MEK is a key modulator for TLR5-induced interleukin-8 and MIP3alpha gene expression in non-transformed human colonic epithelial cells.
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.
Rhee
Sang Hoon
SH
The Division of Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215, USA.
Keates
Andrew C
AC
Moyer
Mary P
MP
Pothoulakis
Charalabos
C
D054418:Chemokine CCL20; D019742:Chemokines, CC / Q000235:genetics*; D003106:Colon / Q000166:cytology / Q000378:metabolism*; D005786:Gene Expression Regulation; D006801:Humans; D016209:Interleukin-8 / Q000235:genetics*; D007413:Intestinal Mucosa / Q000166:cytology / Q000378:metabolism; D048369:MAP Kinase Kinase 1; D019402:Macrophage Inflammatory Proteins / Q000235:genetics*; D008562:Membrane Glycoproteins / Q000502:physiology*; D020929:Mitogen-Activated Protein Kinase Kinases / Q000502:physiology*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D011506:Proteins / Q000502:physiology; D011956:Receptors, Cell Surface / Q000502:physiology*; D015398:Signal Transduction; D048029:TNF Receptor-Associated Factor 6; D051198:Toll-Like Receptor 5; D051193:Toll-Like Receptors
C108355:CCL20 protein, human; D054418:Chemokine CCL20; D019742:Chemokines, CC; D016209:Interleukin-8; D019402:Macrophage Inflammatory Proteins; D008562:Membrane Glycoproteins; D016328:NF-kappa B; D011506:Proteins; D011956:Receptors, Cell Surface; C495346:TLR5 protein, human; D048029:TNF Receptor-Associated Factor 6; D051198:Toll-Like Receptor 5; D051193:Toll-Like Receptors; D020928:Mitogen-Activated Protein Kinases; D048369:MAP Kinase Kinase 1; C482125:MAP2K1 protein, human; D020929:Mitogen-Activated Protein Kinase Kinases