By philipp Boder
The significant influence of mechanical forces on cell behaviour in vivo, such as tension, pressure, gravity, and fluid shear stress, have long been appreciated. However, with the advent of new molecular biology techniques we have begun to depict the signal transduction and genetic changes that occur in response to these events. Our organs have evolved complex tissue organizations as a result of cell cytoskeletal tensions and dynamics in response to the surrounding external force exerted by the extracellular matrix (ECM). By design, mechanical forces can therefore provide informational cues to cells for perceiving topography, environmental disturbances, position and geometry. The mechanisms of mechanoreception on a macroscopic scale are well-understood, with cell-cell and cell-ECM adhesion sites (such as focal adhesions via integrins to the ECM protein fibronectin) altering cytoskeletal protein events, ultimately controlling cell differentiation, proliferation and apoptosis. However, the underlying mechanism that translated mechanical forces to affect a change in gene expression remained a mystery, until the discovery of Yes-associated protein (YAP) and transcription co-activator with PDZ-binding motif (TAZ); both being transcriptional co-regulators that bind to DNA enhancer elements using TEA domain family member (TEAD) factors (Panciera et al., 2017).
FIGURE 1 – Illustration of the effects of mechanical stimuli on cellular YAP/TAZ activity.
A) Active YAP/TAZ transcriptional co-regulators promote gene expression changes via translocation to the nucleus, requiring association with various TEAD factors. B) The left- and right- panels demonstrate the influence of matrix properties and physical conditions which determine the cytoplasmic retention/inactivation or the nuclear localisation/activation of YAP/TAZ, respectively. (Adapted from Panciera et al., 2017).
Interestingly, YAP and TAZ are sensitive to a wide array of biomechanical signals and are able to elicit cell-specific responses depending on the type of mechanical stresses in the present surroundings. Typically, YAP and TAZ have been associated with the Hippo signalling cascade. This regulates organ size in animals through the control of cell proliferation via Linker of Activated T cells kinase (LATK)-dependent phosphorylation of Yap/TAZ, leading to their cytoplasmic degradation. However, cellular mechanotransduction appears to influence YAP and TAZ activity in a LATS-independent manner (Figure 1). YAP/TAZ are inactivated when attached to a soft ECM substrate or a small adhesive area, but become active upon increased mechanical stimulation by a harder ECM or increased cell spread and cytoskeletal tension (Halder et al., 2012). In this regard, a recent study investigated the role of mechanical signals, including ECM rigidity and cell shape, and changes in YAP/TAZ transcriptional activity in dictating epidermal stem cell fate (Totaro et al., 2017).
FIGURE 2- Mechano-responsive YAP/TAZ co-transcriptional regulators influence epidermal stem cell fate.
Confocal immunofluorescence images of neonatal human epidermal keratinocytes (nHEK) plated on large (1024µm2) fibronectin islands stained for the YAP/TAZ proteins (red) and the terminal differentiation marker Involucrin (IVL-green). DAPI staining for nucleus (blue). Scale bar represents 20µm. A) nHEK transfected with siRNA against YAP/TAZ mRNA for knockdown analysis and a non-specific siRNA as a control. B) nHEK cells treated for 24 hours with cytochalasin D (actin polymerisation inhibitor) and a mock control (non-treated). (Adapted from Totaro et al., 2017).
Primary epidermal progenitor cells, specifically neonatal human keratinocytes, were seeded on small (300µm2) and large (1024µm2) microprinted fibronectin islands to determine the effect of cell-ECM adhesiveness on differentiation versus proliferation (stemness) in the context of YAP/TAZ. After 24 hours, immunofluorescence staining for involucrin, a marker for terminal differentiation in keratinocytes, showed a large proportion of differentiated cells on the small islands. These cells adopted a rounded morphology, whereas cells on the larger islands were more spread and undifferentiated. Additionally, staining for YAP/TAZ revealed greater nuclear localisation in large cells. To confirm the role of YAP/TAZ activity in shape-induced epidermal stem cell decisions, YAP/TAZ were inactivated via knock-down with short interfering RNAs (siRNA). Interestingly, these cells exhibited a constant differentiated phenotype and loss of stem cell self-renewal (Figure 2A). This highlights the critical nature of YAP/TAZ in determining epidermal cell fate. Taking into account that cells respond to surrounding physical conditions by modulating actomyosin tension, it would make sense that previous studies have found that epidermal cells adhering to small adhesive areas have an increase in total filamentous actin (F-actin) level (Connelly et al., 2010), that is the polymerised form of actin within cell (globular actin (G-actin) being its monomeric form). From this, it may be hypothesised that higher F-actin concentrations induce cell differentiation, whereby the ratio of F- to G-actin determines epidermal stem cell fate. Unexpectedly, when researchers treated epidermal stem cells with cytochalasin D (Figure 2B), an inhibitor of F-actin polymerisation, keratinocyte differentiation increased significantly even on large adhesive areas, as opposed to non-treated controls (Totaro et al., 2017). In line with these findings, F-actin inhibition also increased cytoplasmic retention of SSYAP/TAZ. Taken together, this suggests F-actin plays a positive role in transducing external mechanical forces, leading to intracellular YAP/TAZ activation and maintenance of epidermal stemness.
In conclusion, it would appear that in the case of epidermal stem cells, YAP/TAZ transcriptional co-regulators act as universal mechanotransducers and mechanoeffectors of physical inputs. However, despite the significant advances made in delineating the YAP/TAZ mechanotransductional pathway, the mechanisms by which they mediate changes on a transcriptional-level remain elusive. Furthermore, it may prove interesting to elucidate any variations in mechanically-activated YAP and TAZ in an in vivo model. Nonetheless, understanding the molecular signalling events in response to mechanical stress has important implications for the design of therapeutic biomaterials, whereby these interactions are exploited to assist healing, for example in severe burn victims requiring epidermal regeneration.