Rapid mitogen-activated protein kinase activation by transforming growth factor alpha in wounded rat intestinal epithelial cells.
OBJECTIVE
To define signaling events initiating healing after intestinal epithelial injury, activation of mitogen-activated protein kinase (MAPK) pathways was assessed after wounding using an in vitro model.


METHODS
Proteins isolated from wounded monolayers of nontransformed intestinal epithelial cells (IEC-6) were analyzed for tyrosine phosphorylation and MAPK expression by Western blot. Extracellular signal-regulated kinase (ERK) 1, ERK2, and Raf-1 activities were assessed by immune complex kinase assays.


RESULTS
Tyrosine phosphorylation of several proteins including ERK1 was substantially increased 5 minutes after injury. Another MAPK, c-Jun-N-terminal protein kinase (JNK), was also activated after wounding. Conditioned medium from wounded but not intact IEC-6 monolayers resulted in increased activity of ERK1, ERK2, and Raf-1 kinase. Wound-conditioned medium stimulated proliferation of subconfluent IEC-6 cells compared with conditioned medium from intact IEC-6 cultures and contained higher amounts of transforming growth factor (TGF)-alpha than supernatants of confluent IEC-6 cultures. Activation of ERK1 and ERK2 was partially inhibited by neutralizing anti-TGF-alpha.


CONCLUSIONS
Wounding of intestinal epithelial cells results in activation of Raf-1, ERK1, ERK2, and JNK1 MAPKs and subsequent cell proliferation in vitro. Activation of ERK1 and ERK2 is mediated in part by TGF-alpha.
Göke
M
M
Gastrointestinal Unit, Department of Medicine, and Center for the Study of Inflammatory Bowel Disease, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114, USA.
Kanai
M
M
Lynch-Devaney
K
K
Podolsky
D K
DK
D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002478:Cells, Cultured; D017077:Culture Media, Conditioned; D004789:Enzyme Activation / Q000187:drug effects; D004847:Epithelial Cells / Q000201:enzymology; D007413:Intestinal Mucosa / Q000201:enzymology*; D048031:JNK Mitogen-Activated Protein Kinases; D048670:MAP Kinase Kinase 4; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020929:Mitogen-Activated Protein Kinase Kinases; D020928:Mitogen-Activated Protein Kinases; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D051381:Rats; D016211:Transforming Growth Factor alpha / Q000494:pharmacology*; D014443:Tyrosine / Q000378:metabolism
D017077:Culture Media, Conditioned; D016211:Transforming Growth Factor alpha; D014443:Tyrosine; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D048670:MAP Kinase Kinase 4; D020929:Mitogen-Activated Protein Kinase Kinases