br Introduction An area of

Introduction An area of growing interest is to understand how extracellular matrix (ECM) and integrins cooperate within the stem cell niche to control the balance between renewal and differentiation. The biological relevance of ECM/integrin interactions in regulating cell functions and signaling has been documented. Crosstalk of integrins with many growth factor receptors (GFRs) and channel proteins has also been investigated (Borges et al., 2000; Miyamoto et al., 1996; Moro et al., 1998; Wu et al., 1998). This represents a functional model in which the same ECM molecule imposes a specific biological response by the juxtaposition of integrins, GFRs, or channel proteins in a confined spatial context, thus rendering ECM proteins a dynamic scaffold for short range paracrine or autocrine growth factor signaling. Fibronectin is a provisional matrix protein that provides guidance cues for directional cell migration during development and disease (Zou et al., 2012). Genetic deletion of fibronectin leads to embryonic death that has been attributed to the indispensible role of fibronectin in developmental branching morphogenesis (George et al., 1993). Moreover, fibronectin modulates the Na+/H+ exchanger, manipulating intracellular pH and then migration (Park et al., 2012). However, fibronectin-dependent mechanisms in glucose transporters remain largely unknown. The mechanisms underlying the interaction between fibronectin and glucose uptake in embryonic stem cells (ESCs) and its related signal mechanisms need to be elucidated. To date, 13 members of the mammalian facilitative glucose transporter (GLUT) family have been identified (Macheda et al., 2005). Among them, GLUT-1 is expressed throughout preimplantation development from oocytes, and increases in developing embryos from the two-cell stage to the blastocyst stage (Morita et al., 1994). Also, GLUT-1 (GLUT-1 −/−) depleted ESCs are non-viable. Therefore, GLUT-1 has been implicated as one of the efficient isoforms to supply glucose to ESCs. It is generally thought that the phosphoinosital-3-kinase (PI3K) pathway induces varying degrees of glucose uptake and GLUT translocation (Farese, 2002; Navarrete Santos et al., 2004; Welsh et al., 2005). However, the sufficiency of this pathway alone to promote glucose uptake remains unproven. Peroxisome proliferator-activated receptor-gamma (PPARγ) regulates glucose metabolism by increasing glucose uptake through facilitative glucose transporter proteins (Armoni et al., 2007). More recently, it has been demonstrated that the administration of the synthetic PPARγ agonist, rosiglitazone, increases glucose uptake in tumor cells (Feng et al., 2011) but reduces glucose uptake in macrophages (Liang et al., 2007). While the correlation between GLUT-1 and PPARγ under the influence of fibronectin is still unclear, GLUT-1 mediates basal glucose transporter expression in ESCs. In addition, an essential role for TC10 (a member of the Rho family of small G proteins) in regulating purchase GSK503 structure and its dynamic rearrangement had emerged from prior studies on the process of GLUT-4 translocation (Khan and Pessin, 2002). Therefore, TC10 also could be a good candidate involved in fibronectin-induced glucose uptake pathways. This study aimed to investigate the effect of fibronectin on GLUT-1 expression, trafficking, and its related signal pathways in mouse embryonic stem cells (mESCs).
Materials and methods
Results
Discussion The present data demonstrate that integrin-bound fibronectin stimulates GLUT-1 synthesis through VEGFR2/Ras/PI3K/Akt and calcium channel/Ca/PKC, which merge at PPARγ, and GLUT-1 trafficking through TC10 and F-actin. A shortage of fibronectin (FN−/−) induces abnormal embryonic development, and finally, embryonic lethality (George et al., 1993). Interestingly, during development, embryos produce fibronectin in the cell itself (Shirai et al., 2005), demonstrating the crucial role of fibronectin during embryogenesis. We found every ECM protein (fibronectin, laminin, collagen I, collagen IV, fibrinogen) increased glucose uptake; among them, fibronectin enhanced glucose uptake the most, which was related with increased GLUT-1 mRNA and protein expression level. In addition, the result shows that α5β1 is a major integrin receptor for fibronectin in mESCs, but that integrin β1 neutralization does not completely block fibronectin-induced 2-DG uptake, raising the possibility that fibronectin-bound integrin signaling is insufficient to increase GLUT-1. Interactions between ECM proteins and growth factors could enhance integrin-growth factor receptor crosstalk and cellular response as well (Borges et al., 2000; Miyamoto et al., 1996; Moro et al., 1998). Among them, VEGF receptors are physically and functionally associated with integrins (Ruoslahti, 2002). Immunoprecipitation and Duolink assay data have provided strong evidence of the integrin β1/VEGFR2 interaction. Presently, the inhibition of both integrin β1 and VEGFR2 blocked 2-DG uptake. Abrogation of fibronectin/integrin β1-induced transactivation of VEGFR by disruption of the non-receptor tyrosine kinase Src, which supports a mechanism in our model whereby Src and other proteins associated with the actin cytoskeleton are required for VEGFR transactivation in response to β1 integrin interaction with fibronectin (Mahabeleshwar et al., 2006, 2007; Soldi et al., 1999). But, such interactions were not experimentally evident in our studies. Although integrin has the ability to activate signal pathways independently (Assoian and Schwartz, 2001), our observation of cross talk between integrin β1 and VEGFR2 provides strong evidence that a juxtamembrane receptor complex assembly promotes integration of the downstream signal pathways. Consistent with a previous study (Rodriguez-Viciana et al., 1996), Ras and PI3K/Akt signaling pathways converged in the cytoplasm. On the other hand, a number of cytoplasmic signaling molecules are potential candidates to interact with the calcium channel (Wu et al., 1998). Our results showed the engagement and clustering of integrin β1 and LTCC by fibronectin, and that this interaction is connected with calcium influx and PKC activation. Yanagida et al. (2004) reported that calcium entry through the plasma membrane is mainly mediated by SOCs, but not by VOCs, in mESCs. However, we found that integrin-bound fibronectin increased the intracellular calcium concentration, which is mediated by calcium entry across the plasma membrane from the outside of the cell, through the release of intracellular VOC and calcium stored through SOC. The signaling mechanism between the integrin and the calcium channel might occur through a membrane-delimited pathway.