The Regulation Of GRK2 On The EP4 Receptor Signaling Pathways Of Human Fibroblast-like Synoviocytes And The Effects Of 6ˊ-Phenylsulfonylpaeoniflorin(CP-25)

Rheumatoid arthritis(RA)is a common autoimmune disease,the pathogenesis of RA is characterized with systemic immune dysfunction and joint synovial inflammation.Fibroblast-like synoviocytes(FLS)abnormal proliferation is one of the important pathological features,FLS as target cells,and effector cells,involved in the inflammatory immune response of RA.Cytokines such as tumor necrosis factor(TNF alpha),prostaglandin 2(PGE2)are participated in this process.PGE2,as an important cytokine,can promote FLS proliferation.It also can regulate the level of c AMP in cells by prostaglandin receptors(EPs)signaling pathway.G protein coupled receptor kinases(GRKs)phosphorylate receptors and promote affinity of binding to arrestins,which uncouple the receptors from G proteins,and target the receptors for desensitization and internalization.However,GRK2 regulates EP4 receptor need to be elucidated.CP-25,as a novel compound phenylsulfinyl-paeoniflorin(paeoniflorin-6’-O-benzene sulfonate)(patent number in China: ZL201210030616.4),is an ester derivatives of Paeoniflorin(Pae)which is the active monomers in total glucosides of paeony(TGP).Our group studies showed that the expression of GRK2 can significantly up-regulate in CIA rats;the level of EP2 m RNA,EP4 m RNA can up-regulate in DCs of AA rats;EP4 agonist CAY10598 can down-regulate the expression EP4 membrane and up-regulate the GRK2 membrane and inhibit the level of c AMP and promote the proliferation of FLS;CP-25 can up-regulate the level of c AMP and inhibit the proliferation of FLS;However,whether GRK2 regulates the signaling of EP4 receptor? CP-25 can regulate GRK2 activity,balance the EP4 receptors signal and improve the function of FLS? The action sites of CP-25 on GRK2? GRK2 to inhibit FLS proliferation remains unclear.In this research,we used FLS and HEK293 cells,and adopted flow cytometry,Co-immunoprecipitation and western blot,and discussed GRK2 regulateing EP4 signaling pathway,the association of EP4 receptor and GRK2 and the effects of CP-25,and the action site of CP-25 on GRK2,which may provide more scientific basis for revealing the mechanism of GRK2 regulateing FLS EP4 signaling pathway and the molecular target of CP-25 in exerting immunoregulatory effects.Objective: 1.To investigate the effects of CP-25 on proliferation of normal human FLS and MH7 A treated by PGE2,which reveal the regulation of EP4 signaling transduction by GRK2 and CP-25 can regulates the signaling of EP4 receptor and GRK2 to inhibit FLS proliferation.2.To observe the interaction between EP4 and GRK2 in HEK293 stimulated by EP4 agonist after pretreatment with PGE2 and the effect of CP-25,which reveal the mechanism of EP4 receptor desensitization,and provide more experiment basis for researching the target of CP-25.Methods: 1.The proliferation of normal human FLS treated by PGE2 or TNF-α were detected by CCK-8;the separation of cytoplasm membrane proteins by ultracentrifugation,the change of EP4 and GRK2 were detected by western blot in normal human FLS cytomembrane.2.The proliferation of MH7 A treated by PGE2 and the effects of CP-25 were detected by CCK-8;the separation of cytoplasm membrane proteins by ultracentrifugation,the change of EP4 and GRK2 were detected by western blot in MH7 A cytomembrane and the effects of CP-25;the expression of EP4 in MH7 A cytomembrane by flow cytometry;the association with EP4 and GRK2 in MH7 A and the effects of CP-25 were detected by co-immunoprecipitation assay;the activity of PKA and c AMP detected by FRET.3.Construction and expression of the eukaryotic expressing vector of p IRES-EGFP-GRK2-C340 S and p IRES-EGFP-GRK2-S670 A mutants recombinant plasmids,the association with EP4 and GRK2 in HEK293 stimulated by EP4 agonist and the effects of CP-25 were detected by Co-immunoprecipitation assay.Results: 1.The effects of PGE2 on normal human FLSCompared with normal group,PGE2(0.35,1.75μM)or TNF-α(10,20,40ng/ml)increased the proliferation of normal human FLS the combination of TNF-α(20 ng/ml)and PGE2(0.35μM)can further promote the proliferation.After stimulated by PGE2 and TNF-α,GRK2 membrane expression in normal human FLS was increased continuously at 10 min,and was down to the basal level at 1h.However,EP4 membrane expression in normal human FLS was decreased at 10 min,andwas up to the basal level at 1h.2.The effects of CP-25 on MH7 A proliferation treated by PGE2PGE2(2,4μM)enhanced MH7 A proliferation in comparison with the control group.Compared with PGE2(2μM),CP-25(10-7,10-6,10-5mol/L)could significantly reduce PGE2-induced MH7 A proliferation.GRK2 membrane in MH7 A was increased continuously after 20 min under PGE2 stimulation.However,EP4 expression in MH7 A was increased at 10 min,and was up to the peak at 1h.After stimulated by PGE2,the flow cytometry detection showed that EP4 membrane expression in MH7 A was decreased at 10 min.GRK2 membrane expression in MH7 A was increased continuously after 10 min under PGE2 stimulation,and was decreased to the basal level at 1h.However,EP4 membrane expression in MH7 A was decreased at 10 min,and was up to the basal level at 1h detected by western blot.EP4 membrane expression was significantly downregulated and GRK2 membrane expression was significantly upregulated after 10 min under PGE2 stimulation,CP-25(1×10-6mol/L)can significantly upregulate EP4 membrane expression and downregulate GRK2 membrane expression.Without PGE2 stimulation,GRK2 association with EP4 on MH7 A was tested by Co-immunoprecipitation assay,PGE2 stimulation clearly increased GRK2 association with EP4 at 10 min versus control.EP4 agonist can improve the activity of c AMP and PKA at a short time,but CP-25 can down-regulate the activity of c AMP and PKA.3.The construction and expression of GRK2 mutant recombinant plasmidspIRES-EGFP-GRK2-C340 S and p IRES-EGFP-GRK2-S670 A mutant recombinant plasmid was successfully constructed,and the recombinant protein expression was correct in HEK293 cell lines after cell transfection.4.The interaction between EP4 and GRK2 in HEK293 stimulated by EP4 agonist after pretreatment with PGE2 and the effects of CP-25p IRES-EGFP-ctr,p IRES-EGFP-GRK2-wt,p IRES-EGFP-GRK2-C340 S and p IRES-EGFP-GRK2-S670 A were successfully constructed,and the recombinant protein expression was correct in HEK293 cell lines after cell transfection.After pretreated for 30 min by PGE2(10μM),CAY10598 stimulation clearly increased GRK2-wt,GRK2-C340 S or GRK2-S670 A association with EP4 in HEK293,CP-25(10-6mol/L)can down-regulate the association of GRK2-wt,GRK2-C340 S or GRK2-S670 A and EP4.p IRES-EGFP-GRK2-wt was successfully constructed,and the recombinant protein expression was correct in HEK293 cell lines after cell transfection.After pretreated for 30 min by PGE2(10μM),CAY10598 stimulation clearly increased the phosphorylation of GRK2-S685,CP-25(10-6mol/L)can down-regulate the phosphorylation of GRK2-S685 and the association of EP4 and p685-GRK2.Conclusions: 1.CP-25 can inhibit PGE2-induced FLS proliferation,which reveals that treatment of CP-25 on RA may through regulating the function of FLS.2.PGE2 increases GRK2 cytomembrane expression and promotes the association of GRK2 and EP4,which results in the enhancement of EP4 desensitization.3.CP-25 can decrease GRK2 membrane expression and inhibit the association of GRK2 and EP4,thus promote EP4 receptor resensitization,the results reveal that CP-25 can inhibit FLS proliferation may through regulating the function of EP4 receptor and GRK2.4.CP-25 can inhibit the phosphorylation of GRK2-S685 and the association of GRK2 and EP4,and regulate EP4 receptor resensitization,so the acting site of CP-25 might be S685 site of GRK2.