Regulation of the cell cycle in the intestinal epithelium

Accumulating evidence points to a role for protein kinase C (PKC) signaling in negative growth regulation in the intestine. Previous studies in this laboratory have shown that PKC activation in the IEC-18 immature, non-transformed rat intestinal crypt cell line results in downregulation of cyclin D1, induction of p21waf1/cip1 expression, and cell cycle withdrawal into G0 (Frey et al., 1997; 2000). The objectives of the current study were: (a) to determine the signal transduction element(s) that link PKC activation to changes in the cell cycle regulatory machinery and (b) to further define the role(s) of individual PKC family members in intestinal epithelial cell cycle regulation by testing the effects of various PKC agonists and inhibitors in the IEC-18 model system. Our studies point to a key role for PKC alpha signaling in PKC agonist-induced cell cycle withdrawal in the intestinal epithelium. Based on evidence that PKC alpha expression is abrogated early during intestinal carcinogenesis, this finding has important implications for the prevention/therapy of intestinal neoplasms. Analysis of the mechanism(s) underlying PKC-mediated cell cycle arrest in intestinal cells points to the involvement of the extracellular signal-related (ERK)/mitogen-activated protein kinase (MAPK) cascade. PKC activation in IEC-18 cells led to the rapid phosphorylation/activation of ERK1/2, and both PKC-mediated alterations in cyclin D1 and p21waf1/cip1 expression and cell cycle arrest were found to be ERK/MAPK-dependent. PKC-mediated ERK1/2 activation was strong and sustained in comparison with that induced by proliferative signals (i.e., growth factors) in this system, and the growth inhibitory effects of the PKC agonist phorbol 12-myristate 13-acetate (PMA) were dominant over pro-proliferative signaling by growth factors when both agents were administered simultaneously. Comparison of the cell cycle-specific effects induced by PKC agonists which differ in their ability to sustain growth arrest in IEC-18 cells showed that maintenance of PKC-mediated cell cycle exit requires sustained activation of the ERK/MAPK pathway. The duration of PKC-mediated growth arrest was found to correlate with the extent and duration of ERK1/2 activation, which in turn correlated with the extent and duration of cyclin D1 downregulation and p21 waf1/cip1 induction. PKC signaling activated both Ras and Raf-1 in IEC-18 cells, suggesting that the PKC and ERK/MAPK pathways intersect at the level of Ras in this system. (Abstract shortened by UMI.)...