Elucidation Of The Roles Of ERK5-KLF4 Axis And BMP9-Leptin Crosstalk In Regulating Osteoblast Proliferation And Osteogenic Differentiation

Objective: For the research of osteogenic lineage cell proliferation,fluid shear stress(FSS)plays a critical role in regulating osteoblast proliferation through extracellular signal-regulated kinase 5(ERK5).Kruppel-like factor 4(KLF4)knockout robustly enhances bone formation due to increased osteoblast differentiation and mineralization.However,the effect of KLF4 on osteoblast proliferation is still unclear.For the research of osteogenic differentiation in stem cells,bone morphogenetic protein 9(BMP9)has been shown to play an important role in regulating osteogenic differentiation and adipogenic differentiation in stem cells.However,the specific mechanism remains to be elucidated.It is unclear concerning the mechanisms underlying BMP9-regulated balance between osteogenic differentiation and adipogenic differentiation in stem cells.Leptin is secreted from adipocyte tissue and plays an important role in regulating energy metabolism.However,it is unclear whether Leptin is involved in BMP9-induced osteogenic differentiation and adipogenic differentiation.Therefore,the purpose of this study was to investigate the effect of KLF4 on osteogenic lineage cell proliferation and the relationship between KLF4 and ERK5;the effect of leptin on BMP9-induced osteogenic differentiation and adipogenic differentiation in stem cells.Methods: For the research of osteogenic lineage cell proliferation,the MC3T3-E1 cells were treated with FSS and/or KLF4 siRNA or ERK5 siRNA,cell viability was accessed by Edu labeling and CCK-8 assay,proliferative genes were assessed by PCR array.Bone marrow stromal cells(BMSCs)were infected with adenovirus expressing KLF4 and/or constitutively active mitogen-activated protein kinase kinase 5(MEK5),cell viability was evaluated by crystal violet staining,colony formation assay,and cell WST1 assay.The levels of KLF4 and ERK5 phosphorylation were identified by qRT-PCR and western blot.For the research of osteogenic differentiation in stem cells,BMSCs and immortalized mouse embryonic fibroblasts(iMEFs)were infected with recombinant adenoviruses expressing BMP9,leptin,or silenced leptin,respectively.ALP staining,ALP activity reading,Alizarin Red S staining,Oil Red O staining,and qRT-PCR assays were performed to analyze ALP activity,calcium deposition,lipid droplet formation and mRNA expression in stem cells.BMSCs and iMEFs were infected with recombinant adenoviruses expressing BMP9,leptin,or silencing leptin respectively,and the cells were collected.The collected cells were injected subcutaneously into the flanks of athymic nude mice,and the mass was extracted after 4 weeks.The micro-CT,H&E staining,and Masson's Trichrome staining were used to detect the mass volume and bone mass density.The JAK1/2 inhibitor ruxolitinib was used to treat BMSCs or iMEFs infected with adenoviruses expressing BMP9,leptin,or silenced leptin.The ALP activity was detected,and the expression of osteogenic and adipogenic genes were detected by qRT-PCR.Results: For the research of osteogenic lineage cell proliferation,KLF4 expression was significantly down-regulated under FSS exposure,however,this was reversed by ERK5 siRNA.KLF4 overexpression inhibited colony formation efficiency and cell viability in BMSCs.Adenoviruses expressing constitutively active MEK5 increased ERK5 phosphorylation,which inhibited KLF4 expression,and promoted cell proliferation in BMSCs.FSS-induced osteoblastic proliferation also involved increased Cyclin B2 and Cdc14 b expression as well as decreased P27 expression.For the research of osteogenic differentiation in stem cells,BMSCs and iMEFs infected with adenovirus expressing BMP9 significantly increased ALP activity,promoted calcium deposition,enhanced lipid droplet formation,increased ectopic bone formation volume and bone mass density,while overexpression of leptin dramatically increased BMP9-induced ALP activity,calcium deposition,lipid droplet formation,ectopic bone formation volume and bone mass density,however,BMP9-promoted lipid droplet formation was inhibited by overexpression of leptin.BMSCs and iMEFs infected with adenovirus expressing silenced leptin showed no significant effect on BMP9-induced ALP activity,calcium deposition,lipid droplet formation,ectopic bone formation volume and bone mass density.BMP9 overexpression in iMEFs promoted the expression of leptin and leptin receptors,while leptin overexpression upregulated the expression of BMP9 in iMEFs.BMP9 overexpression significantly increased ALP activity in iMEFs,however,the effect was reversed by 10?M ruxolitinib.Leptin overexpression significantly increased BMP9-induced ALP activity,and the effect was also reversed by 10?M ruxolitinib.After treatment with 10?M ruxolitinib,BMP9-induced expression of IL6 and OPG were inhibited,and BMP9-downregulated expression of Col10a1 was elevated.Leptin overexpression significantly increased BMP9-induced IL6 and OPG expression,and decreased BMP9-induced Col10a1,PPAR?2,and LPL expression,however,these effects were reversed by 10?M ruxolitinib treatment.Conclusions: ERK5 negatively regulates KLF4 expression and promotes osteogenic lineage cell proliferation in response to FSS or MEK5 overexpression.Leptin synergizes with BMP9-induced osteogenic differentiation,inhibits BMP9-induced adipogenic differentiation in stem cells.Moreover,leptin increases BMP9-induced IL6 and OPG expression,and decreases BMP9-induced Col10a1,PPAR?2 and LPL expression through JAK1/2 signaling pathway.