Fluid Stress Uses Multiple Mechanisms To Enhance The Cell Cycle Progression Of Osteoblastic Cells From G1 To S Phase

OBJECTIVE:To inquire into the cellular mechanism of growth promotion due to shear stress by studying G1-phase events responsible for the suppression of cell transition from the G1 to S phase of the cell cycle,we made an attempt at establishing the most suitable physiological stress to stimulate bone formation.METHODS:The osteoblasts derived from Kunming murine's calvaria were exposed to FSS(12dyn/cm~2 ) for 0,0.25,0.5,1,2,4h,respectively,in the flow chamber in vitro to study its impact on cell proliferation,differentiation and the effection of cell cycle's G1/S checkpoint.The cell proliferation was studied by MTT assay.The cell differentiation was assessed through alkaline phosphatase(ALP) activity assay. Flow-cytometry,Immunofluorescence and RT-PCR techniques were used to evaluate the proportion of S phase in cell cycle,the activity of CDK2,CDK4 and the expression of E2F-1,p27,SIVA-lmRNA,which exposed to demonstrate how FSS underlying multiple mechanisms to enhance the Cell Cycle Progression from G1 to S Phase.RESULTS:FSS increased the proliferation and advanced the time in cell growth curve,but after 1h,2h,4h,the proliferation was inhibited.The FSS also increased the ALP activity,which were significantly stimulated when shear stress was applied after 0.25h and 0.5h,respectively(128%and 158%of control);but the FSS for 1,2,4h decreased ALP activity.The proportion of S phase in cell cycle raised within the early period.The S phase rate increased significantly after 0.5h(P＜0.05),compared with the control.And then the proportion of S phase in cell cycle began to decrease following 0.5h timepoint,and when the time last for 4hs,the rate of S phase decreased significantly(P＜0.05).Shear stress enhanced the activity of cyclin-dependent kinase (cdk) 2 and cdk4,which phosphorylate pRb,and the amount of E2F1 associated with cdk2 also increased.The level of cdk inhibitor p27 and apoptosis-inducing factor SIVA-1,decreased as a result of shear stress.Shear stress markedly elevated the mRNA level ofE2F1 after 0.25h and 0.5h(P＜0.05),this elevation in mRNA faded after 1h.CONCLUSION:These results suggest that the suitable FSS stimulated the transition from the G1 to S phase through the upregulation of CDK2,CDK4 in a way which is similar to the effects of E2F1,except for P27,SIVA-1.This alteration are involved in key biological processes relevant to cell proliferation and differentiation, and such regulation exhibited a pattern of dependence on the time level of shear stress.