Analysis of signal transduction pathway in rat gastric epithelial cells stimulated by interleukin-1β

Mitogen-activated protein kinases (MAPKs), including c-Jun NH2-terminal kinases (JNKs) and extracellular signal-regulated kinases (ERKs), are important intermediates of siganal-transduction pathway from cell suraface to the nucleus in cell proliferation, differentiation, and apoptosis. Recent evidence shows that interleukin-113 (IL-113), a multifunctional cytokine, activates MAPKs in mesangial cells and fibroblasts. However, cellular response of gastric epithelial cells stimulated by IL-113 is not well known. In this study, to analyze the signal transduction pathways, we investigate whether IL-113 activates JNKs and ERKs in rat gastric epithelial ceils (RGM1) using in-gel kinase assays. RGM1 were cultured at 37°C in the optimal media (DMEM + Ham F12) with FCS finally concentrated at 20%. Identification of INKs and ERKs in RGM1 was performed by immunoprecipitation procedure. Recombinant mouse IL-113, at the final dose of 0, 0.1, 1, 10, 102, 103, or 5X103 pg/ml, was added to the culture media and incubated for 20 rain to investigate the dose-dependency of JNKs and ERKs activities in RGM1. IL-113 (100 pg/rnl) was added to the cuture media and incubated for 5, 10, 20, 30, 45, or 60 rain to investigate the time course of these activities. The activation of JNKs and ERKs in RGM1 were dose-dependent with maximal activity attained with 1000 pg/ml of IL-113, respectively. These two maximal activities occurred at 20 rain after stimulation of IL-113. Furthermore, pretreatment with neutralizing antibody against IL-113 inhibited IL-113-induced activation of JNKs and ERKs. The activation of JNKs, but not ERKs, was inhibited by 25% of the maximal levels by pretreatment with genistein, an inhibitor for receptor specific and cytoplasmic tyrosine kinase. On the other hand, Lavendustin A, an inhibitor for receptor specific tyrosine kinase, and GF109203X, an inhibitor for protein kinase C, affected neither activation of JNKs nor ERKs. Thus, the activation of JNKs in RGM1 by IL-113 might be in part mediated by cytoplasmic but not receptor specific tyrosine kinases. In contrast to the activation of JNKs, that of ERKs might not be mediated by neither cytoplasmic nor receptor specific tyrosine kinases. These results suggest that JNKs and ERKs in rat gastric epithelial ceils are activated by IL-l[3, possibly via the specific receptor for IL-[3. Activation of these two kinases may be mediated by some different signaling pathways.