Tu1271 Differential Mechanisms of EGF and GLP-2 Mediated Intestinal Epithelial Cell Proliferation

Abstract

Introduction: Both GLP-2 and EGF can induce intestinal epithelial cell (IEC) growth. We previously showed that both growth factors (GFs) decrease with total parenteral nutrition (TPN) administration, and both exogenous GFs can prevent TPN-associated atrophy. However, the precise mechanisms driving this protective effect remain incompletely understood. This study investigated the role of PI3K/AKT and Wnt/β-catenin signaling mediating EC responses with TPN. Methods: C57BL/6 mice received enteral nutrition (Control) or TPN for 7 days. Wildtype, IEC-ErbB1KO and IEC-pik3r1KO (class IA subunit of PI3K) mice were treated with exogenous EGF or GLP-2. IEC proliferation was measured by PCNA staining. Nuclear and cytoplasm fractions were isolated from jejunal crypt cells and target proteins were measured by western blot. Results: TPN resulted in loss of both GFs expression and decreased EC proliferation. Exogenous EGF or GLP-2 restored IEC proliferation. We then explored differential mechanisms for EGF/GLP-2 responses. ErbB1: Both GFs actions weremarkedly decreased in IEC-ErbB1KOTPNmice, as well as in gefitinib (ErbB1 antagonist) treated TPN mice, demonstrating ErbB1-dependency for these GFs responses. P-AKT: Both GFs restored cytoplasmic p-Akt expression, but only EGF induced sustained nuclear pAKT expression. p-AKT expression was blocked in IEC-pi3kr1KO mice. However, in IECpi3kr1KO mice, GLP-2 induced EC proliferation but EGF. This indicated that GLP-2 acted by a PI3K-pAKT independent mechanism.Wnt-β-catenin: Increased cytoplasmic p-β-catenin (ser33/37) expression was found in TPNmice and both GFs decreased β-catenin ubiquination by preventing this increase. Nuclear p-β-catenin (ser552) expression was down-regulated in TPNmice and both GFs prevented this decline. Cytoplasmic p-GSK3βwas down-regulated in TPN mice and GLP-2 treatment prevented this decline but EGF did not. However, in IEC-pi3kr1KO TPN mice, nuclear p-β-catenin (ser552) and total cytoplasm β-catenin expression were maintained by GLP-2, whereas both β-catenin levels were still diminished in the presence of EGF. Stem cell: A marked loss of LGR5+ crypt cells (1% vs 20% , TPN vs Control) were observed in Lgr5-EGFP knock-in mice with TPN administration. EGF failed to prevent this loss, but LGR5+ was partially protected with GLP-2 (10%). Interestingly, although EC proliferation decreased in GLP-2 treated mice given gefinitib, LGR5+ crypts (10%) were still observed. Conclusions: Reduced EGF and GLP-2 signaling contributed to TPN-associated loss of EC proliferation. Both GFs required an intact ErbB1 signaling for their responses. However, EGF was dependent on PI3K/P-AKT signaling to promote EC proliferation, whereas GLP-2 was partially independent of the PI3K/p-AKT pathway to modulate EC proliferation and appeared to mediate proliferation viaWnt-β-catenin signaling.