Fluid shear stress promotes osteoblast proliferation via the Gαq-ERK5 signaling pathway

Fluid shear stress (FSS) is a ubiquitous mechanical stimulus that potently promotes osteoblast proliferation. Previously, we reported that extracellular signal-regulated kinase 5 (ERK5) is essential for FSS-induced osteoblast proliferation. However, the precise mechanism by which FSS promotes osteoblast proliferation via ERK5 activation is poorly understood. The aim of this study was to determine the critical role of G alpha q in FSS-induced ERK5 phosphorylation and osteoblast proliferation, as well as the downstream targets of the G alpha q-ERK5 pathway. MC3T3-E1 cells were transfected with 50 nM G alpha q siRNA, treated with 5 mM XMD8-92 (a highly selective inhibitor of ERK5 activity), and/or exposed to FSS (12 dyn/cm(2)). Cell proliferation was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The protein expression levels of G alpha q, P-ERK5, ERK5, Cyclin B1, and CDK1 were analyzed by Western blot. Physiological FSS exposure for 60 min remarkably promoted MC3T3-E1 cell proliferation, however, this effect was suppressed by siRNA-mediated G alpha q knockdown or inhibition of ERK5 activity by XMD8-92 treatment, suggesting that G alpha q and ERK5 might modulate FSS-increased osteoblast proliferation. Furthermore, ERK5 phosphorylation was dramatically inhibited by G alpha q siRNA. In addition, our study further revealed that FSS treatment of MC3T3-E1 cells for 60 min markedly upregulated the protein expression levels of Cyclin B1 and CDK1, and this increased expression was predominantly blocked by G alpha q siRNA or XMD8-92 treatment. We propose that FSS acts on the G alpha q-ERK5 signaling pathway to upregulate Cyclin B1 and CDK1 expression, thereby resulting in MC3T3-E1 cell proliferation. Thus, the G alpha q-ERK5 signaling pathway may provide useful information regarding the treatment of bone metabolic disease.