Proteome and Phosphoproteome Analysis in TNF Long Term-Exposed Primary Human Monocytes

Abstract

To better understand the inflammation-associated mechanisms modulating and terminating tumor necrosis factor (TNF-)induced signal transduction and the development of TNF tolerance, we analyzed both the proteome and the phosphoproteome in TNF long term-incubated (i.e., 48 h) primary human monocytes using liquid chromatography-mass spectrometry. Our analyses revealed the presence of a defined set of proteins characterized by reproducible changes in expression and phosphorylation patterns in long term TNF-treated samples. In total, 148 proteins and 569 phosphopeptides were significantly regulated (103 proteins increased, 45 proteins decreased; 377 peptides with increased and 192 peptides with decreased phosphorylation). A variety of these proteins are associated with the non-canonical nuclear factor κB (NF-κB) pathway (nuclear factor κB (NFKB) 2, v-rel reticuloendotheliosis viral oncogene homolog (REL) B, indolamin-2,3-dioxygenase (IDO), kynureninase (KYNU)) or involved in the negative regulation of the canonical NF-κB system. Within the phosphopeptides, binding motifs for specific kinases were identified. Glycogen synthase kinase (GSK) 3 proved to be a promising candidate, since it targets NF-κB inhibiting factors, such as CCAAT/enhancer binding protein (C/EBP) β. Our experiments demonstrate that both proteome and phosphoproteome analysis can be effectively applied to study protein/phosphorylation patterns of primary monocytes. These results provide new regulatory candidates and evidence for a complex network of specific but synergistically acting/cooperating mechanisms enabling the affected cells to resist sustained TNF exposure and resulting in the resolution of inflammation.

GSK3: A Kinase Balancing Promotion and Resolution of Inflammation

“…Further, MAPK-phosphorylated Thr188 may act as a priming site for GSK3β-mediated phosphorylation of Ser184 and Ser179, i.e., phospho-residues favoring dimerization due to conformational changes and enhancing both DNA binding and transactivation activity [40,258]. Decreased C/EBPβ-Thr235 phosphorylation due to GSK3 inactivation was detected in TNF long-term-incubated monocytic cells in the presence of kenpaullone [259] as well as human monocyte-derived DC in response to platelet-activating factor [260]. In contrast, in IL-17-stimulated murine ST2 stromal cells, ERK-mediated priming phosphorylation at Thr188 and subsequent phosphorylation at Ser179 by GSK3 have been described as being repressive for certain C/EBP-dependent genes [261].…”

“…In addition, GSK3 inhibition in TNF-tolerized cells also results in increased binding of p65 at the promoters of tolerizable genes (e.g., IL-6), reduced expression of A20 (a negative regulator of NF-κB signaling, e.g., during TNF incubation [291]), and enhanced chromatin accessibility [288]. Recently, it has further been demonstrated that numerous proteins differentially phosphorylated under conditions inducing high-dose TNF tolerance are potential GSK3 targets [259]. However, further analyses have to elucidate whether additional GSK3-dependent mechanisms are involved in the complex regulation of tolerance formation and the termination of pro-inflammatory signaling.…”

Activation of GSK3 Prevents Termination of TNF-Induced Signaling

“…Preparation of whole-cell and nuclear extracts, determination of protein concentrations, electrophoresis, and Western blotting were performed as described: 20 , 27 cells were lysed in extraction buffer P (8 M urea, 4% 3-[(3-Cholamidopropyl)-dimethylammonio]-propane-sulfonate (Sigma-Aldrich), 30 nM Tris, 1% Nonidet P-40 (AppliChem, Darmstadt, Germany), 1% HALT phosphatase inhibitor (Thermo), and EDTA-free cOmplete protease inhibitor (Sigma-Aldrich) cocktails). To obtain nuclear proteins, cells were additionally sonicated (10 s, 20% power; Bandelin, Berlin, Germany).…”

“…Our recent (phospho-)proteome analysis of long-term TNF-incubated monocytic cells suggests that glycogen synthase kinase (GSK) 3 represents a crucial kinase regulating the phosphorylation status and associated molecular functions orchestrating the termination of inflammation-induced signaling. 20 In addition, by controlling the activation of various transcription factors including NF-κB and C/EBP, GSK3 is strongly involved in the transcriptional regulation of gene expression. 21–23 GSK3 consists of two highly similar paralogues (α and β) and is predominantly controlled via inhibitory phosphorylation at Ser21 (GSK3α) or Ser9 (GSK3β) by kinases such as protein kinase (PK)Cα and β or PKB/Akt.…”

Phosphoproteome profiling uncovers a key role for CDKs in TNF signaling

“…The TNF pathway is a pivotal proinflammatory signaling cascade that relies heavily on protein phosphorylation, the extent of which has been revealed by phosphoproteomic studies (Wagner et al, 2016, Mohideen et al, 2017, Zhong et al, 2014, Krishnan et al, 2015, Welz et al, 2019, Cantin et al, 2006). Much is already known about key mechanistic events and we briefly recapitulate those that are pertinent to our study.…”