86 FAK activity regulates Oct4 expression in teratocarcinomal stem-like cells
Sarah Ali Azouaou 2, Fathi Emhemmed 2, Valérie Schini-Kerth 1, Christian D. Muller 2, Annelise Lobstein 2 and Guy Fuhrmann 1 *
1
UMR 7213 CNRS, Laboratoire de Biophotonique et Pharmacologie, Université de Strasbourg, Faculté de Pharmacie, 74 route du Rhin, 67401 Illkirch, France
2
!"# $%&&# '(")*# +,-./,0.1/2# 34566.7,01.6# 89:/,;2<01=<2*# 6172/>10:# 32# )0/,>-.</?*# Faculté de Pharmacie, 74 route du Rhin, 67401 Illkirch, France
* Corresponding author: Guy Fuhrmann, PhD UMR 7213 CNRS
Laboratoire de Biophotonique et Pharmacologie Faculté de Pharmacie
74 route du Rhin, B.P. 60024, 67401 Illkirch France
Tel: (33) 3 68 85 42 17 Fax: (33) 3 68 85 43 13
87 Abstract
Several publications have highlighted the role of force-dependent cell signaling events in stem cell differentiation. These biophysical signals involve proteins which interact with the extracellular environment and are able to initiate mecanotransduction forces directing stem cell differentiation and determining the cell fate. For instance, the cytoplasmic focal adhesion kinase (FAK), in association with other adhesion molecules, is a central actor of the mecanotransduction machinery which creates biophysic constraints and can lead to phenotypic cell modifications. However the precise impact of FAK in the differentiation of stem cells remains unclear. We therefore investigated the role of FAK during the specification of the poorly differentiated pluripotent teratocarcinomal cell which is a model of Oct4-expressing embryonal cell. We observed that Oct4 downregulation during all trans retinoic acid-inducing differentiation is accompanied by an increase in the phosphorylation of FAK at tyrosine 397 and 861. Moreover, a transient knock-down of the FAK protein accelerates Oct4 gene repression, concomittantly with an increase in the apoptosis rate. These results suggest that counteracting FAK activity during cell differentiation triggers an upheaval of the finely turned regulation of Oct4 gene expression which finally leads to programmed cell death.
88 1. Introduction
The signaling pathways involved in the regulation of stem cell differentiation can be initiated by several processes, including cytokines, growth factors, or even extracellular mechanical activity [Sun et al., 2012]. For example, focal adhesion kinase (FAK) is a signaling mechano-sensing protein at the cell-matrix adhesion sites, which is able to form a complex with Src kinase and to promote cell proliferation, migration and differentiation [Wozniak et al., 2004]. Actually upon growth factor stimulation or cell-matrix interaction, FAK/Src complexes activate MAPK, which induces cell's fate decision [Liao et al., 2013]. However, many details at the initiation of the differentiation process, especially the crosstalk among the different signaling pathways leading to transcription responses, remain largely unclear. For instance, the mechanisms how the differentiation induction factors co-regulate FAK and Src, and how those signals initiate stem cell differentiation have not been fully understood. In embryonic stem cells (ESCs), adhesion signals through FAK/Src are believed to regulate their differentiation to cardiomyocytes [Hakuno et al., 2005]. Meanwhile, FAK is considered a key protein during neurite differentiation and outgrowth in mesenchymal stem cells [Liao et al., 2013]. Interestingly, the inhibition of FAK-induced phosphorylation has been shown to prevent the neurite outgrowth in mesenchymal stem cells [Liao et al., 2013]. Moreover it has been observed that retinoic acid (RA)-inducing differentiation of ESCs is accompanied by an activation of Src and FAK; however their depletion does not interfere with the diffrentiation process, but preferentially promotes an endocrine specification to the detriment of a neural cell fate determination [Afrikanova et al., 2011].
Prosurvival and self-renewal signaling pathways in stem cells share several common molecular components, pointing out the finely tuned balance which controls cell proliferation and differentiation [Konopleva and Jordan, 2011]. As a consequence, it is not surprising that
89 some factors could target specific nodal points of the self-renewal and differentiation machineries [Sarkar et al., 2009]. In highly pluripotent stem cells, i.e. ESCs, it has been observed that induction of differentiation correlates with an arrest of proliferation and a significant increased level of apoptosis. Actually, the process involves huge modifications in the regulation of the expression of the stemness factor Oct4. This transcription factor is known to be essential for pluripotency maintenance and self-renewal. Indeed, Oct4 binds at the promoters and/or regulatory regions of numerous target genes which are associated with proliferation and differentiation processes (Pesce and Schöler, 2001; Jung et al., 2010). To achieve a higher specificity, Oct4 may form protein complexes with other transcriptional regulators, including the homeobox protein Nanog and the SRY-related HMG-box protein Sox2 (Boyer et al., 2005). Finely tuned functional Oct4 levels are crucial for phenotype stability and it is believed that the induction or repression of Oct4 is heavily regulated in order to avoid any deleterious effect of a transient dysfunction. It has been shown that the regulation of Oct4 expression involves different members of the nuclear receptor superfamily, including SF-1 (Steroidogenic Factor 1), LRH-1 (Liver Receptor Homolog-1) and GCNF (Germ Cell Nuclear Factor) [Kellner & Kikyo, 2010]. By means of genetic, molecular, and pharmacological studies, a recent report has demonstrated that a catenin-dependent LRH-1 regulation is required for maintaining steady-state levels of Oct4 [Wagner et al., 2010]. This means that the balance between proliferation and differentiation of pluripotent SCs involves, at least in part, a Wnt/beta-catenin control which can specifically target the upstream regulators of the stemness factor Oct4. Moreover it has been argued that GCNF is able to recruit different MBD (Methylated CpG Binding Domain) proteins to the Oct4 promoter, suggesting a link between Oct4 gene repression and its epigenetic locking [Gu et al., 2011]. A cascade of events from the binding of extracellular signaling molecules to Oct4 gene silencing can therefore be outlined. However it was believed that unknown mechanisms of regulation
90 might also emerge. Recently, it has been postulated that nanotopography affects focal adhesion formation in hESCs, which in turn affects cell@matrix tension, focal adhesion kinase signaling and integrin@growth factor receptor crosstalk, which eventually modulates Oct4 expression in hESCs (Kong et al., 2013). The aim of our work was therefore to evaluate the role of FAK during the differentiation of the human embryonal teratocarcinoma stem cells NT2/D1 (also known as NTERA-2 cl.D1). This cell line, as the pathological counterpart of ESC, is described as a highly pluripotent undifferentiated cell line, a property associated to a strong expression of the stemness regulator Oct4 (Emhemmed et al., 2014; Ali Azouaou et al., 2015). Here we show the FAK activity during cell differentiation can act on the fine-turned regulation of Oct4 gene expression which finally leads to cell decision, either to programmed cell death or to differentiation. Finally our results suggest that FAK signaling plays an important role in regulating all trans retinoic acid (ATRA)-induced neural differantiation of embryonal stem cells.