In addition, in the UACC930 melanoma cell line, where the expression of GRM1 protein is not detectable [24], the induction of Smad2 and Smad3 linker phosphorylation in the presence of riluzole is comparable to that of the other melanoma cell lines that express GRM1 (Figures 3A and 3B). These results suggest that riluzole-induced Smad linker phosphorylation in melanoma cells is not directly related to GRM1 expression.Riluzole Upregulates the Expression of INHBB and PLAU
The positive effect of riluzole on Smad2 and Smad3 linker phosphorylation (Figure 6A) prompted us to investigate the potential action of Riluzole on the expression of TGFb target genes. We used human TGF?BMP Signaling Pathway RT2 ProfilerTM PCR Arrays to identify known TGFb target genes whose expression could be modulated by riluzole. For this purpose, RNAs were isolated from WM793 melanoma cells incubated in the absence or presence of riluzole. cDNAs converted from these RNAs were processed for real-time PCR using one PCR array for the untreated cells-derived cDNA and one array for the riluzole-treated cells-derived cDNA. By comparing the two, we identified four genes whose expression was increased or decreased in the presence of riluzole as compared with untreated WM793 melanoma cells (Table 1). By qPCR, we validated the upregulation of two genes, INHBB and PLAU, in three additional melanoma cell lines (Figure 6B and C). The INHBB gene codes for the inhibin, beta B chain. Two beta B subunits form a homodimer called activin B. Activin is a member of the TGFb superfamily [47]. PLAU codes for the urokinase plasminogen activator (uPA), belonging to the urokinase plasminogen activating system (uPAS). The TGFb signaling positively regulates uPA expression, secretion and stability through the Smad-dependent pathway [48,49]. Therefore riluzole positively regulates the expression of genes associated with the TGFb signaling pathway.

Discussion
In the present study, we described a cross-talk between three pathways involved in melanoma biology, the TGFb signaling pathway [1,2], the AKT/GSK3 pathway [50] and the glutamate signaling [23,24,25,27,42,51]. We demonstrated that the glutamate release inhibitor riluzole inhibited AKT phosphorylation at crucial sites for its activity, and consequently inhibited GSK3b phosphorylation at an AKT site, thereby activating GSK3b. We also provided evidence for the involvement of GSK3 in basal and riluzole-induced linker phosphorylation of Smad2 and Smad3. Indeed, treatment with two types of GSK3 inhibitors and the GSK3a/b knock-down counteracted basal and riluzole-induced Smad2 and Smad3 linker phosphorylation. In addition, in vitro kinase assays confirmed that GSK3b could phosphorylate Smad2 and Smad3 at the cluster of serines 245/250/255 and serine 204 respectively. Interestingly, we also demonstrate riluzole induced Smad linker phosphorylation in the GRM1 negative UACC930 melanoma cell line [24] (this line does not express GRM1 because of a truncation mutation [S. Chen, personal communication]). In this melanoma cell line, like in the others, GSK3 mediated the riluzole-induced Smad linker phosphorylation. However, we previously described that riluzole treatment did not inhibit AKT phosphorylation in UACC930 cells [25]. Therefore, in this line, the activation of GSK3 in the presence of riluzole would involve a different mechanism, such as Wnt signaling inactivation. These results are in apparent contradiction with a study describing riluzole as an enhancer of Wnt/b-catenin signaling and GRM1 as the likely target of riluzole-mediated enhancement of Wnt/b-catenin in the human melanoma cell line A375 and the mouse melanoma cell line B16 [51]. This group also found that riluzole did not induce a decrease in ERK phosphorylation in the A375 melanoma cell line, in contrast to the decrease in phosphorylated ERK in all human melanoma cell lines positive for GRM1 [25,27]. From these results, it is likely that genetic and epigenetic context-dependent responses can be expected when treating melanoma cell lines with riluzole, as already suggested by the mixed responses to riluzole and the failure of some patients to respond to riluzole in clinical trials, independently of GRM1 expression [26,27]. TGFb-induced Smad linker phosphorylation has been described in a wide variety of cellular systems, including melanoma cells. Figure 5. Riluzole-induced Smad linker phosphorylation is independent of GRM1 expression level in melanoma cells. A. Immunoblot with lysates from UACC903-V1 (V1) cells and two clones overexpressing GRM1, called UACC903-G2 and UACC903-G4 (abbreviated as G2 and G4 respectively). B. Riluzole-induced Smad2 and Smad3 linker phosphorylation in V1, G2 and G4 cells. Figure 6. Riluzole upregulates the expression of genes associated with the TGFb signaling pathway. A. Model of riluzole effect on Smad linker phosphorylation. Riluzole inhibits the phosphorylation of AKT at S473 and T308. As a result of AKT inactivation, GSK3 phosphorylation at the AKT site is decreased, resulting in GSK3 activation. GSK3 phosphorylates Smad2 at the cluster of serines 245/250/255 and Smad3 at serine 204. The consequences of these phosphorylation events on Smad2 and Smad3 activities will likely depend on the promoters of the TGFb target genes. B and C. Riluzole treatment increases the expression of the INHBB and PLAU genes respectively in the three independent melanoma cell lines A2058, UACC903 and C8161. mRNA from these three melanoma cell lines untreated or treated with riluzole for 24 hours were analyzed by real-time PCR for the expression of the INHBB and PLAU genes. White bars: untreated cells. Dark bars: Riluzole-treated cells. *, P,0.05, compared with untreated cells (t test). JNK, CDKs, GSK3, depending on the phosphorylation site and the cellular context [8,9,11,17,18,19,20,52]. We have shown that riluzole-induced Smad linker phosphorylation is mechanistically different from the TGFb-induced Smad linker phosphorylation.

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