Schmid Evi, Yan Jing, Nurbaeva Meerim K, Russo Antonella, Yang Wenting, Faggio Caterina, Shumilina Ekaterina, Lang Florian
Department of Physiology, University of Tübingen, Tübingen, Germany.
Department of Biological and Environmental Sciences, University of Messina, S.Agata-Messina, Italy.
PLoS One. 2014 Feb 11;9(2):e88637. doi: 10.1371/journal.pone.0088637. eCollection 2014.
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.
树突状细胞（DCs）是免疫的关键参与者，受糖原合酶激酶GSK3调控。GSK3的活性受到蛋白激酶B（PKB）/Akt和血清糖皮质激素调节激酶（SGK）亚型的抑制，而这些亚型又受到磷脂酰肌醇-3激酶（PI3K）途径的刺激。暴露于细菌脂多糖会增加细胞溶质钙浓度（[Ca²⁺]i），在从表达抗PKB/SGK的GSK3α、β的突变小鼠（gsk3（KI））分离出的DCs中，这种效应会增强。影响[Ca²⁺]i的因素包括细胞内钙库释放钙（CRIS）、通过基质相互作用分子1/2（STIM1/STIM2）调节的Orai1进行的钙库操纵性钙内流（SOCE）、钾离子依赖性钠/钙交换体（NCKX）、钾离子非依赖性钠/钙交换体（NCX）和钙结合蛋白-D28k。本研究探讨了PKB/SGK依赖性GSK3α、β活性是否影响CRIS、SOCE、NCKX、NCX或钙结合蛋白。从gsk3（KI）小鼠和相应的野生型小鼠（gsk3（WT））中分离出DCs，通过Fura2荧光估计[Ca²⁺]i，通过蛋白质印迹法测定Orai1、STIM1、STIM2以及钙结合蛋白-D28k的蛋白丰度，并通过实时聚合酶链反应（PCR）定量mRNA水平。结果显示，毒胡萝卜素诱导的CRIS和SOCE被GSK3抑制剂SB216763（1 - 10 μM，30分钟）或GSK - XIII（10 μM，30分钟）显著减弱，但在gsk3（WT）DCs中比在gsk3（KI）DCs中显著更低。在gsk3（KI）DCs中，Orai1、STIM1和STIM2的蛋白丰度显著低于gsk3（WT）DCs，而钙结合蛋白-D28k的丰度显著更高。在gsk3（KI）DCs中，NCKX和NCX的活性显著高于gsk3（WT）DCs，并被SB216763（1 μM，30分钟）或GSK - XIII（10 μM，30分钟）显著增强。用SB216763（1 μM，4 - 24小时）或GSK - XIII（10 μM，4 - 24小时）处理gsk3（WT）DCs并没有显著改变Orai1、STIM1和STIM2的蛋白丰度。本研究结果表明GSK3在DCs中钙调节方面具有双重作用。急性抑制GSK3减弱了CRIS和SOCE后[Ca²⁺]i的增加，并刺激了NCKX/NCX活性。然而，表达抗PKB/SGK的GSK3α、β下调了CRIS和SOCE后[Ca²⁺]i的增加，这种效应至少部分是由于Orai1、STIM1和STIM2表达的下调以及钠/钙交换体活性和钙结合蛋白D28k表达的上调。
