The Involvement Of Clusterin In Fracture Healing And Osteogenic Differentiation

Objective:Nonunion means delayed fracture repair.This study aims to investigate the roles of Clusterin(CLU)in fracture healing.The CLU levels in serum and callus tissues from nonunion patients were upregulated,compared with that from control cases with normal healing after fracture.Experimental nonunion was induced by a transverse osteotomy of the femoral shaft combined with periosteum removing in rats.Silencing of CLU by orthotopic injection of lentivirus facilitated fracture healing and promoted expression of osteogenic markers.The application of exogenous recombinant CLU protein inhibited osteogenic differentiation of BMSCs.In addition,CLU deactivated Wnt/β-catenin signaling,and the effects of CLU were antagonized by an inhibitor of Wnt/β-catenin signaling,dickkopf-1(DKK1).In conclusion,we demonstrate that CLU knockdown accelerated fracture healing of rats and osteogenic differentiation of BMSCs by downregulating DKK1 expression and activating Wnt/β-catenin signaling.These findings may provide references for treatment of fracture and nonunion in clinic.Methods: Change of CLU expression level in patients with bone nonunion verified at clinical level:Serum samples were collected from 30 patients with nonunion and 12 control patients with normal post-fracture union(for serum isolation and cryopreserved at-80°after preoperative routine blood drawing before internal fixation surgery or re-bone grafting surgery).Callus tissue of 6 nonunion patients and 15 control patients(excess callus of broken end removed during internal fixation after fracture union and ununion callus and sclerotic bone of broken end in nonunion patients during re-bone grafting).After RNA extraction,the expression level of CLU m RNA in callus tissues was detected by Real-time PCR.After protein extraction,the expression level of CLU protein in callus tissue was detected by Western blot.All patients with nonunion and normal fracture union were provided with diagnostic radiographs and CT films with detailed patient information and clinical features.CLU targeting was used to regulate fractureunion in rats at animal level.First,a nonunion model of femur was constructed.Healthy male SD rats at 6-8 weeks of age underwent bone truncation operation in the middle part of femur.Kirschner wire was inserted into bone marrow from the broken end of fracture for loose fixation,and periosteum around the broken end was removed as far as possible.The control group underwent fracture surgery without stripping the periosteum.CLU interfering RNA was synthesized,packed into lentivirus,and injected at multiple points around the fracture site after surgery.At 2,4,6,and 8 weeks after operation,X-ray photographs were taken to observe the fracture healing.The rats were sacrificed 8 weeks after operation,and serum and femur tissue from the fracture site were collected.The proteins in bone tissue were extracted and the expression levels of alkaline phosphatase(ALP),osteocalcin(OCN),osterix(OSX),and Colligation-I were detected.Protein was extracted from bone tissue and CLU,dickkopf1(DKK1)and nuclear β-catenin levels were detected.RNA was extracted from bone tissue,and the expression levels of c-MYC and Axin2 were detected by real-time PCR.The expression of CLU in bone tissue was determined by immunofluorescence.CLU interfering RNA and its negative control were synthesized,packed with lentivirus,and injected around the fracture site one week after the bone amputation operation.After operation,the fracture of rats were taken X-ray photographs to observe the healing of fracture.The rats were sacrificed 8 weeks after operation,and serum and femur tissue from the fracture site were collected.The effect of CLU on Wnt/β-catenin pathway in bone marrow mesenchymal stem cells(BMSCS)was validated by flow cytometry in healthy 4-6-week-old male SD rats,and bone marrow mesenchymal stem cells(BMSC)were isolated.CD44,CD90,CD31 and CD34 were detected by flow cytometry.BMSC was cultured in osteogenic differentiation medium for 7 days to induce osteogenic differentiation(OD),and then treated with recombinant rat CLU protein(5μg/ml).Cell proteins were extracted and the levels of DKK1 and nuclear β-catenin were detected by Western blot.Cell RNA was extracted,and the expression levels of c-MYC and Axin2 were detected by real-time PCR.To explore the effect of CLU on osteogenic differentiation of bone marrow mesenchymal stem cells and the mechanism of synthesis of DKK1 interference sequence,packaging lentivirus,infection of BMSC,3 days after osteogenic differentiation induction for 7 days,at the same time adding recombinant rat CLU protein treatment(5μg/ml),detection: alizarin red staining detection of cell mineralization,and quantitative analysis.Cell ALP activity was detected by the kit.Protein was extracted from cells,and the expression levels of OCN,OSX and collagen-I were detected by Western blot.Cell proteins were extracted and the levels of DKK1 and nuclear β-catenin were detected by Western blot.Cell RNA was extracted,and the expression levels of c-MYC and Axin2 were detected by real-time PCR.DKK1 overexpressed sequence was synthesized,packaged into lentivirus,infected with BMSC,and induced by osteogenic differentiation3 days later for 7 days.The following tests were performed: CLU content in supernatant was verified by ELISA.The protein was extracted and the CLU expression was verified by Western blot.Results:CLU was higly expressed in nonunion patients.We collected the serum and callus tissue specimens of nonunion patients and control cases with normal healing after fracture.As shown in the representative X-ray images,the fractures of controls had completely healed,only slight scars were observed.In nonunion patients,no bony connection was found between the fracture ends.The ELISA results showed that the level of CLU was higher in serum from nonunion patients than that of control cases.Then real-time PCR and western blot results revealed that CLU was upregulated in callus tissues of nonunion patients at transcription and translation levels,compared with that of healthy control.Silencing of CLU promoted fracture healing of rats,nonunion was induced by a transverse osteotomy of the femoral shaft and periosteum removing in rats.As shown in X-ray images,the fracture ends of femur were connected by bony bridge in control rats after fracture for eight weeks,as indicated by red arrow.However,obvious fracture gap existed in nonunion rats until the eighth week post surgery.In the CLU-silenced group,fracture healing was significant at the sixth and eighth week post surgery,similar to the normal control rats.Moreover,bone callus was observed in the fracture site of CLU knockdown rats at the eighth week,as indicated by red arrow.Several osteogenesisrelated molecules were detected in femur of the fracture site.As displayed by western blot results,the expression levels of alkaline phosphatase(ALP),osteocalcin(OCN),osterix(OSX)and collagen-I in the nonunion rats were reduced significantly,suggested osteoblast loss and osteopenia,which were significantly restrained by silencing of CLU.The results in this section suggested that silencing of CLU promoted fracture healing and bone deposition in rats.Knockdown of CLU reduced DKK1 expression and activated Wnt/β-catenin signaling.From immunofluorescence staining and immunoblotting results,we observed that the expression of CLU in femur of control rats was quite low,and it was increased in nonunion rats.The nonunion rats showed elevated DKK1 expression,reduced nuclear level of β-catenin,and inhibited transcription of c-myc and axin2(downstream genes of Wnt/β-catenin signaling),suggesting inactivation of Wnt/β-catenin signaling.The knockdown effectiveness of lentivirus containing CLU short hair sequence was verified by immunofluorescence staining and western blot.The inhibited Wnt/β-catenin signaling was activated by silencing of CLU,evidenced by decrease of DKK1,enhanced nuclear level of β-catenin,and promoted transcription of c-myc and axin2.CLU inhibited osteogenic differentiation in BMSCs.In order to further investigate the roles of CLU in bone development,BMSCs were isolated from femur of rats,and identified by flow cytometry.As exhibited in Fig.4A,BMSCs were identified CD44+,CD90+,CD31-and CD34-.Subsequently,BMSCs were induced osteogenic differentiation for 7 days.Western blot and real-time PCR results revealed that Wnt/β-catenin signaling was activiated after osteogenic differentiation induction in BMSCs,evidenced by reduced DKK1 level,raised nuclear β-catenin level,and accelerated expression of C-MYC and Axin2.Recombinant CLU protein was applied to treat BMSCs suffering from osteogenic differentiation induction.After CLU administration,osteogenic differentiation-induced activation of Wnt/β-catenin signaling was inhibited.Becaused CLU upregulated expression of DKK1,which was a well known inhibitor of Wnt/β-catenin signaling,we hypothesized that CLU may deactivate Wnt/β-catenin signaling by regulating DKK1 expression.In order to confirm this hypothesis,the lentivirus containing interference sequence against DKK1 was used.As exhibited in Fig.5A,osteogeneis differentiation-induced mineralization in BMSCs was attenuated by exogenous CLU addition.However,the effect of CLU on mineralization was antagonized by DKK1 knockdown.Moreover,CLU administration blocked osteogenic differentiation in BMSCs,evidenced by reduced ALP activity and decreased expression of OCN,OSX and collagen-I.The inhibitory effect of CLU was also neutralized by silencing of DKK1.Then Wnt/β-catenin signaling was determined.The data revealed that CLU-induced inactivation of Wnt/β-catenin signaling was recovered by DKK1 knockdown to some degree,evidenced by alterations of β-catenin,C-MYC and Axin2 levels.These results reveled that CLU inhibited osteogenic differentiation by upregulating DKK1 and deactivating Wnt/β-catenin signaling.DKK1 enhanced expression of CLU in BMSCs.Considering that CLU expression was regulated by Wnt/β-catenin signaling,25 we next explored whether CLU was controlled by DKK1.DKK1 was enhanced expressed in BMSCs with osteogenic differentiation,and the overexpression effectiveness was verified by western blot.The immunoblotting results showed that the protein level of CLU in BMSCs was not influenced by DKK1.However,the CLU content in medium supernatant was significantly increased after DKK1 ectopic expression,suggesting that DKK1 promoted the secretion of CLU in BMSCs with osteogenic differentiation.Conclusions:In conclusion,we found that CLU was high expressed in fracture site of nonunion rats,compared with that of normal healing rats.Silencing of CLU by injection of lentivirus promoted fracture repair and bone mass deposition in rats.In BMSCs,CLU administration delayed osteogenic differentiation and mineralization.Moreover,CLU application deactivated Wnt/β-catenin signaling and facilitated the expression of DKK1,an inhibitor of Wnt/β-catenin signaling.The effects of CLU were antagonized by knockdown of DKK1.Additionally,DKK1 overexpression enhanced secretion of CLU protein.These results demonstrated that CLU blocked fracture healing and osteogenic differentiation by upregulating DKK1 and deactivating Wnt/β-catenin signaling pathway.These finding may provide novel potential therapeutically targets for fracture treatment.