Inducible expression of FGF-3 in mouse mammary gland

Abstract

Fibroblast growth factor-3 (FGF-3) is a crucial developmental regulator. Aberrant activation of this gene by mouse mammary tumor virus insertion results in pregnancy-responsive mammary tumorigenesis. To characterize better FGF-3 function in postnatal mammary gland development and cancer initiation/progression, we used a mifepristone (RU486)-inducible regulatory system to express conditionally FGF-3 in the mammary epithelium of transgenic mice. Ectopic overexpression of FGF-3 in pubescent mammary glands elicited severe perturbations in early mammary gland development leading to mammary hyperplasia. Ductal elongation was retarded, multiple cysts persisted in the virgin ducts, and ductal epithelium was expanded and multilayered. The altered ductal architecture and the persistence of hyperplastic multilayered epithelium reflect a defect in growth regulation, which resulted from an imbalance between mitogenic and apoptotic signals. By altering the duration of RU486 treatment, we showed that the persistence of mitogenic signal elicited by FGF-3 is crucial for the initiation, progression, and maintenance of the hyperplastic characteristic of the mammary epithelium. The manifestations elicited by FGF-3 could be reversed by RU486 withdrawal. In addition, synergism between the stimulus from estrogen and FGF-3 mitogenic pathways was evident and likely contributes to the pregnancy-dependent tumorigenesis of FGF-3. Taken together, the mifepristone-inducible regulatory system provides a powerful means for understanding the diverse roles of FGF-3 and its interactions with hormones in mammary gland tumorigenesis.

Fig 1.

Characterization of the RU486-inducible system. (A) The MMTV-KCR-GLp65 construct contains a 1.54-kb MMTV-LTR and GLp65 transactivator linked to KCR as described in Material and Methods. (B) RNase protection assay of 10 μg of RNA from different transgenic mice tissues showed that GLp65 was specifically expressed in the mammary glands (M.Gl). (C) RU486 induction of FGF-3 was seen only in bigenic mammary glands (Bi-Tg: GLp65/UAS_G-FGF-3) and not in monogenic target (UAS_G-FGF-3) or transactivator (MMTV-GLp65) glands given 150 μg/kg of body weight of RU486 or bigenic given placebo treatment for 2 weeks. Expression of GLp65 was seen only in monogenic transactivator and bigenic glands. (D) FGF-3 expression in bigenic mice was induced after 1 day of 150 μg/kg of body weight of RU486 and persisted for the duration of the treatment. (E) Dose-dependent increase in FGF-3 expression was seen only in bigenic mice. FGF-3 expression was not detected in monogenic mice given RU486.

Fig 2.

Induced FGF-3 expression initiates and promotes progression of mammary hyperplasia. Whole-mount analyses of mammary glands from bigenic mice treated with 450 μg/kg of RU486 for 2 (C, D), 4 (E, F), and 6 (G, H) months show that prolonged induction of FGF-3 increased the severity of hyperplasia. Monogenics (Ctrl) treated with RU486 (A, B) and bigenics treated with placebo for 6 months (I, J) manifest normal ductal phenotype. (B, D, F, H, and J are higher magnifications of A, C, E, G, and I, respectively.)

Fig 3.

Histological and in situ analyses of the transgenic mammary gland. (A–C) Sections were stained with hematoxylin and eosin (H&E). Comparison of controls (Ctrl: monogenics or wild types treated with RU486 or bigenics treated with placebo in A) and bigenics (Bi-Tg) given RU486 for 2 months showed that the bigenic glandular epithelium was multilayered and loosely associated (B) in contrast to a uniform singular layer of cells in the control (A). In a more hyperplastic region (C), the bigenic epithelium was completely disorganized and surrounded by dense connective tissue. (D–F) By in situ hybridization (ISH), high levels of FGF-3 expression were detected as blue-black precipitates in the bigenic (E, F) but not in control epithelium of monogenic mice given 2 months of RU486 treatment (D).

Fig 4.

Elevated proliferation of FGF-3-expressing epithelial cells. (A) More prominent nuclei positively staining for BrdUrd were observed in the bigenic (Bi-Tg) than control (Ctrl) sections. The lower two panels are the respective higher magnifications of the upper panels. (B) Quantitation of 300–400 mammary epithelial nuclei per section for BrdUrd staining of RU486- treated monogenic control and bigenic glands. Values are an average fraction of BrdUrd-positive nuclei per section from n = 4 animals per group. (C) RNase protection assay indicates that cyclin D1 is up-regulated in bigenic (Bi-Tg) but not control (Ctrl) glands. Ten micrograms of RNA from three individual RU486-treated monogenic and bigenic mice were hybridized with cyclin D1 and L32 antisense riboprobes.

Fig 5.

FGF-3 down-regulates extracellular matrix factors. More bigenic hyperplastic cells stain positive for phospho-MAPK than control cells. Loss of polarized expression of E-Cadherin occurred in bigenic but not in control cells. Sections were stained with anti-phospho-MAPK and E-cadherin antibodies, and positive signals visualized with Cyanine 3 (red). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (blue).

Fig 6.

FGF-3-elicited mammary gland hyperplasia is reversible. Whole-mount analyses showed that the hyperplastic glands of bigenic mice treated with 450 μg/kg RU486 for 4 months (ON in C and D) can be reversed after a 2-month withdrawal of RU486 (OFF in E and F). Monogenic glands from mice given RU486 displayed normal morphology. (Lower) RPA analyses indicates that the FGF-3 transgene can be turned on and off in the bigenic glands in response to the administration and withdrawal of RU486. FGF-3 was undetectable in the controls.

Fig 7.

Hormonal responsiveness of FGF-3-elicited hyperplasia. Monogenic and bigenic mice were ovariectomized and treated with RU486 (450 μg/kg) for 2 weeks. Subsequently, 50 μg/kg of estrogen or estrogen/progesterone were given for 3 consecutive days. Sesame oil was used as a vehicle control. Histological analyses showed that the bigenic (Bi-Tg) glands given estrogen manifested multilayered cells, which became more severe with estrogen and progesterone cotreatment. In contrast, a uniform layer of epithelium was shown in all monogenic controls (Ctrl). Vehicle treatment does not alter the bigenic ductal morphology.