Quantitative phosphoproteomic analysis of signaling downstream of the prostaglandin e2/g-protein coupled receptor in human synovial fibroblasts: potential antifibrotic networks.

The Prostaglandin E2 (PGE2) signaling mechanism within fibroblasts is of growing interest as it has been shown to prevent numerous fibrotic features of fibroblast activation with limited evidence of downstream pathways. To understand the mechanisms of fibroblasts producing tremendous amounts of PGE2 with autocrine effects, we apply a strategy of combining a wide-screening of PGE2-induced kinases with quantitative phosphoproteomics. Our large-scale proteomic approach identified a PKA signal transmitted through phosphorylation of its substrates harboring the R(R/X)X(S*/T*) motif. We documented 115 substrates, of which 72 had 89 sites with a 2.5-fold phosphorylation difference in PGE2-treated cells than in untreated cells, where approximately half of such sites were defined as being novel. They were compiled by networking software to focus on highlighted activities and to associate them with a functional readout of fibroblasts. The substrates were associated with a variety of cellular functions including cytoskeletal structures (migration/motility), regulators of G-protein coupled receptor function, protein kinases, and transcriptional/translational regulators. For the first time, we extended the PGE2 pathway into an elaborate network of interconnecting phosphoproteins, providing vital information to a once restricted signalosome. These data provide new insights into eicosanoid-initiated cell signaling with regards to the regulation of fibroblast activation and the identification of new targets for evidenced-based pharmacotherapy against fibrosis.