Role of specific CCAAT/enhancer-binding protein isoforms in intestinal epithelial cells.

Intestinal epithelial cells participate in the acute phase response in response to inflammation. We have shown that acute phase protein genes are induced during intestinal acute phase response, and that the CCAAT/enhancer binding protein family of transcription factors are involved. To address the role of specific C/EBP isoforms, we generated IEC-6 rat intestinal epithelial cell lines expressing different C/EBP isoforms, by retroviral infection. Overexpression of C/EBPalpha p30 and C/EBPdelta led to increases in C/EBPbeta LAP and C/EBPbeta LIP endogenous protein levels, as determined by electrophoretic mobility shift assays and Western blot. Inhibition of C/EBP activity with dominant negative C/EBPs (C/EBPbeta LIP, 3hF, 4hF) decreased glucocorticoid-, cAMP- and IL-1 responsiveness of the endogenous haptoglobin gene, while overexpression of each C/EBP isoform increased the responsiveness to these regulators. In contrast, dominant negative C/EBPs or C/EBP isoforms did not alter the expression of alpha-acid glycoprotein in response to dexamethasone and of C/EBPbeta and C/EBPdelta in response to various regulators as assessed by Northern blot. These data show that the three C/EBP isoforms are involved in the regulation of haptoglobin and that C/EBPbeta, C/EBPdelta, and alpha-acid glycoprotein expression are not induced by C/EBP isoforms in contrast to other cell types. C/EBPbeta LAP-expressing cells showed an inhibition of cell growth characterized by a delay in p27(Kip1) decrease in response to serum and a decrease in cyclin D isoforms and cyclin E protein levels. Finally, C/EBP isoforms interact with the E2F4 transcription factor. Thus, specific C/EBP isoforms are involved in the differential expression of acute phase protein genes in response to hormones and cytokines. Furthermore, C/EBP isoforms may play a role in the control of cell cycle progression.

Role of p27(Kip1) in human intestinal cell differentiation.

BACKGROUND & AIMS: Growth arrest and differentiation are generally considered to be temporally and functionally linked phenomena in the intestinal epithelium. METHODS: To delineate the mechanism(s) responsible for the loss of proliferative potential as committed intestinal cells start to differentiate, we have analyzed the regulation of G(1)-phase regulatory proteins in relation to differentiation in the intact epithelium as well as in well-established intestinal cell models that allow the recapitulation of the crypt-villus axis in vitro. RESULTS: With intestinal cell differentiation, we have observed an induction of the cell cycle inhibitors p21(Cip), p27(Kip1), and p57(Kip2) expression with an increased association of p27(Kip1) and p57(Kip2) with cyclin-dependent kinase 2 (Cdk2). At the same time, there was an accumulation of the hypophosphorylated form of the pRb proteins and a strong decline in Cdk2 activity. Stable expression of a p27(Kip1) antisense complementary DNA in Caco-2/15 cells did not prevent growth arrest induced by confluence, but repressed villin, sucrase-isomaltase, and alkaline phosphatase expression. CONCLUSIONS: Our results indicate that the growth arrest that precedes differentiation involves the activation of Rb proteins and the inhibition of Cdk2. Furthermore, intestinal cell differentiation apparently requires a function of p27(Kip1) other than that which leads to inhibition of Cdks.

Requirement of the MAP kinase cascade for cell cycle progression and differentiation of human intestinal cells.

The intracellular signaling pathways responsible for cell cycle arrest and establishment of differentiated cells along the gut axis remain largely unknown. In the present study, we analyzed the regulation of p42/p44 mitogen-activated protein kinase (MAPK) in the process of proliferation and differentiation of human intestinal cells. In vitro studies were done in Caco-2/15 cells, a human colon cancer cell line that spontaneously differentiates into an enterocyte phenotype. In vivo studies were performed on cryostat sections of human fetal intestinal epithelium by indirect immunofluorescence. We found that inhibition of the p42/p44 MAPK signaling by the PD-98059 compound or by ectopic expression of the MAPK phosphatase-1 strongly attenuated E2F-dependent transcriptional activity in Caco-2/15 cells. p42/p44 MAPK activities dramatically decreased as soon as Caco-2/15 cells reached confluence. However, significant levels of activated p42 MAPK were detected in differentiated Caco-2/15 cells. Addition of PD-98059 during differentiation interfered with sustained activation of p42 MAPK and sucrase-isomaltase expression. Although p42/p44 MAPKs were expressed in both the villus tip and crypt cells, their phosphorylated and active forms were detected in the undifferentiated crypt cells. Our results indicate that elevated p42/p44 MAPK activities stimulate cell proliferation of intestinal cells, whereas low sustained levels of MAPK activities correlated with G1 arrest and increased expression of sucrase-isomaltase.