Regulation of nuclear oncogene expression in the exocrine pancreas.

Abstract

Gastrointestinal (GI) hormones and growth factors regulate exocrine pancreatic cell growth and digestive enzyme gene expression. However, the intracellular signals mediating these long-term regulatory effects are poorly understood. To test if nuclear oncogenes, which participate in the regulation of growth and differentiation, could be mediators of long-term exocrine pancreatic regulation, this thesis work addressed the questions: are nuclear oncogenes regulated by GI hormones that have long-term regulatory effect on the exocrine pancreas and if so, what are the mechanisms by which these hormones exert this regulation? Several important regulators of exocrine pancreas were evaluated for their ability to stimulate oncogene expression. To do this rat pancreatic acini were prepared by collagenase digestion, preincubated in HEPES buffered Ringer's medium, and then incubated with GI hormones or other agents. Total RNA was isolated from the acini by modified acid guanidine-thiocyanate-phenol-chloroform extraction. mRNAs of nuclear oncogenes c-fos, c-jun and c-myc were analyzed by Northern or slot blot analysis. It was found that nuclear oncogenes, c-fos, c-jun and c-myc were induced by cholecystokinin (CCK), bombesin, and carbachol, but not secretin, vasoactive intestinal peptide or gastrin. Focusing on CCK, the most important physiologic regulator of pancreatic adaptation, the time and concentration dependence of oncogene induction was studied. CCK-induced c-fos, c-jun and c-myc mRNA expression was found to be rapid, transient, and concentration dependent with a EC50 of 30 nM for CCK octapeptide. The intracellular messengers that mediate CCK induced c-fos expression were identified using inhibitors and activators of intracellular signaling pathways. It was found that inhibitors of intracellular Ca$\sp{++}$, protein kinnase C (PKC) and cAMP pathways alone inhibited CCK induced c-fos mRNA expression. However, activators of each one of the three pathways alone did not significantly induce c-fos mRNA. By contrast, the combination of the activators of the three pathways mimicked the effect of a maximal dose of CCK on c-fos expression. The results suggest that Ca$\sp{++}$, PKC and cAMP acting in concert mediate CCK induced c-fos expression. Nuclear run-on analysis and measurement of mRNA half-life revealed that the activation of c-fos and c-jun mRNA by CCK is mainly transcriptional. However, the elevation of c-myc mRNA by CCK is due to both the increased synthesis of mRNA and decreased degradation. These findings support the hypothesis that nuclear oncogenes could be the nuclear mediators of exocrine pancreatic long-term regulation.