Mechanical Strain Regulates Osteoblast Proliferation Through Ca2+-CaMK-CREB Signal Pathway

ObjectiveTo investigate the effects of mechanical strain on Ca2+-calmodulin dependent kinase (CaMK)-cAMP response element binding protein (CREB) signal pathway and proliferation of osteoblasts.MethodsUsing a four-point bending device, MC3T3-E1 cells were exposed to mechanical tensile strains of 2500 μs and 5000 μs at 0.5 Hz respectively. The intracellular free Ca2+ ([Ca2+]i) concentration and calmodulin activity were assayed by fluorospectrophotometry, CaMK II β, CREB, and phosphorylated (activated) CREB (p-CREB) were assessed by Western blot, and cells proliferation was assayed with MTT. Pretreatment with verapamil was carried out to block Ca2+ channel, and inhibitor U73122 was used to inhibit phospholipase C (PLC).ResultsMechanical strains of 2500 μs and 5000 μs for 1 to 10 minutes both increased [Ca2+]i level of the cells. The 2500 μs strain, a periodicity of 1 h/d for 3 days, activated calmodulin, elevated protein levels of CaMK II β and p-CREB, and promoted cells proliferation, which were attenuated by pretreatment of verapamil or U73122. The effects of 5000 μs strain on calmodulin, CaMK II β, p-CREB and proliferation were contrary to 2500 μs strain.ConclusionThe mechanical strain regulates osteoblasts proliferation through Ca2+-CaMK-CREB signal pathway via Ca2+ channel and PLC/IP3 transduction cascades.