Beta-catenin/TCF4 transactivates miR-30e during intestinal cell differentiation
Y. Liao
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B. Lo nnerdal
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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.
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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)