Activation of GSK3 Prevents Termination of TNF-Induced Signaling

• Published in: Journal of inflammation research Vol. 14; pp. 1717 - 1730
• Main Authors: Welz, Bastian, Bikker, Rolf, Hoffmeister, Leonie, Diekmann, Mareike, Christmann, Martin, Brand, Korbinian, Huber, René
• Format: Journal Article
• Language: English
• Published: New Zealand Dove Medical Press Limited 01.01.2021; Taylor & Francis Ltd; Dove; Dove Medical Press
• Subjects: A20 protein; Antibodies; Enzyme-linked immunosorbent assay; Ethylenediaminetetraacetic acid; Experiments; Flow cytometry; Gene expression; gsk3; il-8; Inflammation; Inflammatory diseases; Interleukin 8; Kinases; Monocytes; nf-κb; NF-κB protein; Original Research; Phosphorylation; pkc; Plasmids; Protein kinase C; Proteins; Proteolysis; Proteomes; Reporter gene; RNA; Sepsis; Signal transduction; siRNA; Staurosporine; termination of inflammation; termination of tnf-induced signaling; tnf; Transcription; Tumor necrosis factor; Tumor necrosis factor-TNF; Canada; Germany; United States > US; Switzerland; NF-κB; staurosporine; GSK3; PKC; termination of inflammation; IL-8; TNF; termination of TNF-induced signaling
• ISSN: 1178-7031; 1178-7031
• DOI: 10.2147/JIR.S300806

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. 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. 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. 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.