Intestinal hyperplasia induced by simian virus 40 large tumor antigen requires E2F2

Abstract

The simian virus 40 large T antigen contributes to neoplastic transformation, in part, by targeting the Rb family of tumor suppressors. There are three known Rb proteins, pRb, p130, and p107, all of which block the cell cycle by preventing the transcription of genes regulated by the E2F family of transcription factors. T antigen interacts directly with Rb proteins and disrupts Rb-E2F complexes both in vitro and in cultured cells. Consequently, T antigen is thought to inhibit transcriptional repression by the Rb family proteins by disrupting their interaction with E2F proteins, thus allowing E2F-dependent transcription and the expression of cellular genes needed for entry into S phase. This model predicts that active E2F-dependent transcription is required for T-antigen-induced transformation. To test this hypothesis, we have examined the status of Rb-E2F complexes in murine enterocytes. Previous studies have shown that T antigen drives enterocytes into S phase, resulting in intestinal hyperplasia, and that the induction of enterocyte proliferation requires T-antigen binding to Rb proteins. In this paper, we show that normal growth-arrested enterocytes contain p130-E2F4 complexes and that T-antigen expression destroys these complexes, most likely by stimulating p130 degradation. Furthermore, unlike their normal counterparts, enterocytes expressing T antigen contain abundant levels of E2F2 and E2F3a. Concomitantly, T-antigen-induced intestinal proliferation is reduced in mice lacking either E2F2 alone or both E2F2 and E2F3a, but not in mice lacking E2F1. These studies support a model in which T antigen eliminates Rb-E2F repressive complexes so that specific activator E2Fs can drive S-phase entry.

FIG. 1.

Domain maps of SV40 T antigens: J domain, Rb protein-binding motif (LXCXE), nuclear localization signal (NLS), origin-binding domain (OBD), Zn domain, ATPase domain, and host range domain (HR). Interactions with cellular components and phenotypic effects of protein expression in intestinal enterocytes are indicated.

FIG. 2.

Expression of T antigen alters specific Rb family protein and transcript levels in villi, and such alteration is dependent on an intact LxCxE motif. (A) Rb family steady-state protein levels in nontransgenic and T-antigen-transgenic villus samples. Protein extracts from villi of nontransgenic and T-antigen-transgenic mice were subjected to immunoblotting for pRb, p130, and p107. Positive (+/+) and negative (−/−) controls refer to normal and KO MEFs, respectively. Protein levels for β-tubulin were used as loading controls. Arrow indicates the position of p130. (B) Rb family steady-state transcript levels in nontransgenic and T-antigen-transgenic villus samples. Equal amounts of cDNAs reverse transcribed from total RNA extracts from villi of nontransgenic and T-antigen-transgenic mice were subjected to PCR amplification using specific primers for pRb, p130, and p107. Transcript levels of Adh5 were used as loading controls.

FIG. 3.

T antigen alters Rb-E2F DNA complexes in villus enterocytes. Protein extracts from villi of nontransgenic and T-antigen-transgenic mice were subjected to E2F EMSA analysis and antibody supershifts. (A) Three major E2F complexes are present in nontransgenic mice, and three new bands appear in TAg^wt intestines. (B) Identification of E2F-containing complexes in control and T-antigen-expressing samples. Antibodies specific for E2F1, E2F2, or E2F3 were added to the EMSA reaction mixtures.

FIG. 4.

(A) Semiquantitative RT-PCR analysis of RNA samples obtained from villus enterocytes from control or T-antigen-expressing mice. Adh was used as a loading control. (B) Steady-state levels of E2F proteins in transgenic intestines. Protein extracts from whole intestines were used to analyze the levels of E2F2 and E2F3a by immunoblotting; β-tubulin was included as a loading control. E2F1 levels were very low and were detected reproducibly only in nuclear extracts; histone H3 was used as a loading control. Lanes labeled (+) and (−) refer, respectively, to normal and KO MEFs used as controls. Levels of the activators E2F2 and E2F3a are higher in intestinal villi expressing TAg^wt or TAg^N136 than in control or TAg³²¹³-expressing intestinal villi.

FIG. 5.

Intermediate proliferative phenotype in the absence of functional E2F2 and E2F3a. Duodenal sections from adult mice were stained for BrdU and counterstained with hematoxylin. In contrast with control mice (A), the expression of T antigen induces ectopic proliferation and intestinal hyperplasia (B), which is ameliorated in the absence of E2F2 alone (D) or in the absence of both E2F2 and E2F3a (E) but not in T-antigen-expressing mice lacking E2F1 (C).

FIG. 6.

(A) The increase in proliferation following expression of T antigen in enterocytes is reduced in the absence of E2F2 alone or of both E2F2 and E2F3a but not in the absence of E2F1. Total numbers of positive BrdU-labeled cells in either the villi or the crypt were counted and averaged for each group of mice and are represented in the graph. Error bars indicate the standard deviation in each group. (B) Ectopic proliferation in TAg^wt; E2F2 KO mice does not correlate with increased E2F1 protein levels. E2F1 expression was measured by Western blotting against a positive control, nontransgenic intestinal samples, or intestinal samples expressing TAg^wt in the presence or absence of functional products of E2F2 alone or of both E2F2 and E2F3a. Depletion of E2F2 was confirmed for the corresponding samples.