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.
McCole
Declan F
DF
Department of Medicine, University of California, School of Medicine, San Diego, California 92103, USA.
Keely
Stephen J
SJ
Coffey
Robert J
RJ
Barrett
Kim E
KE
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