Exogenous mechanical information provides critical regulatory signals to stem cells, biasing the ultimate fate selection of these progenitors [2
]. In the case of bone marrow MSCs, commitment toward adipose or osteoblast lineage appears to be reciprocally regulated [34
]. The bias toward adipocyte differentiation from MSCs, which accompanies conditions such as aging [35
] and anorexia [36
], not only represses osteoblast commitment but also by itself may impair hematopoietic stem cell development [37
]. As such, control of bone marrow adipogenesis is important in determining stem cell decisions of multiple lineages. We have shown that multiple types of mechanical signals limit adipogenesis from MSCs through a β-catenin dependent process [8
]. Understanding how best to activate this inhibitory mechanical signal cascade not only to optimize stem cell fate but also translate the ability of physical signals to suppress obesity as considered in the clinic.
Little is known about the specific features of the mechanical environment, which serve as critical determinants of stem cell fate (e.g., duration, amplitude, and recovery time), or how these signals are ultimately perceived and transduced by the cell. The data reported here support a conclusion that mechanical influences on stem cell decisions saturate quickly, such that the necessary signaling cascade is initiated after only a few mechanical cycles. However, while accumulation of mechanical information is not necessarily critical to defining stem cell outcomes, we found that when the mechanical stimulus is repeated after a rest period of greater than 1 hour, the cell system has not only reestablished its ability to respond but also experiences an amplification of the incoming mechanical signal. To understand this amplification mechanism, we studied those cell processes that are critical in the protection of β-catenin by mechanical signals [8
]. As the mechanical information required is delivered in a manner of minutes, we focused on GSK3β and the kinase that inactivates it, which we have shown here to be Akt, a pleiomorphic kinase, known to be involved in mechanotransduction [25
]. In MSCs, Akt is rapidly activated by mechanical loading, but is refractory to reactivation until a rest period has occurred. Supporting the hypothesis that this signal cascade is crucial to the antiadipogenic potential of mechanical signals, mechanical information delivered following the rest period was amplified, such that Akt activation and subsequent GSK3β phosphorylation were enhanced.
We considered what cellular adaptation induced by mechanical input might enhance Akt-GSK3β signal transduction. FAs represent an intracellular connection between the cell substrate surface and the cytoskeleton and are known to develop tension in response to mechanical perturbation [26
]. These focal contacts can grow in response to applied mechanical force [18
], a process that not only contributes to the enhanced structure of the cell but also influences lineage specification through cytoskeletal structure [14
]. Furthermore, MSCs responsiveness to mechanical signals has been suggested to function on a micrometer scale suggesting interactions between FAs [38
]. In response to the first session of loading, we found that FAs assembled at the edges of the MSCs increased by nearly fivefold in strained cells. Not surprisingly, the increase in FAs was paralleled by an increase in RhoA activity, as RhoA activation promotes adhesion maturation [39
], and RhoA, through Rock-mediated myosin II activation, increases cellular contractility. The resulting tension on integrin triggers protein recruitment and signaling pathway activation leading to strengthening and growth of the adhesion [40
] emphasizing that the cytoskeletal adaptations, while certainly contributing to cell structure, also proactively influence biological processes. In the absence of RhoA activation, FAs failed to form after mechanical loading.
The mechanically mediated assembly of FAs is a striking confirmation that the MSC both recognizes and adapts to exogenous physical stimuli and that these changes influence lineage selection of the precursor. Importantly, the degree to which MSCs perceive and respond to mechanical signals is strongly dependent on the temporal nature of the signal. The cell system, following an initial burst of mechanical signal, must incorporate a rest period before the mechanosensitivity is fully recovered. It appears that this refractory period allows a restructuring of cytoskeletal elements—indicating a transient cellular adaptation— and that this cytoskeletal rearrangement enables downstream amplification of incoming physical signals. As such, it is clear that signal timing, particularly in the context of a 24-hour cycle, is as important as is the magnitude of the signal to optimizing outcomes. As importantly, the temporary nature of the assembled FAs emphasizes yet another dynamic component of the cell response to mechanical signals and may underlie the established benefits of repetitive daily loading [7
Our data emphasize the importance of the cell cytoskeleton in enabling mechanotransduction pathways and that mechanically mediated restructuring of the architecture of the cell allows for amplification of these biological pathways. We believe these data also indicate a role for the cytoskeleton as an active transducer rather than passive scaffold in cellular responsiveness to physical signals. The transient nature of these cytoskeletal adaptations then serves as a short term memory of the physical environment and temporarily facilitates the amplification of further cell responses to downstream challenges. As such, the dynamic cytoskeletal restructuring is not only a means of enhancing signal sensitivity and responsiveness of the cell but also the transient nature of this response indicates that the absence of mechanical signals would ultimately be permissive to the dissolution of mechanosensitivity of the cell system.