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.