Mechanically Induced Focal Adhesion Assembly Amplifies Anti-Adipogenic Pathways in Mesenchymal Stem Cells

Department of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
Stem Cells (Impact Factor: 6.52). 11/2011; 29(11):1829-36. DOI: 10.1002/stem.732
Source: PubMed


The fate of pluripotent mesenchymal stem cells (MSC) is determined through integration of chemical, spatial, and physical signals. The suppression of MSC adipogenesis by mechanical stimuli, which requires Akt-induced inhibition of glycogen synthase kinase 3β (GSK3β) with β-catenin activation, can be enhanced by repetitive dosing within a single day. Here, we demonstrate that reapplication of cyclic strain within a 24-hour period leads to amplification of both Akt activation and its subsequent inhibition of GSK3β, such that total cycle number can be reduced while still inhibiting adipogenesis. Amplification of Akt signaling is facilitated by a dynamic restructuring of the cell in response to mechanical signals, as evidenced by a transient increase in focal adhesion (FA) number and increased RhoA activity. Preventing FA assembly or development of tension blocks activation of Akt by mechanical signals, but not by insulin. This indicates that the FA infrastructure is essential to the physical, but not necessarily the chemical, sensitivity, and responsiveness of the cell. Exploiting the transient nature of cytoskeletal remodeling may represent a process to enhance cell responsiveness to mechanical input and ultimately define the fate of MSCs with a minimal input.

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Available from: Clinton Rubin, Feb 14, 2014
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    • "Focal adhesion assemblies can also provide a platform for numerous other proteins involved in signalling cascades to bind and transmit signals to the nucleus (Clark & Brugge, 1995). Tyrosine kinases such as FAK and paxillin (Burridge et al. 1992), serine-threonine kinases such as MAPK (Chen et al. 1994), GTPases such as Rho (Hall, 1994), and intracellular calcium concentration (Juliano & Haskill, 1993; Kawano et al. 2006; Riddle et al. 2006) are all activated by the formation of focal adhesions, and they, along with their downstream signals, have all been implicated in MSC differentiation (McBeath et al. 2004; Lu et al. 2008; Pala et al. 2008; Kundu et al. 2009; Sen et al. 2011). As mentioned previously , the activity of such proteins is also affected by extrinsic mechanical signalling. "
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    ABSTRACT: Biophysical cues play a key role in directing the lineage commitment of mesenchymal stem cells or multipotent stromal cells (MSCs), but the mechanotransductive mechanisms at play are still not fully understood. This review article first describes the roles of both substrate mechanics (e.g. stiffness and topography) and extrinsic mechanical cues (e.g. fluid flow, compression, hydrostatic pressure, tension) on the differentiation of MSCs. A specific focus is placed on the role of such factors in regulating the osteogenic, chondrogenic, myogenic and adipogenic differentiation of MSCs. Next, the article focuses on the cellular components, specifically integrins, ion channels, focal adhesions and the cytoskeleton, hypothesized to be involved in MSC mechanotransduction. This review aims to illustrate the strides that have been made in elucidating how MSCs sense and respond to their mechanical environment, and also to identify areas where further research is needed.
    Full-text · Article · Nov 2014 · Journal of Anatomy
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    • "Nuclear motions within the cell impose forces on the cytoskeleton, ostensibly initiating mechanotransduction pathways including integrin related signaling. Akt signaling plays an important role in activating cellular sensing involving the cytoskeleton and formation of new focal adhesions and preserving cellular β-catenin levels in response to mechanical stretch [37], [68], [69]. Here, we showed that vibrations increase Akt activation in MLO-Y4 cells. "
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    ABSTRACT: The physical mechanism by which cells sense high-frequency mechanical signals of small magnitude is unknown. During exposure to vibrations, cell populations within a bone are subjected not only to acceleratory motions but also to fluid shear as a result of fluid-cell interactions. We explored displacements of the cell nucleus during exposure to vibrations with a finite element (FE) model and tested in vitro whether vibrations can affect osteocyte communication independent of fluid shear. Osteocyte like MLO-Y4 cells were subjected to vibrations at acceleration magnitudes of 0.15 g and 1 g and frequencies of 30 Hz and 100 Hz. Gap junctional intracellular communication (GJIC) in response to these four individual vibration regimes was investigated. The FE model demonstrated that vibration induced dynamic accelerations caused larger relative nuclear displacement than fluid shear. Across the four regimes, vibrations significantly increased GJIC between osteocytes by 25%. Enhanced GJIC was independent of vibration induced fluid shear; there were no differences in GJIC between the four different vibration regimes even though differences in fluid shear generated by the four regimes varied 23-fold. Vibration induced increases in GJIC were not associated with altered connexin 43 (Cx43) mRNA or protein levels, but were dependent on Akt activation. Combined, the in silico and in vitro experiments suggest that externally applied vibrations caused nuclear motions and that large differences in fluid shear did not influence nuclear motion (<1%) or GJIC, perhaps indicating that vibration induced nuclear motions may directly increase GJIC. Whether the increase in GJIC is instrumental in modulating anabolic and anti-catabolic processes associated with the application of vibrations remains to be determined.
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    • "Actin stress fiber formation increases tension within the cell (Hammerick et al., 2010; Wall et al., 2007), promoting force transfer (Hu et al., 2005; McGarry and Prendergast, 2004) and perhaps modulating a cell's sensitivity to vibrations. At the molecular level, LPA can activate RhoA (Riddick et al., 2008) which, together with the number of mature focal adhesions, can amplify mechanically induced β-catenin signaling in MSCs (Sen et al., 2011) and alter the fate of MSCs. Thus, cellular tension may potentiate the ability of vibrations to promote osteogenesis. "
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    ABSTRACT: Consistent across studies in humans, animals and cells, the application of vibrations can be anabolic and/or anti-catabolic to bone. The physical mechanisms modulating the vibration-induced response have not been identified. Recently, we developed an in vitro model in which candidate parameters including acceleration magnitude and fluid shear can be controlled independently during vibrations. Here, we hypothesized that vibration induced fluid shear does not modulate mesenchymal stem cell (MSC) proliferation and mineralization and that cell's sensitivity to vibrations can be promoted via actin stress fiber formation. Adipose derived human MSCs were subjected to vibration frequencies and acceleration magnitudes that induced fluid shear stress ranging from 0.04Pa to 5Pa. Vibrations were applied at magnitudes of 0.15g, 1g, and 2g using frequencies of both 100Hz and 30Hz. After 14d and under low fluid shear conditions associated with 100Hz oscillations, mineralization was greater in all vibrated groups than in controls. Greater levels of fluid shear produced by 30Hz vibrations enhanced mineralization only in the 2g group. Over 3d, vibrations led to the greatest increase in total cell number with the frequency/acceleration combination that induced the smallest level of fluid shear. Acute experiments showed that actin remodeling was necessary for early mechanical up-regulation of RUNX-2 mRNA levels. During osteogenic differentiation, mechanically induced up-regulation of actin remodeling genes including Wiskott-Aldrich syndrome (WAS) protein, a critical regulator of Arp2/3 complex, was related to the magnitude of the applied acceleration but not to fluid shear. These data demonstrate that fluid shear does not regulate vibration induced proliferation and mineralization and that cytoskeletal remodeling activity may play a role in MSC mechanosensitivity.
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