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less stiff substrates [56]. It is this likely that the increased focal adhesions will affect the differentiation of the osteoblasts, but to what extend is it unclear. We have used paxillin as a measure for focal adhesion size. The cytoplasmic domain of the beta-subunit of integrins (e.g., a5β1) interacts directly with talin, and in turn, talin interacts with both vinculin and paxillin. Focal adhesion kinase (FAK) localizes to focal adhesions because it binds to paxillin [57]. Talin and paxillin may, thus, be more “direct” measures of focal adhesion size than FAK. On the other hand, paxillin and FAK, but not the linker protein talin, actively regulate extra cellular matrix (ECM)-actin linkage through modifications, such as phosphorylation or enzymatic cleavage of proteins that comprise the link. Paxillin, thus, acts as both a linker protein and a mediator of molecular processes, which talin and FAK do not. In addition, paxillin has been associated with the sensing of fluid shear stress in a multitude of cell types, including osteocytes [58]. Osteoblasts appeared to produce more integrin-a5 clusters after PFF. This data is consistent with data from others showing that integrin-a5 gene expression is slightly increased by mechanical loading in human tendon stem/progenitor cells. The mechanical loading-induced changes in actin filaments, paxillin, integrins, microtubulin, and nucleus might directly or indirectly be related to PFF-induced alterations in osteoblast function, such as matrix formation. Moreover, we found that MC3T3-E1 osteoblasts exposed to 1 h PFF enhanced collagen formation at three weeks post-PFF, but alkaline phosphatase (ALP) activity and the formation of mineralized nodules was unchanged (Appendix A4). One hour PFF treatment transiently changed bone cell morphology in for up to 4 h after PFF, and collagen production three weeks later. PFF set into motion a cascade of events, possibly starting with a rearrangement of cytoskeletal fibers and altered nucleus volume. We can speculate that the mechanical stimulus was transduced via nesprins all the way to the nucleus, where transcription of early transcription factors, such as runx2 and osterix may have been up-regulated, which in turn enable transcription of bone matrix genes. Although we did not delineate the sequence of events in the current manuscript, the cascade of events resulted in elevated collagen production up to 3 weeks later. Others have found increased differentiation of MC3T3-E1 osteoblasts several weeks after a single bout of loading is associated with induction of bone morphogenetic protein 2 expression, which then stimulates differentiation [59].
PFF decreased osteoblast cell and nucleus volume. Changes in bone cell nucleus morphology are known to affect gene expression of nuclear matrix and cytoskeletal components in cells cultured on microfabricated substrates [60]. Gene expression in T-cells is also modulated by nuclear shape [61]. It has been shown that changes in bone cell nucleus morphology affect gene expression of nuclear matrix and cytoskeletal components in cells cultured on microfabricated substrates [60]. Therefore, the observed nucleus volume in our study may also have affected gene expression and cell function, although specialized studies
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