Clostridium difficile toxin B activates the EGF receptor and the ERK/MAP kinase pathway in human colonocytes.
OBJECTIVE
Clostridium difficile toxin B (TxB) mediates acute inflammatory diarrhea characterized by neutrophil infiltration and intestinal mucosal injury. In a xenograft animal model, TxB was shown to induce interleukin (IL)-8 gene expression in human colonic epithelium. However, the precise mechanisms of this TxB response are unknown. The aim of this study was to investigate the TxB-mediated proinflammatory pathway in colonocytes.


METHODS
The effect of TxB on epidermal growth factor receptor (EGFR), extracellular signal-regulated kinase (ERK) 1/2 signaling pathway and IL-8 gene expression was assessed in nontransformed human colonic epithelial NCM460 cells. TxB regulation of EGFR-ERK1/2 signaling pathways was determined using immunoblot analysis, confocal microscopy, and enzyme-linked immunosorbent assay, whereas IL-8 gene expression was measured by luciferase promoter assay.


RESULTS
TxB activates EGFR and ERK1/2 phosphorylation with subsequent release of IL-8 from human colonocytes. Pretreatment with either the EGFR tyrosine kinase inhibitor, AG1478, or an EGFR-neutralizing antibody blocked both TxB-induced EGFR and ERK activation. By using neutralizing antibodies against known ligands of EGFR, we found that the activation of EGFR and ERK1/2 phosphorylation was mediated by transforming growth factor-alpha (TGF-alpha). Inhibition of matrix metalloproteinase (MMP) decreased TGF-alpha secretion and TxB-induced EGFR and ERK activation. Inhibition of MMP, EGFR, and ERK activation significantly decreased TxB-induced IL-8 expression.


CONCLUSIONS
TxB signals acute proinflammatory responses in colonocytes by transactivation of the EGFR and activation of the ERK/MAP kinase pathway.
Na
Xi
X
Division of Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA.
Zhao
Dezheng
D
Koon
Hon Wai
HW
Kim
Ho
H
Husmark
Johanna
J
Moyer
Mary P
MP
Pothoulakis
Charalabos
C
LaMont
J Thomas
JT
D000906:Antibodies / Q000494:pharmacology; D001426:Bacterial Proteins / Q000494:pharmacology*; D001427:Bacterial Toxins / Q000494:pharmacology*; D002460:Cell Line; D003106:Colon / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D004789:Enzyme Activation; D066246:ErbB Receptors / Q000187:drug effects*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D019073:HT29 Cells; D006801:Humans; D016209:Interleukin-8 / Q000378:metabolism; D045726:Metalloproteases / Q000037:antagonists & inhibitors / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011480:Protease Inhibitors / Q000494:pharmacology; D016211:Transforming Growth Factor alpha / Q000276:immunology / Q000378:metabolism
D000906:Antibodies; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D016209:Interleukin-8; D011480:Protease Inhibitors; D016211:Transforming Growth Factor alpha; C031815:toxB protein, Clostridium difficile; D066246:ErbB Receptors; D048049:Extracellular Signal-Regulated MAP Kinases; D020928:Mitogen-Activated Protein Kinases; D045726:Metalloproteases