ALBERT

Description: ABSTRACT Prostaglandin E 2 (PGE 2 )-stimulated G-protein coupled receptor (GPCR) activation inhibits pro-fibrotic TGFβ-dependent stimulation of human fibroblast to myofibroblast transition (FMT), though the precise molecular mechanisms are not fully understood. In the present study, we describe the PGE 2 -dependent suppression and reversal of TGFβ-induced events such as α-sma expression, stress fiber formation, and Ras/Raf/ERK/MAPK pathway-dependent activation of myofibroblast migration. In order to elucidate post ligand-receptor signaling pathways, we identified a predominant PKA phosphorylation motif profile in human primary fibroblasts after treatment with exogenous PGE 2 (EC50 30nM, Vmax 100nM), mimicked by the adenyl cyclase activator forskolin (EC50 5µM, Vmax 10µM). We used a global phosphoproteomic approach to identify a 2.5 fold difference in PGE 2 induced phosphorylation of proteins containing the PKA motif. Deducing the signaling pathway of our migration data, we identified Ras inhibitor 1 (RIN1) as a substrate, whereby PGE 2 induced its phosphorylation at Ser291 and at Ser292 by a 5.4- and 4.8-fold increase, respectively. In a series of transient and stable over expression studies in HEK293T and HeLa cells using wild-type (wt) and mutant RIN1 (Ser291/292Ala) or Ras constructs and siRNA knock-down experiments, we showed that PGE 2 -dependent phosphorylation of RIN1 resulted in the abrogation of TGFβ induced Ras/Raf signaling activation and subsequent downstream blockade of cellular migration, emphasizing the importance of such phosphosites in PGE 2 suppression of wound closure. Over expression experiments in tandem with pull-down assays indicated that specific Ser291/292 phosphorylation of RIN1 favoured binding to activated Ras. In principal, understanding PGE 2 -GPCR activated signaling pathways mitigating TGFβ-induced fibrosis may lead to more evidence-based treatments against the disease. This article is protected by copyright. All rights reserved