Prostaglandin E2-Dependent Phosphorylation of RAS Inhibition 1 (RIN1) at Ser 291 and 292 Inhibits Transforming Growth Factor-β-Induced RAS Activation Pathway in Human Synovial Fibroblasts: Role in Cell Migration

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• 作者：Casimiro Gerarduzzi ; QingWen He ; Beibei Zhai ; John Antoniou and John A. Di Battista
• 刊名：Journal of Cellular Physiology
• 出版年：2017
• 出版时间：January 2017
• 年：2017
• 卷：232
• 期：1
• 页码：202-215
• 全文大小：3272K
• ISSN：1097-4652

文摘

Prostaglandin E₂ (PGE₂)-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 PGE₂-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 PGE₂ (EC50 30 nM, Vmax 100 nM), mimicked by the adenyl cyclase activator forskolin (EC50 5 μM, Vmax 10 μM). We used a global phosphoproteomic approach to identify a 2.5-fold difference in PGE₂-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 PGE₂ 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 PGE₂-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 PGE₂ suppression of wound closure. Overexpression experiments in tandem with pull-down assays indicated that specific Ser291/292 phosphorylation of RIN1 favored binding to activated Ras. In principal, understanding PGE₂-GPCR activated signaling pathways mitigating TGFβ-induced fibrosis may lead to more evidence-based treatments against the disease. J. Cell. Physiol. 232: 202–215, 2017.