| Rheumatoid arthritis（RA）is a systemic and chronic autoimmune disease.In addition to causing synovial hyperplasia and erosion of articular cartilage,it affects other important organs such as heart and lungs.The main pathological features of RA are synovitis,articular conformation,synovial hyperplasia,and bone and cartilage damage.Two-thirds of the cells in the proliferated synovial tissue are fibroblast-like synoviocytes（FLS）.They are the key effector cells of RA.Bone and cartilage erosion play an important role in the pathogenesis of RA.It is reported in the literature that there is a large amount of PGE2 in synovial tissue of RA patients.A variety of animal models of arthritis have confirmed that the PGE2-EP4 signaling pathway plays an important role in the occurrence and development of arthritis,causing abnormal FLS proliferation and directly leading to synovial hyperplasia.Guanine nucleotide binding protein,also known as G protein,is a ubiquitous signal molecule that plays an important role in transmembrane signal transduction.It consists of three subunits:α,β,and γ.G protein couples with corresponding G protein-coupled receptors（GPCRs）.When GPCRs are activated,they undergo conformational changes that cause dissociation of G protein α and βy subunits.Gβγ dimers interact with G protein-coupled receptor kinases（GRKs）,PI3K and other downstream effectors.Desensitization and resensitization of GPCRs regulate receptor function.GRKs are associated with GPCRs desensitization.Previous research showed that in RA animal models,PGE2 stimulation caused GRK2 excessive transmembrane,EP4 desensitization,thus promoted abnormal FLS proliferation through the EP4-GRK2-Gαi-cAMP signaling pathway.GPCRs-Gβγ-GRKs complex is crucial for the regulation of receptor function and GRKs catalysis,which has been studied in adrenergic and opioid receptors,but has not been reported in EP4 receptors.Paeoniflorin-6’-oxy-benzenesulfonate（CP-25）is a small molecule monomer obtained by esterifying paeoniflorin,which exerts protective effect in relieving arthritis and bone destruction in adjuvant arthritis,inhibiting GRK2 transmembrane and EP4 desensitization in FLS,and alleviating FLS abnormal proliferation.However,whether Gβγ is involved in the process of CP-25 modulating GRK2-EP4 signaling and its specific mechanism are unknown.Objective:To observe the interaction between EP4,Gβγ and GRK2 in synovial tissue in RA patients and FLS.To clarify the relationship between changes in EP4-Gβγ-GRK2 signaling and abnormal proliferation and migration of FLS.To reveal the role of Gβγand GRK2 interactions in EP4 desensitization and Gβγ downstream signal transduction in FLS.To elucidate the mechanism of CP-25 in inhibiting the abnormal activation of FLS by affecting the interaction between Gβγ and GRK2.Methods:1.Synovial tissue of trauma patients and RA patients was collected and synovial pathology was observed with H＆E staining.The expression of COX-2 was detected by Western blot.The concentration of PGE2 of synovial fluid was measured by ELISA.The expressions of GRK2,EP4 and Gβ were detected by immunohistochemistry and Western blot.The membrane expressions of GRK2,EP4 and Gβ were detected by Western blot.The co-expression of Gβ and GRK2,Gβ and EP4,GRK2 and EP4 were detected by immunofluorescence and co-immunoprecipitation.2.AA model was established with complete Freund’s adjuvant,and AA-FLS was then extracted.RA-FLS was obtained from synovial tissue of RA patients.CP-25 and Gβγinhibitor Gallein were given after PGE2 stimulation in RA-FLS and AA-FLS.Cell proliferation and migration were detected by CCK-8 and Transwell.The membrane expressions of GRK2,EP4 and Gβ were detected by Western blot.Co-expression of Gβand GRK2,Gβ and EP4,GRK2 and EP4 were detected by immunofluorescence and coimmunoprecipitation.3.Stimulated the MH7A cell line with short-term or long-term PGE2.After PGE2 stimulation,CP-25 and Gβγ inhibitor Gallein were given in MH7A cells.Cell proliferation and migration were detected by CCK-8 and Transwell.Membrane expression of GRK2,EP4 and Gβ were detected by Western blot.Co-expression of Gβand GRK2,Gβ and EP4,and GRK2 and EP4 were detected by immunofluorescence and co-immunoprecipitation.4.siRNA was used to silence the Gβ1 subunit of MH7A cells.CCK-8 and Transwell were used to detect the proliferation and migration of MH7A cells under PGE2 stimulation.Western blot was used to detect the membrane expressions of GRK2,EP4 and Gβ.Co-expression of GRK2 and EP4 was detected by immunofluorescence.5.After PGE2 stimulation,Gβγ activator mSIRK and inhibitor GRK2i were administered.Cell proliferation and migration were detected by CCK-8 and Transwell.Membrane expression of GRK2,EP4 and β-arrestin2 were detected by Western blot.Co-expression of Gβ and GRK2,Gβ and EP4,and GRK2 and EP4 were detected by immunofluorescence and co-immunoprecipitation.6.The expression of PI3K,Akt and GSK-3β in RA synovial tissue and MH7A were detected by Western blot.Co-expression of Gβ and PI3K was detected by coimmunoprecipitation.Results:1.Detection of PGE2-EP4-Gβγ-GRK2 signaling in synovial tissue and RA-FLS and the role of CP-25Synovial pathological results showed synovial hyperplasia in RA patients.COX-2 expression was increased in synovial tissue and concentration of PGE2 was high in synovial fluid.Compared with trauma group,total expression of GRK2 and cell membrane expression of GRK2 and Gβ were increased,membrane expression of EP4 was decreased,co-expression of Gβ and GRK2,Gβ and EP4,and GRK2 and EP4 were increased.RA-FLS was extracted and identified positive with Vimentin and CD55 by immunofluorescence.PGE2 promoted the proliferation and migration of RA-FLS,increased membrane expression of GRK2 and Gβ,decreased EP4 membrane expression,increased co-expression of Gβ and GRK2,Gβ and EP4,GRK2 and EP4;CP-25 and Gβγ inhibitor Gallein inhibited PGE2 induced abnormal proliferation and migration,reduced membrane expression of GRK2 and Gβ,increased EP4 membrane expression,and inhibited co-expression of Gβ and GRK2,Gβ and EP4,GRK2 and EP4 in RA-FLS.2.Detection of PGE2-EP4-Gβγ-GRK2 signaling in AA-FLS and role of CP-25The AA model was successfully constructed,and the joints of the rats swelled and the arthritis index increased.AA-FLS was prepared and identified positive with Vimentin and CD55 by immunofluorescence.PGE2 stimulation promoted the proliferation and migration of AA-FLS,increased membrane expression of GRK2 and Gβ,reduced EP4 membrane expression,increased co-expression of Gβ and GRK2,Gβand EP4,GRK2 and EP4;CP-25 and Gallein inhibited PGE2-induced abnormal proliferation and migration,reduce membrane expression of GRK2 and Gβ,increase EP4 membrane expression,and inhibit co-expression of Gβ and GRK2,Gβ and EP4,GRK2 and EP4 in AA-FLS.3.Detection of PGE2-EP4-Gβγ-GRK2 signaling in MH7A Cells and Role of CP-25PGE2 were given in MH7A cells for 10 minutes,total expression of GRK2 and membrane expression of GRK2,Gβ,EP4 were increased.Co-expression of Gβ and GRK2,Gβ and EP4,GRK2 and EP4 were increased.CP-25 inhibited total expression of GRK2 and EP,reduced membrane expression of GRK2,Gβ,EP4,inhibited coexpression of Gβ and GRK2,Gβ and EP4,GRK2 and EP4.When PGE2 was administrated for 24 hours,cell proliferation and migration were promoted.Membrane expression of GRK2 and Gβ were increased,EP4 membrane expression was decreased,co-expression of Gβ and GRK2,Gβ and EP4,GRK2 and EP4 were enhanced.CP-25 and Gallein abrogated PGE2-induced abnormal proliferation and migration of MH7A cells,down-regulated total expression and membrane expression of Gβ and GRK2,up-regulated total expression and membrane expression of EP4,and weakened the co-expression of Gβ and GRK2,Gβ and EP4,GRK2 and EP4.The Gβ1 subunit of MH7A cells was transiently silenced with siRNA,and the PGE2-induced proliferation and migration was inhibited after transfection.Total expression and membrane expression of GRK2,membrane expression of EP4 stayed unchanged.Co-expression of GRK2 and EP4 decreased;After treatment with the Gβγinhibitor GRK2i,PGE2-induced cell proliferation and migration were weakened,membrane expression of GRK2 and β-arrestin2 were decreased,EP4 membrane expression was increased,and the interaction between GRK2 and EP4 was increased;mSIRK administration led to increased cell proliferation and migration induced by PGE2,increased membrane expression of GRK2 and β-arrestin2,decreased membrane expression of EP4,and enhanced interaction between GRK2 and EP4.4.Effects of CP-25 on downstream PI3K/Akt/GSK-3β signaling of GβγThe co-expression of Gβ and PI3K was enhanced,the expression of PI3K was upregulated,and the ratio of phosphorylated Akt and phosphorylated GSK-3β were increased in synovial tissue of RA patients.After PGE2 stimulation,co-expression of Gβ and PI3K in MH7A cells was increased,membrane expression of PI3K was increased,the ratio of phosphorylated Akt and phosphorylated GSK-3β were increased;CP-25 and Gallein down-regulated the co-expression of Gβ and PI3K,decreased membrane expression of PI3K.The ratio of phosphorylated Akt and phosphorylated GSK-3β were also decreased.Conclusion:1.The PGE2-EP4-Gβγ-GRK2 signaling was activated in RA.The enhanced interaction between Gβγ and GRK2,Gβγ and EP4 promoted the interaction of GRK2 and EP4,causing GRK2 transduction and EP4 desensitization,which was related to synovial hyperplasia and abnormal proliferation and migration of FLS in RA.2.Gβγ participated in the abnormal proliferation and migration of FLS induced by PGE2.After inhibiting Gβγ,the interaction of GRK2 and EP4 weakened,which lead to resensitization of EP4 and restoration of normal proliferation and migration in FLS.3.CP-25 re-sensitized EP4 ad inhibited the abnormal proliferation and migration of FLS induced by PGE2 by regulating the EP4-Gβγ-GRK2 signaling.4.The mechanism of CP-25 inhibiting the abnormal proliferation and migration of FLS induced by PGE2,was related to its regulation of PI3K/Akt/GSK-3β signaling downstream of Gβγ. |
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