Transactivation of the epidermal growth factor receptor in colonic epithelial cells by carbachol requires extracellular release of transforming growth factor-alpha.
We have shown previously that the muscarinic agonist, carbachol (CCh), transactivates the epidermal growth factor receptor (EGFr) via calmodulin, Pyk-2, and Src kinase activation. EGFr phosphorylation causes extracellular signal-regulated kinase (ERK) activation and inhibits CCh-stimulated chloride secretion across intestinal epithelial cells. Here we investigated whether CCh-stimulated EGFr transactivation involves EGFr ligand release. Pre-incubation of T(84) cell monolayers with a neutralizing antibody to the EGFr ligand binding domain decreased CCh-induced phosphorylation of EGFr and ERK. CCh-stimulated efflux of (86)Rb+ from T(84) cell monolayers, which parallels changes in chloride secretion, was potentiated by anti-EGFr pre-incubation. Anti-EGFr did not reduce CCh-stimulated Pyk-2 phosphorylation. Co-incubation with the Src kinase inhibitor PP2 and anti-EGFr had an additive inhibitory effect on CCh-induced ERK phosphorylation greater than either inhibitor alone. CCh caused the basolateral release of transforming growth factor alpha (TGF-alpha) into T(84) cell bathing media. A metalloproteinase inhibitor, WAY171318, reduced CCh-induced phosphorylation of ERK and completely blocked EGFr phosphorylation and TGF-alpha release. We conclude that CCh-stimulated EGFr transactivation and subsequent ERK activation, a pathway that limits CCh-induced chloride secretion, is mediated by metalloproteinase-dependent extracellular release of TGF-alpha and intracellular Src activation. These findings have important implications for our understanding of the role of growth factors in regulating epithelial ion secretion.
Department of Medicine, University of California, School of Medicine, San Diego, California 92103, USA.
Declan F
DF
McCole
Stephen J
SJ
Keely
Robert J
RJ
Coffey
Kim E
KE
Barrett
D001665:Binding Sites; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology; D003106:Colon; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000494:pharmacology; D066246:ErbB Receptors / Q000235:genetics*; D006801:Humans; D007413:Intestinal Mucosa / Q000187:drug effects / Q000502:physiology*; D007700:Kinetics; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000378:metabolism; D015533:Transcriptional Activation; D016211:Transforming Growth Factor alpha / Q000494:pharmacology / Q000502:physiology*
D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D016211:Transforming Growth Factor alpha; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D020928:Mitogen-Activated Protein Kinases; D008666:Metalloendopeptidases