Use of normal and transgenic mice to examine the relationship between terminal differentiation of intestinal epithelial cells and accumulation of their cell cycle regulators.

A spatially well organized continuum of proliferation, differentiation, and death is displayed along crypt-villus units in the adult mouse small intestine. This continuum provides an opportunity to examine in vivo the mechanisms by which proliferative status changes as a function of cellular differentiation. Immunohistochemical studies of normal FVB/N mice revealed that as epithelial cells complete their terminal differentiation during a 48-72-h migration up villi, there is a marked and rapid fall in the levels of two important regulators of the G1/S transition, cyclin D1 and cyclin-dependent kinase (cdk) 2. However, cellular levels of their partners, cdk4 and cyclin E, remain unchanged as does the level of pRB. Adult FVB/N transgenic mice were studied that contained an intestinal fatty acid binding protein gene promoter (Fabpi) linked to wild type Simian virus 40 large T antigen (SV40 TAgWt) or a mutant TAg with Lys for Glu substitutions at residues 107 and 108 (SV40 TAgK107/8) that fails to bind pRB and related pocket proteins. Both transgenes are expressed only in villus enterocytes. SV40 TAgWt causes these terminally differentiated cells to re-enter the cycle. Re-entry is accompanied by a reduction in un/hypophosphorylated pRB, an induction of cyclin D1 and cdk2, but no change in cdk4, cyclin E, or E2F-1. In contrast, SV40 TAgK107/8 fails to induce re-entry and does not produce changes in un/hypophosphorylated pRB, cyclin D1, or cdk2 accumulation. These results suggest that un/hypophosphorylated pRB is an important mediator of the cell cycle arrest that normally occurs as enterocytes exit the crypt and complete their differentiation. Fabpi-directed expression of E2F-1 does not cause villus enterocytes to return to the cell cycle, alter their suppression of cyclin D1 or cdk2, or affect their state of differentiation, emphasizing the insensitivity of these cells to the effects of E2F-1. Analyses of p53(-/-) and p53(+/+) mice containing Fabpi-SV40 TAgWt and Fabpi-SV40 TAgK107/8 established that the proliferation induced by SV40 TAgWt does not require p53 and is associated with increased (p53-independent) apoptosis. The presence of cyclin E and cdk4 in differentiating villus enterocytes emphasizes that these cells retain part of their proliferative heritage expressed 24-72 h earlier in the crypt. The data suggest that down-regulation of cdk2 and/or cyclin D1 expression may be important for control of proliferative status and/or execution of terminal differentiation.