NFATc1-ERK5 Pathway Mediates FSS-promoted Osteoblasts Proliferation And The Expression Of BMP7

ObjectiveExtracellular signal-regulated kinase(ERK)5 is a critical cytokine to regulate osteoblast proliferation,but the upstream and downstream of ERK5 are less studied.In this work,the relation between ERK5 and upstream cytokine NFATc1(Nuclear factor of activated T-cells c1)were found,and the way they promotes osteoblasts proliferation.MethodsMC3T3-E1 cells were treated with 12dyn/cm2 FSS(fluid shear stress),5u M XMD8-92(specific ERK5 inhibitor),Cs A(NFATc1 inhibitor)or 20ng/ml EGF(epidermal growth factor).Western blot was used to detect the expression level of NFATc1,ERK5,p-ERK5,E2F2,cyclin E1.And the sub-nuclear location of NFATc1 and p-ERK5 was showed by immunofluorescence.Cells viability was examined by MTT assay.Moreover,PCR array was used to examine m RNA levels of proteins under different intervention.Results12dyn/cm2 FSS can phosphorylate ERK5 to form activated ERK5 which is p-ERK5.At the same time,the expression of NFATc1 was increased.After Cs A and XMD8-92 were applied respectively,Cs A can inhibit both the expression of NFATc1 and the phosphorylation of ERK5,but XMD8-92 only block the phosphorylation of ERK5.Furthermore,NFATc1 and p-ERK5 can be trans-located into the nucleus under the stimulation of FSS,and Cs A suppressed the nuclear translocation of NFATc1 and p-ERK5,but XMD8-92 only inhibited nuclear redistribution of p-ERK5.Obviously,NFATc1 phosphorylated ERK5 and p-ERK5 was trans-located into the nucleus.According to examine proliferation-related cytokines,the expression of E2F2 and cyclin E1 were increased,Cs A and XMD8-92 can inhibit this positive function and cell proliferation through MTT assay.Moreover,when the expression of NFATc1 and phosphorylation of ERK5 were blocked by Cs A and XMD8-92 respectively,the expression level of BMP7 was decreased sharply.ConclusionNFATc1 mediates ERK5 activation by FSS,NFATc1 is an upstream of ERK5,and ERK5 phosphorylation by FSS promotes the nuclear translocation of p-ERK5.Moreover,NFATc1-ERK5 pathway promotes osteoblasts proliferation through E2F2/cyclin E1.And NFATc1-ERK5 signaling pathway mediates the increased expression of BMP7 by FSS.