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 Ca2+-concentration ([Ca2+]i), an effect augmented in DCs isolated from mutant mice expressing PKB/SGK-resistant GSK3α,β (gsk3KI). Factors affecting [Ca2+]i include Ca2+-release from intracellular stores (CRIS), store-operated Ca2+-entry (SOCE) through STIM1/STIM2-regulated Orai1, K+-dependent Na+/Ca2+-exchangers (NCKX), K+-independent Na+/Ca2+-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 gsk3KI mice and respective wild-type mice (gsk3WT), [Ca2+]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 gsk3WT than in gsk3KIDCs. Orai1, STIM1 and STIM2 protein abundance was significantly lower and calbindin-D28k abundance significantly higher in gsk3KI than in gsk3WTDCs. Activity of NCKX and NCX was significantly higher in gsk3KI than in gsk3WTDCs and was significantly increased by SB216763 (1 µM, 30 min) or GSK-XIII (10 µM, 30 min). Treatment of gsk3WT 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 Ca2+ in DCs. Acute inhibition of GSK3 blunted the increase of [Ca2+]i following CRIS and SOCE and stimulated NCKX/NCX activity. However, expression of PKB/SGK-resistant GSK3α, β downregulated the increase of [Ca2+]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+/Ca2+-exchanger activity and calbindin D28k expression.


Ethics Statement
All animal experiments were conducted according to the recommendations of the Guide for Care and Use of Laboratory Animals of the National Institutes of Health as well as the German law for welfare of animals, and the surgical procedures on the mice were reviewed and approved by the respective government authority of the state Baden-Württemberg (Regierungsprä sidium) prior to the start of the study.

Cell Culture
Dendritic cells (DCs) were cultured from bone marrow of 7-12 week old mice. Bone marrow derived cells were flushed out of the cavities from the femur and tibia with PBS. Cells were then washed twice with RPMI and seeded out at a density of 2610 6 cells/10 ml per 60-mm dish. Cells were cultured for 8 days in RPMI 1640 (GIBCO, Carlsbad) containing: 10% FCS, 1% penicillin/streptomycin, 1% glutamine, 1% non-essential amino acids (NEAA) and 0.05% b-mercaptoethanol. Cultures were supplemented with GM-CSF (35 ng/mL, Immunotools) and fed with fresh medium containing GM-CSF on days 3 and 6. Experiments were performed on DCs at days 7-9.

Western Blotting
The protein expression levels were analyzed by Western blotting. In brief, DCs from gsk3 KI or gsk3 WT mice were washed with ice cold phosphate-buffered saline (PBS) and cells were lysed with cell lysis buffer (Cell Signaling Technology, Inc., New England Biolabs). The extracts were centrifuged at 13,000 rpm for 20 min at 4uC and the protein concentration of the supernatant was determined. Total protein (30 mg) was subjected to 10% SDS-PAGE. Proteins were transferred to a nitrocellulose membrane (VWR) and the membranes were then blocked for 1 h at room temperature with 10% non-fat dried milk in tris-buffered saline (TBS) containing 0.1% Tween-20. For immunoblotting the membranes were incubated overnight at 4 uC with antibodies directed against GSK3a/b (D75D3, XP TM antibody, 1:1000, Cell Signaling Technology, Inc., New England Biolabs, 46, 51 kDa), phospho-GSK3a/b (Ser21/9, 1:1000, Cell Signaling Technology, Inc., New England Biolabs, 46, 51 kDa), Orai1 (1:500, Proteintech, Manchester), STIM1 (1:300, Cell Signaling Technology, Inc., New England Biolabs), STIM2 (1:300 Cell Signaling Technology, Inc., New England Biolabs) or calbindin-D28k (1:200, SWANT, Switzerland). A GAPDH antibody (1:1000, Cell Signaling Technology, Inc., New England Biolabs) was used for a loading control. Specific protein bands were visualized after subsequent incubation with a 1:3000 dilution of anti-rabbit IgG conjugated to horseradish peroxidase and a Super Signal Chemiluminescence detection procedure (GE Healthcare, UK). Specific bands were quantified by Quantity one software (Bio rad gel doc system, Chemidoc XRS). Levels of each protein were expressed as the ratio of signal intensity for the target protein relative to that of GAPDH.

Real-time PCR
Total RNA was extracted from mouse dendritic cells in TriFast (Peqlab, Erlangen, Germany) according to the manufacturer's instructions. After DNAse digestion reverse transcription of total RNA was performed using Transcriptor High Fidelity cDNA Synthesis kit (Roche). Polymerase chain reaction (PCR) amplification of the respective genes were set up in a total volume of 20 ml using 40 ng of cDNA, 500 nM forward and reverse primer and 2x GoTaqH qPCR Master Mix SYBR Green (Promega Corporation, Madison, WI, USA) according to the manufacturer's protocol. Cycling conditions were as follows: initial denaturation at 95uC for 2 min, followed by 40 cycles of 95uC for 15 sec, 55uC for 15 sec and 72uC for 20 sec. For the amplification the following primers were used (59-.39orientation): Orai1, fw CATGGTAGCGATGGTGGAAGTC rev TGCT-fCATCGTCTTTAGTGCCT; Orai2, fw ATGGTGGCCATGGTGGAGGT rev ATTGCC-TTCAGCGCCTGCA; STIM1, fw CTTGGCCTGGGATCTCAGAG rev TCAGC-CATTGCCTTCTTGCC; STIM2, fw GCAGGATCTTTAGCCAGAAG rev ACATCT-GCTGTCACGGGTGA; Calbindin, fw TCCCTCACCTAGAGATAGAAGCAGCGC-AG rev AGACAGCAGAATCGAGGAGTCTGCTGCTC; Tbp, fw CAAGCTGGAGGTGATCATCG rev TCCACAG-TGCTCTTGAATTCG.
Specificity of PCR products was confirmed by analysis of a melting curve. Real-time PCR amplifications were performed on a CFX96 Real-Time System (Bio-Rad). All experiments were done in duplicate. Amplification of the house-keeping gene Tbp (TATA binding protein) was performed to standardize the amount of sample RNA. Relative quantification of gene expression was achieved using the Dct method as described earlier [52].

Measurement of intracellular Ca 2+
To determine cytosolic Ca 2+ concentration, the cells were loaded with Fura-2/AM (2 mM, Molecular Probes, Goettingen, Germany) for 15 min at 37uC. Fluorescence measurements were carried out with an inverted phase-contrast microscope (Axiovert 100, Zeiss, Oberkochen, Germany). Cells were excited alternatively at 340 or 380 nm and the light was deflected by a dichroic [Ca 2+ ] i increase upon Ca 2+ release from intracellular stores (upper bars) and upon SOCE (lower bars) in gsk3 WT DCs incubated in the presence and absence of GSK3 inhibitor GSK-XIII (10 mM, 30 min). *(p,0.05), **(p,0.001), unpaired t-test or Mann-Whitney U test. doi:10.1371/journal.pone.0088637.g001 GSK3-Sensitive SOCE PLOS ONE | www.plosone.org mirror into either the objective (Fluar 406/1.30 oil, Zeiss, Oberkochen, Germany) or a camera (Proxitronic, Bensheim, Germany). Emitted fluorescence intensity was recorded at 505 nm and data acquisition was accomplished by using specialized computer software (Metafluor, Universal Imaging Downingtown, USA). The corresponding ratios (F 340 /F 380 ) were used to obtain intracellular Ca 2+ concentrations. The following equation was used: [Ca 2+ ] free = K d x ((R-R min )/(R max -R)) x S f (K d = dissociation constant of Fura-2; R = ratio of emission intensity, exciting at 340 nm, to emission intensity, exciting at 380 nm; R min = ratio at zero free Ca 2+ ; R max = ratio at saturating Ca 2+ ; S f = instrumental constant). As a measure for the increase of cytosolic Ca 2+ concentration, the slope and peak of the changes in intracellular Ca 2+ concentration were determined in each experiment.
To measure SOCE, changes in cytosolic Ca 2+ were monitored upon depletion of the intracellular Ca 2+ stores. Experiments were carried out prior to and during exposure of the cells to the Ca 2+free solution (see below). In the absence of Ca 2+ , the intracellular Ca 2+ stores were depleted by inhibition of the vesicular Ca 2+ pump by thapsigargin (1 mM; Molecular Probes). Readdition of Ca 2+ allowed assessing the SOCE. To monitor the activity of K + dependent (NCKX) or K + independent (NCX) Na + /Ca 2+ exchangers, changes in cytosolic Ca 2+ were determined upon replacement of extracellular Na + by N-methyl-D-glucamine  at pH 7.4 (NaOH). The Na + -free solutions were identical, except that NaCl was replaced by 130 or 90 mM NMDG to measure NCX or NCKX, respectively.

Statistics
Data are provided as means 6 SEM, n represents the number of independent experiments. All data were tested for significance using unpaired Student t-test, Mann-Whitney U test or ANOVA (Kruskal-Wallis Test, Dunnett test). Only results with p,0.05 were considered statistically significant.
In a second series of experiments the increase of [Ca 2+ ] i following intracellular Ca 2+ release and subsequent SOCE was monitored in DCs isolated from mice expressing PKB/Akt and SGK insensitive GSK3a,b (gsk3 KI ) and DCs isolated from wild type mice (gsk3 WT ). As illustrated in Fig. 2, thapsigargin induced an increase of [Ca 2+ ] i due to Ca 2+ release and readdition of Ca 2+ triggered SOCE, effects both significantly blunted in gsk3 KI DCs as compared to gsk3 WT DCs.
Further experiments were performed to elucidate underlying mechanisms. The increase of [Ca 2+ ] i following intracellular Ca 2+ release and/or SOCE is expected to be blunted by stimulation of Ca 2+ extrusion, such as activation of Na + /Ca 2+ exchangers. The increase of [Ca 2+ ] i following removal of extracellular Na + was taken as evidence for Na + /Ca 2+ exchanger activity. In order to discriminate between K + -independent (NCX) and K + -dependent (NCKX) Na + /Ca 2+ exchangers, experiments were performed in the absence (0 mM) or in the presence of high (40 mM) extracellular K + concentrations. In the absence and in the presence of K + , the increase of [Ca 2+ ] i following removal of extracellular Na + was significantly higher in gsk3 KI DCs than in gsk3 WT DCs (Fig. 3). Accordingly, the activity of both, NCX and NCKX was significantly higher in gsk3 KI DCs than in gsk3 WT DCs.
Western blotting and real time PCR were employed to quantify, respectively, the protein and mRNA abundance of the pore forming Ca 2+ release activated Ca 2+ channel (CRAC) moiety Orai1 and its regulators STIM1 and STIM2 in gsk3 WT and gsk3 KI DCs. As illustrated in Fig. 5 (A, B, E), the transcript abundance of Orai1 and the protein abundance of Orai1, STIM1 and STIM2 were all significantly lower in gsk3 KI DCs than in gsk3 WT DCs. The Figure 6. Effects of GSK3 inhibitor SB216763 on Orai1, STIM1, and STIM2 protein abundance in DCs. A. Original western blot showing the protein abundance of Orai1 and respective GAPDH, STIM1 and respective GAPDH, STIM2 and respective GAPDH in DCs without (control) and with SB216763 treatment (1 mM, 4-24 h). Blots were stripped and reprobed with a GAPDH antibody to determine equal protein loading. B. Arithmetic means 6 SEM (n = 4-5 independent experiments) of the relative (to GAPDH) protein abundance of Orai1, STIM1 and STIM2 in DCs without (white bar) and with SB216763 treatment (1 mM, 4-24 h, black bars). doi:10.1371/journal.pone.0088637.g006 GSK3-Sensitive SOCE PLOS ONE | www.plosone.org transcript abundance of STIM1, and STIM2 were not significantly different between genotypes. To confirm that the mutation in gsk3 KI DCs disrupted GSK3a/b phosphorylation, phosphorylated GSK3 and total GSK3 were determined in gsk3 WT and gsk3 KI DCs. Indeed, phosphorylated GSK3 was observed in DCs from gsk3 WT mice but not in DCs from gsk3 KI mice (Fig. 5A).
An increase of [Ca 2+ ] i could further be modified by cytosolic Ca 2+ buffering, a function of Ca 2+ binding proteins such as calbindin-D28k. Accordingly, Western blotting and RT-PCR were employed to quantify calbindin-D28k expression in gsk3 WT and gsk3 KI DCs. As illustrated in Fig. 5 (C-E), the protein, but not transcript abundance of calbindin D28k was significantly higher in gsk3 KI DCs than in gsk3 WT DCs.

Discussion
The present observations reveal a dual role of glycogen synthase kinase 3 (GSK3) in the regulation of dendritic cell (DC) Ca 2+ signaling. At the one hand acute inhibition of GSK3 with the GSK3 inhibitors SB216763 or GSK-XIII blunts the increase of cytosolic Ca 2+ concentration ([Ca 2+ ] i ) following inhibition of the sarco/endoplasmic Ca 2+ ATPase (SERCA) with thapsigargin in the absence of extracellular Ca 2+ as well as the store operated Ca 2+ entry (SOCE) following readdition of extracellular Ca 2+ . On the other hand, disruption of PKB/Akt and SGK dependent GSK3a,b phosphorylation downregulates both, intracellular Ca 2+ release and SOCE. Phosphorylation of GSK3 by PKB/ Akt [40,41] and SGK [42,43] inhibits GSK3 activity. Accordingly, disruption of the PKB/Akt and SGK dependent phosphorylation is expected to prevent the inhibition of GSK3 following stimulation of the phosphoinositide 3 (PI3) kinase pathway [46].
As shown earlier [13], the increase of [Ca 2+ ] i following treatment of DCs with bacterial LPS was virtually abolished in the presence of GSK3 inhibitor SB216763. Those observations are consistent with inhibition of SOCE by the GSK3 inhibitor. Notably, the inhibitor interferes with both, intracellular Ca 2+ release and SOCE. Thus, GSK3 may be involved in the signaling leading to emptying of intracellular stores. Since the GSK3 inhibitor SB216763 was effective within a few minutes it apparently disrupted activation or inactivation of existing proteins. Moreover, as shown in the present study, both K + dependent (NCKX) and K + independent (NCX) Na + /Ca 2+ exchangers are stimulated by short-term inhibition of GSK3 by either SB216763 or GSK-XIII. Enhanced extrusion of Ca 2+ via these transporters could also underlie the reduced SOCE.
In contrast, the decreased SOCE in DCs isolated from gsk3 KI mice is at least partially due to decreased expression of Orai1, STIM1 and STIM2. Moreover, the blunted increase of [Ca 2+ ] i in DCs from gsk3 KI mice during intracellular Ca 2+ release and SOCE are in part due to enhanced Ca 2+ buffering due to increased expression of calbindin-D28k. The differences between DCs from showing the protein abundance of Orai1, STIM2 and respective GAPDH, STIM1 and respective GAPDH, in DCs without (control) and with GSK-XIII treatment (10 mM, 4-24 h). Blots were stripped and reprobed with a GAPDH antibody to determine equal protein loading. B. Arithmetic means 6 SEM (n = 4-5 independent experiments) of the relative (to GAPDH) protein abundance of Orai1, STIM1 and STIM2 in DCs without (white bar) and with (black bars) GSK-XIII treatment (10 mM, 4-24 h). doi:10.1371/journal.pone.0088637.g007 gsk3 KI mice and DCs from gsk3 WT mice is thus partially due to altered gene expression. The blunted increase of [Ca 2+ ] i during intracellular Ca 2+ release and SOCE is further in part due to enhanced activity of both K + dependent (NCKX) and K + independent (NCX) Na + /Ca 2+ exchangers. In contrast, inhibition of GSK3 by either SB216763 or GSK-XIII did not modify expression of Orai1, STIM1 and STIM2.
At least in theory, the differences between DCs from gsk3 KI mice and DCs from gsk3 WT mice may be an indirect result of the altered regulation of GSK3 activity in other cell types, which in turn influence gene expression in DCs. Along those lines PKB/Akt and SGK1 resistance of GSK3 influences steroid hormone release [47], catecholamine release [50], and function of lymphocytes [54]. To the extent that those or further alterations of hormonal or cellular environment do imprint DCs in vivo prior to isolation, the DCs could remain altered following isolation and subsequent culture.
In conclusion, GSK3 plays a dual role in the regulation of cytosolic Ca 2+ concentration in dendritic cells. Acutely, GSK3 activity is required for the full effect of LPS stimulation and SERCA inhibition on cytosolic Ca 2+ activity. On the other hand, disruption of PKB/Akt and SGK dependent phoshorylation of GSK3 downregulates Orai1, STIM1 and STIM2 expression, upregulates calbindin-D28k expression and enhances the activity of K + dependent (NCKX) and K + independent (NCX) Na + /Ca 2+ exchangers in dendritic cells.