Page 74 - Physico-Chemical Niche Conditions for Bone Cells

Shear stress modulates osteoblast cell and nucleus shape
further focusing on the relation between nucleus volume and cell behavior are needed to substantiate such a finding. Moreover, the position of genes with respect to the nuclear envelope affects the regulation of their expression [60,62,63]. In our study, nucleus deformation might have affected gene expression and cell function. Although no significant differences were found in gene expression of paxillin and integrin-a5 after PFF-treatment, nesprin 4 expression was significantly altered. However, it has been shown previously that expression of amongst others insulin-like growth factor-I (IGF-I), vinculin (VCL), and anti- polyclonal (APC) are affected after 1 h PFF-treatment of MC3T3-E1 cells [64–66]. We demonstrated that PFF affects F-actin, integrin, microtubule, cell area, and cell and nucleus volume. Others have shown that Rho GTPases are molecular switches that control various signal transduction pathways in all eukaryotic cells. They are known principally for their pivotal role in regulating the actin cytoskeleton, but their ability to influence cell polarity, microtubule dynamics, membrane transport pathways, and transcription factor activity is probably just as significant [67]. Therefore, it may be hypothesized that PFF changed Rho GTPases, resulting in changes in the actin cytoskeleton in MC3T3-E1 pre-osteoblasts. The Rho GTPases encompass Cdc42, Rac1, and ras homolog family member A (RhoA), of which Rho activation is known to stimulate F-actin fiber formation and focal adhesion formation [68], as we observed after PFF. We did not observe cytoskeletal changes consistent with Rac1 and Cdc42 activation. It is, thus, tantalizing to assume that PFF enhances RhoA activity. We have previously performed RhoA activity measurement (quantified using G-LISATM Small G-protein Activation Assay) in PFF-treated and static human osteoblasts (cultured cells derived from human collagenase-treated bone pieces, three separate donors). RhoA activity was nearly abolished in response to treatment with the Rho inhibitor Y27632, demonstrating the efficiency of the assay. To our great surprise, though, PFF for 1h, comparable in magnitude and frequency to the stimulation in the current manuscript, reduced RhoA activity by 2 to 3-fold (unpublished results). In other words, PFF strongly affects RhoA activity, but either activation of RhoA is transient, and has passed its peak at 1h post-PFF, or some other factor (e.g., small HSPs) is responsible for the actin redistribution observed after PFF. Future studies should explore the mechanisms behind the effect of PFF on nuclear volume, cytoskeletal and cell volume changes in more detail, and try to find a causal relation between PFF-induced changes in cell and nucleus volume and cell behavior.
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