Beta-catenin/TCF4 transactivates miR-30e during intestinal cell differentiation

Cellular and Molecular Life Sciences, Sep 2010

The Wnt/beta-catenin/TCF4 pathway plays critical roles in the maintenance of small intestinal epithelium; however, downstream targets of the beta-catenin/TCF4 complex are not extensively characterized. We identified miR-30e as an immediate target activated by the beta-catenin/TCF4 complex. miR-30e was detected in the peri-nuclear region of the intestinal crypt IEC-6 cells. Bioinformatics analysis revealed clustered beta-catenin/TCF4 binding sites within the miR-30e promoter region. This promoter region was cloned into pGL3-control luciferase reporter vector, with the enhancer region removed. Transfection of pCMV-SPORT6-beta-catenin expression vector dose-dependently increased luciferase activity, and co-transfection of pCMV-SPORT6-TCF4 expression vector further enhanced the promoter activity. Dexamethasone-induced IEC-6 cells differentiation caused a 2.5-fold increase in miR-30e expression, and upon beta-catenin siRNA transfection, miR-30e increased 1.3-fold. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay confirmed the binding between beta-catenin/TCF4 complexes from IEC-6 nuclear extracts and the putative sequences in the miR-30e promoter. These results demonstrate that beta-catenin/TCF4 transactivates miR-30e during intestinal cell differentiation.

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Beta-catenin/TCF4 transactivates miR-30e during intestinal cell differentiation

Y. Liao 0 B. Lo nnerdal 0 0 Y. Liao B. Lonnerdal (&) Department of Nutrition, University of California , One Shields Ave., Davis, CA 95616, USA The Wnt/beta-catenin/TCF4 pathway plays critical roles in the maintenance of small intestinal epithelium; however, downstream targets of the beta-catenin/ TCF4 complex are not extensively characterized. We identified miR-30e as an immediate target activated by the beta-catenin/TCF4 complex. miR-30e was detected in the peri-nuclear region of the intestinal crypt IEC-6 cells. Bioinformatics analysis revealed clustered beta-catenin/ TCF4 binding sites within the miR-30e promoter region. This promoter region was cloned into pGL3-control luciferase reporter vector, with the enhancer region removed. Transfection of pCMV-SPORT6-beta-catenin expression vector dose-dependently increased luciferase activity, and co-transfection of pCMV-SPORT6-TCF4 expression vector further enhanced the promoter activity. Dexamethasoneinduced IEC-6 cells differentiation caused a 2.5-fold increase in miR-30e expression, and upon beta-catenin siRNA transfection, miR-30e increased 1.3-fold. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay confirmed the binding between beta-catenin/TCF4 complexes from IEC-6 nuclear extracts and the putative sequences in the miR-30e promoter. These results demonstrate that beta-catenin/TCF4 transactivates miR-30e during intestinal cell differentiation. - The small intestine has two functionally distinct compartments, villi and crypts of Lieberkuhn (crypts). Villi are finger-like protrusions extending to the intestinal lumen, and contain three types of terminally differentiated cells: (1) enterocytes are the only absorptive cells, whose function is to absorb nutrients from food sources, and comprise the majority of the villi; (2) enteroendocrine cells are local endocrine cells that secret gastrointestinal hormones, such as somatostatin, cholecystokinin and glucagon; and (3) goblet cells which have the sole function to secret mucus, protecting the intestinal barrier from mechanical damage. The crypts contain two types of cells: (1) Paneth cells are also terminally differentiated, and secrete anti-microbial peptides in response to pathogens; and (2) undifferentiated stem cells which reside within crypts, and undergo self-renewal, proliferation, and eventually differentiate into all the above-mentioned terminally differentiated cells [13]. The structural and functional integrity of the small intestine is largely dependent upon a continuous and well-organized flow of fully differentiated cell lineages migrating from the crypts. However, the molecular mechanisms by which the individual cell lineages are derived from the crypts await full characterization. A number of cellular signaling pathways have been suggested to be involved in small intestinal cell proliferation and differentiation, among which the Delta-Notch pathway and Wnt/beta-catenin/T cell factor 4 (TCF4) pathway have drawn most attention [35]. Activation of the highly conserved Delta-Notch signaling pathway is usually considered to maintain the proliferative state of the intestinal progenitor cells, and lead to columnar cell lineages rather than secretory cells [610]; The Wnt/beta-catenin/TCF4 signaling pathway instead plays a key role in maintaining progenitor cells in the proliferative state and making them select towards secretory cells [1115]. Decreased Wnt signaling decreases the size of crypts [12], and aberrant upregulation of the Wnt signaling is associated with local tumorigenesis [16, 17]. However, the molecular determination of individual terminally differentiated cell lineages does not rely solely on either Notch or Wnt signaling but on the coordinate interaction of the two [3, 18, 19]. Wnt signal activates three independent pathways, the canonical Wnt/beta-catenin cascade, the noncanonical planar cell polarity pathway, and the Wnt/Ca2? pathway. The intestinal crypts express members of the canonical Wnt/beta-catenin pathway [20]. The locally expressed Wnt molecule binds to the surface receptor frizzled, and stabilizes cytosolic transcriptional factor beta-catenin from degradation. Subsequently, downstream functional outcomes are elicited by the translocation of beta-catenin to the nucleus, where it forms a nuclear binding complex with TCF4, a member of T-cell factors and specifically expressed in the small intestine, and the complex subsequently regulates transcription of a variety of genes. The end effect of Wnt signaling is a result of gene transactivation by the nuclear beta-catenin/TCF4 complex. To date, genes in the intestine that have been found to be downstream targets of beta-catenin/TCF4 transactivation include PKC [21], p16(INK4a) [22], S100A4 [23], Rad6B [24], c-myc [25], cyclin D1 [26], MMP-7 [27], PPARd [28], and cohesion SMC3 [29], which are mostly involved in tumorigenesis. The downstream targets of beta-catenin/ TCF4 binding complex responsible fo (...truncated)


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Y. Liao, B. Lönnerdal. Beta-catenin/TCF4 transactivates miR-30e during intestinal cell differentiation, Cellular and Molecular Life Sciences, 2010, pp. 2969-2978, Volume 67, Issue 17, DOI: 10.1007/s00018-010-0366-y