Fluid shear stress promotes osteoblast proliferation through the NFATc1-ERK5 pathway

Purpose: Extracellular-regulated kinase 5 (ERK5) is thought to regulate osteoblast proliferation. To further understand how ERK5 signaling regulates osteoblast proliferation induced by fluid shear stress (FSS), we examined some potential signaling targets associated with ERK5 in MC3T3-E1 cells. Methods: MC3T3-E1 cells were treated with XMD8-92 (an ERK5 inhibitor) or Cyclosporin A (CsA, a nuclear factor of activated T cells (NFAT) c1 inhibitor) and/or exposed to 12 dyn/cm(2) FSS. Phosphorylated-ERK5 (p-ERK5) and expression levels of NFATc1, ERK5, E2F2, and cyclin E1 were analyzed by western blot. The mRNA levels of genes associated with cell proliferation were analyzed by Polymerase Chain Reaction (PCR) array. Subcellular localization of p-ERK5 and NFATc1 were determined by immunofluorescence. Cell proliferation was evaluated by MTT assay. Results: NFATc1 expression was up-regulated by FSS. XMD8-92 only blocked ERK5 activation; however, CsA decreased NFATc1 and p-ERK5 levels, including after FSS stimulation. Exposure to NFATc1 inhibitor or ERK5 inhibitor resulted in decreased E2F2 and cyclin E1 expression and proliferation by proliferative MC3T3-E1 cells. Furthermore, immunofluorescence results illustrated that NFATc1 induced ERK5 phosphorylation, resulting in p-ERK5 translocation to the nucleus. Conclusions: Our results reveal that NFATc1 acts as an intermediate to promote the phosphorylation of ERK5 induced by FSS. Moreover, activated NFATc1-ERK5 signaling up-regulates the expression of E2F2 and cyclin E1, which promote osteoblast proliferation.