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 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.
Highlights
The function of dendritic cells (DCs), antigen-presenting cells critically important for primary immune responses and establishment of immunological memory [1,2], is regulated by cytosolic Ca2+ concentration [3]
The present study explored whether glycogen synthase kinase 3 (GSK3) modifies the increase of cytosolic Ca2+ concentration ([Ca2+]i) in dendritic cells (DCs) following stimulation of intracel
The present observations reveal a dual role of glycogen synthase kinase 3 (GSK3) in the regulation of dendritic cell (DC) Ca2+ signaling
Summary
The function of dendritic cells (DCs), antigen-presenting cells critically important for primary immune responses and establishment of immunological memory [1,2], is regulated by cytosolic Ca2+ concentration [3]. [4,5,6,7], which in DCs involves the pore forming channel proteins. The increase of cytosolic Ca2+ concentration is further shaped by cytosolic Ca2+ extrusion mechanisms such as K+-independent (NCX) and K+-dependent (NCKX) Na+/Ca2+ exchangers [10,11]. At least in theory, alterations of free cytosolic Ca2+ concentration could be blunted by cytosolic Ca2+ binding proteins, such as calbindin-D28k [12]. Mechanisms involved in the regulation of cytosolic Ca2+ activity include glycogen synthase kinase 3 (GSK3a,b) [13].
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