Among the repertoire of integrins

Among the repertoire of integrins, the β1-integrin subunit mediates the attachment of hESCs to fibronectin via the α5β1 heterodimer (Baxter et al., 2009), as well as other commonly used ECM (Braam et al., 2008). Although hESCs cultured on ECM have been shown to express active FAK and AKT (Miyazaki et al., 2012; Rodin et al., 2014; Wrighton et al., 2014), the functional contribution of the FAK pathway to hESCs has not been dissected.
Results
Discussion Here we shed light on the events downstream of integrin activation in hESCs and reveal in FAK the mediator of this signaling and a positive regulator of survival, adhesion, and stem cell maintenance. Indeed, hESCs respond to FAK inhibition by exiting the stem cell state through either anoikis or differentiation. We found that integrin activation in hESCs is transduced by FAKY397 to activate AKT and MDM2 and suppress p53 and caspase activation. This FAK-dependent survival pathway is consistent with that reported in adult cell types (Lim et al., 2008), but in the context of hESCs we reveal how integrin signaling supports pluripotency circuits, since AKT and p53 are well known to regulate the balance between self-renewal and differentiation (Singh et al., 2012; Jain et al., 2012). We found no evidence that the loss of FAK signaling in pluripotent PR-619 biases differentiation to a particular lineage, but rather the resultant switch-off of PI3K/AKT and switch-on of p53 can shift hESCs out of pluripotency (Singh et al., 2012; Jain et al., 2012). Our data confirm and extend previous work reporting FAK and AKT phosphorylation in hESCs (Miyazaki et al., 2012; Rodin et al., 2014; Wrighton et al., 2014). However, a recent paper reported that FAK is activated only upon differentiation (Villa-Diaz et al., 2016), similarly to murine ESCs where stem cell maintenance inversely correlates with integrin activation (Toya et al., 2015). This discrepancy with our and previous reports on hESCs may be explained by our data showing that the integrin signaling players are indeed active in hESCs but are only assembled into obvious focal adhesions upon differentiation. Clearly, hESCs manipulate complex integrin machinery for different purposes in different environments. For example, it was shown that ILK inhibition but not FAK inhibition increased endoderm differentiation in the presence of activin A (Wrighton et al., 2014), whereas we saw no effect of ILK on survival or differentiation when added to hESC media. We found that hESCs require FAK for maintenance of substrate adhesion, which is consistent with its role in transmitting forces from integrins to the cytoskeleton (Huveneers and Danen, 2009). Double inhibition of FAK kinase and caspase revealed signs of cytoskeletal hypercontraction, similar to the unique and lethal hESC-response to single-cell dissociation (Ohgushi et al., 2010), but also visible in groups of mutually adhering cells. Thus, our results suggest that integrin signaling is an essential yet distinct cue from cell-cell adhesion for suppression of cytoskeleton contraction and apoptosis. However, integrin signaling may crosstalk with cell-cell adhesion. Indeed, we observed formation of cell aggregates after blocking β1-integrin or FAK knockdown. Finally, we discovered that hESCs possess a nuclear pool of FAK that does not accumulate but reduces after FAK inhibition, unlike in adult cells (Lim et al., 2012). We propose that hESCs utilize non-canonical FAK mechanisms to quickly respond to defective adhesion, which could involve undiscovered roles for nuclear FAK in the context of pluripotent cells.
Experimental Procedures
Author Contributions
Acknowledgments The authors thank Prof. Martin Humphries for the gift of MAB13 and 12g10 antibodies, Dr. Andrew Gilmore for discussion, and Dr. Christoph Ballestrem for microscopy. This work was supported by an MRC CASE studentship (G1000399) in collaboration with Neusentis to L.V., a Turkish Ministry of National Education (MoNE 1416) studentship to B.I., and NW Science fund grant (N0003382) and MRC grant (G0801057) to S.J.K.