RAPID MITOGEN-ACTIVATED PROTEIN-KINASE ACTIVATION BY TRANSFORMING-GROWTH-FACTOR-ALPHA IN WOUNDED RAT INTESTINAL EPITHELIAL-CELLS

Background & Aims: To define signaling events initiating heating 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 nontransf ormed intestinal epithelial cells (IEC-6) were analyzed for tyrosine p hosphorylation and MAPK expression by Western blot. Extracellular sign al-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-1-terminal protein kinase (JNK), was also activated after wounding. Conditioned medium from wounded but no t 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 inta ct IEC-6 cultures and contained higher amounts of transforming growth factor (TGF)-alpha than supernatants of confluent IEC-6 cultures. Acti vation of ERK1 and ERK2 was partially inhibited by neutralizing anti-T GF-alpha. Conclusions: Wounding of intestinal epithelial cells results in activation of Raf-1, ERK1, ERK2, and JNK1 MAPKs and subsequent cel l proliferation in vitro. Activation of ERK1 and ERK2 is mediated in p art by TGF-alpha.