The Role Of GRK2 In The High Expression Of PDE4D In Experimental Arthritic Synovial Cells And The Effect Of Its Inhibitors

Rheumatoid arthritis（RA）is a chronic autoimmune disease that affects the whole system.The clinical manifestations are local swelling,pain and dysfunction of small joints,etc.Abnormal proliferation and activation of fibroblast synovial cells（FLS）leading to the destruction of cartilage and bone tissue are the key pathological features of RA.In normal joints,the FLS in synovial lining that produce hyaluronic acid and lubricating joints,such as lubricant,lubrication and protect joints,and in patients with RA FLS,FLS by generating a large number of matrix metalloproteinases（MMPS）to destruction of cartilage and bone support structure,and by promoting osteoclast formation and inhibit bone repair to promote bone erosion.Therefore,inhibiting the abnormal proliferation of FLS and restoring its normal function are effective strategies for the treatment of RA.Cyclic adenosine monophosphate（cAMP）is a major intracellular second messenger that plays an important role in regulating cell proliferation.Under physiological conditions,a variety of G protein-coupled receptors（GPCRs）coupled to Gαs are expressed on the FLS cell membrane,and ligands such as epinephrine,prostaglandin E2（PGE2）,and adenosine moderately activate the corresponding GPCRs,making the cell memory There is a certain abundance of cAMP,which activates the downstream PKA,promotes the expression of cell cycle inhibition-related proteins,attenuates the activities of extracellular regulated protein kinase（ERK）and cyclin D1/D3 that promote cell proliferation,and keeps FLS in a proliferative quiescent state.The previous study of our group found that in RA,ligands such as epinephrine and PGE2 in the circulation and local joints are pathologically increased,which makes GPCRs on FLS undergo desensitization and endocytosis under the synergistic action of GRK2 and β-arrestin 2.Function is negatively regulated,resulting in decreased levels of cAMP in FLS.At the same time,it has also been reported in the literature that a large amount of phosphodiesterase（PDE）is expressed in inflammatory FLS.PDE can efficiently hydrolyze cAMP,which further reduces the content of cAMP in FLS,which is also an important mechanism leading to abnormal proliferation of FLS-like tumors.Phosphodiesterase（PDE）is a superfamily of enzymes capable of hydrolyzing intracellular cAMP/c GMP.PDE has a total of 11 subtypes（PDE1~11）,and can be divided into: specific hydrolysis of cAMP（PDE4,PDE7,PDE8 family）,specific hydrolysis of c GMP（PDE5,PDE6,PDE9 family）As well as hydrolyzing cAMP/c GMP（PDE1,PDE2,PDE3,PDE10,PDE11）family.It has been reported in the literature that the expression levels of PDE4 isoforms are significantly increased in RA FLS.In vitro stimulation with tumor necrosis factor-α（TNF-α）can induce FLS to express a large amount of PDE4 D,and the use of PDE4 inhibitor Apremilast can significantly inhibit collagen antibody-induced arthritis（CAIA）mice paw swelling,arthritis index,and delay arthritis The onset and improvement of joint pathology suggest that high expression of PDE4 D is an important pathological factor for abnormal proliferation and joint inflammation in FLS,but the pathological mechanism of high expression of PDE4 D in FLS is still unclear.Although PDE4 inhibitors have been clinically used to treat asthma,chronic obstructive pulmonary disease,etc.,their clinical application is limited due to severe and persistent adverse reactions,especially the treatment of chronic diseases makes it difficult for patients to tolerate.Therefore,revealing the molecular mechanism of increased PDE4 D expression in FLS under inflammatory state and reducing its abnormally high expression is a safe and effective strategy for the treatment of RA.A large number of previous studies by the research group found that the effect of TNF-α can reduce the content of cAMP in FLS,which is related to GRK2.It was further found that TNF-α stimulates normal human FLS,which can be stimulated by tumor necrosis factor receptor 2（TNFR2）and tumor necrosis factor receptor.Bodyassociated factor 2（TRAF2）activates GRK2,however,whether TNF-α upregulates PDE4 D expression by activating GRK2,thus leading to cAMP reduction in FLS is unclear.Pre-experiments of the research group showed that TNF-α stimulation in vitro can induce a large amount of PDE4 D in rat FLS,and the use of small interferingRNA（siRNA）to inhibit the expression of GRK2 can significantly inhibit the production of PDE4 D induced by TNF-α,suggesting that GRK2 is indeed involved in inflammatory FLS.Synthesis of PDE4 D in.Existing studies have shown that in addition to negatively regulating GPCRs,GRK2 can mediate a variety of downstream signaling pathways,affecting protein expression and cell function changes,including c-Jun amino-terminal protein kinase（JNK）,p38 mitogen-activated protein kinase（p38MAPK）,phosphatidylinositol-3-kinase（PI3K）-protein kinase B,and nuclear factor kappa B（NF-κB）signaling,etc.The signaling pathway through which GRK2 increases PDE4 D expression remains to be elucidated.On the basis of previous research results and literature reports,this project detected the expression of PDE4 D in FLS of RA patients and CIA rats and in normal FLS induced by TNF-α;The CAIA model established in mice was used to explore the role of GRK2 in the high expression of PDE4 D and abnormal cell proliferation in inflammatory FLS;signaling molecule activity inhibitors were used to clarify the molecular mechanism of GRK2 involved in PDE4 D synthesis in inflammatory FLS;GRK2 inhibitor CP-25 was administered or paroxetine（Par）to treat CIA rats,observe the overall indicators,further test the effect of GRK2 inhibitor on PDE4 D level and related signal transduction in FLS,and reveal its pharmacological mechanism of CIA treatment.This study provides an experimental basis for improving the pathological theory of abnormal proliferation of RA FLS and clarifying the mechanism of GRK2 inhibitor in the treatment of experimental arthritis.Objective: To observe the expression level of PDE4 D in inflammatory FLS and its effect on cell proliferation;to clarify the role and molecular mechanism of GRK2 in inflammatory FLS overexpressing PDE4 D,reducing intracellular cAMP content and causing abnormal cell proliferation;The expression of PDE4 D,the pharmacological effect and mechanism of inhibiting the abnormal proliferation of inflammatory FLS.Methods: In order to reveal the expression level of PDE4 D in inflammatory FLS and its effect on cell proliferation,q RT-PCR and Western blot were used to detect the mRNA and protein expression levels of PDE4 D in FLS of RA patients and CIA rats.The cells were pretreated,and the high-content cell imaging system was used to detect the proliferation of FLS stimulated by TNF-α;in order to clarify the role of GRK2 in inflammatory FLS overexpressing PDE4 D,reducing the content of intracellular cAMP,and causing abnormal cell proliferation,TNF-α was used to stimulate FLS,Western blot was used to detect the expression levels of GRK2 and PDE4 D.siRNA was used to silence GRK2 gene or GRK2 inhibitor to treat TNF-α-stimulated normal rat FLS,and human FLS transfection plasmid was used to overexpress GRK2 gene.Western blot was used to detect GRK2 and PDE4 D.The expression of PDE4 D,the proliferation of FLS was detected by high-content cell imaging system,and the change of cAMP content in FLS was detected by fluorescence resonance energy transfer technology.The CAIA model was established in GRK2+/-mice and compared with the WT mouse model for the overall score of arthritis.The expression of PDE4 D in synovial tissue was observed by immunofluorescence,and the proliferation of joint FLS was shown by H＆E staining.In order to explore the molecular mechanism of GRK2 leading to the high expression of PDE4 D in inflammatory FLS,under the condition of TNF-α-stimulated normal rat FLS,the inhibitor of GRK2-mediated main downstream pathway was pretreated respectively,and the expression of PDE4 D was examined by Western Blot..In order to reveal whether GRK2 inhibitor can reduce the expression of PDE4 D in FLS,inhibit the abnormal proliferation of inflammatory FLS and improve experimental arthritis,a rat CIA model was established,and CP-25（50 mg/kg/d）,Par（15 mg/d）were administered or methotrexate（MTX）（0.5 mg/kg/3d）for 21 days,the overall indicators of arthritis were recorded,H＆E staining was used to evaluate joint pathological changes,and Western blot was used to detect GRK2 in synovial tissue of rats in each group,the expression and activity of p65 and ERK,and the expression level of PDE4 D.Results:1.The expression of PDE4 D was significantly increased in FLS of RA patients and CIA rats,and was positively correlated with abnormal proliferation of FLS of CIA rats.On model d29,the synovial tissue was taken out when the rat CIA reached the peak of inflammation,and FLS was cultivated with the tissue block adherent method.Compared with normal rat FLS,the expression of PDE4 D gene and protein in CIA rat FLS was significantly increased,while the expression of Vimenten,a marker of FLS in CIA rat ankle joint tissue,increased,accompanied by an increase in the mean fluorescence intensity（MFI）of PDE4 D and the MFI of PDE4 D was positively correlated with Vimenten expression.The synovial tissue of normal and RA patients was collected under the premise of ethical and informed consent,and the tissue block adherent method was used to cultivate FLS.Compared with normal human FLS,the expression of PDE4 D protein in FLS of RA patients was significantly increased.It is suggested that the significantly elevated expression of PDE4 D in FLS of RA patients and CIA rats may be related to the abnormal proliferation of FLS.2.PDE4 D plays an important role in promoting the abnormal proliferation of rat FLS stimulated by TNF-α in vitro In vitro stimulation of normal rat FLS with TNF-α can significantly increase the expression of PDE4 D gene and protein in cells,and induce a large number of FLS proliferation.Correlation analysis showed that the MFI of intracellular PDE4 D under the action of TNF-α was positively correlated with the number of cells.Further research found that the chronic effect of TNF-α reduced the production of cAMP in rat FLS under the action of the adenylyl cyclase agonist Foskolin（Fsk）,and the broad-spectrum PDE inhibitor IBMX could restore the ability of Fsk to produce cAMP,suggesting that after TNF-α-α long-term stimulation,the expression of intracellular PDE not only increased,but also significantly enhanced the activity,significantly inhibited the production of intracellular cAMP.Pretreatment with the PDE4 D inhibitor BPN14470 significantly inhibited the pro-proliferative effect of TNF-α on normal rat FLS in vitro.It is suggested that the enhanced activity of PDE4 D and the reduction of intracellular cAMP production capacity are one of the important reasons for the abnormal proliferation of FLS induced by TNF-α.3.TNF-α induces high expression of PDE4 D in normal rat FLS through GRK2 The Western blot assay showed that TNF-α induced high expression of GRK2 in rat FLS.Silencing the expression of GRK2 in rat FLS by siRNA,or inhibiting the activity of GRK2 with GRK2 inhibitors CP-25 or Par,can inhibit the expression of PDE4 D in rat FLS stimulated by TNF-α.Normal human FLS was transfected with GRK2 plasmid,and overexpression of GRK2 could increase the expression of PDE4 D in cells.Further studies found that chronic effects of TNF-α attenuated the ability of prostaglandin E2（PGE2）to stimulate the production of cAMP in rat FLS,and this inhibitory effect could be effectively recovered by pretreatment with the GRK2 inhibitor CP-25.And GRK2 inhibitor CP-25 or Par can significantly inhibit the proliferation of FLS induced by TNF-α.It is suggested that TNF-α induces the high expression of PDE4 D in rat FLS through GRK2 and reduces the level of cAMP production in FLS,thereby mediating the abnormal proliferation of FLS.4.TNF-α may promote the expression of PDE4 D in rat FLS through GRK2-mediated NF-κB and ERK pathways Simultaneously with stimulation of FLS in normal rats with TNF-α,the NF-κB p65 inhibitor JSH-23,JNK inhibitor Bentamapimod,ERK inhibitor Ravoxertinib,P38 MAPK inhibitor Doramapimod,PI3 K inhibitor NVP-BAG956 or CP-25 were administered,detected the expression of PDE4 D in the cells of each group,the results showed that compared with the stimulation group,inhibiting the NF-κB p65,ERK or p38 MAPK signaling pathway could inhibit the expression of PDE4 D to varying degrees and there were statistical differences.We further silenced GRK2 in rat FLS with siRNA and stimulated it with TNF-α,and found that reducing GRK2 expression reduced TNF-α-induced NF-κB p65 and ERK activities,while p38 MAPK activity was not significantly affected.It is suggested that TNF-α can promote the expression of PDE4 D in rat FLS through GRK2-mediated NF-κB and ERK pathways.5.Heterozygous knockout of GRK2 reduces PDE4 D expression and alleviates joint inflammation in FLS of CAIA mice The CAIA model of GRK2 heterozygous knockout（GRK2+/-）mice was established.Compared with the CAIA model of littermate wild type（WT）mice,the arthritis index was significantly reduced,and only slight joint pathological changes appeared,suggesting that the Reducing the overall expression of GRK2 at the genetic level can significantly improve arthritis in CAIA mice.Moreover,the MFI of PDE4 D in the joints of GRK2+/-CAIA mice was significantly lower than that of the WT CAIA group,suggesting that partial knockout of GRK2 in vivo could significantly reduce the expression of PDE4 D in the FLS of the ankle joints of CAIA mice and improve arthritis,thus mediating PDE4 D in FLS.The up-regulation of GRK2 may be an important way for GRK2 to exert pro-inflammatory effect.6.GRK2 inhibitor can reduce the expression of PDE4 D in FLS and effectively improve CIA in rats A CIA rat model was established,and Par,CP-25 or MTX were administered to the CIA rats.The results showed that each administration group could improve the whole body score,arthritis index,arthritis swelling number and secondary paw volume of CIA rats to different degrees,restore the thymus and spleen index,and alleviate joint pathological changes,which had a positive effect on CIA in rats.clear therapeutic effect.It was found that GRK2 inhibitor significantly inhibited the activities of GRK2,NF-κB p65 and ERK and the expression of PDE4 D in the synovial tissue of CIA rats.It is suggested that reducing the expression of NF-κB p65 and ERK-mediated PDE4 D may be one of the important pharmacological effects of GRK2 inhibitor in the effective treatment of experimental arthritis.Conclusion:1.TNF-α activates the NF-κB and ERK pathways by up-regulating GRK2,resulting in the increased expression of PDE4 D,inhibiting the production of intracellular cAMP,and promoting the abnormal proliferation of FLS;2.GRK2 inhibitor can inhibit the excessive proliferation of FLS and improve CIA in rats by reducing the expression of PDE4 D mediated by NF-κB and ERK pathways.