Clostridium difficile toxin A attenuates Wnt/β-catenin signaling in intestinal epithelial cells.
Clostridium difficile toxins A and B (TcdA and TcdB) are homologous glycosyltransferases that inhibit a group of small GTPases within host cells, but several mechanisms underlying their pathogenic activity remain unclear. In this study, we evaluated the effects of TcdA on the Wnt/β-catenin pathway, the major driving force behind the proliferation of epithelial cells in colonic crypts. IEC-6 and RKO cells stimulated with Wnt3a-conditioned medium were incubated with 10, 50, and 100 ng/ml of TcdA for 24 h, resulting in a dose-dependent inhibition of the Wnt signaling, as demonstrated by a T-cell factor (TCF) reporter assay. This was further confirmed by immunofluorescence staining for nuclear localization of β-catenin and Western blotting for β-catenin and c-Myc (encoded by a Wnt target gene). Moreover, our Western blot analysis showed a decrease in the β-catenin protein levels, which was reversed by z-VAD-fmk, a pan-caspase inhibitor. Nonetheless, TcdA was still able to inhibit the Wnt/β-catenin pathway even in the presence of z-VAD-fmk, lithium chloride (a GSK3β inhibitor), or constitutively active β-catenin, as determined by a TCF reporter assay. Furthermore, preincubation of RKO cells with TcdA for 12 h also attenuated Wnt3a-mediated activation of Wnt signaling, suggesting that inactivation of Rho GTPases plays a significant role in that inhibition. Taken together, these findings suggest that attenuation of the Wnt signaling by TcdA is important for TcdA antiproliferative effects.
Departamento de Fisiologia & Farmacologia, Faculdade de Medicina, Universidade Federal do Ceará (UFC), Fortaleza, Brazil.
Bruno
B
Bezerra Lima
Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil.
Bárbara
B
Faria Fonseca
Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil.
Nathália
N
da Graça Amado
Departamento de Fisiologia & Farmacologia, Faculdade de Medicina, Universidade Federal do Ceará (UFC), Fortaleza, Brazil.
Débora
D
Moreira Lima
Departamento de Fisiologia & Farmacologia, Faculdade de Medicina, Universidade Federal do Ceará (UFC), Fortaleza, Brazil.
Ronaldo
R
Albuquerque Ribeiro
Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil.
José
J
Garcia Abreu
Departamento de Fisiologia & Farmacologia, Faculdade de Medicina, Universidade Federal do Ceará (UFC), Fortaleza, Brazil Departamento de Morfologia, Faculdade de Medicina, Universidade Federal do Ceará (UFC), Fortaleza, Brazil gerlybrito@hotmail.com.
Gerly Anne
GA
de Castro Brito
D000818:Animals; D001427:Bacterial Toxins / Q000494:pharmacology*; D045744:Cell Line, Tumor; D004768:Enterotoxins / Q000494:pharmacology*; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D007413:Intestinal Mucosa / Q000166:cytology*; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects; D051153:Wnt Proteins / Q000235:genetics / Q000378:metabolism*; D051176:beta Catenin / Q000235:genetics / Q000378:metabolism*
D001427:Bacterial Toxins; D004768:Enterotoxins; D016271:Proto-Oncogene Proteins c-myc; D051153:Wnt Proteins; D051176:beta Catenin; C041348:tcdA protein, Clostridium difficile