MEK Is a Key Modulator for TLR5-induced Interleukin-8 and MIP3α Gene Expression in Non-transformed Human Colonic Epithelial Cells

Sang Hoon Rhee,Andrew C Keates,Mary P Moyer,Charalabos Pothoulakis

doi:10.1074/jbc.m400967200

Sang Hoon Rhee, Andrew C Keates + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m400967200

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Abstract

Flagellin, a specific ligand for Toll-like receptor 5 (TLR5), is a molecular pattern associated with several bacterial species. Recently, TLR signaling has been intensively studied. However, TLR5-associated signaling in non-transformed colonocytes has not been investigated. Here we studied the expression of cytokines induced by flagellin in non-transformed human colonic NCM460 cells and the signaling mechanisms mediating these responses. Cytokine expression array experiments showed that exposure of the cells to flagellin (100 ng/ml) for 12 h increased the expression of interleukin (IL)-8 and macrophage-inflammatory protein 3alpha (MIP3alpha) in a TLR5-specific manner. Flagellin also activated MAP kinases (ERK1/2, JNK, and p38) and degraded IkappaBalpha. Dominant negative MEK1 (a kinase that activates ERK1/2) blocked flagellin-stimulated IL-8 and MIP3alpha transcriptional activity, while the MEK-specific inhibitors PD98059 and U0126 reduced protein production of these cytokines. Conversely, transfection with a constitutively active MEK1 increased IL-8 and MIP3alpha transcriptional activity in a NFkappaB-independent manner. Furthermore, overexpression of the constitutively active MEK1 induced IL-8 and MIP3alpha protein production. We also demonstrated that C-terminal coiled-coil and TRAF-C domains of TRAF6, unable to mediate NFkappaB activation, are involved in MEK-mediated IL-8 and MIP3alpha expression. Thus, in non-transformed human colonocytes, MEK activation following flagellin/TLR5 engagement is a key modulator for NFkappaB-independent, IL-8 and MIP3alpha expression.

Highlights

Mammalian toll-like receptors (TLRs)1 are a family of type 1 receptors composed of an extracellular leucine-rich repeat do
We present novel evidence indicating that MEK, a kinase involved in ERK1/2 activation, mediates IL-8 and MIP3␣ expression via NF␬B-independent pathway(s) in flagellin/TLR5induced signal transduction pathways
Cytokine Microarray Assay Revealed the Selective Expression of IL-8 and MIP3␣ by Toll-like receptor 5 (TLR5) Engagement in Non-transformed Human Colonocytes—Understanding the expression array of proinflammatory mediators by bacterial flagellin is important to study the pathophysiology of inflammatory responses in the human gut

Summary

Introduction

Mammalian toll-like receptors (TLRs) are a family of type 1 receptors composed of an extracellular leucine-rich repeat do-. Studies on TLR signaling provided information on several adaptor molecules of TLR such as MyD88 (myeloid differentiation factor 88), TIRAP/Mal (TIR domain-containing adapter protein/ MyD88-adapter-like), TRIF (TIR domain-containing adapter inducing interferon ␤), and TRAM (TRIF-related adaptor molecule) [3, 4]. These adaptor molecules mediate NF␬B and MAP kinase activation, leading to proinflammatory cytokine gene expression and immune cell maturation. Among the various pathogen-associated molecular patterns, flagellin, the major component of the flagellar filament, is produced from Gramnegative and Gram-positive bacterial species and stimulate TLR5 leading to cytokine gene expression [5, 6]. MEK Mediates TLR5-induced IL-8 and MIP3␣ Expression proinflammatory cytokine expression by TLR5 engagement in intestinal epithelial cells remain to be elucidated

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