[PubMed] [Google Scholar] 35. Nedd4l in glioma cells. We demonstrated that overexpression and activation by fibronectin of 51 integrin allowed the transactivation of beta-catenin gene targets included in an EMT-like program that induced an increase in cell migration. Hampering of beta catenin activation and cell migration could be similarly achieved by a specific integrin antagonist. In addition we showed that 51 integrin/AKT axis is mainly involved in these processes. However, blockade of beta-catenin by XAV939 (tankyrase inhibitor leading to beta-catenin degradation) did not synergize with p53 activation aiming to cell apoptosis as was the case with integrin antagonists. We therefore propose a dual implication of 51 integrin/AKT axis in glioma cell resistance to therapies and migration each supported by different signaling pathways. Our data thus suggest that 51 integrin may be added to the growing list of beta-catenin modulators and provide new evidences to assign this integrin as a valuable target to fight high grade glioma. < 0,01; ***< 0,005. We then analyzed if 51 integrin activation through binding to fibronectin may enhance beta-catenin activation. For this purpose, U87MG-5 high cells were plated on fibronectin pre-coated wells. The effects of other ECM components (collagen, vitronectin, laminin) were compared to those obtained on non-coated or with poly-L-lysine (a non ECM component) coated wells. As compared to uncoated wells, poly-L-lysine and laminin did not improve the active beta-catenin fraction in U87MG-5 high cells (Figure ?(Figure2A)2A) thus ruling out a role of laminin receptors (14, 64). However, likewise to the increase induced by fibronectin, collagen and vitronectin were both able to similarly enhance the beta-catenin activity suggesting a role of collagen-binding 1 integrins and vitronectin-binding v integrins on these substrates. Our data are in agreement with other studies on non-glioma cells showing that collagen- or vitronectin-related integrins may be able to stimulate the beta-catenin pathway [22, 28]. In order to confirm a specific role of 51 integrin in the fibronectin-dependent activation of beta-catenin, we next compared the activation process in U87MG cells with 5 high or low expression. Fibronectin-dependent beta-catenin activation was strongly enhanced in 5-high cells. In 5-low cells the low basal activity of beta-catenin was enhanced by fibronectin until reaching the basal level in 5-high cells (Figure ?(Figure2B).2B). Similar results were obtained in U373MG cells (Figure ?(Figure2C).2C). Data thus confirmed that on a fibronectin matrix, beta-catenin activation occurs upon fibronectin-linked 5 integrin activation but do not exclude participation of other fibronectin receptors (such as v3 integrin which is also expressed on U87MG and U373MG cells). Open in a separate window Figure 2 Fibronectin matrix triggers active -catenin(A) Western blot analysis of -catenin activation in U87MG-5 high cells plated for 90-min on uncoated (control) or 10 g/ml poly-L-lysine (PLL), fibronectin (Fn), collagen (Coll), vitronectin (Vn) or laminin (Ln) coated wells. GAPDH was used as a loading control. (B) Western blot analysis of fibronectin-induced effects on activation of -catenin in U87MG 5-high and 5-low cells. Cells were plated on fibronectin (10 g/ml)-coated wells for 90-min. (C) Similar experiments as in b) for U373MG 5-high and 5-low cells. One western blot representative of 3 independent experiments is shown. Histograms represent the mean S.E.M. of 3 independent experiments normalized with GAPDH with *< 0,05; **< 0,01; ***< 0,005. Integrin 51 activation increases -catenin transactivation in glioma Haloperidol (Haldol) cells In the former assays, beta-catenin activation was determined by mean of protein level with a specific anti-active beta-catenin antibody [27]. Activation process of beta-catenin was next investigated on the transcriptional activity level. Downstream known targets of beta-catenin transactivation, c-myc, cyclin D1 and axin, were Haloperidol (Haldol) analyzed by real time PCR after cell plating on fibronectin. Interestingly, Haloperidol (Haldol) although basal mRNA level of the 3 genes was not affected by the expression level of 5 integrin, fibronectin clearly enhanced their transcription inside a 5 integrin-dependent manner for both U87MG (Number ?(Figure3A)3A) and U373MG cells (Supplementary Figure S1A). Conversely, inhibition of 51 integrin activity by K34c only affected negatively the mRNA level of the 3 genes in U87MG- and U373MG-5 high cells (Number ?(Number3B3B and Supplementary Number S1B). Data therefore suggested that transcriptional activation of beta-catenin was only obtainable in an 5 integrinCdependent way. To further confirm the implication of the beta-catenin pathway in these effects, U87MG-5 high cells were treated having a tankyrase inhibitor, XAV939, which is known to promote beta-catenin degradation [29] Observe Number ?Number5A).5A). The fibronectin-induced increase of gene transcription was highly and dose-dependently downregulated by XAV939 (Number ?(Number3C).3C). In addition, U87MG-5 high cell treatment with LiCl, a known inducer of beta-catenin transactivation, improved the gene transcription up to the level acquired with fibronectin.