Ectopic expression of FGF-3 results in abnormal prostate and Wolffian duct development

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Abstract

To evaluate the effects of FGF-3 expression in the prostate and male reproductive tract, we employed a bitransgenic system to target FGF-3 to these organs. We present a first study that ectopic FGF-3 expression resulted in exuberant hyperplasia of all bigenic prostatic lobes typified by epithelial stratification, cribiform structures and papillary tufts. These cells displayed increased nuclear-to-cytoplasmic ratios and bromodeoxyuridine (BrdU) proliferative index but retained relatively uniform nuclear androgen receptor (AR) and the tumor suppressor C-CAM1 staining. Furthermore, the dysmorphogenic prostatic cells also resembled PIN (prostatic intraepithelial neoplasia)-like lesions but did not appear to have invaded the basal lamina. In addition to these phenotypes, profound disorders of the bigenic Wolffian duct derivatives were observed. The bigenic ampullary glands and vas deferens were extremely cystic, hypertrophic and hyperplastic; the enlarged epididymi showed a reduction of spermatozoa and the seminal vesicles exhibited a dramatic reduction of seminal secretions. Because of these severe abnormalities, these infertile males presented with diaphragmatic hernias, hemoperitoneum and many secondary abnormalities at sacrifice. Taken together, we show that ectopic FGF-3 expression severely perturbs normal prostate development and our system should be useful for the analyses of early changes in prostatic hyperplasia.

Introduction

Int-2, originally identified as one of six proviral MMTV integration sites in naturally occurring tumors of susceptible mice (Peters et al., 1983; Callahan, 1996; Tekmal and Keshava, 1997), was found to be a member of the fibroblast growth factor (FGF) family, namely FGF-3 (Dickson and Peters, 1987). The FGF family consists of at least 23 members to date (Yamashita et al., 2000); plays many different roles in growth, differentiation, angiogenesis, and tumorigenesis (Burgess and Maciag, 1989; Rifkin and Moscatelli, 1989; Basilico and Moscatelli, 1992); and exerts similar effects on cells other than fibroblasts (Gospodarowicz et al., 1978). The effects of FGFs on cells are mediated by their binding to one or more high affinity FGF receptors (i.e. FGFR1–4), which are tyrosine kinase receptors encoded by four different genes (Johnson and Williams, 1993).

FGF-3 is not expressed in most adult tissues but is detected embryonically in the parietal mesoderm, developing central nervous system, endoderm lining of pharyngeal pouches and the developing tail bud (Jakobovits et al., 1986; Wilkinson et al., 1988, 1989). Gene disruption revealed that FGF-3 plays an important role in embryonic tail and middle ear development (Mansour et al., 1993). When targeted to adult organs (i.e. mammary glands, male reproductive tract, lens of eyes and lungs), FGF-3 induces epithelial hyperplasia (Muller et al., 1990; Ornitz et al., 1991; Stamp et al., 1992; Robinson et al., 1998; Zhao et al., 2001). Taken together, these studies demonstrate that if FGF-3 is inappropriately expressed, it may activate dormant pathways that can then severely perturb normal growth and development.

The MMTV promoter has been used to target transgenes to the mammary glands in transgenic females and reproductive organs in males. Being steroid-responsive (Gunzburg and Salmons, 1992), the MMTV promoter can be regulated by progesterone (Ham et al., 1988), glucocorticoids (Yamamoto, 1985) and androgens (Darbre et al., 1986; Ham et al., 1988). In a previous study, the expression of an MMTV-FGF-3 transgene was detected mainly in the Wolffian duct derivatives including the ampullary glands, vas deferens and seminal vesicles (Muller et al., 1990; Donjacour et al., 1998). Though FGF-3 was also expressed in the dorsolateral lobes of the prostate, its expression failed to elicit any growth perturbations (Donjacour et al., 1998). Because FGFs like FGF-7 and FGF-10 have been established to play a role in prostatic development (reviewed in Thomson, 2001) and the deregulation of the FGF-FGFR axis promotes prostatic tumorigenesis (Yan et al., 1993; Foster et al., 1999), we assessed if ectopic expression of FGF-3 would perturb this signaling cascade and affect prostatic growth and development.

Here, we report that we have successfully developed bigenic mice that express the FGF-3 target transgene under the control of an MMTV-transactivator in the prostate and Wolffian duct derivatives. We establish that ectopic expression of FGF-3 in the prostate results in the formation of cribiform structures and papillary tufts and increases in epithelial stratification and basophilia similar to PIN-like lesions. The prostatic epithelial cells also displayed increased nuclear-to-cytoplasmic ratios, retained uniform AR and C-CAM1 staining but did not appear to have invaded the basal lamina. Disorders of the Wolffian duct derivatives that far exceeded those reported by Donjacour et al. (1998) were also evident. The bigenic males developed diaphragmatic hernia and presented with hemoperitoneum at necropsy. Taken together, these results show that the early and high levels of FGF-3 expression can elicit very profound reproductive tract abnormalities. Most importantly, we have developed an FGF-3 model that is useful for future investigations into early prostatic hyperplasia related to the deregulation of FGF signaling.

Results

Generation of bigenic mice

We used a binary system (Wang et al., 1997a) to study the effects of FGF-3 expression in the male reproductive tract by first creating an MMTV-driven transactivator (HMMB) as described in Materials and methods section and shown in Figure 1a. The transactivator consists of cDNA sequences corresponding to the VP16 activation domain, Gal4 DNA binding domain and a truncated progesterone ligand-binding domain (Wang et al., 1994, 1997b).

Figure 1
figure1

Characterization of bigenic mice. (a) The HMMB transactivator was constructed as described in the Materials and methods section and microinjected to generate transactivator mice (HMMB). Gal4DBD: Gal4 DNA binding domain; PRLBDΔ: truncated progesterone ligand binding domain; VP16: activation domain of herpes simplex virus protein VP16. (b) Northern analyses of 10 μg of total RNA from various tissues of a 11-week-old 9478 transactivator male revealed that the transactivator was expressed predominantly in the urinogenital tract (UGT) consisting of the undissected ampullary glands, prostate and urethra (*). SG: salivary glands. Loading was normalized by 18S levels. Arrowheads represent the position of the HMMB transactivator mRNA. (c) Northern analysis of 5 μg of total RNA isolated from the different lobes of the prostate indicated that the transactivator was moderately detectable in the DLP/VP prostatic lobes and barely expressed in the AP lobes. DLP: dorsolateral prostate; VP: ventral prostate; and AP: anterior prostate. Loading was normalized by 28S levels. Arrowheads denote the position of the HMMB transactivator mRNA. (d) Northern analyses of 10 μg of total RNA isolated from bigenic tissues showed that FGF-3 was detected only in bigenic tissues that expressed the transactivator, namely the Wolffian duct derivatives including the ampullary glands (AG), seminal vesicles (SV), vas deferens (VD) and epididymi (ED) and also the Harderian glands (HG). In bigenic tissues that failed to express the transactivator, namely the mammary glands (MG) and testes, no FGF-3 expression was detected. Loading was normalized by 18S levels. Arrowheads denote the relative positions of the HMMB and FGF-3 mRNAs

For transgenic mice generation, the HMMB transactivator construct was linearized with NotI and Acc65I and microinjected into fertilized B6C3F1 X ICR one-celled embryos. We obtained a transgenic line (9478) that expressed the transactivator predominantly in the mammary glands of females (unpublished data) and at high levels in the urogenital tract (UGT, composed of the ampullary glands and prostate in Figure 1b). Interestingly, the transactivator was expressed at moderate levels in the dorsolateral and ventral prostate (DLP/VP) and at barely detectable levels in the anterior prostate (Figure 1c). As expected, the transactivator was detected at high levels in the ampullary glands (AG) and other Wolffian duct derivatives (Figure 1d under HMMB). Therefore, to expedite our analyses, we expanded the 9478 transactivator line and bred these heterozygous mice to homozygous UASg-FGF-3 target (TGT) mice (Ornitz et al., 1991) to obtain bigenic male mice.

The original objective of this binary system (Wang et al., 1997a) was to develop an inducible model for FGF-3 tumorigenesis. This system is based upon the induction of any UASG-linked target gene by the constitutively expressed transactivator only upon activation by exogenously administered RU486 (Wang et al., 1997a). However, the unexpected perinatal lethality in the bigenic pups between the ages of P4 to P7 in the absence of RU486 suggested to us that this system was already precociously active. Since the focus of our research was to assess the effects of FGF-3 expression in adult tissues, we did not pursue the perinatal lethality any further after circumventing this lethality. By outbreeding the transactivators to ICR mice and using these resulting transactivators (>2 generations in the ICR background) to breed with the FGF-3 targets, we substantially increased the number of surviving bigenic mice (80 bigenic mice from 30 litters vs two from 10 litters initially). Therefore, based on the expression profile of the transactivator in the prostate (Figure 1c) and Wolffian duct derivatives (Figure 1d), we analysed the consequences of FGF-3 expression in these bigenic tissues in the absence of RU486.

FGF-3 expression in bigenic prostates

The expression of the transactivator in the dorsolateral and ventral (DLP/VP) lobes (Figure 1c) prompted us to evaluate if it was able to activate the expression of FGF-3. We performed in situ hybridization analyses on sections derived from all of the prostatic lobes of 8-week-old bigenic mice and compared these sections with those derived from age-matched monogenic mice. FGF-3 was robustly detected by an antisense FGF-3 riboprobe predominantly in the epithelia of each of the bigenic prostatic lobes (arrowheads in Figure 2e,f,k,l,q,r are equivalent points in the respective brightfields and darkfields) but not in the epithelia of the monogenic lobes (Figure 2b,h,n, for FGF-3 target and 2d,j,p for HMMB transactivator) under the conditions used. Therefore, the expressed transactivator is able to induce the expression of the FGF-3 target transgene in the bigenic prostates. Though the transactivator was barely detectable in the anterior prostate by Northerns (Figure 1c), the fact that FGF-3 was detected by in situ hybridization indicated that the transactivator was expressed in this organ albeit at low levels. In addition, the FGF-3 target was not detected in either of the monogenic prostatic sections because of the lack of one or the other transgene (i.e. the monogenic FGF-3 target lacks the HMMB transactivator and vice versa). Having successfully activated FGF-3 in the prostate, we sought to examine the phenotypic consequences of that expression.

Figure 2
figure2

Subcellular localization of FGF-3 expression in prostate. By situ hybridization, FGF-3 was detected predominantly in the stratified epithelial cells in the bigenics (BG in f,l and r) but was not detected in those of the monogenic target (TGT in b,h and n) and transactivator (HMMB in d,j and p) sections under the conditions used. (a,c,e,g,i,k,m,o and q) are brightfield and (b,d,f,h,j,l,n,p and r) are darkfield sections. Prostate: Anterior, AP (af); Dorsolateral, DLP (gl) and Ventral, VP (mr). Sections were counterstained with Hematoxylin. The arrowheads in e and f, k and l and q and r respectively denote exact positions in the brightfield and darkfield sections. The bars denote 100 μm and all sections are at similar magnifications

Histological analyses of bigenic prostates

Since the bigenic prostatic phenotypes were already pronounced at the adult 8-week stage, we concentrated our efforts at this stage. Analyses of each of the bigenic prostatic lobes (anterior, dorsolateral and ventral) histologically (n=4) revealed that these structures were dysmorphogenic (Figures 2e,k,q, and 3c,f,i). The bigenic anterior and dorsolateral prostatic acini (Figure 3c,f respectively) were typified by varying grades of prostatic hyperplasia. These acini were composed of cribiform structures and papillary tufts consisting of stratified epithelia (asterisks in Figure 3c,f). There was also an increase in basophilia characterized by higher numbers of densely-packed nuclei and greater nuclear-to-cytoplasmic (N/C) ratios, which resulted in a considerable loss of luminal space and secretory material in the bigenic organs (compare 3c vs a and b and 3f vs 3d and 3e respectively). In contrast, each of the monogenic (TGT in Figure 3a,d) and HMMB in Figure 3b,e) prostatic lobes contained acini lined by a singular layer of cuboidal-to-columnar secretory epithelium with rounded basally located nuclei characteristic of wildtype structures.

Figure 3
figure3

Histopathology of 8-week-old bigenic prostates. The bigenic (BG) prostatic lobes (c, f and i) displayed epithelial stratification and papillary tufts (*) in contrast to the singular layer of columnar epithelial cells that lined the monogenic (TGT and HMMB) acini (a,b,d,e and g,h respectively). Additionally, more bigenic epithelial cells (c,f and i) incorporated BrdU (arrowheads) than those of monogenics (a,b,d,e and g,h respectively). Prostate: Anterior, AP (ac); dorsolateral, DLP (df); and ventral, VP (gi). Sections were counterstained with Methyl Green. The bar represents 50 μm

The epithelia of the bigenic ventral prostates (VP) were also stratified (asterisk in Figure 3i) and possessed higher N/C ratios. This contrasted starkly with the singular layer of columnar epithelial cells lining the acini of the monogenic prostates (Figure 3g,h). Therefore, the ectopic expression of FGF-3 elicited profound epithelial cell hyperplasia that is morphologically reminiscent of prostatic intraepithelial neoplasia (PIN).

Bigenic prostates show higher proliferation indices

To evaluate if the prostatic hyperplasia was due to the mitogenic effects of FGFs, bromodeoxyuridine (BrdU) incorporation assays were performed after each of the animals were given a single injection of BrdU to specifically label cells in the S phase for 2 h. As shown in the bigenic sections (arrowheads in Figure 3c,f,i indicate stained nuclei), the over-expression of FGF-3 induced a higher proliferative index in the bigenic prostatic epithelia as evidenced by a higher percentage of cells staining positive for BrdU incorporation than monogenic sections (compare Figure 3c,f and i vs a and b,d and e, and g and h respectively). Out of 10 000 epithelial cells counted from four pairs of prostatic lobes, 1.5% of epithelial cells in the bigenic prostates versus 0.2% cells in the monogenic controls stained positive for BrdU. This value is significant as the mature prostate shows an extremely low rate of mitosis (Xue et al., 1997). Taken together, this result shows that FGF-3 is a potent growth factor that can elicit dramatic prostatic hyperplasia by excessively stimulating possibly the FGFR2iiib receptor (Mathieu et al., 1995; Thomson et al., 1997). Our data also demonstrate for the first time that both the FGF-3 mRNA and protein were functional in perturbing prostatic epithelia cell growth.

Androgen receptor and C-CAM1 immunohistochemistry

To assess if ectopic expression of FGF-3 affects the expression of AR, immunohistochemical staining of the prostatic lobes with an antibody specific to AR was performed. As shown in Figure 4, the bigenic sections from 4-month-old mice (Figure 4b,d,f) displayed uniform nuclear AR staining similar to those of the monogenic target (Figure 4a,c,e) and transactivator sections (data not shown). This result indicates that FGF-3 expression does not perturb AR expression or compartmentalization.

Figure 4
figure4

Androgen receptor staining of prostatic sections from 4-month-old bigenic mice. Monogenic target (TGT in a,c and e) and bigenic (BG in b,d and f) prostatic epithelial cells were homogenously stained by an AR antibody (Prins and Birch, 1995). Sections were counterstained with Methyl Green. Prostate: Anterior, AP (ab); Dorsolateral, DLP (cd); Ventral, VP (ef). The bar denotes 50 μm

Since the FGF-3 induced lesions were morphologically similar to PIN, we addressed if these bigenic lesions were progressing towards neoplasia by performing C-CAM1 (CEACAM1) staining. We chose C-CAM1 because it is a marker whose expression begins to diminish from the apical surfaces of prostatic epithelial cells as low grade PIN progresses to high grade PIN; becomes heterogeneous in high grade PIN; and is lost in well-differentiated prostate cancer (Pu et al., 1999). The finding that relatively homogenous C-CAM1 staining at the apical surfaces of epithelial cells from all of the prostatic lobes of both 8-week-old (Figure 5b,d,f) and 4-month-old bigenics (data not shown) were obtained when these were compared with monogenic sections (Figure 5a,c,e) suggested to us that despite resembling PIN, the FGF-3 induced lesions did not appear to be dysplastic. Further analyses of 4-month-old bigenic prostates using peroidic acid schiff (PAS) staining (Figure 6b,d,f) revealed that despite presenting with dysmorphogenic hyperplasia characterized by increased hyperchromasia, compressed nuclei, distorted acini, and a reduction of prostatic secretion, no frank invasion or penetration of basal lamina was evident. In contrast, the age-matched monogenic target prostatic sections appeared normal and showed abundant prostatic secretion (Figure 6a,c,e). Taken together, these results indicate that though FGF-3 may confer a growth advantage to prostatic epithelial cells, additional genetic mutations may be necessary for bona fide neoplasia to occur.

Figure 5
figure5

C-CAM1 staining of prostatic sections from 8-week-old bigenic mice. Relatively uniform C-CAM1 staining at the apical surfaces of the bigenic (BG) prostatic cells (b,d and f) comparable to those of the monogenic target (TGT) were observed (a,c and e). Prostate: Anterior, AP (ab); Dorsolateral, DLP (cd); Ventral, VP (ef). Sections were counterstained with Methyl Green. The bar denotes 50 μm

Figure 6
figure6

Histopathology of prostatic sections from 4-month-old bigenic mice. Though the epithelial of the bigenic (BG) prostatic sections displayed hyperchromatic and compressed nuclei, distorted prostatic acini and epithelial stratification (b,d and f), no frank invasion or penetration of basal lamina was evident. The monogenic target (TGT) sections comprise singular layers of prostatic epithelial cells and appeared normal (a,c and e). Prostate: Anterior, AP (ab); Dorsolateral, DLP (cd); Ventral, VP (ef). Sections were stained with PAS. The bar denotes 50 μm

Gross effects of FGF-3 in bigenic Wolffian duct derivatives

The transactivator also induced FGF-3 expression in the Wolffian duct derivatives (Figure 1d) including the ampullary glands (AG), epididymi (ED), vas deferens (VD) and seminal vesicles (SV) and in the Harderian glands (HG). Bigenic males possessed significantly enlarged scrota that can be easily differentiated from those of the monogenic controls by mere inspection (data not shown). By 8-weeks-of-age, the bigenic reproductive tracts (n=4 animals) were extremely enlarged as typical wet weights consisting of testes, epididymi, vas deferens, bladder, prostate, ampullary glands and urethra were 2.7675±0.0512 g compared to 0.71±0.04 g in aged-matched monogenic control and wildtype structures (n=9 animals). Because FGF-3 expression was also induced in the bigenic Harderian glands (Figure 1d) of both sexes, bilateral exophthalmia were presented (data not shown). Since the monogenic mice were histologically similar to wildtype controls, we subsequently utilized the monogenics as controls.

FGF-3 expression induces abnormal development of Wolffian duct derivative

The effects of FGF-3 expression in 8-week or older bigenic Wolffian duct derivatives were more profound than those reported previously (Donjacour et al., 1998). The cystic bigenic (BG) ampullary glands (Figure 7a) lacked luminal secretions in their expanded acini (Figure 7d), displayed extensive epithelial hypertrophy and hyperplasia (asterisk in Figure 7d) and were also penetrated by hyperplastic projections from the vas deferens (data not shown). In contrast, the monogenic transactivator (Figure 7b and inset) and target (Figure 7c and inset) organs consisted of pseudostratified columnar epithelia with uniform nuclei.

Figure 7
figure7

Histopathology of bigenic Wolffian duct derivatives of 8-week-old bigenic mice. Gross examination revealed that the bigenic (BG) ampullary glands (a), vas deferens (e) and epididymi (i) were larger, more cystic and hemorrhagic (indicated by arrowhead in e) than the corresponding control monogenic target (TGT) and transactivator (HMMB) controls while the bigenic seminal vesicles appeared grossly reddened when compared to the monogenic controls (m). The 5 mm bar in m denotes an equal distance in a,e,i and m. Microscopic analyses of the bigenic ampullary glands (d), vas deferens (h) and epididymi (l) showed profound epithelial stratification (indicated by asterisks in d and h and insets in d,h and l) when compared to the respective monogenic transactivator (b,f and j) and target (c,g and k) controls. Loss of secretion in the bigenic ampullary glands (d), seen as the cheese-like luminal material in the monogenic organs (b and c), and loss of seminal secretions in the bigenic seminal vesicles (p), denoted by pink-colored luminal secretions in the monogenic organs (n and o), were evident. Aspermia was seen in the bigenic vas deferens (arrowhead in h) and epididymi (arrowhead in l) and is in contrast to the abundant spermatozoa found in the monogenic controls (indicated by arrowhead in f and g for vas deferens and j and k for epididymi). The bar in p measures 50 μm and represents an equal distance in (bd, fh, jl and np). Sections in (bd, fh and jl) were stained with hematoxylin and eosin while those in (np) were stained with PAS. The bar in inset sections denotes an equivalent distance of 10 μm in all insets

The bigenic vas deferens contained hemorrhagic fluid-filled cysts (arrowheads in Figure 7e) unlike the normal monogenic organs (HMMB and TGT in Figure 7e,f,g and their respective insets) or the MMTV-Int2TG.NR transgenic organs, which were statistically enlarged only after 8-weeks-old (Donjacour et al., 1998). The bigenic epithelia were basophilic and stratified (asterisk and inset in Figure 7h) and displayed a significantly higher BrdU incorporation index (1.93% in bigenics versus 0.63% in monogenics out of about 3000 cells counted in each case). Furthermore, aspermia (arrowhead in Figure 7h) was evident in most of the bigenic lumens (n=6 animals analysed at 8-weeks or older) in contrast to abundant spermatozoa seen in the monogenic lumens (arrowhead in Figure 7f,g).

Unlike the normal epididymi found in monogenic controls (Figure 7j,k and insets) and the MMTV-TG.NR transgenics due to a lack of FGF-3 expression in these organs (Donjacour et al., 1998), the bigenic (BG) epididymi (n=3 animals) were grossly enlarged (Figure 7i,l). Profound epithelial stratification (Figure 7l and inset) were seen in the bigenic head, body and tail sections as opposed to a singular layer of cuboidal-to-columnar cells in the monogenic organs (Figure 7j,k and insets). Aspermia was also observed in the bigenic lumens (compare arrowheads in bigenics in Figure 7l vs monogenics in 7j,k respectively) and was likely due to spermatozoa blockage at the level of the rete testes or the failure of the hyperplastic epididymal cells to sustain spermatozoa maturation (Figure 7l).

The bigenic seminal vesicles appeared dramatically reddened (Figure 7m) and manifested irregular epithelial stratification and tufting (asterisk in Figure 7p and inset). When stained with PAS, the bigenic epithelial cells showed a considerable absence of secretory vesicles at their apical regions (arrowhead in Figure 7p) resulting in a reduction of seminal secretions and hence to the organ's grossly reddened appearance (Figure 7m). In contrast, abundant apical secretory vesicles in the epithelia (arrowheads in Figure 7n,o) and robust seminal secretions in the monogenic lumens were seen. Therefore, the over-expression of FGF-3 not only induced epithelial hyperplasia but may have also resulted in these bigenic cells losing their secretory function.

Bigenic males are infertile and develop diaphragmatic hernias

Because of these dramatic phenotypes, the bigenic males were infertile. Since the bigenic tests (8-weeks-of age) appeared grossly normal and consisted of abundant spermatozoa when compared to the monogenic controls (data not shown), it is likely that the blockage of spermatozoa transit and the failure of the Wolffian duct derivatives and prostate to function properly resulted in infertility. In older bigenic males (>4-months-old), the Wolffian duct derivatives occupied a significant portion of their distended abdominal cavities and weighed as much as 10% of the total body-weight (i.e. 4.512±1.290 g for n=5 bigenics vs 1.170±0.179 g for n=7 monogenic controls). It is also likely that these changes resulted in bladder obstruction due to compression and hence kidney failure. Because of the abnormally large reproductive tracts, the bigenic males developed diaphragmatic hernia due to increased abdominal pressure while internal hemorrhage (hemoperitoneum) resulted from rupture of the cystic portions of the vas deferens. Furthermore, many secondary effects may have resulted in visceral organ and lymph node enlargements (data not shown). Collectively, these defects contributed to mortality. Taken together, these results indicate that the ectopic expression of FGF-3 can elicit very profound disorders that can severely affect fertility and viability in a fashion more extensive than those reported on the MMTV-Int-2TG.NR mice (Donjacour et al., 1998).

Discussion

Here, we present a first report that the ectopic expression of FGF-3 in the prostate induces profound dysmorphogenic hyperplasia. The bigenic prostatic epithelial cells still retained a fairly homogenous C-CAM1 expression, remained relatively well-differentiated (Gingrich et al., 1999) and did not show frank invasion or penetration of the basal lamina. Consistent with the mitogenic effects of FGF-3, the bigenic prostatic cells displayed a significantly higher proliferation index as assayed by BrdU incorporation when compared to monogenic controls. One of the reasons FGF-3 is able to elicit rapid hyperplasia may be due to its predominant expression in epithelial cells, which could then create an autocrine, paracrine or juxtacrine signaling loop via its cognate receptor FGFR2iiib to deregulate growth. In this fashion, FGF-3 could replace the need for stromal FGFs like FGF-7 and FGF-10 in prostatic growth since all three FGFs can bind the same receptor, i.e. FGFR2iiib (Yan et al., 1993; Ittman and Mansukhani, 1997; Thomson and Cunha, 1999). However, the finding that the prostatic hyperplasia did not seem to become neoplastic at 4 months suggests that additional genetic mutations may be needed to effect this progression. It is also possible that if these bigenic males can survive long enough and do not succumb to hemoperitoneum, their prostatic lesions may eventually progress towards dysplasia. Consistent with this observation, it is intriguing to note that though the ectopic expression of FGF-3 elicited rapid hyperplasia in the virgin mammary glands, spontaneous tumors occurred only after 12 months (unpublished data). Furthermore, the switching of FGF receptor specificity or FGF expression in prostatic epithelial cells is likely required for progression towards malignancy (Yan et al., 1993; Feng et al., 1997; Foster, 1999).

Although these studies were performed using a single transactivator line, the phenotypes presented are not due to integration effects because of similar disorders and expression profiles of the FGF-3 transgenes in the Wolffian duct derivatives in our bigenic system and those of the direct or mono-transgenic systems (Muller et al., 1990; Donjacour et al., 1998). In addition, the use of two separate transgenic lines (transactivator and target) further reduces the possibility. Finally, the finding that the monogenic target or regulator lines did not develop any growth abnormalities implied that these transgenes were not integrated in a locus that is detrimental for growth or is contributing to the neoplastic events.

Though the phenotypes of the Wolffian duct derivatives elicited by FGF-3 here are similar to those of others (Muller et al., 1990; Donjacour et al., 1998), our bigenic mice presented with more dramatic disorders. In addition to exuberant hyperplasia, the bigenic ampullary glands and the vas deferens were cystic and enlarged while the seminal vesicles failed to acquire full secretory function and the epididymi appeared unable to sustain spermatozoa maturation. These pronounced bigenic phenotypes might be explained as follows. An earlier expression profile of FGF-3 may perturb cells when they are more sensitive to changes and/or provide these cells with a FGF-3 ligand to effect autocrine signaling and growth advantage. Secondly, similar to the binary system used by Ornitz et al. (1991), we were most likely able to activate higher levels of FGF-3 to deregulate growth more rapidly. Thirdly, the different genetic background and hence presence of genetic modifiers (Dietrich et al., 1993) could alter the severity of the phenotype. Lastly, a potential interaction between the highly expressed transactivator and FGF-3 target protein with each other, with genetic modifiers, or with other unidentified proteins cannot be discounted.

Despite the ability of FGF-3 to elicit hyperplasia that remained relatively well-differentiated in both the prostate and Wolffian duct derivatives, the extent and severity of these growth abnormalities in these organs were different. The disparate nature of FGF-3 effects could be ascribed to the different anatomical sites of these organs, their relative sizes, levels of FGF-3 expressed, stability of the FGF-3 mRNA or protein in these tissues, and levels of its cognate receptors (i.e. the iiib isoforms of FGFR1 and FGFR2) expressed in these tissues. Furthermore, the presence of different forms of FGF-3 protein (secreted, nuclear and nucleolar forms) in cells (Kiefer et al., 1994) may antagonize each other (Kiefer and Dickson, 1995; Antoine et al., 1997). Finally, the presence of cell-specific growth suppressors may also antagonize the actions of FGF-3.

In summary, we establish that ectopic expression of FGF-3 in prostatic epithelial cells elicits rapid hyperplasia but does not appear to effect progression into malignancy. This suggests that additional genetic mutations (i.e. alterations in the FGF–FGFR signaling axis) are required. That FGF-3 may obviate the need for stromal FGFs and hence confer a growth advantage suggests that the deregulation of FGF axis may be critical for the initiation of prostatic dysplasia. Therefore, by inducing changes in the FGF–FGFR signaling, our FGF-3 prostatic model should allow these early events to be dissected.

Materials and methods

Plasmid constructions

The HMMB transactivator was created as follows. The KCR-transactivator was cloned by inserting a 1.6 kb blunt-ended Asp718-BamHI transactivator composing of cDNA sequences from the Gal4 DNA binding domain (Gal4DBD encoding 1–147 aa), the truncated progesterone ligand binding domain (PRLBDΔ encoding 641–914 aa) and the VP16 activation domain (VP16 encoding 411–487 aa) into the blunt-ended EcoRI–SalI sites of a KCR vector (from Dr Brigid Hogan) containing a bovine growth hormone polyadenylation signal (bGHpA). A 2.4 Kb BamHI MMTV promoter (Sinn et al., 1987) was then ligated into the corresponding sites of the KCR-transactivator to generate the MMTV-transactivator. To minimize integration effects, a NotI (blunt-ended)-Acc65I fragment of the MMTV-KCR-transactivator was subcloned into the BamHI (blunt-ended-Acc65I sites of a vector containing a 2.4 kb hypersensitive site IV (HS4) fragment of the chicken β-globin insulator (Chung et al., 1993) to create the HMMB vector. The HMMB plasmid was linearized with NotI and Acc65I to release a 7.4 kb NotI-Acc65I fragment which was then gel-purified by Qiaex II beads (Qiagen) for microinjection. The FGF-3 template used for the generation of riboprobes was cloned by ligating a 1.0 kb EcoRI-PstI (blunt-ended) FGF-3 fragment (obtained from Dr Susanne Mansour) to the EcoRI-BamHI (blunt-ended) sites of Bluescript KSII vector (Stratagene). To generate an antisense riboprobe, the FGF-3 template was linearized with NotI and T7 RNA polymerase used.

Characterization of transgenic mice

Purified vector-free DNA was microinjected into B6C3FI stud male (Harlan) fertilized one-cell ICR embryos, which were then transferred into pseudo-pregnant ICR recipient mothers (Harlan) to carry the embryos to term. Genotypic analyses by both PCR and Southerns were performed on genomic DNA isolated from tail biopsies using transactivator-specific primers: PR-1 (5′AAG TCA GAG TTG TGA GAG CAC-3′) and PR-2 (5′-ACC TTG ATG AGC TCT CTA ATG-3′) and β-actin internal control primers: SCB1 (5′-GAT GTG CTC CAG GCT AAA GTT-3′) and SCB2 (5′-AGA AAC GGA ATG TTG TGG ATG-3′). A typical PCR consisting of 30 cycles of 94°C for 1 min, 56°C for 40 s and 72°C for 40 s was used to detect both 650 bp transgene-specific and 550 bp β-actin PCR products. Southern analyses were performed using the full-length transactivator cDNA as a probe on BamHI-digested genomic DNA at 60°C (Church and Gilbert, 1984). The UASg-FGF-3 target mice, a gift of Dr Phil Leder, harbors the FGF-3 target gene under the control of a minimum rat collagenase TATA box and four Gal4 DNA binding sites (Ornitz et al., 1991). The Gal4 DNA sites allow the transactivator to bind and activate FGF-3 target transgene expression in bigenic mice. To increase the number of viable bigenics, the transactivators were first outbred for two generations into ICRs and the resulting transactivators (>50% ICR background) were then bred with homozygous FGF-3 targets to yield bigenics. The resulting bigenics were genotyped with both transactivator and FGF-3 specific primers (Int-2F: 5′-ACT CAG GGT CCT GTG GAC AG-3′ and Int-2R: 5′-ACT CAC GGA TTT CTG TTG TG-3′).

Preparation and analyses of RNA

Total RNA was isolated from mouse tissues with the Trizol reagent (Gibco/Life Tech). For Northern analyses, 5–15 μg of total RNA was electrophoresed through a 1.2% agarose formaldehyde gel, transferred and hybridized with random-primed full-length transactivator or FGF-3 cDNA probes to detect the respective mRNAs at 60°C (Church and Gilbert, 1984).

In situ hybridizations

A standard in situ hybridization protocol (Cox et al., 1984) using 0.1–2.5×106 c.p.m. of antisense FGF-3 riboprobe per slide was performed. Following autoradiography on BioMax MR film (Kodak) to determine empirical development time, the slides were dipped in NTB-2 emulsion (Kodak), dried for 6–8 h in a light-tight box, developed, fixed, counterstained in Hematoxylin, coverslipped and photographed.

Histology and immunohistochemistry

The dissected male reproductive tracts were fixed in 4% paraformaldehyde overnight at 4°C, washed and stored in 70% ethanol until ready for processing for paraffin embedding. For histological examination, the slides were stained with Hematoxylin and Eosin or with periodic acid schiff (PAS) according to standard protocols. For BrdU incorporation studies, the mice were first given an intraperitoneal injection of BrdU at a dose of 100 μg/g body-weight 2 h prior to sacrifice to label cells in the S phase and the tissue sections immunostained with a monoclonal BrdU antibody (Zymed) as described previously (Ma et al., 1999). Immunostaining with the Ab669 C-CAM1 (Pu et al., 1999) and PG-21 AR antibodies (Prins and Birch, 1995) were performed accordingly.

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Acknowledgements

We thank Drs Roberto Barrios, Barbara Foster, Paula Kaplan and Fred A Pereira for their valuable help, advice and critical discussions; Drs Brigid Hogan, Jay Chung and Susanne Mansour for their generous gift of the MMTV-KCR, HS4 insulator and murine FGF-3 plasmids respectively; Dr Phil Leder for the FGF-3 target mice and Dr Gail Prins for the AR antibody. We are also grateful to LouAnn Hadsell and John Stockton for their excellent technical support. This work was supported by DOD and NIH grants to SY Tsai, an ACS RPG-95-020-04 grant to FJ DeMayo and a DOD fellowship to Z-Q Ma.

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Correspondence to Sophia Y Tsai.

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Chua, S., Ma, Z., Gong, L. et al. Ectopic expression of FGF-3 results in abnormal prostate and Wolffian duct development. Oncogene 21, 1899–1908 (2002) doi:10.1038/sj.onc.1205096

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Keywords

  • FGF-3
  • transgenic mice
  • prostate
  • Wolffian duct derivatives
  • hyperplasia

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