TUMOR NECROSIS FACTOR ALPHA ACTIVATES MAP KINASES THROUGH TNFα RECEPTOR R1 IN INTESTINAL EPITHELIAL CELLS

Abstract

Our lab has recently shown that high dose TNFα (100 ng/mL) inhibits both intestinal cell proliferation and growth factor-mediated mitogenesis through TNFα receptor R1 (55 kDa), while low dose TNFα (1 ng/mL) stimulates intestinal cell proliferation through TNFα receptor R2 (75 kDa). One mechanism of cytokine and growth factor regulation of cellular function is determined by the kinetics of MAP kinase activation. The purpose of this study was to determine the effects of EGF and TNFα on the kinetics of MAP kinase phosphorylation in intestinal epithelial cells and to determine the independent roles of TNFα receptors R1 and R2 on the signaling events of MAP kinase family members Erk1/Erk2 (p42/p44), JNK/SAPK (p46/p54) and p38. The conditionally immortalized murine colon cell line (YAMC) was studied in a non-immortalized state. Following 24 h incubation at 37°C in media with 0.5% serum, the effects of murine EGF (10 ng/mL) and TNFα (1 and 100 ng/mL) were studied at exposures of 2, 15, 30, 60 and 120 min. Anti-TNFα receptor antibodies were utilized to elucidate TNFα signaling pathways. After treatment with EGF, TNFα or anti-TNFα receptor antibodies, Triton-soluble lysates were obtained and proteins separated by SDS-PAGE. Activated Erk1/Erk2, JNK/SAPK and p38 were determined by Western blot analysis. EGF activates Erk1/Erk2 maximally at 2 min with a return to baseline by 60 min. High dose TNFα (100 ng/mL) activates Erk1/Erk2 maximally at 15 min with sustained activation through 120 min. Similarly, high dose TNFα phosphorylates JNK/SAPK maximally at 15 min. Anti-TNFα receptor R1 agonist antibody activated Erk1/Erk2 and JNK/SAPK. TNFα's activation of Erk1/Erk2 and JNK/SAPK was blocked by anti-TNFα receptor R1 antagonist antibody, while anti-TNFα receptor R2 antagonist antibody had no effect. When low dose TNFα (1 ng/mL) was studied, neither Erk1/Erk2 nor JNK/SAPK were activated. In addition, neither high dose TNFα, low dose TNFα nor EGF appear to activate p38. In summary, TNFα (100 ng/mL) activates both Erk1/Erk2 and JNK/SAPK pathways. Phosphorylation of Erk1/Erk2 by TNFα receptor R1 and EGF showed different patterns of maximal activation and duration. These findings, combined with our previously reported data, suggest that TNFα alters EGF-mediated signaling through TNFα receptor R1 by disrupting MAP kinase activation.