Effects Of TRPV4and Estrogen On Mandibular Condylar Chondrocytes In Rat

Osteoarthritis (OA) is a heterogeneous group of conditions thatcauses joint symptoms and signs, which are associated with defectiveintegrity of articular cartilage in addition to related changes in theunderlying bone at the joint margins. The hallmark of OA is aprogressive degradation of articular cartilage and the general jointstructure. Current available pharma cological interventions mayeffectively handle the joint sy mptoms such as joint pain, tenderness,stiffness, locking, but they have limitations in terms of re versing theaccelerated cartilage degradation. Studies have showed that secretedpro-inflammatory molecules are among the critical mediators of thedisturbed processes implicated in OA pathophysiology. Interventionsmodulating pro-inflamma tory molecules might havechondroprotective role in OA treatment. The temporomandibular joint(TMJ) plays an i mportant role in craniofacial growth and function,and shows a high incidence of OA. M oreover, many studies showedthat fema le had higher incidence and more severe symptoms oftemporomandibular joint disorders（TMD） than male, especially inthose who had signs of (17β-estradiol)E2deficiency, implying thechondroprotective effect of E2. Exactly, there was a growing body ofevidence suggesting that E2had chondroprotective role via itsinhibitory effect on pro-inflammatory cytokines. However, theprecise mechanism of E2chondroprotective effe ct was still wanted.TRPV4is a me mb er of TRP super family of Ca2+-permeablenon-selective cation channels. TRPV4is widely expressed in cochlear hair cells, vibrissal Merkel cells, se nsory ganglia as well as in freenerve endings and cutaneous A and C-fibers terminals, etc. Recentresearch showed that TRPV4might have chondroprotective role inknee joints of the mouse.Although E2and TRPV4have each been reported to bechondroprotective and the correlation of E2and Ca2+influx have alsobeen demonstrated in a wi de variety of cell types, the molecularmechanism between E2and TRPV4was still unknown. In our study,we found that E2could down-regulate NO expr ession by stimulatingTRPV4expression in a post-transcriptional regulating way viainhibiting miR-203, which directly targets TRPV4, in fe male ratmandibular condylar chondrocytes (MCCs). Our findingsdemonstrated the possible molecular mechanism between E2andTRPV4i n MCCs, which was helpful for the identification of thechondroprotective effect of E2.The present study was undertaken to examine alterations inexpression of miR-203and transient receptor potential vanilloid4(TRPV4) in fe male Sprague–Dawley rats MCCs, and analyze theirroles in the down-regulating process of NO by E2. First, we culturedSD rats MCCs. The MCCs were exposure to E2（10-9-10-6M） andLipopolysaccharide (LPS) plus ruthenium red,4α-phorbol12,13-didecanoate(4α-PDD) as well as over-expressed miR-203, thecellular supernatants were analyzed for TNF-α、 IL-1βand NOconcentrations, TRPV4gene and protein level was me asured byRT-PCR and Western blot analysis, respectively.mi R-203gene levelwas measured by quantitative PCR after the treatment with E2（10-9-10-6M）. Dual luciferase activity assay was perfor med toidentify the target gene of miR-203.The results showed that TRPV4was expressed abundantly at theRNA and protein levels. The inhibition effect on NO production under LPS-stimulation by E2（10-9-10-6M）was blocked by RutheniumRed and enhanced by4α-PDD, respectively. Furthermore, E2coulddown-regulate miR-203expression followed with increasing TRPV4expression level in MCCs. Dual luciferase activity assay suggestedthat TRPV4was the direct target gene of miR-203. Enforc ed miR-203reduced TRPV4protein level and blocked the inhibition effect on NOproduction under LPS-stimulation by E2as Ruthenium Re d.Our foundings first suggested that E2might down-regulate NOproduction, at least in part, by inhibiting miR-203via targetingTRPV4.Part Ⅰamounting evidence suggested that TRPV4channel might play animportant role in inflammatory and neuropathic pain. Mimi N. Phanet al. reported that TRPV4was also present in porcine articularchondrocytes.However, there was no research about the location ofTRPV4in MCCs. The aim of the present study was to demo nstrate thepresence of TRPV4in MCCs. In this part, Primary SD rats MCCswere cultured.TRPV4RNA and protein expression was measuredbyRT-PCR、 i mmunolabeling and Western blotting. The resultsshowed that TRPV4was expressed abundantly at the RNA and proteinlevel. Our findings indicate that TRPV4is present in MCCs.Part ⅡOA in TMJ fav ors wo me n over men. It was the fact that mostfe male patients with severe condylar resorption had signs andsymptoms of E2deficiency. However, there was still an unresolved paradox with respect to the exactly role of estrogens in articularcartilage. Currently, increasing evidence indicated that estrogensmight ha ve chondroprotective effect through its anti-inflammationeffect. It was reported that ion channel activity could be regulated bysex hormones, either directly or indirectly via the generation ofintracellular signals. However,the molecular mechanism betweensex hormo nes and TRPV4was still unknown.Our results demonstratedthat E2could up-regulate TRPV4in fe male MCCs. In this part, wemeasured the production of NO、TNF-α、 IL-1βand changes in Ca2+signaling using ELISA and laser cofous microscopy respectively atthe stimulation of E2and TRPV4agonist/antagonists (4α-PDD,Ruthenium Red) in MCCs. I n a situation of LPS(20ng/ml)stimulation,we found that E2（10-9-10-6M）could significantly inhibit NO、T N F-αand IL-1β production. In addition, NO production induced by LPScould significantly increased by TRPV4inhibitor Ruthenium Red,while4αPDD, a TRPV4specific activator could significantlydownregulate NO production. Further more, The inhibition effect onNO production by E2（10-9-10-6M）was blocked by Ruthenium Red andenhanced by4α-PDD, respectively, which suggested that E2mightinhibite NO production in a TRPV4-dependent manner. However, wealso found that The inhibition effect on TNF-α and IL-1β productionby E2（10-9-10-6M）were no relation with TRPV4. Our results providedthe proof of the favorable effect of TRPV4in anti-inflammation onMCCs and suggested that TRPV4was a necessary part in NOinhibition by E2.Part ⅢIn the last few years it had become clear that miR-203wereinvolved in the pathogenesis of various malignant tumors. At the same ti me, evidence for the involvement of miR-203in inflammatorydisorders appeared. Elevated levels of miR-203lead to increasedsecretion of MMP-1and IL-6, indicating that mi R-203might be aproinflammatory and joint-destructive factor. Exactly, in our study,we also found that over-expression of mi R-203resulted in increasedsecretion of NO which was one of the major proinflammat ory factorin OA via targeting TRPV4in MCCs. In this part, Primary SD ratsMCCs were exposure to E2（10-9-10-6M）and LPS plus ruthenium red,4α－PDD as well as over-expressed miR-203, the cellularsupernatants were analyzed for NO concentrations, TRPV4gene andprotein level was measured by RT-PCR and Western blot analysis,respectively.miR-203gene level was measured by quantitative PCRafter the treatment with E2（10-9-10-6M）. Dual luciferase activityassay was performe d to identify the target gene of miR-203.RT-PCR ana lysis showed that TRPV4mR NA level wassignificantly down-regulated after treatment with E2（10-8-10-6M）for72hr, while the protein level was significantly up-regulated afterexposure to E2(10-9-10-6M) for the same time period. Thisinconsistent result suggested that E2might post-transcriptionallyupregulate TRPV4protein level.we hypothesized that E2could inhibit miRNAs which targetedTRPV4.， so we detected miR-203expression level changes in MCCsafter the treatment of E2. Real-time quantification of mi R-203bystem-loop RT-PCR exactly showed that miR-203was significantlydown-regulated in MCCs aft er incubating with E2（10-9-10-6M） for72hr compared with control.TargetScan5.1predicted that TRPV4was the target gene of themi R-203conservely between different species. To explore whetherTRPV4was the target gene of miR-203, we constructed a luciferase reporter vector with the putative TRPV4-3’ UTR target site for themi R-203downstream of the luciferase gene (TRPV4-3’-UTR).Luciferase reporter vector together with the miR-203mi mic or themi RNA mimic control was transfected into293T cells, a significantdecrease in relative luciferase activity was observed whenTRPV4-3’-UTR was co-transfected with the miR-203mimic but n otwith the mi RNA mimic control, suggesting that TRPV4is the directtarget gene of the miR-203.Since E2could inhibit NO pr oduction in a TRPV4-dependentmanner and TRPV4targeted mi R-203was also downregulated by E2,we hypothesized that E2could down-regulate NO production at leastin part by inhibiting miR-203via targeting TRPV4. Exactly, in MCCs,Western blot assay revealed that those transfected with miR-203mi mic exhibited greatly decreased TRPV4protein level comparedwith the mi RNA mimic control transfected cells after the treatmentwith different concentrations of E2. This result proved that miR-203could mo dulate TRPV4expression at the exposure of E2. Asmentioned above, E2could inhibit NO production in LPS-activatedMCCs. The effect could be blocked by TRPV4inhibitor RutheniumRed. In miR-203mi mic transfected cells, we also found this effect.Those miR-203mi mic transfected cells showed significantlyincreased NO production after treatment with E2. Taken all together,our foundings suggested that E2might down-regulate NO production,at least in part, by inhibiting mi R-203via targeting TRPV4.