LPS INFECTION OF INTESTINAL EPITHELIAL CELLS INDUCES EXPRESSION OF COX-2 TROUGH EPIGENETIC MECHANISMS

Introduction: LPS induces the release of COX-2 by intestinal mucosal cells, because it is now emerging that bacteria may induce alteration of epigenetic patterns in host cells, we have investigated whether LPS induced COX-2 activation in human intestinal epithelial cells (HT-29) involves changes of histone modifications and/or DNA methylation at COX-2 gene regulatory region. We hypothesized that LPS infection may have a direct impact on the epigenetic status of COX-2 gene in HT-29 cells and that LPS-induced COX-2 activation may be mediated by rapid changes at COX-2 gene regulatory regions. LPS induces epigenetic events that include both chromatin and DNA methylation modifications after COX-2 activation in HT-29 cells. Material e Methods: HT-29 treated with LPS and not were used for Relative Quantitative expression analysis in real time PCR. Quantitative ChIP analysis was performed on COX-2 promoter for histone modifications analysis. Gene silencing by RNA interference were used for determine the epigenetic mechanism of COX-2 activation LPS-dependent. DNA methylation analysis of COX-2 promoter was performed by MALDI-TOF. Results: We described the chromatin and DNA methylation changes occurring at the COX-2 gene in HT-29 cells as direct consequences of LPS exposure. LPS infection is followed by several expression, chromatin and DNA modifica- tion events including: activation of the COX-2 gene; transient gradual increase of H3K4 dimethylation and decrease of H3K9 dimethylation and H3K27 trimethylation in correspondence of the later 2h peak of COX-2 mRNA expression; rapid cyclical DNA methylation/demethylation events at specific CpG sites at COX-2 gene-regulatory region mainly in concomitance with the early expression gene. Discussion e Conclusion: We have investigated the epigenetic changes occurring at the COX-2 locus during the first 24 h after exposure of human HT29 to LPS and, thus directly attributable to host-parasite interaction. 2h of LPS exposure of HT29 cells induces the JMJD3 binding to COX-2 promoter region and following removing of H3K27me3 associated to expression of COX-2. Most of chromatin marks are then restored in a short time after LPS infection with the exception of LSD1 that no longer bind to COX-2 promoter, possibly responsible of not H3K9me2 demethylation underlying a more condensed chromatin state at the COX-2 gene. In addiction, a cyclic DNA methylation/demethilation of COX-2 promoter, observed in LPS-HT29 cells treatment, can to be associated at stable expression of gene. It will be very important to investigate whether the observed decrease of JMJD3 levels at COX-2 in response to long treatment with LPS may occur in intestinal mucosa and may represent a stable epigenetic mark during long exposition to microorganisms.