Knockdown of laminin α5 stimulates intestinal cell differentiation.

Interactions between cells and the extracellular matrix regulate a wide range of cell processes such as proliferation and differentiation. Laminins are major components of the basement membrane that actively participate in most biological functions via their interactions with a variety of specific cell receptors. The α5-containing laminins (LAMA5) are one of the three main types of laminins identified at the epithelial basal lamina in the adult intestine. The aim of the present study was to investigate the role of α5-containing laminins on intestinal cell proliferation and differentiation. Using an shRNA targeting approach, the effects of knocking down the expression of LAMA5 were investigated in the enterocytic-like Caco-2/15 cell line, a well-characterized model for intestinal cell differentiation. Surprisingly, the abolition of the laminin α5 chain resulted in a drastic increase in the differentiation marker sucrase-isomaltase which was correctly expressed at the apical pole of the cells as observed by indirect immunofluorescence. Transient increases of dipeptidylpeptidase IV, villin, CDX2, HNF-1α, HNF-4α and transepithelial resistance as well as an apparent redistribution of the junctional components ZO-1 and E-cadherin were also observed at early stages of differentiation but no specific effect was observed on cell proliferation as evaluated by BrdU incorporation. Taken together, these data suggest that α5-containing laminins repress intestinal differentiation in its early stages.

Src family kinase inhibitor PP2 accelerates differentiation in human intestinal epithelial cells.

The proto-oncogene Src is an important protein tyrosine kinase involved in signaling pathways that control cell adhesion, growth, migration and survival. Here, we investigated the involvement of Src family kinases (SFKs) in human intestinal cell differentiation. We first observed that Src activity peaked in early stages of Caco-2/15 cell differentiation. Inhibition of SFKs with PP2, a selective SFK inhibitor, accelerated the overall differentiation program. Interestingly, all polarization and terminal differentiation markers tested, including sucrase-isomaltase, lactase-phlorizin hydrolase and E and Li-cadherins were found to be significantly up-regulated after only 3 days of treatment in the newly differentiating cells. Further investigation of the effects of PP2 revealed a significant up-regulation of the two main intestinal epithelial cell-specific transcription factors Cdx2 and HNF1α and a reduction of polycomb PRC2-related epigenetic repressing activity as measured by a decrease in H3K27me3, two events closely related to the control of cell terminal differentiation in the intestine. Taken together, these data suggest that SFKs play a key role in the control of intestinal epithelial cell terminal differentiation.

Polycomb repressive complex 2 impedes intestinal cell terminal differentiation.

The crypt-villus axis constitutes the functional unit of the small intestine, where mature absorptive cells are confined to the villi, and stem cells and transit amplifying and differentiating cells are restricted to the crypts. The polycomb group (PcG) proteins repress differentiation and promote self-renewal in embryonic stem cells. PcGs prevent transcriptional activity by catalysing epigenetic modifications, such as the covalent addition of methyl groups on histone tails, through the action of the polycomb repressive complex 2 (PRC2). Although a role for PcGs in the preservation of stemness characteristics is now well established, recent evidence suggests that they may also be involved in the regulation of differentiation. Using intestinal epithelial cell models that recapitulate the enterocytic differentiation programme, we generated a RNAi-mediated stable knockdown of SUZ12, which constitutes a cornerstone for PRC2 assembly and functionality, in order to analyse intestinal cell proliferation and differentiation. Expression of SUZ12 was also investigated in human intestinal tissues, revealing the presence of SUZ12 in most proliferative epithelial cells of the crypt and an increase in its expression in colorectal cancers. Moreover, PRC2 disruption led to a significant precocious expression of a number of terminal differentiation markers in intestinal cell models. Taken together, our data identified a mechanism whereby PcG proteins participate in the repression of the enterocytic differentiation program, and suggest that a similar mechanism exists in situ to slow down terminal differentiation in the transit amplifying cell population.