TRYPSIN INHIBITOR-INDUCED PANCREATIC GROWTH INVOLVES INDUCTION OF EARLY RESPONSE GENES.:

Pancreatic growth occurs during development and in the adult animal can be regulated by diet and gut hormones such as cholecystokinin (CCK). Pancreatic growth can be induced by feeding the synthetic trypsin inhibitor camostat to increase plasma CCK. It requires functional calcineurin, but a number of other signaling pathways are activated including mTOR, ERK and JNK. Early response genes may be involved in this growth, as they are mediators of short-term signals, which can lead to long-term cellular responses. To evaluate early response genes induction male ICR mice adapted to powder chow were fasted for 18 hours before being refed chow containing 0.1% camostat for 1h, 2h, 4h or 8h. Pancreas was used to isolate RNA and nuclear proteins. Affymetrix gene chips and quantitative real time PCR were utilized to identify and quantitate early response gene mRNA expression after camostat feeding. Gene chip analysis showed a three fold or greater increase for early response genes c-jun, c-fos, junB, ATF3, egr-1, NFIL3, Ier5, Ier3, Ier2. QPCR results confirmed the expression of c-jun, c-fos, ATF-3 and Egr-1, which increased after camostat feeding with a peak at 1 or 2h and then decreased. Western blot analysis revealed that c-Jun, c-Fos, ATF3 and Egr-1 proteins were all increased over a 1–8 hour time course with a peak increase of 10–40 fold at 2h. Camostat feeding also increased c-Jun phosphorylation. In gel shift studies using an AP-1 consensus sequence as the probe, AP-1 DNA binding was enhanced by camostat feeding with a peak at 2h. Super shift studies with specific antibodies revealed c-Jun, c-Fos, and ATF-3 to be the major components of AP-1 induced by camostat feeding. A CCK antagonist IQM-95,333 blocked 90% of c-Jun, and c-Fos protein induction and AP-1 DNA binding activity stimulated by camostat feeding. Our results suggested that early response genes and specifically component of AP-1 may be potential mediators of camostat induced pancreatic growth, which is stimulated by endogenous CCK release.