ITF promotes migration of intestinal epithelial cells through crosstalk between the ERK and JAK/STAT3 pathways.

Juan Le,Duan Y Zhang,Duan Y Zhang,Yong Sun,Peng Wang,Peng Wang,Yong Zhao,Yong Zhao,Wei Qiu,Wei Qiu

doi:10.1038/srep33014

Abstract

Intestinal trefoil factor (ITF), a member of the trefoil factor family, is a “Super-protective factor” for intestinal mucosal protection. This study was designed to explore the mechanism by which ITF promotes intestinal epithelial cell migration. Intestinal epithelial cells were treated with the human ITF (hITF). Phospho-ERK, phospho-STAT3 Tyr705, and phospho-STAT3 Ser727 levels were detected at different time points by western blot. To assess the potential crosstalk between the ERK and JAK/STAT3 pathways, HT-29 cells were treated with the MEK-inhibitor, U0126, and phosphor-STAT3 levels were evaluated. Conversely, cells were treated with the JAK-inhibitor, AG490, and ERK-activity was evaluated. Transwell assay was performed to investigate the effect of the crosstalk on the cell motility. MMP-2 and MMP-9 transcription was analyzed by quantitative real-time PCR. E-cadherin degradation was detected by immunofluorescence. Our results indicate that hITF simultaneously activated the ERK and JAK/STAT3 pathways and a crosstalk was detected between the two pathways. hITF increased cell migration. This effect was abolished by U0126 and AG490 treatment. hITF increased MMP2 and MMP9 mRNA levels and E-cadherin degradation and U0126 and AG490 abolished this effect of hITF. In conclusion, the hITF-induced crosstalk between the ERK and JAK/STAT3 pathways is associated with intestinal epithelial cell migration.

Highlights

In this study, we employed a human intestinal epithelial system, in which HT-29 cell line was cultured in vitro
Our aim was to elucidate the interactions between the ERK and JAK/STAT3 signaling pathways in regulating human intestinal epithelial cell migration promoted by ITF and to lay the foundation for the protection of the intestinal mucosa
HT-29 cells were treated with human intestinal trefoil factor (hITF) at a concentration of 60 μg/mL hITF in the following experiments

Summary

Introduction

We employed a human intestinal epithelial system, in which HT-29 cell line was cultured in vitro. Treatment of HT-29 cells with the ERK inhibitor, U0126, or with the JAK inhibitor, AG490, and evaluation of phosphoSTAT3-level or ERK-activity, respectively, provided information about the crosstalk between ERK and JAK/STAT3 pathways. We performed transwell assays to assess the migratory ability of HT-29 cells after treatment with hITF alone or in the presence of ERK and JAK inhibitors. MMP2 and MMP9 transcription and E-cadherin degradation were analyzed to identify the downstream targets of ITF promoting cell migration. Our aim was to elucidate the interactions between the ERK and JAK/STAT3 signaling pathways in regulating human intestinal epithelial cell migration promoted by ITF and to lay the foundation for the protection of the intestinal mucosa

Objectives

Methods

Results

Conclusion