Activation of GSK3 Prevents Termination of TNF-Induced Signaling.

Background: Termination of TNF-induced signaling plays a key role in the resolution of inflammation with dysregulations leading to severe pathophysiological conditions (sepsis, chronic inflammatory disease, cancer). Since a recent phospho-proteome analysis in human monocytes suggested GSK3 as a relevant kinase during signal termination, we aimed at further elucidating its role in this context.
Materials and Methods: For the analyses, THP-1 monocytic cells and primary human monocytes were used. Staurosporine (Stauro) was applied to activate GSK3 by inhibiting kinases that mediate inhibitory GSK3α/β-Ser21/9 phosphorylation (eg, PKC). For GSK3 inhibition, Kenpaulone (Ken) was used. GSK3- and PKC-siRNAs were applied for knockdown experiments. Protein expression and phosphorylation were assessed by Western blot or ELISA and mRNA expression by qPCR. NF-κB activation was addressed using reporter gene assays.
Results: Constitutive GSK3β and PKCβ expression and GSK3α/β-Ser21/9 and PKCα/βII-Thr638/641 phosphorylation were not altered during TNF long-term incubation. Stauro-induced GSK3 activation (demonstrated by Bcl3 reduction) prevented termination of TNF-induced signaling as reflected by strongly elevated IL-8 expression (used as an indicator) following TNF long-term incubation. A similar increase was observed in TNF short-term-exposed cells, and this effect was inhibited by Ken. PKCα/β-knockdown modestly increased, whereas GSK3α/β-knockdown inhibited TNF-induced IL-8 expression. TNF-dependent activation of two NF-κB-dependent indicator plasmids was enhanced by Stauro, demonstrating transcriptional effects. A TNF-induced increase in p65-Ser536 phosphorylation was further enhanced by Stauro, whereas IκBα proteolysis and IKKα/β-Ser176/180 phosphorylation were not affected. Moreover, PKCβ-knockdown reduced levels of Bcl3. A20 and IκBα mRNA, both coding for signaling inhibitors, were dramatically less affected under our conditions when compared to IL-8, suggesting differential transcriptional effects.
Conclusion: Our results suggest that GSK3 activation is involved in preventing the termination of TNF-induced signaling. Our data demonstrate that activation of GSK3 - either pathophysiologically or pharmacologically induced - may destroy the finely balanced condition necessary for the termination of inflammation-associated signaling.
Competing Interests: Dr Bastian Welz reports grants from DGKL; Stiftung für Pathobiochemie und Molekulare Diagnostik (2017/20), during the conduct of the study. Dr Rolf Bikker reports grants from DGKL; Stiftung für Pathobiochemie und Molekulare Diagnostik (2020/21), during the conduct of the study. Ms Leonie Hoffmeister reports PhD scholarship from Hannover Biomedical Research School - MD/PhD program Molecular Medicine, during the conduct of the study. Dr Martin Christmann reports grants from DGKL; Stiftung für Pathobiochemie und Molekulare Diagnostik (2017/19) and DFG (391096506), during the conduct of the study. The donor foundations had no role in the design of the study; in the conduct of the experiments; in the collection, analysis, or interpretation of the data; in the writing or revision of the manuscript; or in the decision to publish the results. The authors report no other conflicts of interest in this work.
(© 2021 Welz et al.)