| Rheumatoid arthritis (RA) is a chronic, systemic inflammatory disorder that may affectmany tissues and organs, but principally attacks the joints producing an inflammatorysynovitis that often progresses to destruction of the articular cartilage and ankylosis ofthe joints. In particular, this joint degradation has long been explained by theover-production of inflammatory cytokines in synovial tissue, such as IL-1, TNF-α andprostaglandin E2(PGE2). PGE2mediates its biological activities through four differentreceptors, termed EP1-4, which couple to different guanine-nucleotide binding(G)-proteins and downstream signal transduction systems. Experiment studies suggestedthat EP2and EP4are involved in the pathophysiology of RA. Both EP2and EP4receptors are G protein-coupled receptors (GPCR), which all couple to the Gαs protein.Beta-arrestin1(arrestin2) and β-arrestin2(arrestin3) are cytosolic proteins involved inthe termination of signaling by activated GPCRs with G-protein-coupled receptorkinases (GRKs). However, the effect of β-arrestins in regulating EP receptor need to beelucidated. Paeoniflorin (Pae), a monoterpene glucoside, is one of the main bioactive componentsof total glucosides of paeony (TGP) extracted from the root of Paeonia lactiflora thathas been used for gynaecological problems and for cramp, pain and giddiness for over1500years in Chinese medicine. Anti-inflammatory, analgesia, antioxidative,antihepatic injury and immunoregulatory activities of TGP have been extensivelyproved in our laboratory for many years. TGP not only inhibits secondary inflammatoryreaction, bone destruction and ultrastructure change of synoviocytes in adjuvant inducedarthritis (AA) rats, but also significantly decreases the expression of Gi protein, restorescyclic adenosine monophosphate level and protein kinase A activity. Whether theregulation of β-arrestins expressions by TGP have interrelationship with its inhibitoryeffect on FLS proliferation is still not clear. In present work, we use rat and humanfibroblast like synoviocytes (FLSs), PGE2, EP2receptor specific agonist Butaprost andsmall interfering RNA (siRNA) to investigate the role of β-arrestin2on regulating EP2signal pathway in arthrosynovitis inhibition effect of Pae.OBJECTIVE This work was carried out to investigate the potential molecularmechanism of PGE2in inducing FLS hyperplasy and the effect of Pae throughobserving the expression and distribution change process of EP2and β-arrestin2. Theexpression dynamic changes of β-arrestin2, EP2and cAMP level in human FLSstimulated by Butaprost and the effect of β-arrestin2in EP2receptor internalization andcell proliferation were also elucidated. This work may reveal the mechanisms of Pae inattenuating synovitis and open up a field for investigating possible therapeutic strategies,including modulation of β-arrestin activity.METHODS Normal FLS were isolated from primary synovial tissue obtained from ratsand patients. The expression of vimentin was examined immunohistochemically toverificate the cell morphology. The proliferation of FLS was detected by MTT assayand3H-TdR incorporation assay. Cyclic-AMP concentration was determined by radioimmunity. Membrane EP2expression was determined through flow cytometry,western blot and laser scanning confocal microscopy. The expression of total EP2,β-arrestin2and Gαs in FLS and membrane β-arrestin2level were detected by westernblot. Co-immunoprecipitation assay was applied to evaluate the activity of Gαs.Delivery of siRNA into human FLS using the Lipofectamin2000system. SiRNA wastranfected into human FLS effectively after culture for48h following remove ofsiRNA/Lipofectamin2000compounds. The efficiency of β-arrestin2gene silence wasevaluated by RT-PCR, real time fluorescent quantitation PCR and western blot. EP2and β-arrestin2mRNA level was determined by RT-PCR and real time fluorescentquantitation PCR.RESULTS1. The role of β-arrestin2on regulating EP2internalization and FLS proliferation1.1The expression of β-arrestin2and EP2as well as cAMP levels in FLS weredynamicly changed in response to Butaprost activation.MTT result suggested that the suboptimal concentration of Butaprost was1×10-7mol· L-1and optimum time point was24h for FLS proliferation. After stimulated byButaprost, a selective EP2agonist, EP2membrane expression in hFLS was increasedgradually till20min then decreased continuously, and EP2membrane expression waseven lower than that of control level at24h. The western blot detection showed thatmembrane EP2expression was up to the peak at20min in response to Butaprosttreatment, and then decreased to control level at2h after stimulation as was found byflow cytometry. However, membrane β-arrestin2expression was increased continuouslyafter20min under Butaprost induction. The whole fluorescence intensity of FLSstimulated by Butaprost was enhanced in response to Butaprost1×10-7mol· L-1activation. At about20min, fluorescence intensity of FLS membrane was increasedmarkedly then decreased gradually indicated an internalizationprocess of EP2. Finally, at120min, fluorescence intensity of both membrane and cytolymph attenuatedsuggested a low level of EP2expression. Cyclic-AMP concentration was upregulatedsequentially till about30min once upon butaprost activation, and then was reducedsignificantly at about2h. Twenty four hours after Butaprost treatment, cAMP level wasdecreased to even lower than that of control FLS. The data suggested that on earlierperiod of EP2activation, cAMP level was increased and act as a protective effector forFLS, but after unremitting stimulation by Butaprost, cAMP concentration was reducedand promoted FLS hyperplasy.1.2The role of β-arrestin2in controling EP2intenalization and cell proliferation.The Lipofectamin2000system was used to transfect siRNA into human FLS. Smallinterfering RNA was tranfected into human FLS effectively after culture for48hfollowing remove of siRNA/Lipofectamin2000compounds. The results of RT-PCR,real time fluorescent quantitation PCR and western blot confirmed thatArrb2-homo-1293siRNA is efficient for β-arrestin2gene silencing. The sequence ofArrb2-homo-1293is: Sense:'-CCAACCUCAUUGAAU UUGATT-3'; Anti-sense:5'-UCAAAUUCAAUGAGGUUGGTT-3'. After stimulated by Butaprost, EP2membrane expression in human FLS transfected with β-arrestin2siRNA was increasedgradually, after1hour, the membrane EP2expressions were significantly higher thanthat of control. Βeta-arrestin2gene silencing FLSs showed no obviously response uponPGE2stimulation compared to normal cells. These results demonstrate that β-arrestin2plays an important role in EP2receptoe intenalization and cell proliferation.2. Pae inhibites FLS hyperplasy induced by PGE2via regulating β-arrestin2andEP2mediated pathway.2.1Pae blocked the decreased EP2membrane expression induced by PGE2in FLSThe suboptimal concentration of PGE2was125ng· ml-1and optimum time point was 24h for FLS proliferation. Rat FLS proliferation was evoked by PGE2125μg· L-1, andwas significantly inhibited by the administration of Pae at concentrations of1×10-8,1×10-7,1×10-6,1×10-5mol· L-1by different degrees. Cycle-AMP concentration wasreduced upon PGE2activation for24h, but was restored by Pae administration of1×10-7,1×10-6,1×10-5mol· L-1. As detected by flow cytometry, the membrane EP2expression was downregulated in response to PGE2induction, Pae1×10-6,1×10-5mol· L-1ameliorated membrane EP2level obviously. Total EP2expression wasobviously increased after PGE2stimulation compared with the normal FLS, Pae1×10-6,1×10-5mol· L-1decreased total EP2expression. Human FLS proliferation was evokedby PGE2compared with the normal FLS, and was significantly inhibited by theadministration of Pae at concentrations of1×10-7,1×10-6,1×10-5mol· L-1. EP2mRNAlevel was increased by PGE2stimulation and decreased by Pae1×10-8,1×10-7,1×10-6,1×10-5mol· L-1in vitro. Total expression of EP2in human FLS was upregulated byPGE2125μg· L-1stimulation, but was reduced by Pae1×10-6,1×10-5mol· L-1in vitro;Membrane EP2level of human FLS was downregulated in response to PGE2activation,but was restored by Pae1×10-6,1×10-5mol· L-1. These data suggest Pae inhibites FLShyperplasy induced by PGE2via blocking EP2abnormal internalization and restoringcAMP concentration.2.2Pae reduced total and membrane β-arrestin2expression in PGE2induced FLSBoth total and membrane β-arrestin2levels were obviously increased after PGE2stimulation compared with the normal rat FLS, Pae1×10-6,1×10-5mol· L-1decreasedtotal β-arrestin2expression, and Pae1×10-7,1×10-6,1×10-5mol· L-1administrationreduced membrane β-arrestin2level in rat FLS. Real time fluorescent quantitation PCRshowed a high level of β-arrestin2mRNA in PGE2-induced human FLS, Paesubstantially restored β-arrestin2mRNA expression at the concentrations of1×10-6,1×10-5mol· L-1. Total and membrane surface expression of β-arrestin2in human FLS were elevated to the same extent by PGE2, Pae1×10-6,1×10-5mol· L-1showedinhibitory effects on both expressions.2.3Pae restored Gαs activity inhibited by PGE2Interestingly, total Gαs protein level in hFLS was not obviously changed by either PGE2stimulation or Pae administration. Gαs activity was upregulated by PGE2induction, Pae1×10-6,1×10-5mol· L-1administration in vitro obviously attenuated Gαs activity. Thedata indicate that Pae inhibites FLS hyperplasy induced by PGE2via raising the activityof Gαs and mentaining the level of cAMP.CONCLUSIONS1. At about20min after Butaprost stimulation, EP2membrane expression wasincreased markedly then decreased gradually indicated an internalization process, at120min, both membrane and cytolymph EP2expressionwere reduced. On earlierperiod of EP2activation (within30min), cAMP level was increased and act as aprotective effector for FLS, but after unremitting stimulation by Butaprost, cAMPconcentration was reduced and promoted FLS hyperplasy. The time dependentchanges of EP2and cAMP were associated with the consecutively increasedexpression of β-arrestin2(significantly increased at about30min).2. After stimulated by Butaprost, EP2membrane expression in human FLS transfectedwith β-arrestin2siRNA was increased gradually, after1hour, the membrane EP2expressions were higher than that of control. Βeta-arrestin2gene silencing FLSsshowed no obviously response upon PGE2stimulation compared to normal cells.Beta-arrestin2played an important role in EP2receptoe intenalization and cellproliferation.3. Pae significantly inhibited human fibroblast-like synoviocytes hyperplasy induced byPGE2via decreasing β-arrestin2gene and protein expression and β-arrestin2membrane distribution, reducing total EP2expression, but increasing EP2membrane level, restraining EP2receptor internalization, and finally raising the activity of Gαs.Similar results were observed in both human and rat FLS, which indicated that bothcells have homoioplasticly biological responses.
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