RESUMEN
Mutations in the human adenomatous polyposis coli (APC) gene are thought to initiate colorectal tumorigenesis. The tumor suppressor function of APC is attributed primarily to its ability to regulate the WNT pathway by targeting the destruction of beta-catenin. We report here a novel role for APC in regulating degradation of the transcriptional co-repressor C-terminal-binding protein-1 (CtBP1) through a proteasome-dependent process. Further, CtBP1 suppresses the expression of intestinal retinol dehydrogenases, which are required for retinoic acid production and intestinal differentiation. In support of a role for CtBP1 in initiation of colorectal cancer, adenomas taken from individuals with familial adenomatous polyposis contain high levels of CtBP1 protein in comparison with matched, uninvolved tissue. The relationship between APC and CtBP1 is conserved between humans and zebrafish and provides a mechanistic model explaining APC control of intestinal retinoic acid biosynthesis.
Asunto(s)
Oxidorreductasas de Alcohol/metabolismo , Proteínas de Unión al ADN/metabolismo , Genes APC , Mucosa Intestinal/metabolismo , Adenoma/genética , Adenoma/metabolismo , Poliposis Adenomatosa del Colon/genética , Poliposis Adenomatosa del Colon/metabolismo , Oxidorreductasas de Alcohol/antagonistas & inhibidores , Oxidorreductasas de Alcohol/genética , Animales , Secuencia de Bases , Línea Celular Tumoral , Neoplasias del Colon/genética , Neoplasias del Colon/metabolismo , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Humanos , Técnicas In Vitro , Modelos Biológicos , Mutación , Complejo de la Endopetidasa Proteasomal/metabolismo , ARN Interferente Pequeño/genética , Especificidad de la Especie , Tretinoina/metabolismo , Pez Cebra/embriología , Pez Cebra/genética , Pez Cebra/metabolismo , beta Catenina/antagonistas & inhibidores , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
Congenital hypertrophy/hyperplasia of the retinal pigmented epithelium is an ocular lesion found in patients harboring mutations in the adenomatous polyposis coli (APC) tumor suppressor gene. We report that Apc-deficient zebrafish display developmental abnormalities of both the lens and retina. Injection of dominant-negative Lef reduced Wnt signaling in the lens but did not rescue retinal differentiation defects. In contrast, treatment of apc mutants with all-trans retinoic acid rescued retinal differentiation defects but had no apparent effect on the lens. We identified Rdh5 as a retina-specific retinol dehydrogenase controlled by APC. Morpholino knockdown of Rdh5 phenocopied the apc mutant retinal differentiation defects and was rescued by treatment with exogenous all-trans retinoic acid. Microarray analyses of apc mutants and Rdh5 morphants revealed a profound overlap in the transcriptional profile of these embryos. These findings support a model wherein Apc serves a dual role in regulating Wnt and retinoic acid signaling within the eye and suggest retinoic acid deficiency as an explanation for APC mutation-associated retinal defects such as congenital hypertrophy/hyperplasia of the retinal pigmented epithelium.
Asunto(s)
Proteína de la Poliposis Adenomatosa del Colon/metabolismo , Ojo/embriología , Tretinoina/metabolismo , Proteínas Wnt/metabolismo , Pez Cebra/embriología , Proteína de la Poliposis Adenomatosa del Colon/deficiencia , Proteína de la Poliposis Adenomatosa del Colon/genética , Animales , Ojo/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica , Homocigoto , Microinyecciones , Datos de Secuencia Molecular , Mutación , Oligonucleótidos Antisentido/farmacología , Tretinoina/farmacología , Pez Cebra/genéticaRESUMEN
Mutations in the adenomatous polyposis coli (APC) gene result in uncontrolled proliferation of intestinal epithelial cells and are associated with the earliest stages of colorectal carcinogenesis. Cyclooxygenase-2 (COX-2) is elevated in human colorectal cancers and plays an important role in colorectal tumorigenesis; however, the mechanisms by which APC mutations result in increased COX-2 expression remain unclear. We utilized APC mutant zebrafish and human cancer cells to investigate how APC modulates COX-2 expression. We report that COX-2 is up-regulated in APC mutant zebrafish because of a deficiency in retinoic acid biosynthesis. Treatment of both APC mutant zebrafish and human carcinoma cell lines with retinoic acid significantly reduces COX-2 expression. Retinoic acid regulates COX-2 levels by a mechanism that involves participation of the transcription factor C/EBP-beta. APC mutant zebrafish express higher levels of C/EBP-beta than wild-type animals, and retinoic acid supplementation reduces C/EBP-beta expression to basal levels. Both morpholino knockdown of C/EBP-beta in APC mutant zebrafish and silencing of C/EBP-beta using small interfering RNA in HT29 colon cancer cells robustly decrease COX-2 expression. Our findings support a sequence of events in which mutations in APC result in impaired retinoic acid biosynthesis, elevated levels of C/EBP-beta, up-regulation of COX-2, increased prostaglandin E(2) accumulation, and activation of Wnt target genes.
Asunto(s)
Ciclooxigenasa 2/genética , Tretinoina/fisiología , Empalme Alternativo , Animales , Secuencia de Bases , Línea Celular , Línea Celular Tumoral , Cartilla de ADN , Dinoprostona/metabolismo , Regulación Enzimológica de la Expresión Génica , Genes APC , Humanos , Morfolinas , ARN Mensajero/genética , Proteínas Wnt/genética , Pez Cebra , beta Catenina/fisiologíaRESUMEN
Development of normal colon epithelial cells proceeds through a systematic differentiation of cells that emerge from stem cells within the base of colon crypts. Genetic mutations in the adenomatous polyposis coli (APC) gene are thought to cause colon adenoma and carcinoma formation by enhancing colonocyte proliferation and impairing differentiation. We currently have a limited understanding of the cellular mechanisms that promote colonocyte differentiation. Herein, we present evidence supporting a lack of retinoic acid biosynthesis as a mechanism contributing to the development of colon adenomas and carcinomas. Microarray and reverse transcriptase-PCR analyses revealed reduced expression of two retinoid biosynthesis genes: retinol dehydrogenase 5 (RDH5) and retinol dehydrogenase L (RDHL) in colon adenomas and carcinomas as compared with normal colon. Consistent with the adenoma and carcinomas samples, seven colon carcinoma cell lines also lacked expression of RDH5 and RDHL. Assessment of RDH enzymatic activity within these seven cell lines showed poor conversion of retinol into retinoic acid when compared with normal cells such as normal human mammary epithelial cells. Reintroduction of wild type APC into an APC-deficient colon carcinoma cell line (HT29) resulted in increased expression of RDHL without affecting RDH5. APC-mediated induction of RDHL was paralleled by increased production of retinoic acid. Investigations into the mechanism responsible for APC induction of RDHL indicated that beta-catenin fails to repress RDHL. The colon-specific transcription factor CDX2, however, activated an RDHL promoter construct and induced endogenous RDHL. Finally, the induction of RDHL by APC appears dependent on the presence of CDX2. We propose a novel role for APC and CDX2 in controlling retinoic acid biosynthesis and in promoting a retinoid-induced program of colonocyte differentiation.