Page 70 - Physico-Chemical Niche Conditions for Bone Cells

Shear stress modulates osteoblast cell and nucleus shape
and gene expression of nesprins were affected by PFF. Interestingly, PFF did not change the expression of actin-linked nesprin 1 and nesprin 2, while the expression of microtubule-linked nesprin 4 was down-regulated by PFF. It is a tantalizing thought that PFF-induced mechanical stimuli on the cell membrane are transferred via the cytoskeleton to the nucloskeleton via nesprins, where it mediates a change in gene expression of nesprin 1 and nesprin 2 in a feedback loop. However, such causal relations need to be established in future studies.
In our study, we demonstrated that mechanical loading affects bone cell area and nucleus volume. We also found changes in gene expression for nesprin 4. Increasing evidence demonstrates that nuclear mechanotransduction plays a critical role in physiology and diseases, and that the response of cells to mechanical stimuli is associated with alterations of nuclear transport, unfolding or modifications of nuclear proteins, and/or changes of chromatin organization and nuclear mechanics [36–38]. Therefore, it is very well possible that the PFF-induced changes in bone cell and nucleus volume result in epigenetic changes.
PFF of 6.5 Pa/s shear force was used to treat the cells, since we have found earlier that the response of MC3T3-E1 pre-osteoblasts is linearly dependent on the rate of fluid shear stress, which depends on the amplitude and frequency of stress [39,40]. The fluid shear stress amplitudes and frequencies in bone have been determined by a combination of experiments and computer models, where the peak fluid shear stress around mouse osteocytes in situ has been estimated to range up to 5 Pa [28]. That this range of fluid shear stress is enough to stimulate bone cells were confirmed by in vitro studies [6,9].
Changes in cell and nucleus volume with PFF coincided with alterations in the F-actin network. Cells subjected to PFF clearly showed F-actin stress fibers. PFF also affected cell body shape, i.e., the cell body was more extended with a polygonal osteoblast-like shape compared to the more cuboidal control cells, as well as cell pseudopodia (Figure 7A). Our results are consistent with findings by others, also showing that during fluid shear stress- treatment of MC3T3-E1 osteoblasts, the F-actin develops into prominent stress fibers, which are oriented parallel to the cell long axis [27,41]. In addition, the tensegrity model describing the architectural basis of cells indicates that cytomembrane deformation is sensed initially as a change in the tension of F-actin stress fibers [42]. F-actin directly or indirectly interacts with the nucleus [16]. In this study, PFF affected F-actin, integrin, and microtubules, which coincided with changes in cell and nuclear area and volume. Evidence is accumulating that points to a more specific role for small heat shock proteins (sHSPs) in protecting proteins from mechanical stress. Small HSPs functions and pathways are involved in sensing and responding to mechanical cues [43]. In addition, PGE2 and transforming growth factor-b (TGF- b) can induce the expression of sHSPs in osteoblasts [44]. Previously we already showed that PFF stimulates PGE2 and TGF-b release within minutes after the start of PFF [45,46].
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