Ruolo della via di segnale di Wnt5a nel fenotipo dei miofibroblasti sub-epiteliali intestinali in corso di Colite Ulcerosa

ABSTRACT Background: Wnts are a highly conserved family of secreted glicoproteins involved in several physiological and pathological processes. Intestinal subepithelial myofibroblasts (ISEMFs), a cellular population releasing growth factors and cytokines involved in tissue remodelling, have been recently identified as a source of Wnts ligands. Since Wnt5a, the main ligand of the non-canonical pathway, plays a pivotal role in intestinal epithelial repair and is up-regulated in chronic inflammatory disorders such as rheumatoid arthritis, the aim of our study was to evaluate, in ISEMFs derived from normal subjects and ulcerative colitis (UC) patients, the expression of Wnt5a and its receptors and determine its autocrine role on ISEMFs. Methods: ISEMFs were isolated from colonic mucosa of healthy subjects (n=6) or from patients with UC (n=10). Wnt5a and Frzd1, 2, 4, 5 expressions was evaluated by quantitative RT-Time PCR and Western Blot. To determine the autocrine effect of Wnt5a, we performed functional studies using Wnt5a KO by shRNA or exposing ISEMFs to human recombinant Wnt5a. Following 96 hours culture, cell proliferation was quantitated by 3H-tymidine incorporation and cell cycle analysis. To evaluate anti-apoptotic role of Wnt5a, we performed functional studies exposing ISEMFs to human recombinant Wnt5a and staurosporin. Following 24 hours culture Annexin V and caspase-3 assay was estimated. To identify pathway induced by Wnt5a signal, functional studies using ?-catenin KO by shRNA or exposing ISEMFs to Ca2+ inhibitors was performed and CamKII protein levels was measured by Western blot. Results: UC-derived ISEMFs showed a significantly higher Wnt5a expression at mRNA and protein level (p<0.05) in basal condition as compared to controls. Furthermore, as compared to controls UC-derived ISEMFs produced significantly higher amounts of Wnt5a following exposure to LPS or TGF? (p<0.05). ISEMFs derived both normal and UC patients showed a similar pattern of Wnts receptors. Wnt5a silencing in UC-derived ISEMFs and in control ISEMFs, caused a significant reduction in cell proliferation (p<0.05). Indeed, rWnt5a supplementation stimulated cell proliferation and resistance to staurosporin induced-apoptosis. Wnt5a signal is ?-catenin independent because supplementation of rWnt5a in KO ?-catenin ISEMFs led to a significative increase of cell proliferation (p<0.05). On the contrary,Wnt5a signal seems to be mediated by CamKII, promoting an increase of its phosphorilation. Conclusions: ISEMFs from UC patients produce an excess of Wnt5a that is able to stimulate cellular proliferation, resistance to apoptosis and that acts by CamKII activation. We speculate that Wnt5a plays a key role to expand the ISEMFs population in UC patients and might contribute to tissue damage and remodeling during chronic inflammation.