Activated Wnt3a/beta-catenin Signalinhibits Proliferation And Chondrocyte Differentiation Of Articular Cartilage Mesenchymal Progenitor Cells Through P53pathway

BackgroundCurrently, the number of primary osteoarthritis (Osteoarthritis, OA) is growingup tobecome to the second leading cause of disability in the elderly, but its pathogenesis is notclear. Recent studies have shown that: the aging of the stem cell tissue and organ agingcould be the important internal factors. In2004, Dowthwaite GP found that human articularcartilage existin mesenchymal progenitor cells (MPCs).On the other hand, Alsalamehhavefound the number of MPCs in normal articular cartilage is more than primary OAarticular cartilage. Our previous study also implies that: the number of MPCs in OAarticular cartilage per unitissignificantly less than the normal group, and their proliferationand differentiate to chondrocytes is also significantly lowerthan the normal group. We mayinfer that the MPCs in articular cartilage play an important role in the repair of cartilagedamage, thus, the MPCs aging have a particular relation to degeneration of articularcartilage in OA,Unfortunatelythe research of mechanism of MPCs aging in articularcartilages on going and not clear yet. Recently data have shownthat OA articular cartilageexist the expression of Wnt proteins. In mechanical injury of articular cartilage, expressionof Wnt signal target genes Axin-2and c-JUN raised, and secreted frizzled protein isdownerregulated, it suggests that Wnt signal is active. Another recent study found that thereis a relationship between the Wnt signal pathway and p53/p21pathway. Target genes of theWnt signal pathway is TCF4, which is also the target gene of p53. Over expression ofβ-catenin in retinoblastoma cell cytoplasm can increase the transcriptional activity of p53.In the present study, a clear increase in p53and p21expression was observed in theORS(old rat serum) group compared with it in the YRS(young rat serum) group. After the100ng/mL DKK1and si-β-catenin treatment, the expression of p53and p21wassubstantially decreased. These results imply that the activated Wnt/β-catenin signal have cross-talk with p53/p21pathways.In view of the p53/p21is an important pathway as cellaging, so we inferred that, activation of the Wnt signal may induced MPCs aging throughthe p53/p21pathway, and promote the process of OA.ObjectiveTo explore the expressing of Wnt3a in normal MPCs and OA MPCs, then, observe theproliferation of normal human MPCs,and how the Wnt3a/β-catenin signal is activated orblocking. In order to discuss the possibility effect of Wnt signal on MPCs aging, weexamine the effect of activated Wnt signal on p53expression in MPCs through p53Luciferase, while use siRNA technology to inhibit the expression of p53, to block Wntsignalactivated induced MPCs aging, and explain the p53/p21pathway where promote theMPCs aging with activated Wnt signal, to explore the mechanism of Wnt signal on theMPCs aging regulation, provide a preliminary basis for the deeper study of OA pathologicalmechanisms.Method:1. The effect of activated Wnt signal on the MPCs aging1.1Obtain the specimens of articular cartilage and MPCs1.1.1Source of specimen: Take the OA cartilage from who had taken total kneearthroplasty from2009-2011.The donor site is the posterior of femur condyles. There are8male and16female in this group, whose average age is (52.5±3.0) years old. While take thefemur head normal cartilage from who had taken total hip arthroplasty due to the femurneck fracture which is taken as control. There are11male and19female in this group,whose average age is (58.0±5.2) years old.Choose the specimen which look like a normalobservation of articular cartilage without abnormalities and fibrosis by Outbridge gradingstandards. All the patients have had signed the informed consent pre-operation.1.1.2Obtain the specimens of articular cartilage MPCs1.1.2.1Enzyme digestion and separation of the articular chondrocytesCut normal articular cartilage fragments prepared about1mm3, digestion by series ofenzymatic, separation of the articular cartilage cells into single cell suspension and trypanblue staining. Count the number of survived cells.1.1.2.2Quantitative analysis of articular cartilage MPCs 100μl cell suspension (containing1×106cells) were added to the sample tube and thecontrol tube, sample tube plus fluorescently labeled monoclonal antibody to CD105, CD166(1:100dilution), control tubes joined isotype control antibody,4°C incubated in the dark30minutes. Add PBS+1%FBs+0.1%NaN31ml500g centrifuged for5minutes, thesupernatant is aspirated carefully to terminate the reaction. Add PBS+1%FBs+0.1%NaN31ml, centrifuged for5minutes at500g. Washing the cells, carefully aspiratesupernatant to join PBS400μlr shake detected by flow cytometry. Take statistical analysis ofthe amount of MPCs.1.1.2.3Immunomagnetic bead separate articular cartilage MPCsAccording to our previous modified immunomagnetic bead separation method. Themagnetic coupling of mouse anti-human CD105monoclonal antibody and anti-mousesecondary antibody was incubated for20minutes at4℃after the antigen-antibodycomplex is washed3times, suspended in1ml of culture solution, adding an enzymedigestion and separation of articular cartilage cells, total particulate Cells ratio of50:1cells-particles complex gently oscillating at4°C for20min, cells-bead complexes weresuspended in chymopapain solution immunomagnetic bead separation after10minutes, andincubated for10-20minutes using a similar approach to destroy the antigen-antibodyreaction, and CD105-positive cells, at37°C,5%CO2incubator for3-5days of culture;immunomagnetic bead separation and then the mouse anti-human CD166monoclonalantibody binding CD105-positive cells, obtained CD105/CD166positive cells at37°C,5%CO2incubator culture, some cells were detected by flow cytometry CD105/CD166positivecell ratio.1.2The expression of Wnt signal in normal and OA articular cartilage MPCs.1.2.1Source of specimen: same as above.1.2.2Obtain the specimens of articular cartilage MPCs: same as above.1.2.3Expression of Wnt signal in the articular cartilage MPCs:1.2.3.1Immunity class of articular cartilage Wnt1, Wnt3a, Wnt5a, β-cateninRespectively take normal cartilage and primary OA articular cartilage,0.05%proteaseXIV of the pretreatment, fixed in10%formalin; dehydrated paraffin-embedded sectionsunderwent Wnt1, Wnt3a, Wnt5a, β-catenin staining, and thenimmunity class.1.2.3.2Expression of Wnt1, Wnt3a, Wnt5a, β-catenin in normal cartilage and OA articular cartilage MPCs1.2.3.2.1Real-time PCR detection of normal and primary OA articular cartilage MPCsin Wnt1, Wnt3a, Wnt5a, the amount of β-catenin expression.1.2.3.2.2Western blotting assay β-catenin expression in normal cartilage and primaryOA articular cartilage MPCs.MPCs cells were seeded into24-well plates, adding a final concentration of30mmol/L,LiCl, collected after overnight Cell Lysates, after centrifugation, the supernatant was boiled inLaemmli sample buffer for denaturing electrophoretic separation, transferred to amembrane.The first antibody is the antibody of anti-β-cateninprotein, secondary antibodies isthe IgG antibody which contain horseradish peroxidase in mouse. Take β-actin as control.1.2.4ActivatedWnt signaleffect on proliferation, chondrocyte differentiation of MPCs1.2.4.1ActivatedWnt signal conditioned medium preparation: preparation of twoproducts containing Wnt3a conditioned medium. Wnt3a conditioned medium with purifiedWnt3a (R&D systems) preparation (concentration100ng/ml), cultured in the packet is a1/10ECM gum8.1.2.4.2Activated Wnt signal effect on cell proliferationThe MPCs were divided into four groups: MPCs+common culture of MPCs+Wnt3aconditioned medium of MPCs+ordinary medium+Wnt signal blocking conditionedmedium of MPCs+Wnt3a conditioned medium+Wnt signal blocking conditionedmedium. Dubbed in a cell suspension of2.0×104cells/ml take each group of cells, eachhole shop100μl, laying two96-well plates. After4days of culture joined cartilage celldifferentiation-inducing agent (1mM sodium pyruvate,1x10-7M to DEX,1%ITS,10ng/ml rhTGF-β3and antibiotics), and cultured for1week, with the real-time PCRdetection MPCs type II collagen, aggrecan, SOX9mRNA expression levels. Make the ODvalue of the measured growth curve.1.2.4.3Activated Wnt signal effect on chondrocyte differentiation of MPCsCartilage cell differentiation-inducing agent in Wnt3a conditioned medium was added(1mmol/L sodium pyruvate,1×10-7mol/LDexamethasone,1%ITS,10ng/ml rhTGF-β3andantibiotics), and cultured for1week later, by real-time the PCR detection amount mRNAexpression of collagen Ⅱ,aggrecan and SOX9in MPCs.Take normal cartilage celldifferentiation-inducing medium as control. 1.2.5The effect of block Wnt signal on MPCs proliferation, chondrocyte differentiation1.2.5.1Wnt signal block condition medium preparation: ordinary medium and Wnt3aconditioned medium were added sFRP3(100ng/ml) of DKK-1(100ng/ml). MPCs werecultured in the above medium, the normal medium take as control.1.2.5.2Block Wnt signal cell proliferation, chondrocyte differentiationThe methods of MPCs blocking conditioned medium in normal and Wnt signal, cellproliferation, chondrocyte differentiation are the same as above.1.2.6Activated Wnt signal plus blocking effect on MPCs proliferation, chondrocytedifferentiation1.2.6.1ActivatedWnt signal conditioned medium preparation: preparation of twoproducts containing Wnt3a conditioned medium. Wnt3a conditioned medium with purifiedWnt3a (R&D systems) preparation (concentration100ng/ml), cultured in the packet is a1/10ECM gum8.1.2.6.2Wnt signal block condition medium preparation: ordinary medium and Wnt3aconditioned medium were added sFRP3(100ng/ml) of DKK-1(100ng/ml). MPCs werecultured in the above medium, the normal medium take as control.1.2.6.3The methods of MPCs activatedand block conditioned medium in normal andWnt signal, cell proliferation, chondrocyte differentiation are the same as above.2. The role of p53/p21pathway play in the activated of Wnt signal promote MPCsaging2.1The effect of activated Wnt signal on p53expression in MPCs2.1.1p53fluorescent reporter virus vector and transfected MPCspLVX-P53starts sub-EGFP-3FLAG CMV promoter is replaced p53promoter ofpLVX-EGFP-3FLAG carrier obtained (Mlu I and Xho I restriction site points), theexpression vector with EcoR and Xba I double enzyme cut, cut the EGFP-3FLAG fragmentreplaced the luciferase gene. Double digestion with Mlu and Xho I of the expression vector,the truncated CMV promoter fragment, approximately7.1kb vector fragment was recoveredafter electrophoresis of the digestion products. p53start sub homologous recombinationinto the expression vector was transformed into the recipient strain positive clones wereidentified. LUC start sub homologous recombination into the expression vector wastransformed into the recipient strain positive clones were identified; LUC plasmid positive clones were sequenced. Viral packagedvirus concentrated and purified.2.1.2Wnt signal is activated MPCs on p53expressionThe MPCs is divided into four groups: A: of MPCs+ordinary medium, B: of MPCs+ordinary medium+Wnt signal blocking conditioned medium C: of MPCs+Wnt3a D: ofMPCs+Wnt3a conditioned medium+Wnt signal blocking conditioned medium. P53primer sequence information is as follows:F: CCCAAGCAATGGATGATTTGAR: GGCATTCTGGGAGCTTCATCTThe fluorescence intensity of the fluorescence detector detects two culture conditionsMPCs, compare the expression of p53.After one week of culture, RT-PCR was used to detect the amount of mRNAexpression of the MPCs in type II collagen, aggrecan, SOX9’s. D (570) the measured valuesare plotted as a growth curve.2.2Wnt signalactivated interference lentiviral transfection of p53MPCs proliferation,differentiationInterfere with the lentiviral the MPCs transfected p53cultured cells, Wnt signalactivated conditioned medium configuration with before. Cell proliferation, to cartilagedifferentiation method is the same as above. Take normal medium take as control.Results1.The mRNA expression of Wnt1and Wnt3a abnormal patients is significant lowerthan OA.The mRNA expression of β-catenin in normal patients is no difference with OA.The protein of β-catenin is significant lower than OA. As the significant addition of Wnt1,Wnt3a and β-catenin protein, it implies that Wnt signal is active in OA.2.When this Wnt signal is active, the proliferative capacity of MPCs in articularcartilage is drop, vice versa. When put the blocking activated of Wnt signal, the MPCs’ issomewhat recovery. When this Wnt signal is active, the mount of CollagenII、aggrecan、SOX9is significant lower than Wnt normal express which induced by chondrocytedifferentiation in MPCs. When we block the Wnt signal, the mount of CollagenII、aggrecan、SOX9is no difference with normal Wnt express. When we plus the blocking ofactivated Wnt, the mount is recover. When this Wnt signal is active, the mount of β-cateninprotein is significant higher than normal Wnt express. When we block the Wnt signal, the mount of β-catenin protein is lower than normal. When we blocked the activated Wnt, themount of β-catenin protein is the same as the normal.3.When we block the Wnt signal, the mount of p53and protein express is significantlower than normal Wnt signal. When this Wnt signal is active, the mount of p53and proteinexpress is significant higher than normal. When we plus the blocking of activated Wnt, themount is recover, but the protein of p53is lower than the normal Wnt signal.4.When this Wnt signal is active, the express of p53start luciferase is significanthigher than normal Wnt signal. When we block the Wnt signal, the express of p53startluciferase is significant lower than normal Wnt signal. When we plus the blocking ofactivated Wnt, the mount is a little bit lower than the normal, but no difference.5.Normal medium and in the MPCs Wnt3a induction medium significantly acceleratedproliferation of p53RNAi interference, there is a significant difference. The MPCs innormal medium and Wnt3a induction medium enhanced p53RNAi interferencechondroinductive collagenⅡ, aggrecan andSOX9mRNA expression.Conclusion:1. OA articular cartilage in patients with MPCs Wnt1and Wnt3a and β-catenin proteinexpression increased significantly, indicating that its existence of activated Wnt signal.2. Wnt signal affect the proliferation of the MPCs in articular cartilage, the activatedWnt signal will decline the MPCs proliferation; also inhibit the cartilage differentiation ofMPCs. This suggests that activated of the Wnt signal will reduce the ability of repairdamage cartilage by articular cartilage MPCs.3. Articular cartilage MPCs P53gene and protein expression is association withWntsignal activated. When the Wnt signal is activated, the P53expression in MPCs ofarticular cartilage is increased, while its proliferation and ability to differentiate intochondrocytes decreased. However, when we block the expression of p53, activeWnt signalshows no effect on activated articular cartilage MPCs proliferation and cartilagedifferentiation. This suggests that the activatedWnt signalinducesthe aging of MPCs inarticular cartilage through the p53/p21pathway. Therefore, the Wnt signal or p53expression may have a role to regulatethe repair of articular cartilage injury.