Morphogenic protein epimorphin protects intestinal epithelial cells from oxidative stress by the activation of EGF receptor and MEK/ERK, PI3 kinase/Akt signals

Abstract

Epimorphin is a mesenchymal protein that regulates morphogenesis of epithelial cells. Our preliminary study suggested a novel function of epimorphin in enhancing survival of intestinal epithelial cells (IEC). Oxidative stress leads to cell injury and death and is suggested to be a key contributor to pathogenesis of inflammatory bowel disease. This study was conducted to determine whether epimorphin protects IEC from oxidative stress. Rat intestinal epithelial cell line IEC-6 was cultured with epimorphin (10 and 20 mug/ml), and the life span of IEC was assessed. The mean life span of IEC-6 cells was prolonged 1.9-fold (P < 0.0006) by treatment with epimorphin. We then examined the epimorphin signaling pathways. Epimorphin phosphorylated epidermal growth factor (EGF) receptor, activated the MEK/extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase and phosphatidylinositol 3 (PI3) kinase/Akt pathways, phosphorylated Bad, and induced Bcl-X(L) and survivin. Hydrogen peroxide (1 mM) induced cell death in 92% of IEC-6 cells, but epimorphin dramatically diminished (88.7%) cell death induced by hydrogen peroxide (P < 0.0001). This protective effect of epimorphin was significantly attenuated by inhibitors of MEK and PI3 kinase (P < 0.0001) or EGF receptor-neutralizing antibody (P = 0.0007). In wound assays, the number of migrated cells in the wound area decreased (72.5%) by treatment with 30 muM hydrogen peroxide, but epimorphin increased the number of migrated cells 3.18-fold (P < 0.0001). These results support a novel function of epimorphin in protecting IEC from oxidative stress. This anti-oxidative function of epimorphin is dramatic and is likely mediated by the activation of EGF receptors and the MEK/extracellular signal-regulated kinase and PI3 kinase/Akt signaling pathways and through the induction of anti-apoptotic factors.

Non-classical export of epimorphin and its adhesion to αv-integrin in regulation of epithelial morphogenesis

“…Syb, synaptobrevin, Epm, epimorphin. extracellular supplementation of epimorphin prolongs the lifetime of nutrient-deprived intestinal cells and also attenuates apoptosis induced by cytotoxic reagents (Iizuka et al, 2007). Consistent with this, we found that exposure to soluble extracellular epimorphin was sufficient to confer apoptosis resistance in HMECs that were maintained in unsupplemented medium, even after 4 weeks (see supplementary material Fig.…”

“…We defined ␣v-integrin as the core component of the epimorphin receptor using cell adhesion assays, pulldown assays, immunoprecipitation analysis and the 3D morphogenesis assay, whereas recent work also implicated involvement of EGF receptors in the cell-epimorphin interaction in intestinal epithelia (Iizuka et al, 2007). ␣v-Integrin and its downstream effectors FAK and ERK (which also acts as an effector of EGFR family members) are known to play important roles in the developmental processes of many organ systems (Bader et al, 1998;Bayless et al, 2000;Eliceiri and Cheresh, 2000).…”

Involvement of epimorphin in the repair of experimental renal fibrosis in mice

“…Recombinant epimorphin (final concentration of 20 mg/ml) or vehicle was added to the culture medium, and the supernatants were collected at various time points (8, 16 and 24 h) after treatment. The recombinant epimorphin was prepared as described previously by Hirai et al 31 The collected mediums were concentrated 15-fold with centrifugal filter units (Ultrafree-MC; Millipore, Bedford, MA, USA). MMP activities in each concentrated sample were analyzed by gel zymography similar to that described above except that 1 mg/ml gelatin instead of casein was added to the gel and the substrate buffer was 50 mM Tris and 20 mM CaCl 2 (pH 7.6).…”

Extracellularly Extruded Syntaxin-4 Is a Potent Cornification Regulator of Epidermal Keratinocytes

“…We also detected extracellularly effluent syntaxin-3 from keratinocytes undergoing necrotic/apoptotic cell death (22). Although these syntaxins may execute distinctive functions after being exposed extracellularly, they have been commonly shown to elicit survival activity from adjacent cells (11,12,22,34). This function may account for the thickened lower epidermal layers in organ culture and the mice model, the cells of which otherwise progress with an active anoikis program, which may be often driven by the extracellular expression of syntaxin-4.…”