No abstract

By having demonstrated previously that p27Kip1 , a potent inhibitor of G 1 cyclin-cyclin-dependent kinases complexes, increases markedly during intestinal epithelial cell differentiation, we examined the effect of p27Kip1 on the activity of the transcription factor CDX2. The present results revealed the following. 1) p27 Kip1 interacts with the CDX2 transcription factor. 2) In contrast to CDX2 mRNA levels, CDX2 protein expression levels significantly increased as soon as Caco-2/15 cells reached confluence, slowed their proliferation, and began their differentiation. The mechanism of CDX2 regulation is primarily related to protein stability, because inhibition of proteasome activity increased CDX2 levels.

Normal cellular models able to efficiently recapitulate intestinal epithelial cell differentiation in culture are not yet available. The aim of this work was to establish and genetically characterize a mesenchymal-epithelial coculture system to identify transcriptional regulators involved in this process. The deposition of rat intestinal epithelial cells on human intestinal mesenchymal cells led to the formation of clustered structures that expanded shortly after seeding. These structures were composed of polarized epithelial cells with brush borders and cell junction complexes. A rat GeneChip statistical analysis performed at different time points during this process identified hepatocyte nuclear factor-4α (HNF-4α) and hepatocyte nuclear factor-1α (HNF-1α) as being induced coincidently with the apparition of polarized epithelial structures. Stable introduction of HNF-4α in undifferentiated epithelial cells alone led to the rapid induction of HNF-1α and several intestinal-specific markers and metabolism-related genes for which mRNA was identified to be upregulated during epithelial differentiation. HNF-4α was capable to transactivate the calbindin 3 gene promoter, a process that was synergistically increased in the presence of HNF-1α. When HNF-4α-expressing cells were plated on mesenchymal cells, an epithelial monolayer formed rapidly with the apparition of dome structures that are characteristics of vectorial ion transport. Forced expression of HNF-1α alone did not result in dome structures formation. In sum, this novel coculture system functionally identified for the first time HNF-4α as an important modulator of intestinal epithelial differentiation and offers an innovative opportunity to investigate molecular mechanisms involved in this process.