Glucocorticoid receptor signalling and the effect of interleukin 1 beta on glucocorticoid mediated gene expression in intestinal epithelial cell lines Caco-2 and IEC-6
von Szilvia Toth
Datum der mündl. Prüfung:2000-10-31
Betreuer:Prof. Dr. Dr. h.c. Giuliano Ramadori
Gutachter:Prof. Dr. Rüdiger Hardeland
Gutachter:Prof. Dr. Kurt Jungermann
EnglischBackground and aims: It has been reported that epithelial cells are involved in the process of inflammatory bowel disease (IBD) producing proinflammatory cytokines and chemokines. Glucocorticoids, being the most effective drugs for IBD patients, seem to have influence not only on the infiltrated inflammatory cells but also on epithelial cells. It is a clinical observation that about 20 % of IBD patients are resistant to glucocorticoids and requires alternative treatment. Since there are only few data regarding influence of glucocorticoids on intestinal epithelial cells, the aim of this work was to characterize the glucocorticoid receptor signalling pathway in the human colon carcinoma cell line Caco-2 and rat intestinal cell line IEC-6. The second aim of this study was to test the hypothesis that IL-1b, a major cytokine in IBD may inhibit glucocorticoid receptor action. Methods: The subcellular distribution of glucocorticoid receptor was studied by Western blot and immunostaining procedures using antibodies against rat and human GR. Its quantity was determined by radiobinding assay in both cell lines. The regulatory (activating and repressing) effects of dexamethasone on gene transcription in presence or absence of IL-1b were analyzed by reporter gene assay using either glucocorticoid receptor- (pGRE-SEAP) or nuclear factor kappa B responsive element (pNF-kB-SEAP) carrying constructs. The effect of IL-1b on receptor translocation was investigated by immunostaining. The repressing effect on gene transcription was further investigated by RT-PCR for TNFa, a cytokine typically down-regulated by glucocorticoids. In Caco-2 GR quantity was increased by the GR overexpression vector pRShGRa. Results: The subcellular distribution of GR was found to be different in the human and rat cells. In Caco-2 we observed mostly nuclear staining in both Dex-treated and not treated cells, in IEC-6 the unliganded form of the receptor was located in the cytoplasm, incubation with Dex resulted its translocation to the nucleus. IL-1b could repress this process. The two cell lines were different regarding their receptor amount; we detected significant more specific binding sites in IEC-6 than in Caco-2. Dexamethasone induced GR mediated transcriptional activity of the reporter gene in dose-dependent manner, and this process could be repressed by IL-1b in both cell lines. We detected only functional inhibition of GR, IL-1b did not change GR quantity apparently. The proinflammatory cytokine IL-1b could increase NF-kB mediated transcription in each cell line. Glucocorticoids could inhibit IL-1b induced NF-kB activation only in IEC-6. IL-1b increased TNFa-mRNA expression, too. Dex had repressive effect only in case of IEC-6 but not in Caco-2. Overexpressing GR using pRShGRa vector, both transactivation and transrepression by Dex could be restored in Caco-2. Blocking the NF-kB-pathway by a dominant negative IkB vector could not restore glucocorticoid receptor action disturbed by IL-1b. Conclusions: Our data suggest that proinflammatory cytokines may influence the effect of corticosteroids in intestinal epithelial cells. The quantity of glucocorticoid receptor is crucial for responsiveness. The receptor number under a certain threshold may render cells unresponsive with respect to transrepressive effects, while transactivation is intact. IL-1b inhibits GR signalling. This inhibition seems to be independent on NF-kB activation since blocking the NF-kB pathway by IkB overexpression does not counteract the IL-1b effects on glucocorticoid receptor action.
Keywords: glucocorticoid receptor action; interleukin 1 beta; nuclear factor kappa B