Study Of Molecular Mechanisms Of Osteoblastic Cell Proliferation Induced By Fluid Shear Stress

Objective:The incidence of osteoporosis increased along with the grievous aged population in China, and osteoporosis has become a remarkable public health issue. In the treatment of osteoporosis, proper exercise is essential, in addition to rational drug use. However, the mechanism of mechanotranduction of bone cells has not been fully understood, which lead to difficulties for clinicians guiding patients exercise to promote their recovery. This study use self-made fluid shear stress loading system to investigate role of ERK5in mechanotransduction of MC3T3-E1pre-osteoblastic cells in vitro. The results would be useful for the development of novel therapeutic agents for osteoporosis and bone fracture.Methods:After loaded with fluid shear stress, osteoblastic cells were subjected to Westernblot and Immunofluorescence experiment to investigate the mechanism of ERK5phosphorylation induced by fluid shear stress. Then, we use MEK5inhibitors, BIX02188and BIX02189, gifts form Boehringer Ingelheim, to block EKR5phosphorylation, the mechanism of proliferation of osteoblastic cells under cyclic fluid shear stress was investigated by MTT, Westernblot and Immunofluorescence.Results:MC3T3-E1cells were subjected to12dyn/cm2fluid laminar flow for different time periods, the results showed that ERK5was significantly phosphorylated in MC3T3-E1pre-osteoblastic cells under FSS. Serum-starved cells unloaded with FSS only exhibited a faint ERK5phosphorylation, but phosphorylation of ERK5was significantly upregulated after15min and reached peak at45min. Similar results were obtained in MG-63osteoblast-like cell line. We also subjected MC3T3-E1osteoblastic cells to FSS at different intensities (6,12and18dyn/cm2) for30,45and60min, respectively. The results showed that FSS caused ERK5activation in an intensity-and time-dependent manner, with12dyn/cm2FSS as the strongest stimuli for ERK5in these groups. Before loaded with FSS, actin filaments were poorly reorganized, after subjection to FSS at12dyn/cm2, actin filaments rapidly (within5min) reorganized into thick parallel bundles of stress fiber in the cells, the percentage of stress fiber positive cells was increased with the loading time and reached a maximum by around90min, when stress fiber positive cells were nearly four folds more than that in unloaded control. But after90min, more cells detached from the glass coverslips without morphological change and cytoskeleton of cells began to disrupt, which lead to stress fiber positive cells decreased to69.02±4.71%when loaded for120min. Subjecting1μM cytochalasin D before or during fluid loading lead to downregulation of cytoskeletal reorganization and ERK5phosphorylation, while, incubating cells with0.1μM nocodazole only downregulated ERK5activation, but had no effect on cytoskeleton reorganization. Further more, pre-treating cells with both cytochalasin D and nocodazole before and during loading nearly blocked ERK5phosphorylation. Activation of FAK was also investigated in this experiment and the results revealed that cytoskeleton disruption downregulated FAK phosphorylation, which was similar to ERK5. After loaded with fluid shear stress, phosphorylated ERK5translocated into nucleus, while, cytoskeletal disruption had no effect on phosphorylated ERK5translocation. Both BIX02188and BIX02189inhibited ERK5phosphorylation in a time-dependent manner, and reached peak at2h incubation, after2h, phosphorylation of ERK5was nearly completely blocked. After2h incubation with BIX02188or BIX02189, cells were cultured in DMEM/F12without serum for different time periods, then cells were activated with200μM H2O2for5min, we found only faint ERK5phosphorylation was detected after4h culture, which suggested that the inhibition of both inhibitors lasted for at least4h. Results from MTT test revealed that cyclic fluid shear stress significantly promoted osteoblastic cells proliferation, inhibition of ERK5phosphorylation lead to suppression of cellular proliferation, similar results were obtained from BrdU incorporation. Cyclic fluid shear stress promoted osteoblastic cells proliferation via ERK5activation, and upregulated its downstream AP-1and cyclin D1expression, but inhibited p-16expression, while inhibition of ERK5phosphorylation reversed this effect. Compared with continuous fluid shear stress, cyclic fluid shear stress was superior in promoting ERK5phosphorylation and cyclin D1expression.Conclusion:Fluid shear stress activates ERK5, in which, cytoskeletal reorganization is essential, cytoskeletal reorganization dependent FAK phosphorylation induced by fluid shear stress may be involved in this mechanism. Disruption of cytoskeletal reorganization downregualtes ERK5activation, but has no effect on ERK5translocation. Cytoskeleton in cells could quickly reorganize into stress fibers in response to fluid shear stress, while, long time mechanical stimulation, even at physiological level, lead to cytoskeleton disruption. Cyclic fluid shear stress promotes ERK5activation, which mediates AP-1and cyclin D1expression and suppresses p-16expression, to promote osteoblastic cells proliferation. Compared with continuous fluid shear stress, cyclic fluid shear stress is superior in promoting cellular proliferation. Both BIX02188and BIX02189significantly inhibit ERK5phosphorylation and this inhibition last for at least4h. Collectively, ERK5is a critical target molecule in proliferation of osteoblastic cells induced by fluid shear stress, further study on its role in mechanotransduction of osteoblasts would be helpful for treatment of osteoporosis.