Figure 10. N-cadherin engagement down-regulates Bim-EL protein levels in a Mek1/2 dependent manner. Serum-starved cells were seeded at medium density on PL or N-cad and incubated with or without 20 mM U0126 for 24 hours (A) or 4 hours (C), then protein were extracted and analyzed by western blotting with either anti-Bim, anti-phospho-Erk1/2, anti-Erk1/2, or anti a-tubulin antibodies. Relative densities of Bim-EL were normalized to a-tubulin. As a positive control of Bim-EL mobility shift, adherent cells were treated with 10 ng/ ml of PMA for 1 hour (B). Represented data are the mean of three independent experiments. Asterisks indicate a significant difference (P,0.05) as compared with no treatment. Arrows show Bim-EL mobility shift. doi:10.1371/journal.pone.0033206.g010 sufficient to sustain cell survival when they are deprived of other survival factors such as integrin interactions with the ECM. We further report that N-cadherin engagement mediates cell survival by enhancing the Erk1/2 kinase pathway and down-regulating the pro-apoptotic protein Bim-EL (Fig. 12). Bim-EL has been extensively involved in apoptotic pathways in neurons upon NGF deprivation or after K+ withdrawal [16]. Thus, we propose that N-cadherin engagement may antagonize a common apoptotic pathway activated in neurons upon various stress conditions. The involvement of cadherins in cell survival has been described previously in different cell types either normal or tumoral [3,7,42,43,44]. However, the molecular mechanism whereby cadherin adhesion contributes to cell survival is still poorly N-Cadherin Anti-Apoptotic Signaling understood and controversial. N-cadherin engagement has been shown to promote survival through the activation of the PI3- kinase/Akt and subsequent phosphorylation of the pro-apoptotic protein Bad in metastatic cancer cells [7] and in vein smooth muscle cells [45]. The PI3-kinase pathway has also been implicated in E-cadherin-mediated cell survival in Ewing Tumor cells [8] and in epithelial cells [4,5]. Therefore, we examined the role of PI3-kinase/Akt pathway. N-cadherin ligation did not affect Akt and Bad phosphorylation. This was observed at different time points from 30 min to 24 hours and using two different approaches, calcium switch assay and biomimetic substrates. In addition, the inhibitor of PI3-kinase, LY294002, did not block the GT1-7 cell survival after N-cadherin engagement. In agreement with our results, Skaper et al. showed that the inhibition of PI3- kinase/Akt pathway failed to block the survival of hippocampal and cerebellar granule neurons induced by N-cadherin agonist [46]. N- and E-cadherin engagements have been shown to up- regulate Bcl-2 protein [7,8,42]. However, in our model, the levels of Bcl-2 protein were not affected by N-cadherin engagement. These results suggest that signaling pathways that affect cell survival in response to N-cadherin engagement in neuronal cells are independent of the PI3-kinase and Bcl-2 proteins and may involve alternative pathways such as MAP kinases. Among the MAP kinases, Erk1 and 2 have been widely associated with neuronal cell survival, while JNK and p38 are often implicated in cell death [47]. Several studies suggest that Erk1/2 mediates neuroprotective activity of extracellular factors such as integrins and neurotrophins, as well as during survival of damaged neurons [5,48]. We show that N-cadherin adhesion induces an increase of Erk1/2 phosphorylation and a subsequent inhibition of GT1-7 cell death. Pharmacological inhibition of the MAP kinase pathway using U0126 resulted in the inhibition of Erk1/2 phosphorylation and an increased number of apoptotic cells on N-cadherin. Therefore our results suggest that N-cadherin protects neurons against cell death by activating specifically Erk1/ 2. Similar observations were reported in ovarian carcinoma cells [49] and in epithelial cells [50] where E-cadherin engagement activates the Ras/Erk1/2 cascade but not the PI3-kinase/Akt pathway. One critical regulator of apoptosis and target of the MAP kinase pathway is the pro-apoptotic protein Bim. The regulation of Bim activity is complex, involving both transcriptional and posttran- scriptional mechanisms. Bim is normally expressed at low levels, and its pro-apoptotic activity kept in check by rapid phosphory- lation by Erk1/2 resulting in its ubiquitination and proteosomal degradation. Reginato et al. have shown that attached MCF-10A cells contain low levels of Bim-EL and undetectable levels of Bim- L and Bim-S. All three isoforms were highly up-regulated after cell detachment from the ECM [14]. Fukazawa et al. reported that Bim-EL is the only form expressed in human HBC4 and MDA- MB231 cancer cell lines [51]. They showed that treatment with MAP Kinase inhibitors, which prone cells for apoptosis in the absence of cell anchorage, induced a reduction of Bim-EL phosphorylation together with an increase of Bim-EL protein levels. Similarly, we found that Bim-EL was the only form of Bim expressed, albeit at low level, in GT1-7 cells. Upon cell deprivation in cell-matrix and cell-cell contacts (PL substrate) Bim-EL was up- regulated. Conversely, N-cadherin engagement down-regulated Bim-EL protein levels in association with its phosphorylation as revealed by mobility shift [33]. Moreover, the Mek1/2 inhibitor U0126 prevented this phosphorylation, indicating that Bim-EL undergoes Erk1/2 dependent phosphorylation upon N-cadherin engagement, which may be responsible for its down-regulation. Interestingly, plating GT1-7 cells on fibronectin also elicited the PLoS ONE | www.plosone.org 12 March 2012 | Volume 7 | Issue 3 | e33206