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Endocrinology, doi:10.1210/en.2004-0526
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Endocrinology Vol. 145, No. 9 4292-4300
Copyright © 2004 by The Endocrine Society

Differential Effects of Transforming Growth Factor-ß1 on Cellular Proliferation in the Developing Prostate

Darren C. Tomlinson, Sarah H. Freestone, O. Cathal Grace and Axel A. Thomson

Medical Research Council Human Reproductive Sciences Unit, Center for Reproductive Biology, University of Edinburgh, Edinburgh, Scotland, United Kingdom EH16 4SB

Address all correspondence and requests for reprints to: Dr. Axel A. Thomson, Medical Research Council Human Reproductive Sciences Unit, Center for Reproductive Biology, University of Edinburgh, Chancellor’s Building, 49 Little France Crescent, Old Dalkeith Road, Edinburgh, Scotland, United Kingdom EH16 4SB. E-mail: axel.thomson{at}hrsu.mrc.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
TGFß1 plays an important role in the growth of the prostate and has been reported to stimulate or inhibit the proliferation of prostatic epithelia. We show here that Tgfß1, Tgfß2, and Tgfß3 mRNA expression correlated with developmental growth of the prostate. Recombinant TGFß1 inhibited the growth of the prostate when added to cultures of ventral prostate (VP) organs grown in vitro. Interestingly, TGFß1 had contrasting effects on cellular proliferation; it stimulated proliferation at the periphery of the organs (distal to urethra), but inhibited proliferation in the center of the organs (proximal to urethra). We speculate that differential effects on proliferation may be determined by the level of cellular differentiation, because cells at the periphery are undifferentiated whereas those in the center are more highly differentiated. TGFß1 also stimulated branching morphogenesis at growing ductal tips at the perimeter of the VP. To investigate potential mechanisms of TGFß1 action, we examined the three-dimensional distribution of smooth muscle in prostatic organs after treatment with TGFß1. TGFß1 showed a significant effect on the distribution of smooth muscle within VPs, which may mediate part of its effect on proliferation. Finally, we addressed how testosterone and TGFß1 might affect gene expression in our developmental system. Testosterone repressed the expression of Tgfß2 mRNA in the prostate, whereas TGFß1 showed a modest repression of fibroblast growth factor-10 mRNA. It appeared that the effects of these factors were more pronounced in a model of prostatic mesenchyme devoid of epithelia than in prostatic organs (containing epithelia).


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ANDROGENS INDUCE THE development of the prostate in the urogenital tract (UGT), stimulate its growth during embryonic and neonatal life, and maintain the prostate in adult life. Numerous growth factors have been identified as regulators of prostatic growth as well as mediating mesenchymal/epithelial interactions involved in the control of proliferation and differentiation. These include members of the fibroblast growth factor (FGF), Wnt, Hedgehog, and TGFß families of molecules. Members of the TGFß superfamily have been implicated in biological processes ranging from specification of cell fate during embryogenesis to regulation of cell proliferation during development and adulthood. In mammals there are three Tgfß genes, Tgfß1, Tgfß2, and Tgfß3, which function through the same receptor (1).

During urogenital development, both male and female rodents contain a condensed pad of mesenchyme located below the bladder, called the ventral mesenchymal pad (VMP), which is involved in the development of the ventral prostate (VP) (2, 3). The VMP constitutively expresses factors involved in prostatic development such as FGF10, which functions as a mesenchymal paracrine regulator of prostatic epithelia (4) and is essential for the formation of the prostate (5). Recently, TGFß1 has been shown to repress the expression of Fgf10 mRNA in primary mesenchyme involved in prostatic organogenesis (6). This suggests that TGFß1 might inhibit prostatic growth by down-regulating molecules that stimulate prostatic growth.

The expression and function of TGFßs in the prostate have been examined in numerous studies and model systems, ranging from normal to tumorigenic cells both in vitro and in vivo (reviewed in Ref. 7). TGFßs have been shown to affect a wide variety of cellular activities and can stimulate or inhibit proliferation, apoptosis, and differentiation. Many studies have used cell lines in vitro, suggesting that TGFßs elicit their effects directly. However, other studies, such as cocultures or cells grown as xenografts, permit the observation of indirect effects that are mediated via cell types other than those in which effects are observed. A recent study has highlighted the importance of stromal TGFß action in prostate tumorigenesis, showing that the loss of TGFß receptor signaling in prostatic stroma leads to neoplastic lesions (8).

TGFß1 is expressed in the developing prostate (9, 10, 11, 12, 13) and can inhibit organ growth in vitro (14). TGFß1 and its receptor proteins have been localized to both stromal and epithelial tissue compartments (9, 10, 11, 12, 13, 15), suggesting that there could be both direct and indirect effects on cells within the prostate. TGFß1 inhibits the proliferation and induces cell death of both normal prostatic epithelial and stromal cells derived from rodents and humans (16, 17, 18). However, other studies have reported that low concentrations of TGFß1 can increase the proliferation of prostatic cells (19, 20, 21). This suggests that TGFß1 may play a differential role in the regulation of proliferation of prostatic cells, and it is possible that different levels of cellular differentiation may account for some of the variable effects on cell growth. Ablation of androgens by castration has suggested a role for TGFßs during prostate regression (22, 23). However, androgen-mediated repression of Tgfß isoforms was not observed in isolated prostatic epithelial or stromal cells (14), suggesting that stromal-epithelial interactions were required for the repression of Tgfß transcripts by androgens.

TGFß1 has been shown to have significant effects on the differentiation of stromal cells in the prostate and stimulates the expression of smooth muscle (SM) markers in cells grown in vitro (24, 25, 26). SM plays a key role in prostatic organogenesis and regulates interactions between the VMP and nascent prostatic buds (3). During subsequent development of the prostate, there is an asymmetric distribution of SM surrounding prostatic ducts; there is little or no SM at growing epithelial tips and increased SM differentiation surrounding maturing ducts proximal to the urethra (27).

It is clear that TGFßs can have several diverse activities in the prostate, and we have examined the effects of TGFß1 on developing prostate organs in vitro to study cells in their correct context and to examine potential regulatory loops that control organogenesis. We have investigated the expression of Tgfß isoform transcripts during early embryonic and neonatal prostate development and have demonstrated that their expression correlates with the growth and development of the prostate. TGFß1 appeared to inhibit prostate growth, but, surprisingly, promoted branching morphogenesis. Also, TGFß1 had different effects on cell proliferation depending on the position of the cell within the organ. SM distribution was affected by TGFß1, which led to better defined SM adjacent to epithelial ducts and may mediate part of the effect of TGFß1 on proliferation. Testosterone was shown to repress Tgfß transcript levels, although the repression was more robust in the absence of prostatic epithelia. Similarly, TGFß1 also caused repression of Fgf10 transcript levels, but the level of repression seemed to be reduced by the presence of epithelia.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissue isolation
Whole UGTs, VMPs, and VPs were microdissected from outbred Wistar rats, where the day of copulatory plug observation was taken as embryonic d 0.5 (e0.5), and the day of birth was designated postnatal d 0 (P0). Animals were maintained and killed in accordance with United Kingdom Home Office guidelines and legislation. Total RNA was prepared as described previously (28). For ontogeny studies, tissues were pooled from 15–20 specimens for embryonic tissues and from 10–25 specimens for postnatal tissues (depending on size).

Analysis of transcript levels
A DNA template for Fgf10 was made as previously described (4). DNA templates for Tgfß1, Tgfß2, and Tgfß3 riboprobes were synthesized by RT-PCR from P0 UGT cDNA and subcloned into pBluescript KSII+ (Stratagene, La Jolla, CA) as previously described (6). DNA templates for cyclophilin (Cphn) and 28S were obtained from Ambion, Inc. (Austin, TX). 32P-Labeled antisense riboprobes were transcribed, and ribonuclease (RNase) protection assay performed as previously described (4). RNase-protected products for Fgf10, Tgfß1, Tgfß2, Tgfß3, 28S, and Cphn were 323, 261, 245, 324, 155, and 103 nucleotides (nt), respectively. The Cphn internal control was observed to yield a number of probe-specific fragments of between 90–100 nt in addition to the 105 nt band. These additional bands were included for quantification and normalization. Gels were imaged using a Storm PhosphorImager (Molecular Dynamics, Sunnyvale, CA), and transcript abundance was determined from the intensity of bands, calculated using ImageQuant V.1.2 (Molecular Dynamics). Transcript abundance was normalized to Cphn internal standards.

Organ culture
Serum-free organ cultures of P0 Wistar rat VPs and VMPs were performed as previously described (29). Culture medium was supplemented with 10–8 M testosterone and/or 10 ng/ml TGFß1 for the desired length of treatment. Cultured organ rudiments were examined and imaged using an MZ6 dissection microscope (Leica, Deerfield, IL) and an ICA camera (Leica) using Photoshop software (Adobe Systems, San Jose, CA). The two-dimensional area and perimeter were measured on captured images using NIH Image software.

Histology and immunohistochemistry
The histology sections of cultured VPs were examined after immunohistochemical staining. Immunohistochemistry for 5-bromo-2'deoxyuridine (BrdU) and pan-cytokeratin colocalization was performed as previously described (30). The antibodies were diluted 1:300 for anti-BrdU (Fitzgerald Industries International, Inc., Concord, MA) and 1:200 for anti-pan-cytokeratin (Sigma-Aldrich Corp., Poole, UK). BrdU was detected with Alexofluor 488 (Molecular Probes, Inc., Eugene, OR), and pan-cytokeratin was detected with Cy5 (Amersham Pharmacia Biotech, Little Chalfont, UK).

Whole-mount immunohistochemistry was performed as follows. Organs were fixed in 4% paraformaldehyde for 2–4 h at 4 C, followed by 48 h of washing in PBS (with several changes). Organs were washed in PBS with 0.5% Triton X-100 (PBST) for 2 h and incubated for 6 h in PBST with 2% normal goat serum at room temperature. Organs were incubated overnight at 4 C with SM {alpha}-actin antibody (Sigma-Aldrich Corp.) at a 1:5000 dilution in PBST and 2% normal goat serum, followed by 7 h of washing in PBST at room temperature. Next, organs were incubated overnight at 4 C with antimouse fluorescein isothiocyanate-conjugated antibody (Sigma-Aldrich Corp.) at a 1:50 dilution in PBST and 2% normal goat serum, followed by three 1-h washes with PBST. Organs were counterstained with propidium iodide (Sigma-Aldrich Corp.) at a final concentration of 10 µg/ml in PBST for 20 min, followed by three 1-h washes with PBST. Organs were mounted on cavity slides in Permafluor (Beckman Coulter, Buckinghamshire, UK) and viewed on a Leica MZFLIII stereomicroscope, and images were acquired using a Coolsnap CCD camera with Coolsnap software (Nikon, Melville, NY) and a Mac G4 computer (Apple Computer, Cupertino, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Tgfß isoform transcript expression in male UGTs and VPs
Tgfß transcript levels were analyzed in male UGTs (Fig. 1AGo) and VPs (Fig. 1BGo) by RNase protection assay. In the male UGT, Tgfß1 and Tgfß3 transcript levels were low on e17.5, increased to maximum levels on P2, and were expressed at low levels on P6. Tgfß2 transcript levels decreased from e17.5 to e19.5, but increased to maximum levels on P2 and decreased again to low levels on P6. Analysis of transcript levels in embryonic VPs was not practical before P0 due to difficulties in precise microdissection of VPs. In the postnatal VP, Tgfß transcripts decreased with increasing age. Maximum transcript levels were observed on P0 for Tgfß2 and on P2 for Tgfß1 and Tgfß3. All transcript levels had decreased between 4- and 10-fold by P6.



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  		FIG. 1. Expression of Tgfß transcripts in the male UGT and VP by RNase protection assay. Five micrograms of RNA were hybridized with 32P-labeled antisense riboprobes for Tgfß1, Tgfß2, and Tgfß3; 28S was used as an internal standard. Transcript levels were normalized to 28S. Numbers below autoradiographs show the percent transcript abundance relative to P0 UGT (A) or P0 VP (B) and are the average of two independent experiments using tissues pooled from several specimens. A, Tgfß mRNA levels in embryonic (e17.5 and e19.5) and neonatal (P0, P2, and P6) UGT. B, Tgfß mRNA levels in the neonatal VP (P0, P2, P4, and P6).

 
Effect of TGFß1 on VP size and branching morphogenesis
P0 VPs were cultured in the presence or absence of TGFß1 and testosterone for 6 d to address the effect of TGFß1 on the developing prostate. Whole-mount images of VPs were captured at the end of culture (Fig. 2AGo). The images were imported into NIH Image 1.62f, and the two-dimensional area of VPs was measured (in pixels) to provide an estimate of organ size. The data were calculated from 30 organs from each treatment group and four independent experiments. Average sizes of organs were as follows (±SEM); –T, 9,500 ± 404 pixels; –T+TGFß1, 7,500 ± 230 pixels; +T, 15,100 ± 552 pixels; and +T+TGFß1, 10,300 ± 404 pixels. The two-dimensional area of VPs cultured with TGFß1 was significantly lower in both the presence and absence of testosterone after 6 d [–T+TGFß1, 21% reduction compared with –T (by t test, P = 4.79e–4); +T+TGFß1, 32% reduction compared with +T (by t test, P = 4.03e–9); n = 4]. TGFß1 inhibited 86% of testosterone-induced growth (Fig. 2BGo). These data indicate that TGFß1 inhibits the growth of prostatic organs in vitro, using organ size as an indicator of growth.



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  		FIG. 2. The effect of TGFß1 on growth and branching of the prostate. P0 rat VPs were grown in serum-free culture for 6 d in the presence or absence of 10–8 M testosterone with or without TGFß1 (10 ng/ml). A, Whole-mount live images of VPs on d 6 of culture. Scale bar, 1 mm. B, Graph showing the two-dimensional area of cultured VPs represented as relative size in pixels. A statistically significant difference in size was observed after the addition of TGFß1 (denoted by asterisks). C, Graph showing the mean number of epithelial buds around the periphery of cultured VPs, expressed as a ratio to organ perimeter (mean number per 1000 pixels of perimeter ± SEM). A statistically significant difference in the number of peripheral epithelial tips was observed after the addition of TGFß1 (denoted by asterisks).

 
Next, we investigated the effects of TGFß1 on branching morphogenesis by counting the number of epithelial branch tips at the periphery of organs grown in vitro (Fig. 2CGo). Previous analysis of the effect of TGFß1 on branching morphogenesis of VPs counted the number of branched tips per square millimeter over the whole organ (14), but this was impossible in our system because individual buds within the VP images were difficult to distinguish to ensure that only tips, and not bends or curves, were counted. Thus, we counted the number of buds around the periphery of the organs. The number of tips was expressed as a ratio to organ perimeter (tip number per 1000 pixels of perimeter; using NIH Image) so that organs of different sizes could be compared and to control for treatment effects on organ size. The number of tips per 1000 pixels ± SEM of perimeter were; –T, 36.6 ± 0.7; –T+TGFß1, 46.6 ± 0.9; +T, 51.7 ± 0.9; and +T+TGFß1, 56 ± 1.2. TGFß1 significantly increased the number of tips per 1000 pixels of perimeter in VPs cultured with or without testosterone (–T compared with –T+TGFß1 by t test, P = 7.89e–6; +T compared with +T+TGFß1 by t test, P = 8.30e–4; n = 4).

Although the two-dimensional area of VPs grown with TGFß1 decreased (Fig. 2BGo), the number of buds at the organ periphery increased (Fig. 2CGo). This was somewhat surprising and led us to investigate whether this increased branching might be accompanied by increased proliferation. To test this idea, the effect of TGFß1 on the proliferation rate of cells in different regions within the VP was examined.

Effect of TGFß1 on cell proliferation
To determine the effect of TGFß1 on cell proliferation in the prostate, VPs were grown in the presence or absence of TGFß1 and in the presence or absence of testosterone. The data were calculated from 18 measurements of nine organs from each treatment group and three independent experiments. TGFß1 reduced the proliferation of both stromal and epithelial cells in the center of the VP (proximal to urethra) in the presence or absence of testosterone (Fig. 3Go, A and B). Rates of proliferation, measured by BrdU incorporation, were as follows (±SEM): epithelia: –T, 10.4 ± 0.9%; –T+TGFß1, 7.3 ± 0.6%; +T, 7.3 ± 0.8%; +T+TGFß1, 2.4 ± 0.5%; stroma: –T, 7.8 ± 0.6%; –T+TGFß1, 3.9 ± 0.5%; +T, 5.9 ± 1.0%; and +T+TGFß1, 1.8 ± 0.6%. Statistical differences between treatment groups were as follows: epithelia: –T compared with –T+TGFß1 by t test, P = 3.15e–3; +T compared with +T+TGFß1 by t test, P = 2.83e–6; stroma; –T compared with –T+TGFß1 by t test, P = 1.38e–5; and +T compared with +T+TGFß1 by t test, P = 4.63e–4 (n = 3). Testosterone also reduced the level of BrdU incorporation in the proximal region compared with that in untreated organs, and the addition of both testosterone and TGFß1 caused greater repression of proliferation.



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  		FIG. 3. The effect of TGFß1 on cellular proliferation in the distal and proximal regions of the prostate. P0 rat VPs were grown in serum-free culture for 6 d in the presence or absence of 10–8 M testosterone with or without TGFß1 (10 ng/ml); on d 6, BrdU was added to the culture medium for 2 h. BrdU incorporation was visualized by immunohistochemistry (green staining, which appears yellow when colocalized with nuclear propidium iodide staining) and localized to the epithelium by immunohistochemistry for pan-cytokeratin (blue); nuclei were stained with propidium iodide (red). A, Immunohistochemistry of VP epithelial ducts proximal to the urethra (note the presence of ductal lumens) under different treatment conditions (–T, +T, –T+TGFß1, and +T+TGFß1). B, Graph of BrdU incorporation (±SEM), illustrating rates of proliferation of stroma and epithelia in the proximal region (center of the organ). TGFß1 elicited a reduction in both stromal and epithelial proliferation in the presence or absence of testosterone (statistically significant differences are marked with asterisks). C, Immunohistochemistry of VP epithelial ducts distal to the urethra (note the edge of the organ shown in each image) under different treatment conditions (–T, +T, –T+TGFß1, and +T+TGFß1). D, Graph of BrdU incorporation (±SEM), illustrating the rates of proliferation of stroma and epithelia in the distal region (periphery of the organ). TGFß1 elicited an increase in both stromal and epithelial proliferation in the absence of testosterone (statistically significant differences are marked with asterisks).

 
In contrast, in the peripheral region (distal to the urethra), TGFß1 significantly increased epithelial and stromal proliferation in the absence of testosterone (Fig. 3Go, C and D). Rates of cellular proliferation, measured by BrdU incorporation, were as follows: epithelium: –T, 13 ± 1.1; –T+TGFß1, 24.6 ± 0.7; +T, 26.4 ± 1.4; +T+TGFß1, 26.3 ± 1.1); stroma: –T, 9.6 ± 1.4; –T+TGFß1, 13.4 ± 0.7; +T, 12.5 ± 1.1; and +T+TGFß1, 10.8 ± 1.0. Statistical differences in proliferative rates were: epithelium: –T compared with –T+TGFß1 by t test, P = 1.11e–7; and stroma: –T compared with –T+TGFß1 by t test, P = 2.49e–5 (n = 3). TGFß1 had no significant effect in the distal region on epithelial or stromal proliferation in the presence of testosterone (Fig. 3Go, C and D). This may be due to testosterone stimulating cell growth maximally; therefore, TGFß1 may not be able to induce any further increase in proliferation. Our results show that TGFß1 can both stimulate and inhibit growth within the prostate, and that the location of the cell within the organ is important with regard to its response to TGFß. The differential response to TGFß is likely to be due to differences in cellular differentiation, because, in general, cells in the center of the prostate are more highly differentiated than cells at the periphery. Additionally, cells at the periphery have been associated with higher proliferative rates (31).

Effect of TGFß1 on SM distribution
Because TGFß1 showed significant effects on cellular growth, and previous studies have shown that it can regulate SM differentiation in vitro (24, 25, 26), we examined the effect of TGFß1 on the SM distribution in organs grown in vitro (n = 4, comprising a total of ~30 organs). Our hypothesis was that part of the effect of TGFß1 on growth may be mediated by the differentiation of mesenchyme into SM and a resulting change in the local growth factor profile. SM differentiation is affected by androgens and TGFß1 and shows a proximal to distal gradient along epithelial ducts. The asymmetric distribution of SM along prostatic ducts suggested a possible role in mediating part of the differential effect of TGFß1 on ductal growth. VPs were grown with or without testosterone and with testosterone and TGFß1, followed by fixation and whole-mount immunohistochemistry for SM {alpha}-actin (Fig. 4Go). SM actin is an early marker of smooth muscle differentiation and is observed in mesenchmye/stroma juxtaposed to epithelia. In the absence of testosterone, SM actin was visible surrounding the epithelial buds at the periphery as well as throughout the ductal network (Fig. 4AGo). In the presence of testosterone, SM actin was somewhat reduced at the distal epithelial tips; some tips appeared almost entirely free of SM actin, although some tips showed moderate SM actin (Fig. 4BGo). Overall, there was less robust smooth muscle distribution at epithelial tips in the presence of testosterone. In organs grown in the presence of testosterone and TGFß1, there appeared to be a clear effect on SM distribution. SM actin was observed at ductal tips as well as in structures outlining the ductal network (Fig. 4CGo). In particular, the periductal SM distribution in the presence of TGFß1 was more pronounced, suggesting an effect of TGFß1 on SM distribution in organs grown in vitro. Furthermore, the change in SM distribution correlated with the effect on growth and thus may mediate part of the differential effect of TGFß1 on epithelial proliferation. It is important to note that these observations are correlative and do not define SM as a functional mediator of TGFß1. However, these data extend in vitro observations on the effects of TGFß1 on SM differentiation to stromal cells in vitro in their correct tissue environment.



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  		FIG. 4. Effects of TGFß1 on SM distribution in the prostate. VPs were grown in vitro in the absence of testosterone, the presence of testosterone, and the presence of testosterone and TGFß1, followed by whole-mount immunohistochemistry for SM actin. SM actin was stained in green, and nuclei were stained with propidium iodide (red). On the left of the figure whole organs are shown (x5 magnification); on the right, higher magnification (x10) images of the same organs are shown. The data shown are representative images drawn from four independent experiments and a total of 31 organs. A, Organs grown in the absence of testosterone. B, Organs grown in the presence of testosterone. C, Organs grown in the presence of testosterone and TGFß1. Green arrows highlight buds or ducts showing SM actin staining of note; red arrows highlight buds showing little SM actin staining.

 
Regulation of Tgfß and Fgf10 transcript levels
Next, we examined the effects of testosterone and TGFß1 on the expression of transcripts for Tgfß and Fgf10, respectively. Our hypothesis was that the presence of epithelia may alter the response of mesenchyme to testosterone or TGFß1. To test this hypothesis, we compared the effects of testosterone and TGFß1 on transcripts for Tgfß and Fgf10, respectively, in prostatic rudiments containing or lacking epithelia. These rudiments correspond to VPs (which contain epithelia) and VMPs (which lack epithelia). The VMP forms in both males and females and is composed of condensed mesenchyme involved in prostatic induction. In males, the presence of testosterone leads to the induction of epithelial prostatic buds by the VMP, and the VMP becomes part of the VP as it develops. In females, there are lower testosterone levels and no (or very few) prostatic buds, and the VMP persists into neonatal life before disappearing (2, 3). Both VP and VMP contain mesenchyme that regulates prostatic growth as well as transcripts for Tgfßs and Fgf10. Testosterone has been shown to repress Tgfß mRNA levels in adult prostate (22), whereas TGFß has been shown to repress Fgf10 transcripts in developing prostatic mesenchyme (6). During prostate development, the regulation of Tgfß by testosterone is unknown. Testosterone does not appear to regulate Tgfß transcripts in primary cultures of prostatic epithelium or stroma, suggesting that stromal/epithelial interactions may be important for correct regulation by TGFßs (14). Testosterone regulation of Tgfß may be a mechanism of controlling prostatic organogenesis and may indirectly regulate growth via repression of Fgf10. Thus, we decided to examine the response of Tgfß transcripts to testosterone in the developing prostate to test this hypothesis. P0 VP and VMP (VMP rudiments consisting of VMP and associated UR; Fig. 5AGo) were cultured for 3 d in the absence or presence of testosterone (n = 2, ~20 organs in each group for each experiment). No change in Tgfß1 and Tgfß3 transcript levels was observed in VPs in the presence of testosterone. However, Tgfß2 transcript levels were lower in VPs cultured with testosterone. In the VMP, testosterone repressed Tgfß1 and Tgfß3 transcript levels by 2- and 3-fold, respectively (Fig. 5AGo). Our results suggest more robust regulation of Tgfß transcript expression in the VMP (–epithelium) than VP (+epithelium) and different responses of Tgfß transcripts to testosterone in VP and VMP.



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  		FIG. 5. Regulation of Tgfß and Fgf10 transcripts in the prostate. The regulation of Tgfß transcripts by testosterone in VP and VMP organ rudiments was examined by RNase protection assay (A). The regulation of Fgf10 transcripts by TGFß1 in VP organs grown in vitro was examined by RNase protection assay (B). A, P0 rat VPs and VMPs were grown in serum-free culture for 3 d in the presence or absence of 10–8 M testosterone. Five micrograms of RNA were hybridized with 32P-labeled riboprobes for Tgfß1, Tgfß2, Tgfß3, and Cphn as an internal standard. Transcript levels were normalized to Cphn. Numbers below autoradiographs show the percent transcript abundance relative to untreated VP or VMP and are the average of two experiments (40 organs in total). B, P0 rat VPs were grown in serum-free culture for 3 or 7 h in the presence or absence of TGFß1 (10 ng/ml). Five micrograms of RNA were hybridized with 32P-labeled antisense riboprobes for Fgf10 and Cphn as internal standards. Transcript levels were normalized to Cphn. Numbers below autoradiographs show the percent transcript abundance relative to untreated VP and are the average of two experiments (30 organs in total).

 
TGFß1 has been shown to cause a rapid and reversible repression of Fgf10 mRNA in primary ventral mesenchymal pad cells (a model of prostatic mesenchyme) (6). Maximum repression of Fgf10 transcript levels was observed at 3 h, with levels returning to normal by 48 h. A 3-fold down-regulation of Fgf10 transcripts was observed in VMP organ rudiments grown in vitro, after treatment with TGFß1. To determine whether this down-regulation occurred in the prostate, VPs were cultured in vitro in the absence or presence of TGFß1 for 3 or 7 h (Fig. 5BGo; n = 2, ~20 organs in each group for each experiment). The addition of TGFß1 caused a small, but consistent, repression of Fgf10 transcript levels after 3 h (Fig. 5BGo). To determine whether this effect was due to poor penetration of the TGFß1 protein in the VP, organs were cultured for 7 h. Again, the repression of Fgf10 transcript levels was small, but consistent (Fig. 5BGo). Given the relatively short period of exposure to TGFß1, it is very likely that the changes in Fgf10 mRNA levels are due to gene regulatory events, rather than changes in the proportion of cells that express Fgf10. Our results suggest that TGFß1 does not regulate Fgf10 in VPs to the same extent as that observed in a model of primary prostatic mesenchyme (6). We propose that the epithelium in the VP inhibits or reduces TGFß1 repression of mesenchymal Fgf10 expression, in contrast to the more robust regulation observed in the VMP. This is supported by the observation that Tgfß transcripts showed a more robust down-regulation in VMP (devoid of epithelium) compared with VP. We suggest that mesenchymal/epithelial interactions have an important effect on gene regulation in the prostate.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The role of TGFß1 in the growth and development of neonatal VP was investigated using an in vitro organ culture system. TGFß1 was shown to reduce the area of VPs, confirming TGFß1 as an inhibitor of prostate growth, but surprisingly, TGFß1 also increased the number of epithelial buds around the periphery of the organs. This suggested that TGFß1 might have multiple roles in prostate growth and development. Inhibition of growth of the VP and seminal vesicle by TGFß1 has been previously demonstrated (14, 32). Itoh et al. (14) also demonstrated the ability of TGFß to inhibit branching morphogenesis of the VP. However, in our study TGFß1 was found to promote the number of branched epithelial tips, suggesting that TGFß1 might stimulate branching morphogenesis. This difference is probably due to analysis of different areas of the prostate. Itoh et al. (14) examined the number of branching ducts throughout the organs, whereas our data determined the number of buds around the periphery of the organs. We chose to examine the periphery for two reasons; we found it too difficult to precisely determine the number of branching ducts in center of the organ due to its complexity (i.e. confidently differentiate between a bud tip and a sharp ductal bend), and we speculated that there may be different effects at the periphery because this is where most proliferation and branching morphogenesis occur (31). We conclude that TGFß1 stimulated branching morphogenesis at the periphery of the growing prostate (in vitro), but are not able to determine whether this effect is due to the direct action of TGFß1 on branching epithelia or is mediated via indirect effects from the mesenchyme.

In the lung, TGFß1 has been shown to induce cleft formation and thus stimulate branching morphogenesis (33). Whether increased proliferation is associated or required for branching is unclear, because proliferation is not required for lung bud formation (34), although it usually follows it. Additionally, we have recently shown that inhibition of Hedgehog signaling led to increased branching at the periphery of the prostate as well as a reduction in epithelial proliferation. Addition of recombinant sonic hedgehog led to decreased branching as well as decreased epithelial proliferation (but increased mesenchymal proliferation) (30). This indicates that proliferation and branching morphogenesis in the prostate may be independent, and we suggest that it is unlikely that the effect of TGFß1 on branching is due to increased proliferation.

TGFß1 has been shown to inhibit cell proliferation (18, 35, 36, 37, 38), but in some circumstances may increase the rate of cellular proliferation (19, 20, 21). We have demonstrated that in prostate organs grown in vitro, TGFß1 increased the proliferation rate of epithelial and stromal cells in the distal region and inhibited the proliferation of cells in the proximal region. These data established that TGFß could have opposite effects on cellular proliferation within the same organ, and we suggest that these different effects may be dependent upon the level of cellular differentiation of the target cell. Epithelial and stromal differentiation occurs in a proximal to distal fashion during prostate development (27, 39), and thus the prostate contains relatively undifferentiated cells at the periphery (distal to urethra) compared with more differentiated cells in the middle (proximal to urethra). Testosterone promotes differentiation of prostate epithelial and stromal cells (27, 39), and it appeared that testosterone slightly decreased proliferation in the proximal region (30). We observed that the inhibition of proliferation in the proximal region by TGFß1 was greater in organs cultured with testosterone than in those cultured without testosterone. The addition of testosterone would have increased differentiation in the proximal region. This observation supports the hypothesis that TGFß1 inhibits the proliferation of differentiated cell types while promoting proliferation of undifferentiated cells in the prostate.

There are complex interactions between androgens and TGFß signaling in the prostate. Androgens may regulate the expression of TGFßs and their receptors, and it is possible that other cross-talk between these pathways may occur. In vitro, it has been shown that ligand-bound androgen receptor inhibits TGFß-mediated transcriptional responses (40). This would suggest that androgens might inhibit the effects of TGFß. We did not observe this; we found effects of TGFß1 in the presence or absence of testosterone as well as additive effects in some circumstances. It is important to note that our observations are for cellular proliferation and differentiation, and it may be that these processes do not involve the gene regulatory mechanisms reported by Chipuk et al. (40).

We have previously demonstrated that TGFß1 represses Fgf10 transcript levels in prostatic mesenchyme (6); however, the same level of Fgf10 transcript repression was not observed in the studies presented here. The major difference between our previous studies and the data presented here is that, unlike the VMP, the VP contains epithelium and differentiated stroma. The presence of epithelium or differentiated stroma may inhibit the regulation of Fgf10 transcript levels by TGFß1, because epithelial and stromal cells have been proposed to produce factors that control Fgf10 transcript levels in the mesenchyme of the lung (33, 41, 42). The regulatory mechanism of branching in the lung, which may show some similarities to the prostate, involves numerous factors, including FGF10 and TGFß1. TGFß1 represses Fgf10 transcript levels in the mesenchyme at the epithelial tip in the lung, resulting in up-regulation of Fgf10 at adjacent sites. The expression of Fgf10 at new sites induces formation of new branches (33). Because our studies examined the amount of Fgf10 mRNA throughout the organ, it is possible that local repression of Fgf10 transcripts was masked by the increase in Fgf10 transcripts at other sites. Another possible explanation for the modest repression of Fgf10 by TGFß1 in the VP is that there was a clear stimulation of branching morphogenesis. Thus, it is possible that there was a direct down-regulation of Fgf10 by TGFß1, but an indirect up-regulation in response to increased branching morphogenesis. We propose that it is likely that epithelial factors play an active role in Fgf10 expression, which might explain the different responses of mesenchyme in the presence (VP) or absence (VMP) of epithelium, and that regulation of Fgf10 in pure mesenchyme is simpler than that in the presence of complex mesenchymal/epithelial interactions.

Previous studies have demonstrated that testosterone may regulate Tgfß expression in the adult prostate (14, 22, 23). In contrast, Itoh et al. (14) demonstrated that Tgfß transcript levels were not regulated in isolated stromal or epithelial cells by testosterone, suggesting that the regulation of Tgfß transcript levels by testosterone in our organ system requires stromal-epithelial interactions. In data presented here, testosterone only slightly repressed Tgfß2 transcript levels in the VP, suggesting that Tgfß transcript levels may be regulated by different mechanisms in developing compared with adult VPs. Also, this could suggest that the mesenchymal-epithelial interactions observed in the developing prostate are different from the stromal-epithelial interactions in the adult prostate. Interestingly, Tgfß1 and Tgfß3 transcript levels were repressed in the VMP by testosterone, which suggests that regulation may be more robust in the absence of epithelium; this is supported by our observation that Fgf10 shows more dramatic regulation in the absence of epithelia.

A schematic diagram of the effects of TGFß1 on prostatic growth during development is shown in Fig. 6Go. TGFß1 appears to have differential roles in regulating epithelial growth and promotes epithelial proliferation in the distal region of the VP, but inhibits epithelial proliferation in the proximal region. TGFß1 may also act as an inducer of branch formation by playing a role in cleft formation at the end of the epithelial buds, an effect that has been observed in the lung (33). Although TGFß1 increases branching of prostatic epithelia, it is not clear whether this effect arises from the direct action of TGFß1 on epithelia or via indirect action via the mesenchyme. Additionally, we do not know whether the proliferative effects of TGFß are directly or indirectly mediated. We propose that some of the effects of TGFß on proliferation may be indirectly mediated via the increased differentiation of mesenchyme into SM; however, we can only speculate as to what this proportion might be. The stimulation of SM differentiation in prostatic stroma by TGFß has been established in vitro (24, 26), and our data suggest that this also occurs in stroma within prostatic organs. The balance between growth stimulatory mesenchyme and growth inhibitory SM may play a key role in the control of epithelial proliferation in the prostate. Androgens may also control this balance (43). There are several ways in which the response of cells to TGFß may be determined. For example, the epithelial response to TGFß may be controlled by its own level of differentiation or the differentiation of adjacent mesenchyme or stroma. Our experiments do not address this question, but this might be an area for future investigation. In conclusion, the data presented here show that TGFß can have opposing effects on cellular proliferation in the prostate, and it will be important to determine what proportions of these effects are direct or indirect.



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  		FIG. 6. A model of the effects of TGFß1 on prostate growth. A schematic diagram of single prostatic duct is shown to illustrate the effects of TGFß1 at different locations within the organ as well as on different cellular compartments. At growing ductal tips (at the periphery of the prostate), both mesenchyme and epithelium are undifferentiated, and there are high rates of proliferation. Additionally, the epithelium is undergoing branching morphogenesis. Androgens stimulate epithelial growth via the mesenchymal compartment, and many growth factors control mesenchymal/epithelial interactions that are essential for growth and differentiation. In the distal region, TGFß1 stimulates the proliferation of the mesenchyme and epithelium as well as increasing epithelial branching. TGFß1 also stimulates the differentiation of mesenchyme into SM, which leads to loss of undifferentiated mesenchyme and its paracrine stimulators of the epithelial compartment. Additionally, there is an increase in levels of SM and constitutively expressed inhibitors of epithelial proliferation. In mature prostatic ducts proximal to the urethra (the center of the organ), there are lower rates of proliferation and increased levels of differentiation. Prostatic mesenchyme has differentiated into stroma consisting of fibroblasts, SM, and vasculature. In the proximal region, TGFß1 inhibits the proliferation of stroma and epithelia. The balance between stimulatory mesenchyme and inhibitory SM may be controlled by androgens and TGFß1. Androgens have been shown to inhibit SM differentiation, whereas TGFß1 has been shown to stimulate SM differentiation. Although TGFß1 has numerous effects in the prostate, it is unclear which of these are due to direct or indirect action of TGFß1. For example, TGFß1 stimulates branching of epithelial tips, yet it is not known whether this effect is mediated through receptors on epithelia, mesenchyme, or both cell types.

 


   Acknowledgments
 
We thank Denis Doogan and James MacDonald for technical assistance. We thank Dr. Griet Vanpoucke for comments upon the manuscript, and members of the laboratory for encouragement and suggestions.


   Footnotes
 
This work was supported by the Medical Research Council (United Kingdom) and the Congressionally Directed Medical Research Program for Prostate Cancer Research (DAMD17-00-01-0034; to A.A.T.).

Abbreviations: BrdU, 5-Bromo-2'-deoxyuridine; Cphn, cyclophilin; e, embryonic day; Fgf, fibroblast growth factor; nt, nucleotide; P, postnatal day; PBST, PBS with 0.5% Triton X-100; RNase, ribonuclease; SM, smooth muscle; UGT, urogenital tract; VMP, ventral mesenchymal pad; VP, ventral prostate.

Received April 23, 2004.

Accepted for publication June 4, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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