Mechanical loading by fluid shear is sufficient to alter the cytoskeletal composition of osteoblastic cells

Many structural modifications have been observed as a part of the cellular response to mechanical loading in a variety of cell types. Although changes in morphology and cytoskeletal rearrangement have been widely reported, few studies have investigated the change in cytoskeletal composition. Measuring how the amounts of specific structural proteins in the cytoskeleton change in response to mechanical loading will help to elucidate cellular mechanisms of functional adaptation to the applied forces. Therefore, the overall hypothesis of this study was that osteoblasts would respond to fluid shear stress by altering the amount of specific cross-linking proteins in the composition of the cytoskeleton. Mouse osteoblats cell line MC3T3-E1 and human fetal osteoblasts (hFOB) were exposed to 2 Pa of steady fluid shear for 2 h in a parallel plate flow chamber, and then the amount of actin, vimentin, alpha-actinin, filamin, and talin in the cytoskeleton was measured using Western blot analyses. After mechanical loading, there was no change in the amount of actin monomers in the cytoskeleton, but the cross-linking proteins alpha-actinin and filamin that cofractionated with the cytoskeleton increased by 29% (P < 0.01) and 18% (P < 0.02), respectively. Localization of the cross-linking proteins by fluorescent microscopy revealed that they were more widely distributed throughout the cell after exposure to fluid shear. The amount of vimentin in the cytoskeleton also increased by 15% (P < 0.01). These results indicate that osteoblasts responded to mechanical loading by altering the cytoskeletal composition, which included an increase in specific proteins that would likely enhance the mechanical resistance of the cytoskeleton.