OSTEOBLAST CYTOSKELETAL MODULATION IN RESPONSE TO MECHANICAL STRAIN IN-VITRO

The structural integrity of microfilaments has been shown to be necess ary for the signal transduction of mechanical stimuli within osteoblas ts. Qualitative and quantitative changes within the cytoskeleton of os teoblasts may therefore be crucial components of the signal transducti on processes of these cells in response to mechanical stimulation. Avi an osteoblasts were strained with a device that deforms a flexible, ce ll-laden membrane at a defined frequency and intensity in a uniform bi axial manner. We examined the effects of mechanical strain on the accu mulation of protein and the expression of the major cytoskeletal eleme nts and specific integrin-binding (arginine-glycine-aspartic acid) pro teins of these cells. Mechanical strain increased the level of total e xtracellular matrix-accumulated fibronectin by approximately 150% and decreased that of osteopontin by approximately 60% but had no quantifi able effect on the accumulation of pi integrin subunit or collagen typ e I. An examination of the major elements of the cytoskeleton demonstr ated that neither the level of actin nor that of the intermediate fila ment protein vimentin changed; however, the amount of tubulin decrease d by approximately 75% and the amount of vinculin, a major protein of focal adhesion complexes, increased by approximately 250%. An analysis of protein synthesis by two-dimensional gel electrophoresis of [S-35] methionine-labeled cytoskeletal proteins demonstrated that the changes in the accumulation of vinculin and tubulin resulted from their alter ed synthesis. Messenger RNA analysis confirmed that the changes in acc umulation and protein synthesis observed for vinculin, fibronectin, an d osteopontin were controlled at a pretranslational level. Immunofluor escent microscopy demonstrated that mechanical strain led to increased formation and thickening of actin stress fibers, with a commensurate dissociation in microtubules and a clear increase in levels of vinculi n at the peripheral edges of the cells. In conclusion, the elevated ra te of synthesis and the increased accumulation of vinculin and fibrone ctin, as well as the increase in the number and size of stress fibers and focal adhesion complexes, suggest that mechanical strain leads to a coordinated change both in the cytoskeleton and in extracellular mat rix proteins that will facilitate tighter adhesion of an osteoblast to its extracellular matrix.