The Mechanisms Of Dual Specificity Phosphatase 5 In The Osteogenic Differentiation Of Bone Marrow Mesenchymal Stem Cells

Background:Bone marrow mesenchymal stem cells(BMMSCs)have the potential for multi-lineage commitment,and promoting osteogenic differentiation of BMMSCs is the key to bone regeneration and the treatment of bone metabolic diseases.Multiple signaling pathways are involved in the regulation of BMMSC osteogenesis,and protein phosphorylation plays an important role.Protein phosphorylation is a reversible dynamic process catalyzed by kinases and phosphatases.Dual specificity phosphatases(DUSPs)can dephosphorylate both tyrosine and serine/threonine residues,exhibiting essential role in a variety of physiological and pathological processes,including bone homeostasis.DUSP5,a member of DUSPs superfamily,has been reported to alleviate arthritis by inhibiting osteoclastogenesis.However,the role and regulatory mechanisms of DUSP5 in osteogenic differentiation of BMMSCs are still unclear.Objective:This study aims to illustrate the role and mechanisms of DUSP5 in osteogenic differentiation of BMMSCs and to explore the therapeutic effect of DUSP5 in osteoporosis.Materials and methods:(1)human BMMSCs(hBMMSCs)were cultured in proliferative medium(PM)and osteogenic medium(OM)for 7 days and 14 days,and alkaline phosphatase(ALP)and alizarin red S(ARS)staining and quantitative analysis proved good osteogenic potential of hBMMSCs.qRT-PCR and western blot were used to determine DUSP5 level.(2)DUSP5 knockdown,DUSP5-overexpressing and DUSP5 rescued hBMMSCs cell lines were constructed by lentivirus.After osteogenic induction,ALP and ARS staining and quantitative analysis were performed to detect the osteogenic differentiation capability.qRT-PCR and western blot were used to detect OSX and RUNX2 expressions.In addition,hBMMSCs mixed with ?-tricalcium phosphate(?-TCP)scaffold were implanted under the skin of the back of nude mice,and the mice were sacrificed after 8 weeks.H&E staining and immunohistochemistry for osteocalcin(OCN)were used to detect the ectopic bone formation in vivo.(3)Western blot was used to examine the expression levels of SMAD2/3 and SMAD1 signaling key indicators:p-SMAD2 and p-SMAD3;p-SMAD 1/5/9,p-SMAD1/5 and p-SMAD1 in the control and DUSP5 knockdown/overexpressing/rescued hBMMSCs.To explore whether the promotive effect of DUSP5 on hBMMSCs osteogenesis was mediated by SMAD1 signaling,we inhibited SMAD1 expression in DUSP5overexpressing hBMMSCs.After osteogenic induction,ALP and ARS staining and quantification,qRT-PCR and western blot were used to detect the osteogenesis capability of hBMMSCs.(4)Protein immunoprecipitation(Co-IP)was performed to confirm the interaction between DUSP5 and SMAD1 phosphatases,including PPM1A,PP1,PDP and SCP1/2.After then,GST-tagged DUSP5 protein was expressed in BL21 E.coli and purified.GST pull-down assay was conducted to further verify the direct combination between DUSP5 and SMAD1 phosphatases.The phosphorylated levels of SMAD1 in DUSP5 knockdown,overexpression and rescued hBMMSCs were tested by western blot.Next,SCP1/2 were suppressed in DUSP5 deficiency hBMMSCs and were upregulated in DUSP5 overexpression hBMMSCs,and protein expressions of p-SMAD 1/5/9,p-SMAD 1/5 and p-SMAD 1 were tested by western blot.(5)DUSP5 and SCP1 fragments plasmids were designed and Co-IP assay was conducted to determine the domains that are responsible for the interaction between DUSP5 and SCP1 fragments.(6)DUSP5 was knocked down or overexpressed in HEK293T,and the interaction between SCP1/2 and SMAD1 was detected by Co-IP.(7)Ovariectomized-induced(OVX)osteoporosis and SHAM mouse models were constructed.3 months later,the femurs were obtained for micro-CT scan,morphological analysis,H&E staining and imnunohistochemistry of OCN.Mouse bone mesenchymal stromal cells(mBMMSCs)were flushed and cultured,and the expression of DUSP5 was tested by qRT-PCR and western blot.In addition,3 months after the establishment of the osteoporosis model,Dusp5 overexpressing lentivirus(Dusp5)or the control lentivirus(vector)were injected through the tail vein.After 1 month,the bone mass of femur was examined by micro-CT,morphological analysis,H&E staining and immunohistochemistry of OCN.mBMMSCs of the SHAM,OVX,OVX+vector and OVX+Dusp5 groups were cultured in PM and OM,and the osteogenic differentiation potential was determined by ALP and ARS staining and qualification,qRT-PCR and western blot.Results:(1)DUSP5 was significantly induced during the process of hBMMSCs osteogenic differentiation.(2)DUSP5 promoted hBMMSCs osteogenic differentiation both in vitro and in vivo.DUSP5 knockdown inhibited the osteogenic differentiation of hBMMSCs both in vitro and in vivo,whereas DUSP5 overexpression exhibited the opposite effect.Moreover,the osteogenic potential of DUSP5 rescued hBMMSCs was obviously restored.(3)DUSP5 promoted osteoblastic commitment of hBMMSCs by activating SMAD1 signaling.DUSP5 did not influence the phosphorylation of SMAD2/3 signaling,whereas the phosphorylation levels of key indicators of SMAD1 pathway,including p-SMAD 1/5/9,pSMAD1/5 and p-SMAD1 were positively correlated with DUSP5 expression.SMAD1 deficiency eliminated the promotive effect of DUSP5 on hBMMSCs osteogenesis.(4)DUSP5 activated SMAD1 signaling in a SCP1/2-dependent manner.There was no interaction between DUSP5 and PPM1A,PP1 or PDP,but DUSP5 interacted with SCP1/2.Knocking down SCP1/2 in DUSP5 deficiency hBMMSCs and upregulating SCP1/2 in DUSP5-overexpressing hBMMSCs both reversed the phosphorylation levels of SMAD1.(5)The linker region of DUSP5 interacted with the phosphatase domain of SCP1/2.Co-IP assay confirmed that DUSP5 segments containing the linker region can be combined with SCP1/2,and the SCP1/2 fragments containing the phosphatase domain can be immunoprecipitated by DUSP5.(6)DUSP5 inhibited the interaction of SCP1/2 and SAMD1.The interaction between SCP1/2 and SMAD1 was enhanced by knocking down of DUSP5,while DUSP5 overexpression inhibited the interaction between SCP1/2 and SMAD1.(7)Dusp5 overexpression efficiently alleviated osteoporosis.The bone mass of the OVX mice femur was significantly lower than that of the SHAM group,and the expression of DUSP5 in the OVX group was significantly decreased.Importantly,osteoporosis of the OVX mice was significantly improved after the injection with Dusp5 lentivirus,and the corresponding mBMMSCs possessed better osteogenic capability than the OVX group.Conclusion:This study uncovers a new role of DUSP5 to promote osteogenic differentiation of BMMSCs.Mechanically,the linker region of DUSP5 interacts with the phosphatase domain of SCP1/2 and inhibits the dephosphorylation effect of SCP1/2 on SMAD1,thereby activating SMAD1 pathway and promoting the osteogenic differentiation of BMMSCs.Dusp5 overexpression effectively alleviates bone loss of osteoporosis.Collectively,this study provides theoretical basis for bone regeneration and the treatment of bone metabolic diseases based on BMMSCs by small compounds targeting the linker region of DUSP5.