Use of Normal and Transgenic Mice to Examine the Relationship between Terminal Differentiation of Intestinal Epithelial Cells and Accumulation of Their Cell Cycle Regulators*

Abstract

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 TAg^Wt) or a mutant TAg with Lys for Glu substitutions at residues 107 and 108 (SV40 TAg^K107/8) that fails to bind pRB and related pocket proteins. Both transgenes are expressed only in villus enterocytes. SV40 TAg^Wt 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 TAg^K107/8 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 TAg^Wt and Fabpi-SV40 TAg^K107/8 established that the proliferation induced by SV40 TAg^Wt 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.