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Abstract

Introduction: Epidermal growth factor receptor (EGFR) expression and signaling can induce cellular protection after intestinal inflammation. L-glutamine (GLN) is known to prevent apoptosis after intestinal injury (e.g. heat stress (HS)) by activating mitogen activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3-K)/ Akt pathways. Hypothesis: We assessed if EGFR expression and signaling are essential in GLN’s cell protective mechanism in intestinal epithelial-6 (IEC-6) cells after HS, and investigated whether PI-3K and MAPK pathways are involved in GLN-mediated EGFR signaling. Methods: GLN-starved IEC-6 cells were treated with GLN with/ without EGFR siRNA (20nM), EGFR tyrosine kinase inhibitor AG1478 (20µM), ERK1/2 inhibitor PD98059 (50µM), p38MAPK inhibitor SB203580 (1µM), or PI3-K/ Akt inhibitor LY294002 (25µM). GLN-mediated cell survival was measured via MTS assay 24h post-HS (44°Cx50min). Total, cytoplasmatic and nuclear levels of EGFR were determined via Western blot under basal and stressed conditions (37°Cx45min; 43°Cx45min). Furthermore, phosphorylated and/or total levels of cleaved caspase-3, PARP1, ERK1/2, p38 MAPK, and Akt were assessed by Western blots under basal conditions (37°Cx45min) and non-lethal HS (43°Cx45min). Statistics were calculated via student’s t-test. Results: Here, we show that HS attenuated total, cytoplasmatic, and nuclear EGFR levels in IEC-6 cells. GLN prevented the decrease of total, cytoplasmatic and nuclear EGFR levels, which attenuated apoptosis, shown via siEGFR. Furthermore, EGFR kinase activity inhibition by AG1478, ERK1/2 kinase inhibition by PD98059 and PI-3 kinase inhibitor LY294002 lessened GLN’s protective effect, but not p38MAPK inhibitor SB203580. AG1478 attenuated GLN-mediated increases in ERK1/2 and decreases in p38MAPK phosphorylation. However, AG1478 had no effect on GLN-mediated augmentations in Akt phosphorylation. Conclusions: In summary, not only EGFR expression was important in GLN’s protective mechanism, but also GLN’s activation of EGFR tyrosine kinase activity. GLN-mediated EGFR signaling activated ERK1/2 and decreased p38MAPK signaling. However, GLN-mediated Akt phosphorylation after HS seems not to be activated via EGFR signaling.