Phosphoproteomics highlights complex resource management upon inflammatory stimulation of fibroblasts

The stimulation of cells by inflammatory mediators gives rise to intricate signaling cascades, inducing specific biological functions. The role of kinase activation for the establishment of specific inflammatory functions has only scarcely been established. A time-course analysis of human fetal fibroblasts stimulated with Interleukin-1β (IL-1β) and/or Dexamethasone (Dex) was conducted using mass spectrometry-based proteomics and phosphoproteomics in conjunction with lysolipid and oxylipin profiling. The IL-1β induced proteome alterations indicated metabolic, transcriptional, and translational activation, including inflammatory marker proteins such as NOS1, THBS1, and STING1. The induction of mitochondrial proteins and the formation of numerous lysolipids indicated an increase in beta-oxidation. In addition to the NF-κB and STAT signaling pathways, which are characteristic of inflammatory activation, the MAP and AKT kinase signaling pathways were found to be strongly induced. Six hours after treatment, the observed signaling events exhibited a notable decline, nearly returning to their initial states after 24 hours. It is noteworthy that nearly all of these signaling activities were also observed in cells treated with Dex alone. Additionally, the proteome exhibited transient alterations, which included proteins otherwise characteristic of an inflammatory response, such as MMP3 and NFKB2. Activation of the kinase PIKFYVE was apparently specific for dexamethasone but constituted a minority of the total phosphorylation events. Only after 24 hours was the induction of proteins characteristic of glucocorticoids, such as TSC22D3 and MAOA, observed. The analysis of the effects of dexamethasone on the background of established inflammatory signaling verified the known inhibitory functions and resulted in the expression of anti-inflammatory proteins and oxylipins, but hardly affected the signaling events involving MAP and AKT kinases. In conclusion, this data demonstrates that a majority of inflammation-associated signaling events in fibroblasts needs to be attributed to resource and stress management rather than the establishment of specific inflammatory effector functions.