Epidermal growth factor receptor-mediated proliferation of enterocytes requires p21waf1/cip1 expression.
OBJECTIVE
Epidermal growth factor receptor (EGFR)-mediated increase in enterocyte proliferation following massive resection is a major mechanism by which the small intestine adapts to the loss of its mucosal surface area. In addition, expression of the cyclin-dependent kinase inhibitor p21(waf1/cip1) is required for resection-induced enterocyte proliferation. This study sought to establish a mechanistic link between EGFR-mediated intestinal epithelial cell proliferation and p21(waf1/cip1) expression.


METHODS
EGF was used to stimulate IEC-6 and HCA-7 cells. P21(waf1/cip1) messenger RNA (mRNA) and protein expression were measured by real-time polymerase chain reaction and Western blot, respectively. P21(waf1/cip1) promoter studies were performed using p21(waf1/cip1) promoter-driven luciferase assay. Pharmacologic inhibitors of PI3-kinase and mitogen activated protein kinase (MAPK) were used to block these pathways downstream of the activated EGFR. Constitutively active Ras, Raf, or MEK-1 constructs were transfected into cells for overexpression studies. Cell proliferation was measured by bromodeoxyuridine incorporation following p21(waf1/cip1) silencing with RNAi. Finally, Cyclin D(1)/Cdk interaction was evaluated by immunoprecipitation.


RESULTS
EGFR activation in intestinal epithelial cells induced the expression of p21(waf1/cip1) mRNA and protein This event was transcriptionally regulated via a 50-bp segment of the p21(waf1/cip1) promoter as a result of MAPK activation. Exogenous EGF failed to induce proliferation in p21(waf1/cip1)-silenced cells and adaptive proliferation after intestinal resection in p21(waf1/cip1)-null mice. Functionally, p21(waf1/cip1) up-regulation was required for stabilizing Cyclin D/Cdk 4 complexes and intestinal cell proliferation.


CONCLUSIONS
EGFR-mediated induction of enterocyte proliferation requires MAPK-dependent increase in p21(waf1/cip1) expression in intestinal epithelial cells. These studies elucidate an important mechanism for resection-induced enterocyte proliferation during intestinal adaptation.
Sheng
George
G
Department of Surgery, University of Cincinnati College of Medicine, Ohio, USA.
Bernabe
Kathryn Q
KQ
Guo
Jun
J
Warner
Brad W
BW
D000818:Animals; D015153:Blotting, Western; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D003110:Colonic Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D020895:Enterocytes / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D066246:ErbB Receptors / Q000378:metabolism*; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D066298:In Vitro Techniques; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D012334:RNA, Neoplasm / Q000235:genetics*; D051381:Rats
C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D012334:RNA, Neoplasm; D066246:ErbB Receptors; D020928:Mitogen-Activated Protein Kinases