A 14-day Dexamethasone timecourse exposure in Caco-2 monolayers results in differential expression of tight-junction and cytoskeleton regulatory pathway genes

Abstract

Glucocorticoid (GC) hormones are modulators of endogenous stress responses and are important pharmaceuticals for inflammatory and autoimmune diseases. The gastrointestinal epithelium is a significant tissue target of Glucocorticoids; perturbation of epithelial barrier function during the endogenous stress response plays a major role in the pathophysiology of inflammatory bowel disease. Epithelial permeability and barrier function are mediated by the tight junction protein complex, with a network of molecular/cellular interactions occurring between the actin cytoskeleton, RhoGTPase, Akt/PI3K and growth factor receptor, and inflammatory cytokine signaling, influencing the establishment of a partial EMT phenotype. To improve our understanding of GC-responsive gene expression in a gastrointestinal epithelial context, we tested polarized Caco-2 monolayer cultures during at 30-day timecourse, with 15-days of continuous Dexamethasone exposure. Trans-epithelial resistance (TEER) was recorded to provide a physiological quantification of barrier function during the timecourse treatments. Presence of intracellular glucocorticoid hormone activates the human glucocorticoid receptor (GCR, human NR3C1 gene) transcription factor, resulting in transcriptional activation and repression of various GCR-responsive genes. We tested for differential gene expression with a multiplexed panel of 250 gene expression panel using the Nanostring nCounter(R) system. Gene panel selection was based on membership of genes in canonical KEGG pathways for tight-junction, adherens junction, focal adhesion, actin cytoskeleton regulation, and colorectal cancer. Our TEER results confirm, as previously reported, that long-term Dexamethasone exposure results in decreased permeability in Caco-2 monolayers ~day 20-25. Culture age and Dexamethasone exposure both contributed to differential gene expression for cell-cell junction, protein kinases, survival and cancer associated genes, reported here in the context of their corresponding KEGG pathway representations. The findings give evidence for GC and time-associated patterns of transcriptional response and provide further insight into long-term glucocorticoid-associated physiological effects on the gastrointestinal epithelia. Future research utilizing more advanced cell culture methods will use this data-set as a reference.