RESUMEN
Variations of protein kinase C (PKC) expression greatly influence the proliferation-to-differentiation transition (PDT) of intestinal epithelial cells and might have an important impact on intestinal tumorigenesis. We demonstrate here that the expression of PKCalpha in proliferating intestinal epithelial cells is repressed both in vitro and in vivo by the SOX9 transcription factor. This repression does not require DNA binding of the SOX9 high-mobility group (HMG) domain but is mediated through a new mechanism of SOX9 action requiring the central and highly conserved region of SOXE members. Because SOX9 expression is itself upregulated by Wnt-APC signaling in intestinal epithelial cells, the present study points out this transcription factor as a molecular link between the Wnt-APC pathway and PKCalpha. These results provide a potential explanation for the decrease of PKCalpha expression in colorectal cancers with constitutive activation of the Wnt-APC pathway.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Mucosa Intestinal/metabolismo , Proteína Quinasa C-alfa/metabolismo , Factor de Transcripción SOX9/metabolismo , Proteína de la Poliposis Adenomatosa del Colon/metabolismo , Animales , Línea Celular , Células Epiteliales/citología , Células Epiteliales/metabolismo , Humanos , Mucosa Intestinal/citología , Proteína Quinasa C-alfa/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Factor de Transcripción SOX9/genética , Transducción de Señal/fisiología , Factor de Transcripción Sp1/genética , Factor de Transcripción Sp1/metabolismo , Transcripción Genética , Proteínas Wnt/metabolismoRESUMEN
Tight junctions have recently emerged as essential signaling regulators of proliferation and differentiation in epithelial tissues. Here, we aimed to identify the factors regulating claudin-7 expression in the colon, and analyzed the consequences of claudin-7 overexpression in colorectal carcinoma (CRC). In healthy human colonic crypts, claudin-7 expression was found to be low in the stem/progenitor cell compartment, where Tcf-4 activity is high, but strong in differentiated and postmitotic cells, where Tcf-4 is inactive. In contrast, claudin-7 was overexpressed in areas with high Tcf-4 target gene levels in CRC samples. In vitro, Tcf-4 was able to repress claudin-7 expression, and the high mobility group-box transcription factor Sox-9 was identified as an essential mediator of this effect. Claudin-7 was strongly expressed in the intestine of Sox-9-deficient mice and in CRC cells with low Sox transcriptional activity. Sox-9 overexpression in these cells reinstated claudin-7 repression, and residual claudin-7 was no longer localized along the basolateral membrane, but was instead restricted to tight junctions. Using HT-29Cl.16E CRC cell spheroids, we found that Sox-9-induced polarization was completely reversed after virus-mediated claudin-7 overexpression. Claudin-7 overexpression in this context increased Tcf-4 target gene expression, proliferation, and tumorigenicity after injection in nude mice. Our results indicate that Tcf-4 maintains low levels of claudin-7 at the bottom of colonic crypts, acting via Sox-9. This negative regulation seems to be defective in CRC, possibly due to decreased Sox-9 activity, and the resulting claudin-7 overexpression promotes a loss of tumor cell polarization and contributes to tumorigenesis.
Asunto(s)
Polaridad Celular/fisiología , Neoplasias Colorrectales/patología , Proteínas del Grupo de Alta Movilidad/fisiología , Proteínas de la Membrana/genética , Factores de Transcripción TCF/fisiología , Factores de Transcripción/fisiología , Western Blotting , Línea Celular Tumoral , Claudinas , Neoplasias Colorrectales/genética , Humanos , Microscopía Confocal , Microscopía Fluorescente , ARN Interferente Pequeño , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción SOX9 , Proteína 2 Similar al Factor de Transcripción 7RESUMEN
The HMG-box transcription factor Sox9 is expressed in the intestinal epithelium, specifically, in stem/progenitor cells and in Paneth cells. Sox9 expression requires an active beta-catenin-Tcf complex, the transcriptional effector of the Wnt pathway. This pathway is critical for numerous aspects of the intestinal epithelium physiopathology, but processes that specify the cell response to such multipotential signals still remain to be identified. We inactivated the Sox9 gene in the intestinal epithelium to analyze its physiological function. Sox9 inactivation affected differentiation throughout the intestinal epithelium, with a disappearance of Paneth cells and a decrease of the goblet cell lineage. Additionally, the morphology of the colon epithelium was severely altered. We detected general hyperplasia and local crypt dysplasia in the intestine, and Wnt pathway target genes were up-regulated. These results highlight the central position of Sox9 as both a transcriptional target and a regulator of the Wnt pathway in the regulation of intestinal epithelium homeostasis.