The signaling pathways mediating human intestinal epithelial cell differentiation remain largely undefined. Phosphatidylinositol 3-kinase (PI3K) is an important modulator of extracellular signals, including those elicited by E-cadherin-mediated cell-cell adhesion, which plays an important role in maintenance of the structural and functional integrity of epithelia. In this study, we analyzed the involvement of PI3K in the differentiation of human intestinal epithelial cells. We showed that inhibition of PI3K signaling in Caco-2/15 cells repressed sucrase-isomaltase and villin protein expression. Morphological differentiation of enterocyte-like features in Caco-2/15 cells such as epithelial cell polarity and brushborder formation were strongly attenuated by PI3K inhibition. Immunofluorescence and immunoprecipitation experiments revealed that PI3K was recruited to and activated by E-cadherin-mediated cell-cell contacts in confluent Caco-2/15 cells, and this activation appears to be essential for the integrity of adherens junctions and association with the cytoskeleton. We provide evidence that the assembly of calcium-dependent adherens junctions led to a rapid and remarkable increase in the state of activation of Akt and p38 MAPK pathways and that this increase was blocked in the presence of anti-E-cadherin antibodies and PI3K inhibitor. Therefore, our results indicate that PI3K promotes assembly of adherens junctions, which, in turn, control p38 MAPK activation and enterocyte differentiation.

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Progression of eukaryotic cells through the cell cycle is governed by the sequential formation, activation, and subsequent inactivation of a series of cyclin-dependent kinase (Cdk) complexes. p27Kip1 (p27) is a Cdk inhibitor that blocks, in vitro, the activity of cyclin D-Cdk4, cyclin D-Cdk6, cyclin E-Cdk2 as well as cyclin A-Cdk2, a complex active during S phase. The level of p27 protein expression, usually high in G 0 /G 1 resting cells, declines as cells progress toward S phase and enforced expression of p27 in fibroblasts causes G 1 arrest. This situation prevails in CCL39, a Chinese hamster lung fibroblast cell line (this report). However, in addition to p27, several other Cdk inhibitors known to alter G 1 progression coexist in most mammalian cells. To investigate the specific contribution of p27 in the control of the mitogensensitive G 0 /G 1 arrest, we specifically reduced its synthesis by expressing a full-length p27 antisense cDNA in CCL39 cells. Interestingly, reduction of up to 90% of p27 protein expression increased both basal and serumstimulated gene transcription of cyclin D1, cyclin A, dihydrofolate reductase, and DNA synthesis reinitiation. Moreover, overexpression of this antisense allows cells to grow for several generations in a serum-free medium supplemented with insulin and transferrin only, thus suggesting that p27-depleted cells cannot exit the cell cycle. These effects were fully reversed by coexpression of a plasmid encoding p27 sense. We conclude that p27, by setting the level of growth factor requirement, plays a pivotal role in controlling cell cycle exit, a fundamental step in growth control.