CCKB/gastrin receptor mediates synergistic stimulation of DNA synthesis and cyclin D1, D3, and E expression in Swiss 3T3 cells

In order to develop a model system for identifying signaling pathways and c ell cycle events involved in gastrin-mediated mitogenesis, we have used hig h efficiency retroviral-mediated transfection of cholecystokinin (CCK)(B)/g astrin receptor into Swiss 3T3 cells. The retrovirally-transfected CCKB/gas trin receptor binds I-125-CCK-8 with high affinity (Kd = 1.1 nM) and is fun ctionally coupled to intracellular signaling pathways including rapid and t ransient increase in Ca2+ fluxes, protein kinase C-dependent protein kinase D activation, and MEK-dependent ERK1/2 activation. In the presence of insu lin, CCK-8 or gastrin induced a 66.5 +/- 8.8-fold (mean +/- SEM, n = 24 in eight independent experiments) increase in cellular DNA synthesis, reaching a level similar to that achieved by stimulation with a saturating concentr ation of fresh serum, and much greater than the response to each agonist ad ded alone. CCK-8 also induced a striking increase in the expression of cycl ins D1, D3, and E and hyperphosphorylation of Rb acting synergistically wit h insulin. Similar effects were observed when CCKB/gastrin receptor was act ivated in the presence of EGF or bombesin. Our results demonstrate that act ivation of CCKB/gastrin receptor retrovirally-transfected into Swiss 3T3 in duces a potent synergistic effect on DNA synthesis, accumulation of cyclins D1, D3, and E and hyperphosphorylation of Rb in combination with insulin, EGF, or bombesin. Thus, the CCKB/gastrin receptor transfected into Swiss 3T 3 cells provides a novel model system to elucidate mitogenic signal transdu ction pathways and cell cycle events activated via this receptor. (C) 2001 Wiley-Liss, Inc.