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

Schmid E, Yan J, Nurbaeva MK, Russo A, Yang W, Faggio C, Shumilina E, Lang F - PLoS ONE (2014)

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Tübingen, Tübingen, Germany.

ABSTRACT
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.

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Effects of GSK3 inhibitors SB216763 or GSK-XIII on K+ independent (NCX) and K+ dependent (NCKX) Na+/Ca2+ exchanger activity in DCs.A,B. Representative original tracings showing [Ca2+]i in Fura-2/AM loaded gsk3WT without (control, open circles) or with (closed circles) SB216763 treatment (1 µM, 30 min) DCs prior to and following removal of external Na+ (0 Na+) at 0 mM K+ (A) and at 40 mM K+ (B). C,D. Arithmetic means ± SEM of the peak (left) and slope (right) values of [Ca2+]i increase following removal of external Na+ at 0 mM K+ (C, n = 24–51) and at 40 mM K+ (D, n = 43–46) in gsk3WT DCs, without (white bars) or with (black bars) SB216763 treatment (1 µM, 30 min). *(p<0.05), unpaired t-test. E,F. Arithmetic means ± SEM of the peak (left) and slope (right) values of [Ca2+]i increase following removal of external Na+ at 0 mM K+ (E, n = 47–82) and at 40 mM K+ (F, n = 43–46) in gsk3WT DCs without (white bars) or with (black bars) GSK-XIII treatment (10 µM, 30 min). *(p<0.05), unpaired t-test or Mann–Whitney U test.
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pone-0088637-g004: Effects of GSK3 inhibitors SB216763 or GSK-XIII on K+ independent (NCX) and K+ dependent (NCKX) Na+/Ca2+ exchanger activity in DCs.A,B. Representative original tracings showing [Ca2+]i in Fura-2/AM loaded gsk3WT without (control, open circles) or with (closed circles) SB216763 treatment (1 µM, 30 min) DCs prior to and following removal of external Na+ (0 Na+) at 0 mM K+ (A) and at 40 mM K+ (B). C,D. Arithmetic means ± SEM of the peak (left) and slope (right) values of [Ca2+]i increase following removal of external Na+ at 0 mM K+ (C, n = 24–51) and at 40 mM K+ (D, n = 43–46) in gsk3WT DCs, without (white bars) or with (black bars) SB216763 treatment (1 µM, 30 min). *(p<0.05), unpaired t-test. E,F. Arithmetic means ± SEM of the peak (left) and slope (right) values of [Ca2+]i increase following removal of external Na+ at 0 mM K+ (E, n = 47–82) and at 40 mM K+ (F, n = 43–46) in gsk3WT DCs without (white bars) or with (black bars) GSK-XIII treatment (10 µM, 30 min). *(p<0.05), unpaired t-test or Mann–Whitney U test.

Mentions: A short-term inhibition of GSK3 by either SB216763 (1 µM, 30 min, Fig. 4A–D) or GSK-XIII (10 µM, 30 min, Fig. 4E, F) also resulted in a significant upregulation of NCX and NCKX activity.


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

Schmid E, Yan J, Nurbaeva MK, Russo A, Yang W, Faggio C, Shumilina E, Lang F - PLoS ONE (2014)

Effects of GSK3 inhibitors SB216763 or GSK-XIII on K+ independent (NCX) and K+ dependent (NCKX) Na+/Ca2+ exchanger activity in DCs.A,B. Representative original tracings showing [Ca2+]i in Fura-2/AM loaded gsk3WT without (control, open circles) or with (closed circles) SB216763 treatment (1 µM, 30 min) DCs prior to and following removal of external Na+ (0 Na+) at 0 mM K+ (A) and at 40 mM K+ (B). C,D. Arithmetic means ± SEM of the peak (left) and slope (right) values of [Ca2+]i increase following removal of external Na+ at 0 mM K+ (C, n = 24–51) and at 40 mM K+ (D, n = 43–46) in gsk3WT DCs, without (white bars) or with (black bars) SB216763 treatment (1 µM, 30 min). *(p<0.05), unpaired t-test. E,F. Arithmetic means ± SEM of the peak (left) and slope (right) values of [Ca2+]i increase following removal of external Na+ at 0 mM K+ (E, n = 47–82) and at 40 mM K+ (F, n = 43–46) in gsk3WT DCs without (white bars) or with (black bars) GSK-XIII treatment (10 µM, 30 min). *(p<0.05), unpaired t-test or Mann–Whitney U test.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3921210&req=5

pone-0088637-g004: Effects of GSK3 inhibitors SB216763 or GSK-XIII on K+ independent (NCX) and K+ dependent (NCKX) Na+/Ca2+ exchanger activity in DCs.A,B. Representative original tracings showing [Ca2+]i in Fura-2/AM loaded gsk3WT without (control, open circles) or with (closed circles) SB216763 treatment (1 µM, 30 min) DCs prior to and following removal of external Na+ (0 Na+) at 0 mM K+ (A) and at 40 mM K+ (B). C,D. Arithmetic means ± SEM of the peak (left) and slope (right) values of [Ca2+]i increase following removal of external Na+ at 0 mM K+ (C, n = 24–51) and at 40 mM K+ (D, n = 43–46) in gsk3WT DCs, without (white bars) or with (black bars) SB216763 treatment (1 µM, 30 min). *(p<0.05), unpaired t-test. E,F. Arithmetic means ± SEM of the peak (left) and slope (right) values of [Ca2+]i increase following removal of external Na+ at 0 mM K+ (E, n = 47–82) and at 40 mM K+ (F, n = 43–46) in gsk3WT DCs without (white bars) or with (black bars) GSK-XIII treatment (10 µM, 30 min). *(p<0.05), unpaired t-test or Mann–Whitney U test.
Mentions: A short-term inhibition of GSK3 by either SB216763 (1 µM, 30 min, Fig. 4A–D) or GSK-XIII (10 µM, 30 min, Fig. 4E, F) also resulted in a significant upregulation of NCX and NCKX activity.

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Tübingen, Tübingen, Germany.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus