ca(2+) (CRAC) channels. As patients with mutations in the CRAC channel genes STIM1 and ORAI1 show abnormal enamel mineralization, we hypothesized that CRAC channels might be an important Ca(2+) uptake mechanism in enamel cells. Investigating primary murine enamel cells, we found that key components of CRAC channels (ORAI1, ORAI2, ORAI3, STIM1, STIM2) were expressed and most abundant during the maturation stage of enamel development. Furthermore, inositol 1,4,5-trisphosphate receptor (IP3R) but not ryanodine receptor (RyR) expression was high in enamel cells suggesting that IP3Rs are the main ER Ca(2+) release mechanism. Passive depletion of ER Ca(2+) stores with thapsigargin resulted in a significant raise in [Ca(2+)]i consistent with SOCE. In cells pre-treated with the CRAC channel blocker Synta-66 Ca(2+) entry was significantly inhibited. These data demonstrate that enamel cells have SOCE mediated by CRAC channels and implicate them as a mechanism for Ca(2+) uptake in enamel formation

K⁺ independent (NCX) and K⁺ dependent (NCKX) Na⁺/Ca²⁺ exchanger activity in DCs from <i>gsk3^KI</i> and <i>gsk3^WT</i> mice.

<p><b>A,B.</b> Representative original tracings showing [Ca²⁺]_i in Fura-2/AM loaded <i>gsk3^WT</i> (open diamonds) and <i>gsk3^KI</i> (closed circles) DCs prior to and following removal of external Na⁺ (0 Na⁺) at 0 mM K⁺ (<b>A</b>) and at 40 mM K⁺ (<b>B</b>). <b>C,D.</b> Arithmetic means ± SEM of the peak (left) and slope (right) values of [Ca²⁺]_i increase following removal of external Na⁺ at 0 mM K⁺ (<b>C</b>, n = 95–129) and at 40 mM K⁺ (<b>D</b>, n = 27–34) in <i>gsk3^WT</i> DCs (white bars) and <i>gsk3^KI</i> DCs (black bars). *(p<0.05), ***(p<0.001), unpaired <i>t</i>-test.</p