RESUMEN
Mice lacking junctional adhesion molecule A (JAM-A, encoded by F11r) exhibit enhanced intestinal epithelial permeability, bacterial translocation, and elevated colonic lymphocyte numbers, yet do not develop colitis. To investigate the contribution of adaptive immune compensation in response to increased intestinal epithelial permeability, we examined the susceptibility of F11r(-/-)Rag1(-/-) mice to acute colitis. Although negligible contributions of adaptive immunity in F11r(+/+)Rag1(-/-) mice were observed, F11r(-/-)Rag1(-/-) mice exhibited increased microflora-dependent colitis. Elimination of T cell subsets and cytokine analyses revealed a protective role for TGF-ß-producing CD4(+) T cells in F11r(-/-) mice. Additionally, loss of JAM-A resulted in elevated mucosal and serum IgA that was dependent upon CD4(+) T cells and TGF-ß. Absence of IgA in F11r(+/+)Igha(-/-) mice did not affect disease, whereas F11r(-/-)Igha(-/-) mice displayed markedly increased susceptibility to acute injury-induced colitis. These data establish a role for adaptive immune-mediated protection from acute colitis under conditions of intestinal epithelial barrier compromise.
Asunto(s)
Inmunidad Adaptativa/inmunología , Colitis/inmunología , Mucosa Intestinal/inmunología , Intestinos/inmunología , Inmunidad Adaptativa/genética , Animales , Traslocación Bacteriana/genética , Traslocación Bacteriana/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/inmunología , Colitis/inducido químicamente , Colitis/genética , Sulfato de Dextran , Epitelio/inmunología , Epitelio/metabolismo , Femenino , Citometría de Flujo , Expresión Génica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/inmunología , Inmunoglobulina A/genética , Inmunoglobulina A/inmunología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , Intestinos/microbiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Permeabilidad , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/inmunología , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Inflammatory cytokines have been proposed to regulate epithelial homeostasis during intestinal inflammation. We report here that interferon-gamma (IFN-gamma) regulates the crucial homeostatic functions of cell proliferation and apoptosis through serine-threonine protein kinase AKT-beta-catenin and Wingless-Int (Wnt)-beta-catenin signaling pathways. Short-term exposure of intestinal epithelial cells to IFN-gamma resulted in activation of beta-catenin through AKT, followed by induction of the secreted Wnt inhibitor Dkk1. Consequently, we observed an increase in Dkk1-mediated apoptosis upon extended IFN-gamma treatment and reduced proliferation through depletion of the Wnt coreceptor LRP6. These effects were enhanced by tumor necrosis factor-alpha (TNF-alpha), suggesting synergism between the two cytokines. Consistent with these results, colitis in vivo was associated with decreased beta-catenin-T cell factor (TCF) signaling, loss of plasma membrane-associated LRP6, and reduced epithelial cell proliferation. Proliferation was partially restored in IFN-gamma-deficient mice. Thus, we propose that IFN-gamma regulates intestinal epithelial homeostasis by sequential regulation of converging beta-catenin signaling pathways.
Asunto(s)
Células Epiteliales/inmunología , Homeostasis , Interferón gamma/inmunología , Intestinos/inmunología , Transducción de Señal , beta Catenina/metabolismo , Animales , Apoptosis , Línea Celular , Proliferación Celular , Colitis/genética , Colitis/inmunología , Colitis/metabolismo , Colitis/patología , Células Epiteliales/citología , Células Epiteliales/metabolismo , Interferón gamma/deficiencia , Interferón gamma/metabolismo , Mucosa Intestinal/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Wnt/metabolismoRESUMEN
Expression of the tight junction protein junctional adhesion molecule-A (JAM-A) has been linked to proliferation and tumour progression. However, a direct role for JAM-A in regulating proliferative processes has not been shown. By using complementary in vivo and in vitro approaches, we demonstrate that JAM-A restricts intestinal epithelial cell (IEC) proliferation in a dimerization-dependent manner, by inhibiting Akt-dependent ß-catenin activation. Furthermore, IECs from transgenic JAM-A(-/-)/ß-catenin/T-cell factor reporter mice showed enhanced ß-catenin-dependent transcription. Finally, inhibition of Akt reversed colonic crypt hyperproliferation in JAM-A-deficient mice. These data establish a new link between JAM-A and IEC homeostasis.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Células Epiteliales/citología , Células Epiteliales/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores de Superficie Celular/metabolismo , Transducción de Señal/fisiología , beta Catenina/metabolismo , Animales , Moléculas de Adhesión Celular/genética , Línea Celular , Proliferación Celular/efectos de los fármacos , Humanos , Immunoblotting , Ratones , Ratones Mutantes , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Receptores de Superficie Celular/genética , Ribonucleósidos/farmacología , Transducción de Señal/genética , Uniones Estrechas/genética , Uniones Estrechas/metabolismo , beta Catenina/genéticaRESUMEN
BACKGROUND & AIMS: Dkk1 is a secreted antagonist of the Wnt/ß-catenin signaling pathway. It is induced by inflammatory cytokines during colitis and exacerbates tissue damage by promoting apoptosis of epithelial cells. However, little is known about the physiologic role of Dkk1 in normal intestinal homeostasis and during wound repair following mucosal injury. We investigated whether inhibition of Dkk1 affects the morphology and function of the adult intestine. METHODS: We used doubleridge mice (Dkk1d/d), which have reduced expression of Dkk1, and an inhibitory Dkk1 antibody to modulate Wnt/ß-catenin signaling in the intestine. Intestinal inflammation was induced with dextran sulfate sodium (DSS), followed by a recovery period in which mice were given regular drinking water. Animals were killed before, during, or after DSS administration; epithelial homeostasis and the activity of major signaling pathways were investigated by morphometric analysis, bromo-2'-deoxyuridine incorporation, and immunostaining. RESULTS: Reduced expression of Dkk1 increased proliferation of epithelial cells and lengthened crypts in the large intestine, which was associated with increased transcriptional activity of ß-catenin. Crypt extension was particularly striking when Dkk1 was inhibited during acute colitis. Dkk1d/d mice recovered significantly faster from intestinal inflammation but exhibited crypt architectural irregularities and epithelial hyperproliferation compared with wild-type mice. Survival signaling pathways were concurrently up-regulated in Dkk1d/d mice, including the AKT/ß-catenin, ERK/Elk-1, and c-Jun pathways. CONCLUSIONS: Dkk1, an antagonist of Wnt/ß-catenin signaling, regulates intestinal epithelial homeostasis under physiologic conditions and during inflammation. Depletion of Dkk1 induces a strong proliferative response that promotes wound repair after colitis.
Asunto(s)
Colitis/metabolismo , Colon/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Mucosa Intestinal/metabolismo , Proteínas Wnt/metabolismo , Cicatrización de Heridas , Enfermedad Aguda , Animales , Anticuerpos Monoclonales/administración & dosificación , Azoximetano , Proliferación Celular , Células Cultivadas , Colitis/inducido químicamente , Colitis/genética , Colitis/patología , Colon/patología , Neoplasias Colorrectales/inducido químicamente , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Sulfato de Dextran , Modelos Animales de Enfermedad , Regulación hacia Abajo , Homeostasis , Inyecciones Intraperitoneales , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/inmunología , Mucosa Intestinal/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Transducción de Señal , Factores de Tiempo , Proteínas Wnt/antagonistas & inhibidores , beta Catenina/metabolismoRESUMEN
Intestinal homeostasis is regulated in-part by reactive oxygen species (ROS) that are generated in the colonic mucosa following contact with certain lactobacilli. Mechanistically, ROS can modulate protein function through the oxidation of cysteine residues within proteins. Recent advances in cysteine labeling by the Isotope Coded Affinity Tags (ICATs) technique has facilitated the identification of cysteine thiol modifications in response to stimuli. Here, we used ICATs to map the redox protein network oxidized upon initial contact of the colonic mucosa with Lactobacillus rhamnosus GG (LGG). We detected significant LGG-specific redox changes in over 450 proteins, many of which are implicated to function in cellular processes such as endosomal trafficking, epithelial cell junctions, barrier integrity, and cytoskeleton maintenance and formation. We particularly noted the LGG-specific oxidation of Rac1, which is a pleiotropic regulator of many cellular processes. Together, these data reveal new insights into lactobacilli-induced and redox-dependent networks involved in intestinal homeostasis.