Mechanistic Study On GRK2 Mediated Turnoff Of Hippo-YAP Signaling In Promoting The Hyperplasia Of Fibroblast-like Synoviocytes

Rheumatoid arthritis（RA）is an autoimmune disease caused by the complex interaction between environmental factors and genetics,and its main pathological feature is synovitis.Fibroblast-like synoviocytes（FLS）are one of the main cell types in the synovial tissue of RA patients,and they play an important role in the pathogenesis of RA.In normal joints,FLS produces joint lubricants and various matrix components to shape the extracellular matrix（ECM）.In the joints of RA patients,the excessive proliferation and activation of FLS,on the one hand,produces a large number of matrix metalloenzymes,which decompose cartilage and bone tissue.On the other hand,tumor-like hyperplasia of FLS forms pannus with new blood vessels,which promotes bone erosion.Therefore,inhibiting the excessive proliferation and activation of FLS has become a promising RA treatment strategy.The proliferation of FLS is regulated by a variety of signaling pathways.A variety of G protein-coupled receptors（GPCRs）are expressed on FLS.Physiological concentrations of GPCRs,such as epinephrine,prostaglandin E2（PGE₂）,and adenosine,moderately activate Gαs-coupled GPCRs to maintain the steady-state of cyclic adenosine monophosphate（c AMP）in FLS.c AMP reduces the expression of cyclin through protein kinase A and keeps cells in the G1 phase,thereby inhibiting the proliferation of FLS.In the RA inflammatory environment,the pathologically high concentration of GPCRs ligands,such as adrenaline and PGE₂,is enriched in the joint microenvironment,which overactive the corresponding receptors on FLS.This initiates the negative signal mediated by G protein-coupled receptor kinase 2（GRK2）andβ-arrestin 2,receptor desensitization and endocytosis,resulting in a decrease in intracellular c AMP and the elimination of the anti-proliferation effect of GPCRs.With the deepening of research,the key role of GRK2 in the abnormal proliferation of FLS has been clarified,and inhibition of its activity can significantly improve joint inflammation.GRK2 may be a new target for RA treatment,but its pathological mechanism remains to be elucidated.Studies have found that in the abnormal proliferation of FLS,GRK2 not only affects the function of GPCRs but also mediates a variety of unique downstream signals to play a pathological role,such as phosphatidylinositide 3-kinases/AKT（PI3K/AKT）,extracellular regulated protein kinases（ERK）,and nuclear factor kappa-B（NF-κB）pathways.However,these cannot fully explain the mechanism of GRK2 mediating the abnormal proliferation of FLS.The Hippo pathway is an evolutionarily highly conserved pathway,which plays a central role in regulating cell proliferation and cell fate to control organ growth and regeneration.In mammals,the core components of the Hippo pathway include mammalian sterile 20-like kinases 1/2（MST1/2）,large tumour suppressor 1/2（Lats1/2）and its downstream effectors,and Yes-associated protein/transcriptional coactivator with PDZ-binding motif（YAP/TAZ）.Under normal circumstances,MST1/2 phosphorylate and activate Lats1/2,which in turn phosphorylates and inhibits YAP/TAZ.Phosphorylation of YAP/TAZ by Lats causes the latter to bind to 14-3-3 protein,which is confined to the cytoplasm,resulting in functional inactivation and degradation.Under pathological conditions,the phosphorylation of YAP/TAZ decreases and transfers to the nucleus in a large amount of unphosphorylated state,and combines with the TEA domain transcription factor（TEAD）to initiate the transcription of genes that induce cell proliferation and migration.The abnormal activation of YAP has been reported many times in human cancer research,and it promotes the occurrence and development of tumors through its function of promoting cell proliferation and growth.YAP is also associated with some fibrotic diseases,such as systemic scleroderma and pulmonary fibrosis.These diseases are related to abnormal activation of fibroblasts and excessive ECM deposition,leading to abnormal cell function and tissue sclerosis.The latest study found that inhibiting YAP can improve the condition of RA animal models,suggesting that YAP is involved in the pathological mechanism of RA,but the specific mechanism has not been reported.Because the abnormal proliferation of FLS is the core of the pathological changes of RA,combined with the key role of GRK2 in the abnormal proliferation of FLS,it is not clear whether there is a connection between GRK2 and the Hippo pathway in FLS.This subject studied the subcellular localization of YAP in the synovial tissues of RA patients and CIA rats and clarified the changes of Hippo-YAP signal in FLS under inflammatory conditions and its role in the abnormal proliferation of FLS.GRK2^+/-mice were used to establish a collagen-antibody induced arthritis（CAIA）model in vivo.The FLS was stimulated with PGE₂ in vitro to explore the regulation effect of GRK2 in the Hippo-YAP signal and its influence on joint inflammation.Based on the Hippo-YAP signal to further reveal the pharmacological mechanism of GRK2 inhibitors in the treatment of CIA,the GRK2 inhibitor paroxetine was given to treat CIA in rats.This topic provides an experimental basis for in-depth improvement of the pathological mechanism of the abnormal proliferation of FLS in RA and the pharmacological effects of GRK2inhibitors.Objective:It clarifies the role of Hippo-YAP signaling in the abnormal activation of FLS in the CIA.It elucidates the molecular mechanism by which GRK2 in CIA FLS turns off Hippo-YAP signal,promotes nuclear translocation of YAP,and causes abnormal proliferation of FLS.It is revealed that GRK2 inhibitors can prevent the abnormal proliferation of FLS and improve the mechanism of arthritis by restarting the Hippo-YAP signal.Methods:In order to clarify the role of the Hippo-YAP signal in the abnormal activation of CIA FLS,the immunofluorescence method was used to detect YAP nuclear translocation in the Vimentin-positive FLS in the synovial tissue of RA patients and CIA rats.Western blot method was used to detect the expression and activity of YAP in CIA FLS.The YAP-TEAD inhibitor VP was used to treat CIA FLS in vitro,the nuclear translocation of YAP was observed by immunofluorescence method,the m RNA level of downstream target genes of YAP was detected by q RT-PCR method,and the proliferation and migration ability of FLS was detected by HCI method and Transwell plate method.To clarify the molecular mechanism of GRK2 shutting down Hippo-YAP signal in CIA FLS,promoting nuclear translocation of YAP and causing abnormal proliferation of FLS.The FLS was stimulated by PGE_2,and the expression level of GRK2 was detected by Western blot.The si RNA silencing GRK2 gene or GRK2 inhibitors were used to treat FLS stimulated by PGE₂ in rats.The nuclear translocation of YAP was observed by the immunofluorescence method,and the expression and activity of Lats1 and YAP were detected by Western blot.GRK2^+/-mice were used to establish a CAIA model.Immunofluorescence was used to observe the expression of Vimentin in synovial tissues in response to FLS proliferation,YAP expression,and YAP nuclear translocation in Vimentin-positive FLS.To reveal that GRK2 inhibitors can prevent the abnormal proliferation of FLS and improve the mechanism of arthritis by restarting Hippo-YAP signals.The CCII emulsion was used to induce SD rats to establish a CIA model.After successful modeling,indomethacin（2.5mg/kg/d）,paroxetine（15mg/kg/d）,and methotrexate（0.5/mg/3d）were treated for 21 days in CIA rats.The overall indicators were recorded and evaluated for the efficacy of the drug.H&E staining and X-ray images were used to evaluate the pathological changes of joints.Western blot method was used to detect the expression and activity of YAP,Lats1,and GRK2 in the FLS of each group.The subcellular localization of YAP in the FLS of each group was observed by the immunofluorescence method.Results:1.The Hippo-YAP pathway in RA FLS is closedCompared with normal human synovial tissue,the MFI of Vimentin and YAP in the synovial tissue of RA patients was significantly increased,and the nucleus distribution of YAP in RA FLS was significantly higher than that in normal FLS.The synovial tissue was taken when the CIA reached the peak of inflammation on the 29th day of the modeling.It was found that compared with normal rats,the MFI of Vimentin and YAP in the synovial tissue of the CIA group was significantly increased,and the YAP nucleus in FLS Increased translocation.The FLS was cultured by the tissue block adherence method,and 2-3 generations of FLS were tested.Compared with normal FLS,the expression of YAP in FLS in the CIA group increased significantly,while the ratio of p-YAP^S127/YAP decreased significantly.It is suggested that in the inflammatory state of CIA,YAP is expressed in large amounts but mostly in an unphosphorylated state,which increases nuclear translocation and closes the Hippo pathway.2.Blocking YAP-TEAD combination inhibits abnormal proliferation and migration of FLS in CIA ratsImmunofluorescence detection revealed that YAP in about 79%of normal FLS was mainly distributed in the cytoplasm,and YAP in about 80%of CIA rat FLS was mainly located in the nucleus.The q RT-PCR method found that compared with normal FLS,CIA FLS significantly increased the m RNA levels of YAP’s downstream target genes CTGF and Cyr61,and the proliferation and migration ability of CIA FLS was significantly enhanced.After treating FLS of CIA rats with Verteporfin,an inhibitor of YAP-TEAD,YAP was mainly located in the nucleus in about 28%of cells,and the number was significantly reduced.Compared with FLS in the CIA group,VP treatment in vitro significantly reduced the expression levels of CTGF and Cyr61,and significantly inhibited the proliferation and migration of CIA FLS.The above results prove that the Hippo-YAP signal is involved in the regulation of abnormal proliferation and migration of CIA FLS,but the specific regulation mechanism is still unclear.3.High-concentration PGE₂ stimulation in vitro promotes the closure of the Hippo-YAP signal and induces abnormal proliferation and migration of FLSCompared with the control FLS,high-concentration PGE₂（5μM）stimulation made FLS proliferate abnormally active.It was found that the total expression of MST1 and the ratio of p-MST1^T183/T180/MST1 were not significantly different.The total expression of Lats1 and YAP were significantly increased,and the ratios of p-Lats1/2^S909/Lats1 and p-YAP^S127/YAP were significantly decreased.Under PGE₂ stimulation,YAP in about 70%of rat FLS was located in the nucleus,which was significantly higher than the 18%of normal group FLS.Separate the cytoplasmic and nuclear components of FLS stimulated by PGE₂for 24h in vitro.Western blot results showed that after PGE₂ stimulation,the expression of YAP in the cytoplasm was significantly reduced,while the expression in the nucleus was significantly increased.Further research found that compared with the PGE2 stimulation group,the Lats1 agonist C19 treatment of PGE₂ induced in vitro FLS in rats can significantly increase the levels of p-Lats1/2^S909/Lats1 and p-YAP^S127/YAP,making YAP in about 42%of the cells localized in the nucleus,lower than 66%of the PGE₂ stimulation group.Besides,C19 treatment in vitro significantly reduced the transcription of CTGF and Cyr61 caused by PGE₂ stimulation and inhibited the excessive proliferation and migration of cells.The above results suggest that high-concentration PGE₂ stimulation can significantly promote the proliferation and migration of rat FLS,inhibit Lats1 activity and reduce YAP phosphorylation,thereby promoting the latter to enter the nucleus and induce downstream gene transcription.4.PGE₂ down-regulates Lats1 phosphorylation through GRK2 and turns off Hippo-YAP signalingThe test found that the expression of GRK2 in the FLS of rats induced by PGE₂increased significantly.Silencing the expression of GRK2 in rat FLS by si RNA,or inhibiting the activity of GRK2 by PAR,can significantly increase the ratio of p-Lats1/2^S909/Lats1,p-YAP^S127/YAP under PGE₂ stimulation.At the same time,the expression of GRK2 and YAP were significantly reduced,and there was no significant effect on the expression and activity of MST1.GRK2-si RNA or PAR significantly reduced YAP nuclear translocation induced by PGE₂.GRK2^+/-mice were used to establish the CAIA model,and the mouse ankle joints were taken for immunofluorescence staining.Compared with the WT-Control group,the MFI of Vimentin and YAP in the synovial tissue of WT-CAIA mice was significantly increased.Compared with the WT-CAIA group,the MFI of Vimentin and YAP in the synovial tissue of GRK2^+/--CAIA mice was significantly reduced.Count the position of YAP in the FLS of each group.The proportion of cells located in the nucleus of YAP in the total cells were approximate:24%（WT-Control group）,71%（WT-CAIA group）,23%(GRK2^+/--Control group),26%(GRK2^+/--CAIA group).The above results suggest that GRK2 is expressed in large amounts under the action of PGE₂,which shuts down the Hippo-YAP signal by inhibiting the phosphorylation of Lats1 and promotes nuclear translocation of YAP,thereby mediating the abnormal proliferation of FLS.5.GRK2 inhibitors can restart Hippo-YAP signaling,inhibit FLS proliferation,and reduce the overall indicators of CIA ratsEstablish a CIA rat model,and give GRK2 inhibitor paroxetine,indomethacin,and MTX to treat CIA rats.Compared with the Normal group,the body weight of the CIA-Veh group began to drop on d14,reached the lowest on d20～d23,and then began to slowly increase.The globle assessment,arthritis index,swollen joints count,and secondary paw swelling of the CIA-Veh group increased with the development of the disease,reaching the highest around d23～d26,and then began to decline.The spleen index of the CIA-Veh group increased significantly,while the thymus index decreased significantly.The ankle joint and toes were swollen,and the X-ray image showed that the bones of the ankle joint were severely eroded.The H&E image of the ankle joint in the CIA-Veh group showed that the excessive proliferation of synovial cells resulted in the narrowing of the joint cavity,a large number of immune cells eroded to the joint capsule,accompanied by a large area of pannus formation,obvious cartilage erosion and a large number of white blood cell aggregation.Besides,the FLS cultured with tissue block adherence method,compared with the Normal group,the CIA-Veh group significantly reduced the expression of p-Lats1/2^S909/Lats1 and p-YAP^S127/YAP,and the expression of GRK2 was significantly increased.Compared with the CIA-Veh group,the body weight of the drug treatment groups fluctuated during the administration period from d14 to d20,but during the administration period from d14 to d35,the overall weight was higher than that of the CIA-Veh group.The globle assessment,arthritis index,swollen joints count,and secondary paw swelling of the drug groups kept decreasing.The three drugs can significantly reduce the spleen index,significantly increase the thymus index,and effectively eliminate the swelling of the ankle and toes,and inhibit bone erosion.Besides,H&E staining showed that drug treatment can significantly inhibit joint tissue inflammation,inflammatory cell infiltration,and damage to bone tissue.Among them,paroxetine and MTX significantly inhibit the formation of pannus and abnormal proliferation of synovial cells,while indomethacin cannot effectively inhibit the proliferation of synovial cells and the formation of pannus.Tissue block adherence to culture FLS,compared with the CIA-Veh group,the drug group can significantly reduce the expression of GRK2,and increase the expression of p-Lats1/2^S909/Lats1 and p-YAP^S127/YAP.The location of YAP was detected by the immunofluorescence method.The proportion of cells located in the nucleus of YAP in the total cells were approximate:25%（Normal group）,83%（CIA-Veh group）,60%（CIA-Indo group）,39%（CIA-PAR group）,and 51%（CIA-MTX group）.According to the above results,it is suggested that paroxetine can increase the phosphorylation level of Lat s1 by inhibiting GRK2,thereby phosphorylating YAP and inhibiting its nuclear translocation,inhibiting the abnormal proliferation of FLS,thereby improving the condition of CIA rats.Conclusions:1.The closure of the Hippo-YAP signal in RA FLS leads to increased nuclear localization of YAP and induces gene transcription,which is an important mechanism for abnormal proliferation and migration of FLS.2.GRK2 up-regulated by PGE₂ promotes abnormal proliferation of FLS by inhibiting the phosphorylation of Lats1 and closing the Hippo-YAP pathway.\3.GRK2 inhibitors can reduce YAP nuclear translocation by restarting Hippo-YAP signaling,inhibit FLS proliferation,and improve the signs of CIA rats.