Hepatocyte nuclear factor 4alpha (HNF4alpha) is a regulator of hepatocyte and pancreatic transcription. Hnf4alpha deletion in the mouse is embryonically lethal with severe defects in visceral endoderm formation. It has been concluded in the past that the role of Hnf4alpha in the developing colon was much less important than in the liver. However, the precise role of Hnf4alpha in the homeostasis of the small intestinal epithelium remains unclear. Our aim was to evaluate the potential of Hnf4alpha to support an intestinal epithelial phenotype. First, Hnf4alpha potential to dictate this phenotype was assessed in nonintestinal cell lines in vitro. Forced expression of Hnf4alpha in fibroblasts showed an induction of features normally restricted to epithelial cells. Combinatory expression of Hnf4alpha with specific transcriptional regulators of the intestine resulted in the induction of intestinal epithelial genes in this context. Second, the importance of Hnf4alpha in maintaining the homeostasis of the intestinal epithelium was investigated in mice. Mice conditionally deficient for intestinal Hnf4alpha developed normally throughout adulthood with an epithelium displaying normal morphological and functional structures with minor alterations. Subtle but statistical differences were observed at the proliferation and the cytodifferentiation levels. Hnf4alpha mutant mice displayed an increase in the number of goblet and enteroendocrine cells compared with controls. Given the fundamental role of this transcription factor in other tissues, these findings dispute the crucial role for this regulator in the maintenance of intestinal epithelial cell function at a period of time that follows cytodifferentiation but may suggest a functional role in instructing cells to become specific to the intestinal epithelium.

Background. Integrins are transmembrane αβ heterodimer receptors that function as structural and functional bridges between the cytoskeleton and ECM (extracellular matrix) molecules. The RGD (arginine-glycine-aspartate tripeptide motif)-dependent integrin α8β1 has been shown to be involved in various cell functions in neuronal and mesenchymal-derived cell types. Its role in epithelial cells remains unknown.Results. Integrin α8β1 was found to be expressed in the crypt cell population of the human intestine but was absent from differentiating and mature epithelial cells of the villus. The function of α8β1 in epithelial crypt cells was investigated at the cellular level using normal HIECs (human intestinal epithelial cells). Specific knockdown of α8 subunit expression using an shRNA (small-hairpin RNA) approach showed that α8β1 plays important roles in RGD-dependent cell adhesion, migration and proliferation via a RhoA/ROCK (Rho-associated kinase)-dependent mechanism as demonstrated by active RhoA quantification and pharmacological inhibition of ROCK. Moreover, loss of α8β1, through RhoA/ROCK, impairs FA (focal adhesion) complex integrity as demonstrated by faulty vinculin recruitment.Conclusions. Integrin α8β1 is expressed in epithelial cells. In intestinal crypt cells, α8β1 is closely involved in the regulation of adhesion, migration and cell proliferation via a predominant RhoA/ROCK-dependent mechanism. These results suggest an important role for this integrin in intestinal crypt cell homoeostasis.

Intestinal epithelial integrity and polarity are maintained by cohesive interactions between cells via the formation of tight junctions. Irregularities in tight junctions have only recently been found to be associated with the initiation and progression of intestinal neoplasia. The claudin family of proteins is integral to the structure and function of the tight junction but little is known of the molecular events that regulate the expression of these components. The present report identifies cathepsin L, classically a lysosomal cysteine protease, as being induced during intestinal epithelial cell polarization and differentiation. Inhibition of intracellular cathepsin L activity results in the accumulation of disorganized cell layers and a decline in the expression of differentiation markers in cultured intestinal epithelial cells. This coincides with a rapid up-regulation of claudin-1 protein accumulation. Mutant mice defective in cathepsin L activity (furless) display an elevated level of intestinal claudin-1 and claudin-2 expression. Loss of cathepsin L activity leads to a marked increase in tumor multiplicity in the intestine of Apc(Min) mice. Given the traditionally viewed biological role of cathepsin L in the processing of lysosomal content as well as in pathological extracellular matrix remodeling, the results here demonstrate an as yet unsuspected intracellular role for this protease in normal intestinal epithelial polarization and initiation of neoplasia.

A central hallmark of cancer cells is the reprogramming of cellular metabolism to meet the bioenergetic and biosynthetic demands of malignant growth. Here, we report that the miR-17∼92 microRNA (miRNA) cluster is an oncogenic driver of tumor metabolic reprogramming. Loss of miR-17∼92 in Myc(+) tumor cells leads to a global decrease in tumor cell metabolism, affecting both glycolytic and mitochondrial metabolism, whereas increased miR-17∼92 expression is sufficient to drive increased nutrient usage by tumor cells. We mapped the metabolic control element of miR-17∼92 to the miR-17 seed family, which influences cellular metabolism and mammalian target of rapamycin complex 1 (mTORC1) signaling through negative regulation of the LKB1 tumor suppressor. miR-17-dependent tuning of LKB1 levels regulates both the metabolic potential of Myc(+) lymphomas and tumor growth in vivo. Our results establish metabolic reprogramming as a central function of the oncogenic miR-17∼92 miRNA cluster that drives the progression of MYC-dependent tumors.

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.

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Fine regulation of the phosphatase and tensin homologue (PTEN) phosphatase dosage is critical for homeostasis and tumour suppression. The 3′-untranslated region (3′-UTR) of Pten mRNA was extensively linked to post-transcriptional regulation by microRNAs (miRNAs). In spite of this critical regulatory role, alternative 3′-UTRs of Pten have not been systematically characterized. Here, we reveal an important diversity of Pten mRNA isoforms generated by alternative polyadenylation sites. Several 3′-UTRs are co-expressed and their relative expression is dynamically regulated. In spite of encoding multiple validated miRNA-binding sites, longer isoforms are largely refractory to miRNA-mediated silencing, are more stable and contribute to the bulk of PTEN protein and signalling functions. Taken together, our results warrant a mechanistic re-interpretation of the post-transcriptional mechanisms involving Pten mRNAs and raise concerns on how miRNA-binding sites are being validated.

Hepatocyte nuclear factor-1α (HNF1α) is a transcription factor expressed in tissues of endoderm origin. Mutations in HNF1A are associated with maturity-onset diabetes of the young 3 (MODY3). Mice deficient for Hnf1α are hyperglycemic, with their pancreatic β-cells being defective in glucose-sensing insulin secretion. The specific mechanisms involved in this defect are unclear. Gut hormones control glucose homeostasis. Our objective was to explore whether changes in these hormones play a role in glucose homeostasis in the absence of Hnf1α. An increase in ghrelin gene transcript and a decrease in glucose-dependent insulinotropic polypeptide (GIP) gene transcripts were observed in the gut of Hnf1α-null mice. These changes correlated with an increase of ghrelin and a decrease of GIP-labeled cells. Ghrelin serological levels were significantly induced in Hnf1α-null mice. Paradoxically, GIP levels were also induced in these mice. Treatment of Hnf1α-null mice with a ghrelin antagonist led to a recovery of the diabetic symptoms. We conclude that upregulation of ghrelin in the absence of Hnf1α impairs insulin secretion and can be reversed by pharmacological inhibition of ghrelin/GHS-R interaction. These observations open up on future strategies to counteract ghrelin action in a program that could become beneficial in controlling non–insulin-dependent diabetes.