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We show that Edar inhibit Wnt/(-catenin signalling. We confirm the results previously reported by Chaudarry et al., 2004. Wnt/(-catenin is involved in both cell adhesion and cellular signalling as a mediator of Wingless/Wnt-dependent signal transduction (ref). Axin/Conductin, in cooperation with the tumor suppressor gene product APC ( ) promote (-catenin degradation, whichinvolves serine-threonine phosphorylation on the N-terminus of (-catenin by GSK3( and subsequent ubiquitination and degradation. (-catenin/Lef-TCF signalling is initiated by binding of extracellular Wnt ligands to their respective receptors of the Frizzled (Fz) family, using LRP membrane-bound co-receptors, members of the low density lipoprotein receptor-related protein family. This binding prevent(-catenin degradation which accumulates and translocate into nucleus where it interact with transcription factor of the T cell factor/Lymphoid enhancer factor family (TCF/Lef) to activate the target Wnt-responsive genes (Gordon and Nusse, 2006). In our experiments, we used a (-catenin form (S45Y) which is mutated in serine 45, the phosphorylation target site. This (-catenin form in not phosphorylated,escape degradation, is present continuously into the nucleus and is constitutively active. The transfection of this mutated form together with Edar or its adapter EDARADD inhibit severely its transcriptional activity. We further confirmed the inhibition of Wnt/(-catenin signalling by Edar in two cancerous cell lines where the endogenous (-catenin is constitutively active. HepG2 are human hepaticcarcinoma-derived cells which carry a deletion in (-catenin gene which removes the NH-2 terminal region (amino acids 25-140) containing the GSK-3( phosphorylation site (De La Coste A et al., 1998). SW480 are human colorectal tumor-derived cells which carry a mutation in the APC gene. In both cell lines, the transfection of Edar, Edaradd or both is able to significantly inhibit the transcriptionalactivity of (-catenin. To avoid any experimental artefact due to the constitutive active state of (-catenin and its continuous presence in the nucleus, we attempted to mimic the activation of (-catenin via Wnt ligand stimulation at the cell surface level. For this we used a fusion construct (Wnt/Fz) which was proven to activate the cytoplasmic (-catenin (REF). Interestingly, in these conditionsEdar is also able to severely inhibit the endogenous (-catenin activity. Taken together, all these result demonstrate strongly that Edar down regulate (-catenin/Lef-TCF signaling in vitro.
As many TNF-receptor members, in addition to NF-kB, Edar may activate other signaling pathways as JNK signaling (REF). In order to identify by which pathway Edar inhibit Wnt/(-catenin activity, we used a potentNF-kB inhibitor, the Ikbalpha supper repressor. IkBalpha is the major NF-kB inhibitor which retains NF-kB in the cytoplasm in resting cells. After any stimulation, IkBalpha proteins are phosphorylated on the two critical serine (32 and 36) which target them to ubiqutination and degradation rending NF-kB free to translocates into the nucleus to achieve its transcriptional activity. As expected, weshow that transfection of the ?????? Completely abolished the activity of NF-kB. Interestingly, in the absence of NF-kB activity, Edar become enable to down regulate the Wnt/(-catenin signaling pathway in HEK293T, HepG2 and SW480 cells. Moreover, pharmacological inhibition of NF-kB using?????? drug leaded to the same results (data not shown). These result demonstrated strongly that Wnt/(-catenininhibition by Edar is NF-kB dependent. (-catenin has multiples roles in various developmental processes (Clevers H, 2006), in some self-renewing tissues in mammals, in bone, gut, haematopoietic system and skin appendages development. In another hand the Edar/NF-kB signalling has reported to play more specific functions in skin appendages formation which is strongly attested by mouse models and by...
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