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Focal Adhesion Kinase Regulation of Human Embryonic Stem Cells
[Thesis]. Manchester, UK: The University of Manchester; 2013.
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Abstract
Undifferentiated human embryonic stem cells (hESCs) grow on the extracellular matrix (ECM) substrate fibronectin (FN) in defined feeder-free conditions. The ECM is part of the hESCs pluripotent niche and supports their maintenance, but the contribution to survival remains to be elucidated. Understanding the mechanism of survival is particularly crucial in hESCs, since it affects their expansion in cell culture and ultimately translation of research to the clinic. HESCs bind to FN mainly via α5β1- integrin, known to be upstream of important survival cascades in other cell types. However, it is not understood if and how FN/integrin binding supports those molecular pathways in the context of pluripotent hESCs. The aim of this work was to elucidate the survival cascade downstream of the FN/integrin interaction in hESCs. Initially, when hESCs were cultured on a non-integrin activating substrate they initiated an apoptotic response that also occurred when β1-integrin was selectively blocked with antibody, leading the cells to detach from FN. Integrin activation is generally transduced within cells via a complex adhesome of scaffold and kinase proteins, among which the focal adhesion kinase (FAK) plays a key role. Indeed, blocking β1-integrin resulted in dephosphorylation of endogenous FAK in hESCs. When FAK kinase activity was directly inhibited (with small molecule inhibitors), hESCs responded by detaching from FN and activating caspase-3, leading to an increase in apoptosis. Furthermore, flow cytometry analysis showed that the population of hESCs that underwent apoptosis still retained the pluripotency-associated marker NANOG. FAK is a convergent point between growth factor signaling and the PI3K/Akt pathway, with a well-reported role in the maintenance of hESCs. Consistently, FN activated both AKT and its target the ubiquitin ligase MDM2 at the protein levels, while pAkt was reduced after β1-integrin blocking and FAK inhibition. Cell imaging showed that MDM2, which regulates p53 degradation in the nucleus, displayed reduced nuclear localisation after FAK inhibition, opening the possibility for a change in the p53 balance in hESCs. In fact, p53 protein increases after FAK inhibition corresponding also to caspase activation. Further investigation explored if FAK-dependent pathways are also implicated in the maintenance of hESC pluripotency. Inhibition of FAK led the cells that survived apoptosis to lose stem cell morphology, decrease pluripotency-associated markers and change nuclear shape. Moreover, a small pool of FAK was found in the nucleus of hESCs cultured on FN, but decreased after FAK inhibition. FAK was also co- immunoprecipitated with NANOG protein in standard hESC culture while NANOG decreased after sustained FAK inhibition. This data suggests that nuclear roles of FAK could support, together with the cytoplasmic activation of the PI3K cascade, both survival and pluripotency pathways requiring further investigation. In conclusion, the original contribution of this work is to identify in FAK the downstream survival effector of the FN/β1-integrin interaction in hESCs. HESCs survival is maintained by the binding of β1-integrin to FN and activation of FAK kinase and downstream PI3K/Akt, leading to the suppression of p53 and caspase activation. In parallel, promotion of these pathways by FAK is suggested also to support the key pluripotency circuitry, feeding into NANOG. Overall, FAK is proposed here as an important regulator of hESC survival and fate.