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 by activating MAPK and phosphatidylinositol 3-kinase (PI3-K)/Akt pathways. However, the role of EGFR expression and signaling in GLN-mediated cellular protection in intestinal epithelial-6 (IEC-6) cells after heat stress (HS) is unknown. To address the role of EGFR in GLN-mediated protection, IEC-6 cells were treated with GLN in the presence or absence of EGFR small interfering RNA, the EGFR tyrosine kinase inhibitor AG1478, the ERK1/2 inhibitor PD98059, the p38MAPK inhibitor SB203580, or the PI3-K/Akt inhibitor LY294002 under basal and HS conditions. GLN-mediated cell survival was measured using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay. Phosphorylated and/or total levels of EGFR, cleaved caspase-3, poly(ADP-ribose) polymerase-1, ERK1/2, p38MAPK, and Akt were assessed by Western blotting. We showed that HS induced a decrease in total, cytoplasmic, and nuclear EGFR levels in IEC-6 cells, which was prevented by GLN supplementation, leading to attenuated apoptosis via EGFR small interfering RNA. Furthermore, the protective effect of GLN was lessened by AG1478, PD98059, and LY294002 but was not affected by 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. In summary, EGFR expression was important in the protective mechanism of GLN, as well as GLN-mediated 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 to be independent of EGFR signaling.

BackgroundExtracellular matrix (ECM) stabilization and fibronectin (FN)-Integrin signaling can mediate cellular protection. L-glutamine (GLN) is known to prevent apoptosis after injury. However, it is currently unknown if ECM stabilization and FN-Integrin osmosensing pathways are related to GLN’s cell protective mechanism in the intestine.MethodsIEC-6 cells were treated with GLN with or without FN siRNA, integrin inhibitor GRGDSP, control peptide GRGESP or ERK1/2 inhibitors PD98059 and UO126 under basal and stressed conditions. Cell survival measured via MTS assay. Phosphorylated and/or total levels of cleaved caspase-3, cleaved PARP, Bax, Bcl-2, heat shock proteins (HSPs), ERK1/2 and transcription factor HSF-1 assessed via Western blotting. Cell size and F-actin morphology quantified by confocal fluorescence microscopy and intracellular GLN concentration by LC-MS/MS.ResultsGLN’s prevention of FN degradation after hyperthermia attenuated apoptosis. Additionally, inhibition of FN-Integrin interaction by GRGDSP and ERK1/2 kinase inhibition by PD98059 inhibited GLN’s protective effect. GRGDSP attenuated GLN-mediated increases in ERK1/2 phosphorylation and HSF-1 levels. PD98059 and GRGDSP also decreased HSP levels after GLN treatment. Finally, GRGDSP attenuated GLN-mediated increases in cell area size and disrupted F-actin assembly, but had no effect on intracellular GLN concentrations.ConclusionTaken together, this data suggests that prevention of FN degradation and the FN-Integrin signaling play a key role in GLN-mediated cellular protection. GLN’s signaling via the FN-Integrin pathway is associated with HSP induction via ERK1/2 and HSF-1 activation leading to reduced apoptosis after gut injury.

Objective Osmotically acting amino acids can be cytoprotective following injury. As threonine (THR) induces osmotic cell-swelling, our aim was to investigate the potential for THR to induce cellular protection in intestinal epithelial cells and evaluate possible mechanisms of protection. Methods Cells treated with a range of THR doses were evaluated following heat stress (HS) injury. Alpha-aminoisobutyric acid (AIB), a non-metabolizable amino acid analog, was used as an osmotic control. MTS assays were used to assess cell survival. Heat shock protein (HSP) expression and cleaved caspase-3 (CC3) were evaluated via western blot. Cell morphology and cell size sanalyzed via microscopy. Results Following HS, THR treatment increased cell viability versus CT in a dose-dependent fashion from 5 to 20 mM. The non-metabolized amino acid analogue, Alpha-aminoisobutyric acid (AIB) also increased cell survival in HS cells versus HS-CT. HSP70 and HSP25 expression increased with THR and AIB treatment versus HS-CT. THR also increased HSP25 in non-stressed cells. Microscopic evaluation revealed both THR and AIB preserved structural integrity of the actin cytoskeleton in HS-cells versus HS-CT. THR, but not AIB, enhanced nuclear translocation of HSP25 during HS. This nuclear translocation, was associated with a 60% decrease in apoptosis in HS cells with THR. No anti-apoptotic effect was observed with AIB. Conclusions This is the first demonstration THR increases HSP70 and HSP 25 and protects cells from HS. THR’s mechanism of protection may involve cytoskeletal stabilization, HSP up-regulation and nuclear translocation, and decreased apoptosis. THR’s protection appears to involve both cell swelling-dependent and independent processes.

Background This ongoing academic collaboration was initiated for providing support to set up, validate, and maintain everolimus therapeutic drug monitoring (TDM) assays and to study long-term inter- laboratory performance. Methods This study was based on EDTA whole blood samples collected from transplant patients treated with everolimus in a prospective clinical trial. Samples were handled under controlled conditions during collection, storage, and were shipped on dry ice to minimize freeze-thaw cycles. For more than 1.5 years participating laboratories received a set of 3 blinded samples on a monthly basis. Among others, these samples included individual patient samples, patient sample pools to assess long-term performance and patient samples pools enriched with isolated everolimus metabolites. Results The results between LC-MS/MS and the everolimus Quantitative Microsphere System (QMS, Thermo Fisher) assay were comparable. The monthly inter-laboratory variability (CV%) for cross validation samples ranged from 6.5 – 23.2% (average of 14.8%) for LC-MS/MS and 4.2 – 26.4% (average of 11.1%) for laboratories using the QMS assay. A blinded long-term pool sample was sent to the laboratories for 13 months. The result was 5.31 ± 0.86 ng/mL (range 2.9–7.8 ng/mL) for the LC-MS/MS and 5.20 ± 0.54 ng/mL (range 4.0–6.8 ng/mL) for QMS laboratories. Conclusions Enrichment of patient sample pools with 5–25 ng/mL of purified everolimus metabolites (46-hydroxy everolimus and 39-O-desmethyl everolimus) did not affect the results of either LC-MS/MS or QMS assays. Both LC-MS/MS and QMS assays gave similar results and showed similar performance, albeit with a trend towards higher inter-laboratory variability among laboratories using LC-MS/MS than the QMS assay.