|Full text PDF:||http://hdl.handle.net/11858/00-1735-0000-0028-86AB-B|
Mycophenolic acid (MPA) is an important immunosuppressive drug (ISD) prescribed to prevent graft rejection in the organ transplanted patients. However, its use is sporadically linked to the leak flux diarrhea and other gastrointestinal (GI) disturbances in around 75% patients through an unknown mechanism. Recently, we reported increased tight junctions (TJs) permeability through p38MAPK mediated MLCK pathway in MPA treated Caco-2 cells monolayer. On the other hand, certain drugs are known to alter gene expression via epigenetic changes causing un-expected side effects. Whether MPA-induced GI complication, especially diarrhea, has any link with alteration at epigenetic level is unknown. In the present study, we investigated epigenetic changes and possible involvement of midkine dependent PI3K pathway in alteration of TJs under MPA treatment. Caco-2 cells were grown in monolayers to develop the TJs and were treated for 72 hours with MPA or MPA+PI3K inhibitors (/LY/AMG) or MPA+midkine inhibitors (iMDK) and or DMSO. ChIP-O-Proteomics was performed to identify proteins associated with active (H3K4me3) and repressive (H3K27me3) chromatin histone modifications after MPA treatment. The role of appropriate candidate(s) in the context of epithelial monolayer integrity maintenance was analyzed. Caco-2 monolayer integrity was assessed by TEER and FITC-dextran value. We identified a total of 333 and 306 proteins associated with active and repressive histone modification marks, respectively. However, a number of novel proteins have also been identified including midkine. Midkine is a growth factor which is involved in the regulation of various diseases of inflammatory background. Our further analysis identified that midkine functions as the upstream regulator and alters the expression of various genes involved in the TJs maintenance. Epigenetic analysis shows that MPA treatment increases the global histone acetylation levels as well as the enrichment for transcriptional active histone modification mark (H3K4me3) not only at the promoter region of midkine, but also at the promoter regions of midkine effector genes such as PI3K, cdx-2, claudin-2, p38MAPK, ATF-2, MLCK and MLC-2. In contrast, the promoter region of occludin and claudin-1 was enriched for transcriptional repressive histone modification mark (H3K27me3) after MPA treatment. In line with the chromatin status, MPA treatment increased the expression of midkine, PI3K, Cdx-2, claudin-2, p38MAPK, ATF-2, MLCK, and MLC-2 both at transcriptional and translational level, while occludin and claudin-1 expression were negatively influenced. Interestingly, the MPA induced gene expression changes and functional properties of Caco-2 cells could be preserved by the inhibition of midkine mediated PI3K pathway using a chemical inhibitor (PI3K inhibitors or iMDK inhibitors). Collectively, our results highlight that MPA disrupts the structure of TJs via midkine dependent activation of PI3K pathway that results in decreased integrity of Caco-2 monolayer. These results led us to suggest that… Advisors/Committee Members: Groß, Uwe (advisor), Pöggeler, Stefanie (referee), Asif, Abdul Rahman (referee), Dressel, Ralf (referee), Kramer, Wilfried (referee), Rohlfs, Marko (referee), Wimmer, Ernst A. (referee).