Abstract
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.
Highlights
Intestinal epithelial cells form a critical mucosal barrier between the host’s internal milieu and the external environment and serve as an integral component of the mucosal immune system [1]
Induction of Haptoglobin after Inflammation—We have previously shown that the acute phase protein gene haptoglobin was expressed and regulated in IEC-6 cells [7, 8, 16]
Effect of C/EBP Isoform Expression on C/EBP Endogenous Protein Levels—To determine the role of specific C/EBP isoforms in intestinal epithelial cells, we isolated different cell clones expressing various C/EBP isoforms after retroviral infection and puromycin selection. 10 clones were isolated for each infection
Summary
Intestinal epithelial cells form a critical mucosal barrier between the host’s internal milieu and the external environment and serve as an integral component of the mucosal immune system [1]. The CCAAT/enhancer-binding protein family of transcription factors is one class of transactivators involved in the regulation of APP expression in various tissues, including the intestine [9] These genes belong to a class of transcription factors with adjacent leucine zipper dimerization and basic DNA-binding domains that enable the various C/EBP isoforms (␣, , ␦, ␥, ⑀, gadd153/CHOP) to form homo- or heterodimers [10]. Role of Specific CCAAT/Enhancer-binding Protein Isoforms expression of specific C/EBP isoforms or to inhibit C/EBP activity in the rat intestinal epithelial cell line IEC-6 and to assess the phenotype of these cell lines according to the regulation of the acute phase response as well as of cell proliferation
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