GSK-3 mediates differentiation and activation of proinflammatory dendritic cells

Universität Heidelberg, Heidelburg, Baden-Württemberg, Germany
Blood (Impact Factor: 10.45). 03/2007; 109(4):1584-92. DOI: 10.1182/blood-2006-06-028951
Source: PubMed


The key components of the intracellular molecular network required for the expression of a specific function of dendritic cells (DCs) are as yet undefined. Using an in vitro model of human monocyte-derived DC differentiation, this study investigates the role of glycogen synthase kinase 3 (GSK-3), a multifunctional enzyme critical for cellular differentiation, apoptosis, self-renewal, and motility, in this context. We demonstrate that GSK-3 (1) inhibits macrophage development during differentiation of DCs, (2) is constitutively active in immature DCs and suppresses spontaneous maturation, and (3) acquires a proinflammatory functional status mediating high levels of IL-12, IL-6, and TNF-alpha secretion, and partially inhibits IL-10 in the context of DC activation. In particular, GSK-3 enhances IL-12p35 mRNA expression and thus the production of the proinflammatory cytokine IL-12p70 by integrating the activities of other kinases priming GSK-3 targets and the inhibitory effects of Akt-1. GSK-3 may therefore act as a key integrator of activating and inhibitory pathways involved in proinflammatory DC differentiation and activation.

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Available from: Anthony Ho, Apr 30, 2015
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    • "In A and D, single and double asterisk represents P < 0.05 and P < 0.005, respectively. JOURNAL OF CELLULAR BIOCHEMISTRY GLYCOGEN SYNTHASE KINASE 3 INCREASES IL-1B INDUCED NO with other cell types [Martin et al., 2005; Rodionova et al., 2007; Rådinger et al., 2009] "
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    ABSTRACT: Excessive nitric oxide from the inducible nitric oxide synthase (iNOS) increases shock-induced hepatic injury, hepatic dysfunction, inflammation, and mortality in animal models. Cytokines increase the expression of iNOS in hepatocytes, but the signaling mechanisms involved are not completely understood. We have previously demonstrated that Akt mediates the inhibitory effect of cAMP and insulin on cytokine-induced hepatocyte iNOS expression. We hypothesized that glycogen synthase kinase 3 (GSK3), a target of Akt phosphorylation, would regulate hepatocyte iNOS expression. In cultured rat hepatocytes, GSK3 inhibitors decreased IL-1β mediated nitric oxide (NO) production and iNOS protein expression, while the phosphatidylinositol 3-kinase (PI3K)/Akt pathway inhibitor LY294002 increased the cytokine-mediated NO production and iNOS expression. Over-expression of the constitutively active form of GSK3β enhanced IL-1β-mediated iNOS expression. GSK3 catalyzes the phosphorylation of c-Jun at the c-terminal Thr239 that facilitates c-Jun degradation. Inhibition of GSK3 with SB216763 and lithium chloride significantly reduced, whereas blocking PI3K/Akt increased phosphorylation of c-Jun at Thr239. The levels of total-c-Jun and c-Jun phosphorylated at Ser63 inversely correlated with c-Jun phosphorylated at Thr239, GSK3 activation and iNOS expression. Over-expression of a dominant negative c-Jun not only caused an increase in iNOS promoter activity and iNOS protein expression but was also able to reverse the SB216763-mediated suppression of iNOS. These results demonstrate that GSK3, a downstream target of Akt, regulates IL-1β-stimulated iNOS expression in hepatocytes by directly phosphorylating c-Jun in an inhibitory manner. J. Cell. Biochem. © 2014 Wiley Periodicals, Inc.
    Journal of Cellular Biochemistry 01/2015; 116(1). DOI:10.1002/jcb.24951 · 3.26 Impact Factor
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    • "The kinase is transiently phosphorylated upon lipopolysaccharide (LPS) stimulation [18], [19] but still contributes to the development of a proinflammatory phenotype of DCs [20]. Pharmacological inhibition of GSK3 has previously been shown to attenuate IL-12 [18]–[20], IL-6 and TNFα [20], [53] production and to enhance IL-10 production [18], [19]. Moreover, GSK3 inhibitors blunt the increase of IL12p70 secretion from monocyte-derived DCs following PI3 kinase inhibition with wortmannin [20]. "
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    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.
    PLoS ONE 02/2014; 9(2):e88637. DOI:10.1371/journal.pone.0088637 · 3.23 Impact Factor
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    • "GSK3 is constitutively active in DCs and suppresses their spontaneous maturation, as shown by increased expression of costimulatory molecules CD80, CD83 and CD86 and higher levels of IL-6 secretion upon pharmacological inhibition of GSK3 with LiCl [27]. Upon DC activation in response to a variety of TLR agonists, GSK3 attains a proinflammatory status mediating production of proinflammatory IL-12, IL-6, TNFa, IL-1b, and IFNc and negatively regulating the production of anti-inflammatory IL-10 [11] [17] [26] [27]. Accordingly, administration of a GSK3 inhibitor has been shown to suppress a Th1-mediated immune response against Contents lists available at SciVerse ScienceDirect "
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    ABSTRACT: The function of dendritic cells (DCs) is modified by glycogen synthase kinase GSK3 and GSK3 inhibitors have been shown to protect against inflammatory disease. Regulators of GSK3 include the phosphoinositide 3 kinase (PI3K) pathway leading to activation of protein kinase B (PKB/Akt) and serum and glucocorticoid inducible kinase (SGK) isoforms, which in turn phosphorylate and thus inhibit GSK3. The present study explored, whether PKB/SGK-dependent inhibition of GSK3 contributes to the regulation of cytosolic Ca(2+) concentration following stimulation with bacterial lipopolydasscharide (LPS). To this end DCs from mutant mice, in which PKB/SGK-dependent GSK3α,ß regulation was disrupted by replacement of the serine residues in the respective SGK/PKB-phosphorylation consensus sequence by alanine (gsk3(KI)), were compared to DCs from respective wild type mice (gsk3(WT)). According to Western blotting, GSK3 phosphorylation was indeed absent in gsk3(KI) DCs. According to flow cytometry, expression of antigen-presenting molecule major histocompatibility complex II (MHCII) and costimulatory molecule CD86, was similar in unstimulated and LPS (1 μg/ml, 24 h)-stimulated gsk3(WT) and gsk3(KI) DCs. Moreover, production of cytokines IL-6, IL-10, IL-12 and TNFα was not significantly different in gsk3(KI) and gsk3(WT) DCs. In gsk3(WT) DCs, stimulation with LPS (1 μg/ml) within 10 min led to transient phosphorylation of GSK3. According to Fura2 fluorescence, bacterial lipopolysaccharides (LPS, 1 μg/ml) increased cytosolic Ca(2+) concentration, an effect significantly more pronounced in gsk3(KI) DCs than in gsk3(WT) DCs. Conversely, GSK3 inhibitor SB216763 (3-[2,4-Dichlorophenyl]-4-[1-methyl-1H-indol-3-yl]-1H-pyrrole-2,5-dione, 10 μM, 30 min) significantly blunted the increase of cytosolic Ca(2+) concentration following LPS exposure. In conclusion, PKB/SGK-dependent GSK3α,ß activity participates in the regulation of Ca(2+) signaling in dendritic cells.
    Biochemical and Biophysical Research Communications 06/2013; 437(3). DOI:10.1016/j.bbrc.2013.06.075 · 2.30 Impact Factor
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