Year-end Sale % OFF 
Abstract
p40, a Lactobacillus rhamnosus GG (LGG)-derived soluble protein, ameliorates intestinal injury and colitis, reduces apoptosis, and preserves barrier function by transactivation of the EGF receptor (EGFR) in intestinal epithelial cells. The aim of this study is to determine the mechanisms by which p40 transactivates the EGFR in intestinal epithelial cells. Here we show that p40-conditioned medium activates EGFR in young adult mouse colon epithelial cells and human colonic epithelial cell line, T84 cells. p40 up-regulates a disintegrin and metalloproteinase domain-containing protein 17 (ADAM17) catalytic activity, and broad spectrum metalloproteinase inhibitors block EGFR transactivation by p40 in these two cell lines. In ADAM17-deficient mouse colonic epithelial (ADAM17(-/-) MCE) cells, p40 transactivation of EGFR is blocked, but can be rescued by re-expression with WT ADAM17. Furthermore, p40 stimulates release of heparin binding (HB)-EGF, but not transforming growth factor (TGF)α or amphiregulin, in young adult mouse colon cells and ADAM17(-/-) MCE cells overexpressing WT ADAM17. Knockdown of HB-EGF expression by siRNA suppresses p40 effects on transactivating EGFR and Akt, preventing apoptosis, and preserving tight junction function. The effects of p40 on HB-EGF release and ADAM17 activation in vivo are examined after administration of p40-containing pectin/zein hydrogel beads to mice. p40 stimulates ADAM17 activity and EGFR activation in colonic epithelial cells and increases HB-EGF levels in blood from WT mice, but not from mice with intestinal epithelial cell-specific ADAM17 deletion. Thus, these data define a mechanism of a probiotic-derived soluble protein in modulating intestinal epithelial cell homeostasis through ADAM17-mediated HB-EGF release, leading to transactivation of EGFR.
Highlights
Background: p40 is a Lactobacillus rhamnosus GG-derived protein
Effects of p40 on heparin binding (HB)-EGF release and ADAM17 activation in vivo are examined after administration of p40-containing pectin/zein hydrogel beads to mice. p40 stimulates ADAM17 activity and EGF receptor (EGFR) activation in colonic epithelial cells and increases HB-EGF levels in blood from WT mice, but not from mice with intestinal epithelial cell-specific ADAM17 deletion
We found that p40 prevented TNF and cycloheximide-induced apoptosis detected by Western blot analysis of cleaved PARP and Caspase-3 in ADAM17Ϫ/Ϫ mouse colonic epithelial (MCE) cells expressing WT ADAM17, but not with control vector (Fig. 3B)
Summary
Cell Culture—T84 cells (ATCC, CCL-248), a human colonic adenocarcinoma cell line, were cultured in 1:1 mixture of Ham’s F12 medium and DMEM with 2.5 mM glutamine, 5% fetal bovine serum (FBS), and 100 units/ml penicillin and streptomycin at 37 °C with 5% CO2. ADAM17 activity was detected using a InnoZymeTM TACE Activity Kit (Calbiochem) according to the manufacturer’s instructions using recombinant human ADAM17 as the standard This assay was designed to detect human ADAM17 activity, our analysis of ADAM17Ϫ/Ϫ MCE cells transduced with WT mouse ADAM17, catalytically inactive ADAM17 EϾA, or vector alone indicated that mouse ADAM17 could be detected. Cellular proteins isolated from cultured cells and colonic epithelial cells were mixed with Laemmli sample buffer and separated by SDSpolyacrylamide gel electrophoresis for Western blot analysis using antibodies against total EGFR (Millipore), phosphoEGFR (Tyr-1068) (Cell Signaling Technology), phospho-Akt (Ser-473) (Cell Signaling Technology), total Akt (Cell Signaling Technology), PARP (which identifies both full-length and cleaved PARP) (Cell Signaling Technology), Caspase-3 (which identifies both full-length and cleaved Caspase-3) (Cell Signaling Technology), ADAM17 (Cell Signaling Technology, 3976), and -actin (Sigma-Aldrich).
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
