Deregulated E2F activity induces hyperplasia and senescence-like features in the mouse pituitary gland

Abstract

The retinoblastoma gene, RB1, is one of the most frequently mutated genes in human cancer. Rb heterozygous mice develop pituitary tumors with 100% incidence, and the E2F transcription factors are required for this. To assess whether deregulated E2F activity is sufficient to induce pituitary tumors, we generated transgenic mice expressing an inducible E2F3 protein in the intermediate lobe of the pituitary gland. We found that short-term deregulation of E2F activity, similar to the earliest stages of Rb loss, is able to induce abnormal proliferation of otherwise quiescent melanotrophs. However, while long-term exposure to deregulated E2F activity results in hyperplasia of the intermediate lobe, it did not lead to tumor formation. In fact, melanotrophs become insensitive to sustained E2F stimulation and enter an irreversible senescence-like state. Thus, although deregulated E2F activity results in hyperproliferation, it is not sufficient to mimic loss of Rb, sustain proliferation of melanotrophs, and ultimately induce pituitary tumors. Similarly, we found that primary cells in tissue culture become insensitive to sustained E2F3 activation and undergo premature senescence in a pRB-, p16Ink4a-, and p19Arf-dependent manner. Thus, we conclude that deregulated E2F activity is not sufficient to fully mimic loss of Rb due to the engagement of a senescence response.

FIG. 1.

POMC ER-E2F3 transgenic mice. (A) Schematic representation and detection of the ER-E2F3 transgenic construct. BglII restriction sites, the POMC probe, and the 1.6-kb BglII fragment are indicated. By Southern blot analysis, we detected a transgene-specific band (1.6 kb) and a band corresponding to the endogenous POMC gene, confirming the presence of the transgene in the founder mice (F₀) and the F₁ litter. (B) ER-E2F3 expression as determined by immunoblotting on total pituitary lysates with an estrogen receptor (ER)-specific antibody. (C) (Left panel) Relative expression of E2F3 mRNA in transgenic (dashed bar) and wild-type (black bar) pituitaries. (Right panel) Expression of murine E2f3 in a wild-type intermediate lobe (black bar) and in intermediate lobe pituitary tumors (ILPT) derived from Rb wild-type and mutant mice. (D) ER-E2F3 expression in situ. Immunohistochemistry with an antibody specific for human E2F3 on pituitaries derived from wild-type (WT) and transgenic (Tg) mice treated for 3 days with tamoxifen (+TAM). NL, neural lobe; IL, intermediate lobe; AL, anterior lobe. (E) ER-E2F3 is functional and induces unscheduled proliferation in vivo. PCNA and MCM2 expression and bromodeoxyuridine (BrdU) incorporation measured by immunohistochemistry in the intermediate lobe of wild-type (WT) animals treated with tamoxifen and transgenic animals (Tg) treated witheither tamoxifen or sunflower oil as a control .

FIG. 2.

Deregulated E2F activity induces hyperplasia. (A) Experimental design. Schematic representation of tumorigenic steps leading to intermediate lobe tumors in Rb^+/− mice (upper panel) and the experimental approach used in this study (lower panel). (B) Activation of E2F3 results in hyperplasia. Hematoxylin and eosin staining of wild-type and transgenic pituitaries treated for 0, 1, 3, and 6 months with daily injections of tamoxifen (TAM). Note the enlargement of the intermediate lobe in transgenic mice treated for 3 months and 6 months (bar). (C) Relative thickness of the intermediate lobe. The bar graph indicates the relative thickness of the intermediate lobe upontamoxifen treatment for the indicated times in transgenic animals relative to wild-type animals and the standard deviation of the mean. For each time point, the thickness of the intermediate lobe was measured in at least three different sections derived from five mice per genotype. The difference in size of the intermediate lobe in transgenic mice compared to wild-type (WT) mice is statistically significant upon 3 and 6 months of tamoxifen (TAM) treatment (Student t test; P = 0.0003 and P = 0.00001, respectively) (D) Neoplastic foci in Rb heterozygous mice. Pituitaries were stained as indicated for panel B. Note the appearance of neoplastic foci in 5-month-old mice and the progression towards tumor development in 8-month-old mice.

FIG. 3.

Melanotrophs become resistant to sustained increased E2F activity. (A) Decreased proliferation following sustained E2F activation. Bromodeoxyuridine incorporation measured by immunohistochemistry in the intermediate lobe of wild-type (WT) and transgenic (Tg) animals treated for the indicated time with daily tamoxifen (TAM) injections. The bar graph indicates the percentage of bromodeoxyuridine-positive melanotrophs in the intermediate lobe and the standard deviation of the mean. (B) Expression of ER-E2F3 protein is retained in tamoxifen-treated mice. Immunohistochemistry analysis of ER-E2F3 expression in the intermediate lobe of wild-type (WT) and transgenic (Tg) animals treated for the indicated times with daily tamoxifen injections. The bar graph indicates the percentage of E2F3-positive melanotrophs in the intermediate lobe and the standard deviation of the mean. (C) Sustained E2F activity triggers melanotrophs to become refractory to E2F stimulation. From the top: bromodeoxyuridine incorporation, MCM2 expression, and hematoxylin and eosin staining of melanotrophs after 3 days of tamoxifen treatment of 9-month-old transgenic mice either not previously exposed to tamoxifen (left panel) or treated for 6 months followed by 1 month of withdrawal (right panel). Note that the hyperplastic morphology of the intermediate lobe (bar) of the long-term-induced transgenic animals does not regress upon tamoxifen withdrawal (right panel). (D) Percentage of bromodeoxyuridine-positive melanotrophs. Treatment of 9-month-old transgenic animals leads to the same extent of S-phase induction achieved in 3-month-old transgenic animals (compare second lane from left with last on the right). Sustained E2F activity prevents S-phase induction in melanotrophs (fourth and sixth lanes from the left).

FIG. 4.

MEFs exhibit a biphasic response to E2F3 stimulation. (A) E2F3 induction enhances S-phase entry in cycling MEFs. The percentage of MEFs incorporating bromodeoxyuridine upon E2F3 induction is shown. NI, not infected; ER-E2F3, infected cells not induced;ER-E2F3 + OHT, infected cells induced with 4-hydroxy tamoxifen. (B) Transient induction of E2F targets upon E2F stimulation. Expression levels of the E2F targets PCNA, cyclin E1 (CCNE1), and CDC6 upon E2F3 induction was assessed by quantitative PCR, normalized against GAPDH, and the not-induced samples (t = 0) set as 1.0. (C) Expression of the ER-E2F3 fusion protein throughout the assay was assessed at the indicated times, and vinculin was used as a loading control.

FIG. 5.

pRB is recruited to E2F dependent promoters in response to E2F3 activation. (A) Transient induction of E2F targets upon E2F3 activation. Expression levels of the E2F targets EZH2, CDC2, and cyclin E1 (CCNE1) after E2F3 activation was assessed by quantitative PCR, normalized against GAPDH, and not induced (t = 0) was set as 1.0. (B) ER-E2F3 binds to E2F-responsive promoters and recruits pRB upon activation. Enrichment, relative to the input, of the EZH2, CDC2, and GAPDH promoters following E2F3, pRB, or Flag chromatin immunoprecipitation (ChIP) is shown.

FIG. 6.

Sustained E2F activity induces pRB-dependent senescence-like features. (A) Morphological changes as a consequence of E2F activity. Hematoxylin- and eosin-stained intermediate lobe of wild-type and transgenic mice treated for 6 months with tamoxifen. Note the increased nucleoplasmic-cytoplasmic ratio in transgenic animals compared to the wild type. (B) Accumulation of senescence markers upon sustained E2F activity. Panels show staining of either pituitary gland sections derived from wild-type (WT) and transgenic (Tg) animals or pituitary tumors derived from Rb^+/− mice. (Upper panel) Two magnifications of DAPI staining showing the formation of senescence-associated heterochromatic foci (SAHFs) in the intermediate lobe of transgenic animals upon sustained E2F activity. Note the formation of foci in transgenic animals treated for 6 months. (Lower panels) Immunohistochemistry analysis of p16^Ink4a and p19^Arf in the intermediate lobe. (C) Percentage of p16^Ink4a and senescence-associated heterochromatic focus-positive melanotrophs in the intermediate lobe of wild-type and transgenic mice treated for the indicated times with tamoxifen (TAM) and in the intermediate lobe tumor of Rb^+/− mice.

FIG. 7.

E2F-induced senenescence is dependent on pRB, p16^Ink4a and p19^Arf. (A) E2F-induced senescence is pRB dependent. MEFs were passaged following a 3T3 protocol. Genotypes and treatments of cells were as follows: uninfected (RBloxP), ER-E2F3 infected (ER-E2F3, RB loxP), ER-E2F3 infected and induced with tamoxifen (ER-E2F3, RB loxP:OHT), Adeno-CRE infected (RB lox P: CRE), Adeno-CRE and ER-E2F3 infected and induced with tamoxifen (ER-E2F3, RB loxP: OHT and CRE). Tamoxifen administration and Adeno-CRE infection were performed at passage 5 and passage 6, respectively, as indicated by the arrows. (Inset) Protein expression levels of pRB in MEFs of the indicated genotype either infected with Adeno-CRE or not infected. The expression of vinculin was used as a loading control. (B) E2F3-induced senescence is partly dependent on p16^Ink4a. MEFswere passaged following a 3T3 protocol. Genotypes and treatments of cells were as follows: wild-type, ER-E2F3 infected (ER-E2F3, Ink4+/+), wild-type ER-E2F3 infected and induced with tamoxifen (ER-E2F3, Ink4+/+:OHT), p16^Ink4a null MEFs ER-E2F3 infected (ER-E2F3, Ink4*/*) p16^Ink4a null MEFs ER-E2F3 infected and induced with tamoxifen (ER-E2F3, Ink4*/*: OHT). Tamoxifen administration was performed at the indicated passage (arrow). (C) E2F-induced senescence is ARF dependent. MEFs were passaged following a 3T3 protocol. Genotypes and treatments of cells as follows: wild-type, ER-E2F3 infected (ER-E2F3, ARF+/+), wild-type ER-E2F3 infected and induced with tamoxifen (ER-E2F3, ARF+/+: OHT), ARF^−/− MEFs ER-E2F3 infected (ER-E2F3, ARF−/−), and ARF^−/− MEFs ER-E2F3 infected and induced with tamoxifen (ER-E2F3, ARF+/+: OHT). Tamoxifen administration was performed at the indicated passage (arrow).

FIG. 8.

Model for the biphasic response to E2F deregulation. See text for discussion of the model.