Session: SAT 1-25-Glucocorticoid Actions & HPA Axis
Poster Board SAT-19
Aims: To develop an in vitro model for evaluation of the GRβ-related transcriptional regulation relevant in the development of steroid resistance.
Material and Methods: Caco-2 intestinal cell line was used for developing of the GRß stable expressing cell line (Caco-Grβ). GRß was cloned form the GRa isoform. Clonal selection was performed with neomycin treatment. The GRβ expression was confirmed by quantitative real-time PCR and Western blot analysis. The glucocorticoid-mediated signaling transduction pathway was evaluated using pGRE-SEAP (Clontech) system after treatment of the basic Caco-2 and Caco-GRβ cell lines with dexamethasone (Dex, 100 nmol). Gene expression profiles of both the basic Caco-2 and the Caco-2-GRß were evaluated using Agilent44K cDNA microarrays under basal condition and after Dex treatment. Pathways affected by differentially expressed genes were evaluated by Ingenuity pathway analysis (IPA).
Results: The GRα/GRβ ratios on the mRNA level were 1/0.6 and 1/0.001 in Caco-2GRβ cells and wild type Caco-2 cells, respectively. Luciferase activity measured after Dex treatment in Caco-GRβ cell lines was approximately 50% of that measured in basic Caco-2 cells. Dex treatment affected the expression of 151 transcripts (88 up and 63 down) in basic Caco-2 and only 16 transcripts in Caco-GRβ cells. 1182 transcripts were differentially expressed (279 under- and 903 overexpressed) in Caco-2-GRß cells compared to the basic Caco-2 cell line. IPA revealed that these transcripts are involved in the following pathways: “cell death and survival”, “cell morphology”, “cancer”, “cell-to-cell signaling”, “connective tissue disorders”, “cell mediated immune response”, “gastrointestinal and endocrine and immunological diseases”.
Conclusions: In Caco-2 cells, similarly to HeLa cells, GRβ may posses a dominant-negative effect of GRα mediated gene transcription. Therefore, Caco-2GRβ cell line may be a useful model for studying the pathomechanism of steroid resistance in inflammatory bowel disease. Forced expression of GRβ may affect the sort of cells through GR-independent mechanisms.
Nothing to Disclose: BT, IL, KF, HB, KR, AP
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