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Departments of Urology (W.Y.C., L.B., C.W., G.S.P.) and Physiology and Biophysics (G.S.P.), University of Illinois College of Medicine, Chicago, Illinois 60612; and New York University Medical School (L.I.G.), New York, New York 10016
Address all correspondence and requests for reprints to: Gail S. Prins, Ph.D., Department of Urology, M/C 955, 820 South Wood Street, Chicago, Illinois 60612. E-mail: gprins{at}uic.edu
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Introduction |
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Estrogen action is mediated through estrogen receptor 
(ER) and
ERß in the prostate gland. In the normal developing prostate, ER
expression is confined to periductal mesenchymal cells surrounding the
proximal ducts, whereas ERß is expressed in low levels in the
undifferentiated epithelial cells (9, 10). Although neonatal estrogen
exposure did not initially alter the expression of ERß (10), there
was an immediate up-regulation of ER expression within periductal
stromal cells along the length of the developing ducts, which allows
for amplification of estrogenic effects in those cells (9). Thus,
although some of the effects of neonatal estrogens on the developing
prostatic epithelium may be directly mediated through low levels of
ERß, careful consideration must be given to perturbations in
stromal-derived paracrine factors mediated through elevated ER
levels in those cells.
An important family of growth factors that may in part mediate estrogen’s effects on the developing prostate gland is transforming growth factor-ß (TGFß). In a variety of systems, TGFßs have been shown to inhibit epithelial cell proliferation, alter cell differentiation, and stimulate fibroblast growth, all of which are prostatic responses to neonatal estrogen exposure (11, 12). TGFß1 has also been shown to be a critical factor in determining branching morphogenesis of the mammary gland and lung (13, 14). Although the developing rodent prostate has been shown to express TGFß1, -ß2, and -ß3 messenger RNAs (mRNAs) in a developmentally specific and cell-specific manner (15, 16, 17, 18), its role during prostate morphogenesis has not been clearly defined. In addition, although the adult rat prostate has been shown to express the TGFß receptors type I (RI) and type II (RII) (19, 20, 21), their localization in the developing prostate gland has not been characterized.
There are three isoforms of TGFß (TGFß1, -ß2, and –ß3) identified in mammalian tissues, and all possess similar biological activities (22, 23, 24). Each TGFß isoform is secreted as a homodimer bound noncovalently to ß1-, ß2-, or ß3-specific latency-associated peptide (LAP) and is biologically inactive. Activation of TGFß takes place extracellularly and is believed to occur through proteolytic dissociation of LAP from TGFß (25, 26). Transduction of the TGFß signal requires coordination between two transmembrane serine-threonine kinase receptors, designated TGFß-RI and -RII (27). Upon ligand binding, TGFß-RII binds to and transphosphorylates TGFß-RI, which, in turn, phosphorylates the Smad2 and -3 transcription factors. Phosphorylated Smad2/3 associate with Smad4, and the complex translocates to the nucleus, where it interacts with specific response elements on regulated genes (28).
Neonatal estrogen exposure could affect TGFß signaling by augmenting or interfering with TGFß production or its signaling molecules at one or several sites in the TGFß mechanism of action and thereby permanently alter prostate growth and differentiation. To determine whether this occurs, we initially characterized the ontogeny of latent and active TGFß1, total TGFß2, total TGFß3, TGFß-RI and -RII, and Smad2/3 in the developing rat prostate gland using immunocytochemical techniques. Alterations in these TGFß signaling molecules were then examined in the developing prostate after neonatal exposure to estradiol benzoate. We observed that both TGFß isoform production as well as TGFß receptors are altered by estrogen exposure and provide evidence that this interrupts TGFß-driven differentiation pathways in the developing prostate gland.
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Materials and Methods |
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Immunocytochemistry
Immunocytochemistry was performed according to previously
published methods (29). Briefly, frozen prostatic complexes or
individual lobes were mounted on precooled chucks (-20 C) in a
Reichert-Jung cryostat (Leica, Inc., Deerfield,
IL), and sections (6 µm) were thaw-mounted on gelatin-coated
glass slides. Whenever possible, individual lobes were sectioned
longitudinally to reveal the proximal-distal orientation. At 4 C, the
sections were fixed in 2% paraformaldehyde, rinsed, incubated with
appropriate 2% blocking serum (goat or horse), and subsequently
incubated overnight with primary antibody. The specific antibodies,
sources, and concentrations used are presented in Table 1
. The primary antibody was reacted with
biotinylated secondary antibody (Vector Laboratories, Inc., Burlingame, CA), and biotin was detected with an
avidin-biotin peroxidase kit (ABC-Elite, Vector Laboratories, Inc.), using diaminobenzidine tetrachloride as a chromagen. The
sections were stained with Gill’s no. 3 hematoxylin (1:4) as a blue
nuclear counterstain. As a final step, the sections were dehydrated
gradually with alcohol, cleared with xylene, and coverslipped with
Permount (Fisher Scientific, Itasca, IL). For AR, a
fluorescein (cy3)-labeled antirabbit secondary antibody was used. The
slides were mounted with Vectashield containing DAPI (Vector Laboratories, Inc.) to visualize nuclei. Photographs of
diaminobenzidine tetrachloride-stained slides were taken with an
Olympus microscope system (Olympus Corp., New Hyde Park,
NY) using Kodak Ektachrome Elite 100 film (Eastman Kodak Co., Rochester, NY). Photographs of the fluorescein-labeled
slides were taken using a Zeiss Axioskop (Carl Zeiss, New
York, NY) and a Princeton Instruments Microview digital
camera (Princeton Instruments, Trenton, NJ).
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RT-PCR
TGFß1 and RPL19, a ubiquitous ribosomal RNA, were reverse
transcribed and coamplified to obtain semiquantitative results. Total
RNA was isolated from prostate tissue using guanidium
thiocyanate-chloroform extraction (RNA STAT-60, Tel-Test,
Friendswood, TX). Two micrograms of total RNA were reverse transcribed
at 48 C for 50 min in 100 µl PCR reaction buffer (Perkin Elmer, Norwalk, CT) with 10 mM deoxy-NTPs and 25
mM MgCl2 through use of the reverse primers
(see below) and 400 U murine leukemia virus reverse transcriptase
(Promega Corp., Madison, WI). The forward primers (see
below), 2.5 U Taq DNA polymerase (Perkin Elmer), and 10 µCi[-32P]deoxy-CTP
(Amersham) were added to the reaction mix, and the sample
was overlaid with light mineral oil. Amplification was carried out for
30 cycles by incubation at 94 C for 1 min, 55 C for 2 min, and 72 C for
3 min, with a final extension at 72 C for 4 min in a Perkin Elmer 9600 thermal cycler. The radiolabeled complementary DNAs
were separated on a 4% NuSieve/agarose (3:1) gel (FMC, Rockland, ME),
and specific radioactive bands were quantitated on a PhosphorImager
(Molecular Dynamics, Inc., Sunnyvale, CA) after transfer
of emitted radioactivity on a phosphor plate. The intensity of the
TGFß1 signal was normalized to that of the ribosomal protein RPL19
internal control.
For TGFß1, the forward primer (TGFß1 5'-primer, 5'-CTTCAGCTCCACAGAGAAGAACTGC-3') and reverse primer (TGFß1 3'-primer, CACGATCATGTTGGACAACTGCTCC-3') produced a 298-bp product corresponding to nucleotides +1267 to +1564 (30). For RPL19, the forward primer (RPL19 5'-primer, 5'-CTGAAGGTCAAAGGGAATGTG-3') and reverse primer (RPL19 3'-primer, 5'-GGACAGAGTCTTGATGATCTC-3') produced a 195-bp product corresponding to nucleotides +401 to +595 (31). It was determined for each product that amplification for 30 cycles fell within the linear range with respect to the amount of input RNA.
RT-PCR was performed on ventral prostate tissue from days 6 and 30 from oil control and estrogen-treated rats in five to eight replicates. Comparisons between oil and estrogen treatments were performed in parallel. The mean ± SEM of the relative values of TGFß1 mRNA (normalized to RPL19) were obtained for each time point and treatment, and ANOVA followed by the Schiff test was used to determine statistical significance.
Western blotting
Cellular proteins (cytoplasmic and nuclear) were isolated by
homogenizing ventral prostates in a high salt buffer containing
protease inhibitors [10 mM Tris-HCl and 400 mM
NaCl (pH 7.4) containing 0.1 mM leupeptin, 1 trypsin
inhibitor unit/ml aprotinin, and 2 mM
phenyl-methylsulfonylfluoride]. Cellular debris was removed by
centrifugation at 10,000 x g for 10 min at 4 C, and
proteins were denatured by boiling in SDS sample buffer [50
mM Tris (pH 6.8), 2% SDS, 5% 2-mercaptoethanol, 10%
glycerol, and 0.005% bromophenol blue]. The protein concentration was
quantified by the Bradford assay (Bio-Rad Laboratories, Inc., Hercules, CA). Proteins (50 µg each) were separated by
SDS-PAGE and electrotransferred onto a nitrocellulose membrane as
previously described (29). The membrane was immunoblotted with an
antibody that recognized both Smad2 and -3 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Biotinylated antigoat
secondary antibody (Vector Laboratories, Inc.) was
detected using a peroxidase kit (ABC-Elite, Vector Laboratories, Inc.) and the Vector TMB substrate kit.
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Results |
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). Labeling of
adjacent sections with antibody against -actin revealed that the
appearance of TGFß1 immunostain in the ECM was coincident with the
initiation of smooth muscle differentiation from the periductal
mesenchyme as revealed by the appearance of -actin (Fig. 1B). By day
5, differentiating smooth muscle cells surrounded the length of the
prostatic ducts, and the associated ECM stained weakly to moderately
for active TGFß1 (Fig. 1, C and D). On day 10, the periductal stain
for active TGFß1 was strong and extended from the proximal to the
distal ducts with no apparent gradient in staining intensity (Fig. 1E).
However, the periductal smooth muscle layer and the TGFß1-stained
region were thicker around the proximal ducts than around the distal
ducts. By day 15, the TGFß1 stain surrounding the central and distal
ducts thinned considerably and became discontinuous around the
lumenized ducts (Fig. 1G). This pattern correlated with the periductal
smooth muscle layer, which had thinned to a one- to two-cell thickness
(Fig. 1H). In the proximal portions of the day 15 ducts, the periductal
TGFß1 immunostain was thick and continuous, which also directly
correlated with the thickness of the smooth muscle in that region. The
day 25–30 prostates exhibited weak to moderate TGFß1 stain in a
discontinuous pattern associated with the smooth muscle cells
surrounding the ducts. The immunostain for active TGFß1 was weaker on
day 45. The overall temporal staining pattern was similar in the dorsal
and lateral lobes, except that it was delayed by 3–4 days.
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). On
day 6, estrogenized prostates showed a marked increase in staining
intensity for ECM-associated active TGFß1 when prostate sections were
stained side by side with control tissues (Fig. 2, A and B). However,
this stain was confined to the periductal smooth muscle layer, which
was separated from the basement membrane and epithelial ducts by a
multicell layer of fibroblasts as previously described for estrogenized
prostates (10) (Fig. 2, C and D). The ECM associated with the
periductal fibroblasts immediately adjacent to the basement membrane
was negative for active TGFß1. Similar findings were observed in the
day 10 prostates. In contrast to the control prostates where TGFß1
declined with ductal development, active TGFß1 associated with the
thickened periductal smooth muscle layer remained strong through day 30
in estrogenized prostates (Fig. 2, E–G). Thus, estrogen treatment
resulted in both an increased TGFß1 immunostain and an extension of
the temporal pattern normally observed in the developing prostate.
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, H–J). In the
estrogenized prostates, the immunostain for LAP was stronger than that
in the control tissue on day 6 (in a side by side comparison), and the
peak extracellular intensity did not decline between days 10–30 (Fig. 2, K—M). These data indicate that the increase in active TGFß1
observed in estrogenized prostates reflects increased production and/or
secretion of TGFß1 by smooth muscle cells.
Semiquantitative RT-PCR was used to determine whether increased TGFß1
in the estrogenized prostates was due to increased levels of TGFß1
mRNA. No differences in TGFß1 mRNA levels were observed on either day
6 or day 30 between the control and estrogenized prostates (Fig. 3).
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). Stromal
cells continued to stain positively, but not to the degree observed in
the epithelium. By day 30, the apical stain remained strong in the
cytoplasm of the luminal epithelium, but overall appeared decreased
compared with that in day 15 prostates on side by side comparison.
Competition studies with immunogenic peptide eliminated all signal in
the epithelial and stromal cells (Fig. 4C). TGFß3 immunostain was
restricted to the epithelial cells in the developing prostate. On day
6, TGFß3 signal was weak, and epithelial immunostain increased
markedly as cells differentiated and ducts lumenized between days
10–15 (Fig. 4D). Day 30 epithelial cells retained strong TGFß3
signal localized to the supranuclear clear zone and the apical border.
Competition studies with immunogenic peptide eliminated all signal in
the epithelial cells (Fig. 4F).
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, B and
E). By day 30, signal for both growth factors had returned to
near-control levels in the central to distal regions of the glands,
whereas the proximal duct epithelium remained negative.
TGFß receptors
The ontogeny and cell localization of TGFß-RI and TGFß-RII
were examined in control and estrogenized developing prostates by
immunocytochemistry using specific antibodies to each receptor. In day
6 control prostates, many, but not all, epithelial cell nuclei were
darkly stained for TGFß-RI in addition to weaker cytoplasmic signal.
The periductal stromal cells also showed moderate staining for
TGFß-RI, whereas the interductal fibroblasts were negative. As the
prostates developed, the epithelial nuclear signal declined to
approximately 20% frequency by day 10 (Fig. 4G) and was only sporadic
by day 15. During this time, the cytoplasmic signal in epithelial cells
markedly increased. Both cytoplasmic and nuclear signals were
eliminated at these time points by competition with excess antigenic
peptide, indicating specificity for TGFß-RI (Fig. 4I).
In day 6 control prostates, epithelial cells were weakly positive for
TGFß-RII in the distal aspects of the developing gland, whereas the
proximal duct epithelium was negative. An increase in epithelial
cytoplasmic stain was observed as ducts began to lumenize between days
10–15 (Fig. 4J, arrow). In the proximal ducts, basal cells
stood out with a dark specific stain (arrowhead). Periductal
smooth muscle cells exhibited specific stain for RII (open
arrowhead), whereas fibroblasts were negative. This pattern of
stain remained through day 30. Specificity of stain was confirmed with
competition experiments where all signal was absent in the presence of
a 10-fold excess of antigenic peptide (Fig. 4L).
In estrogen-exposed rats, TGFß-RI was weak or absent in epithelial
cells and fibroblasts, but was present in periductal smooth muscle on
days 6 and 10 (Fig. 4H). By days 15–30, the epithelial cytoplasmic
signal was comparable to that in oil-treated control prostates,
indicating that the estrogen effect on TGFß-RI was transient. In
contrast, estrogen exposure had no effect on the TGFß-RII staining
pattern (Fig. 4K).
Smad2
The expression of Smad2, the intracellular signal transducer of
TGFß-RI, was examined by immunocytochemistry and Western analysis.
Western blots stained with an antibody that recognizes both Smad2 and
-3 indicated by mol wt that only Smad2 (52 kDa) was present in the
developing prostates on day 10 (Fig. 5C).
Immunocytochemical staining revealed that on day 10, the basally
located epithelial cells exhibited strong cytoplasmic stain for Smad2
(Fig. 5A, arrowhead). In nonlumenized ducts where all cells
express basal cell cytokeratins 5/15 (36), only those in contact with
the basement membrane were positive for Smad2. This dark basal cell
stain persisted throughout development up to day 30. As epithelial
cells began to differentiate, luminal cells began to stain positively
for Smad2 (Fig. 5A, arrow). On day 15, this was weak, but
the stain was stronger on day 30, when it was concentrated in the
supranuclear clear zone. Exposure to neonatal estrogen had no effect on
the staining pattern or quantity of Smad2 in the ventral prostate (Fig. 5B). The slight increase in the density of the 52-kDa Smad2 band of the
estrogenized day 10 prostate compared with that of the control lane
(Fig. 5B) is due to the relative increase in basal cells in the
estrogenized prostates at that time (36).
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). By day 10, the incidence of
epithelial cell nuclei positive for p21 signal had increased to about
50% and was most frequent in ducts that had begun to lumenize (Fig. 6B). p21 signal peaked on day 15 when most all epithelial cells were
positive (Fig. 6C). By day 30, little or no p21 signal remained in the
epithelial cell nuclei (Fig. 6D). In rats exposed to neonatal
estrogens, transient expression of p21 was not observed. Rather, the
ventral prostate glands were negative for p21 on days 6, 10, 15, and 30
(Fig. 6, E–H). This finding was repeated in three sets of tissues at
each time point.
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). In rats exposed to neonatal
estrogens, AR was down-regulated to low or absent levels in epithelial
cells, as previously reported (36). However, in periductal stromal
cells, AR was primarily localized to the cytoplasm (Fig. 7B).
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Discussion |
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-actin showed that TGFß1 always colocalized to the smooth muscle
cells and its associated ECM, which supports the previous findings that
the smooth muscle cells are the source of TGFß1 in the rat prostate
gland (15, 17). Interestingly, a temporal expression pattern for latent
and active TGFß1 was observed whereby levels were initially low,
peaked on day 10 of development, and declined thereafter to a weak and
discontinuous stain. As both total as well as active TGFß1 exhibited
this temporal pattern, the data suggest that it is related to increased
production of this growth factor. As recently shown by Itoh et
al. (16), TGFß1 mRNA levels in the present study did not change
over this period, indicating that changes in production levels are
mediated posttranscriptionally. TGFß2 was initially observed in periductal smooth muscle cells by day 5 of development; however, in contrast to TGFß1, the immunostain levels did not change over time. TGFß2 and TGFß3 immunostain appeared in epithelial cells as they began to differentiate into secretory cells, and their primary localization in the supranuclear clear zone suggests that they are secreted products. Previously, TGFß3 mRNA was shown to increase between days 1 and 20 in the rat ventral prostate, which corresponds to the increased protein levels observed herein (16). However, the observed developmental decrease in TGFß2 mRNA (16) was not observed at the protein level in this study, which indicates that there may not be a direct relationship between mRNA and protein levels for TGFß2.
Exposure to estradiol during the neonatal period differentially altered the levels of the TGFß isoforms in the developing prostate. Estrogens altered the temporal expression pattern of TGFß1, such that levels were increased compared with control levels as early as day 4 of life and did not decline over time until after day 30. As results with TGFß1-LAP revealed that total as well as active TGFß1 increased, they suggest that estrogens increased the production and/or secretion of TGFß1 and did not act merely by increasing activation of the latent form. As these changes were not reflected at the mRNA level, it appears that the estrogen-induced alterations are posttranscriptional in nature. Previous reports have shown that estrogens can alter TGFß1 expression in other systems; however, the specific effect may depend on cell type and context (39, 40, 41).
Epithelial expression of TGFß2 and TGFß3 was significantly delayed by early exposure to estrogen. When these factors appeared in estrogenized prostates on day 30, they were confined to the distal regions of the prostatic ducts, which is consistent with our previous work demonstrating that estrogenization leads to permanent differentiation defects of the epithelium in the proximal and central ducts (4, 36). As TGFß2 and TGFß3 appear to be secretory products of a differentiated epithelium, their delay in expression could be viewed as a result of developmental estrogenization rather than a cause of the phenomenon.
In the present study, both TGFß-RI and TGFß-RII were present in the developing prostate epithelial cells as well as the periductal smooth muscle, indicating that both cell types are potential targets of TGFß action. The type of intracellular staining observed in these cells was similar to what has been previously described in adult rat and human prostate (20, 21, 42). In contrast to the adult rat prostate where estrogen treatment had no effect on receptor expression (21), neonatal estrogen exposure was found to transiently decrease TGFß-RI in the epithelial cells between days 6–15, whereas TGFß-RII appeared unchanged. As the TGFß mechanism of action requires the coordinate expression and action of TGFß-RI and TGFß-RII, transient loss of TGFß-RI would lead to loss of TGFß action within epithelial cells during this critical developmental period.
Recently, Smads have been identified as the intracellular molecules that mediate TGFß action (28). In the present study, we identified the presence of Smad2 in the developing prostate epithelium, further indicating that these cells are targets of TGFß action during development. Neonatal estrogen did not alter the levels of Smad2 in the prostate gland, which indicates that Smad regulation is not a target for estrogen action. That the smooth muscle cells did not immunostain for Smad2 or -3 suggests either that those cells cannot respond to TGFß or that the levels are lower than the sensitivity of this technique. Evidence that the periductal smooth muscle cells are, in fact, responding to TGFß comes from the observation that AR was localized to the cytoplasm of periductal stromal cells in the day 6 estrogenized prostate. This observation is significant because it was recently reported that TGFß1 is capable of redistributing AR from its normal nuclear location to the cytoplasm in rat prostate stromal cells specifically (38). That we observed this phenomenon in the present study suggests that the elevated TGFß1 produced by smooth muscle cells in estrogenized prostates may have a direct autocrine effect on those cells. In this context, it is important to note that TGFß-RI did not decline in the smooth muscle after neonatal estrogen exposure; therefore, TGFß signaling was intact in those cells.
One of the known genes that is regulated by TGFß1 is p21waf-1/cip1, a cyclin-dependent kinase inhibitor that blocks the activity of cyclin E/cdk2 (43). Elevation in p21 levels induced by TGFß1 inhibits the passage of cells from G1 to S in the cell cycle, thus restricting cell proliferation. This system has recently been shown to be intact in human prostate cells, where it is thought to mediate the negative growth effects of TGFß1 by arresting cells in G1 and driving them into an apoptotic pathway (37). We chose to use p21waf-1/cip1 as a potential marker of TGFß1 activity in the developing prostate and followed its expression over time. In control rats, transient expression of p21 was observed in epithelial nuclei between days 10–15, which directly coincides with the temporal expression pattern of TGFß1 by periductal smooth muscle cells. Importantly, this transient expression of p21 also temporally coincides with terminal differentiation of prostatic stem cells into basal and luminal epithelial cells as previously determined with differentiation markers (36, 44). The expression of p21waf-1/cip1 has been found to correlate with terminal differentiation of multiple cell lineages, leading to the proposal that it contributes to cell cycle exit and differentiation during development (45, 46, 47). More recently, it was shown that elevated expression of p21waf-1/cip1 correlated with the onset of the terminally differentiated phenotype in mouse keratinocytes and that a subsequent drop in p21 levels permitted progression of these cells to the late stages of differentiation (48). These researchers hypothesized that in addition to playing a positive role in the commitment to differentiate, decreased expression of p21 is necessary for terminal differentiation. Taken together, we propose that transiently expressed TGFß1 by smooth muscle cells in the developing prostate acts as a paracrine factor that transiently induces p21 expression in the adjacent epithelial cells. In so doing, TGFß1 plays a role in stimulating the entry of epithelial cells into a differentiation pathway. The subsequent decrease in epithelial p21, perhaps related to decreased TGFß1 from smooth muscle cells, would allow the prostatic epithelium to terminally differentiate. This sequence of events would normally occur during a discreet window of opportunity, and an interruption in these events could result in permanent developmental defects of the prostate gland.
This theory receives support from the developmental events observed in the estrogenized prostates. In those tissues, p21waf-1/cip1 expression was absent at all time points examined, which would account for the differentiation defects observed in the prostatic epithelium throughout the life of these animals (4, 5, 6, 36). The data presented herein indicate that the absence of p21 in the developing epithelium of estrogenized rats is related to an interruption in TGFß signaling. There are several, not mutually exclusive, mechanisms by which the TGFß signal may be interrupted in the estrogenized prostate. Although there is an early increase and extension of TGFß1 production by smooth muscle cells, the proliferation of a thick layer of fibroblasts adjacent to the basement membrane after estrogen exposure may act as a physical barrier that blocks TGFß1 paracrine communication between smooth muscle and epithelial cells (8). Indeed, immunostaining shows that ECM in the fibroblast zone is negative for active TGFß1 in estrogenized prostates and that the TGFß1 located in the smooth muscle/ECM does not make contact with the epithelium. Secondly, there is a transient decrease in TGFß-RI in the estrogenized epithelium, which would temporarily block transduction of TGFß signals in those cells. Finally, it is possible that epithelial TGFß2 and TGFß3 function as autocrine factors that are involved with epithelial cell differentiation. As neonatal estrogens delay the early expression of epithelial TGFß2 and TGFß3, TGFß signaling may be interrupted at this level. The TGFß mechanism described herein may explain how early exposure to estrogens sets into motion the accumulation of undifferentiated stem cells or those with differentiation defects that can serve as precursors cells for dysplastic foci and tumor formation in the aging prostate (5, 49, 50). This is highly significant in view of the basal/stem cell theory of tumor biology, which proposes that carcinomas arise from arrested differentiation of tissue-determined stem cells (51).
We have consistently observed a marked retardation in prostatic ductal
branching in the estrogenized prostate (5) which may be a consequence
of the observed elevation in active TGFß1 levels. TGFß1 has been
shown to inhibit branching morphogenesis in the developing lung and
mammary gland (13, 14). Importantly, a biphasic effect of TGFß1 on
mammary epithelium has been demonstrated by which low concentrations
induced ductal elongation and branching and high concentrations were
inhibitory (52). In a recent study, surfactant protein C-TGFß1
transgenic mice were generated, which targets high expression of
TGFß1 to the developing lung (53). An abnormal distribution of
-actin/myofibroblasts was observed around the leading edges of lung
epithelial tubes, which resulted in arrest of lung morphogenesis at the
pseudoglandular stage of development. Those researchers proposed that
the abnormal stromal cells that accumulated around the epithelium in
response to TGFß1 may tether the distal airways and inhibit expansion
of the terminal lung buds. This is highly significant in light of our
recent demonstration that neonatal estrogen stimulates the
proliferation of a layer of fibroblasts between the basement membrane
of prostatic buds and the periductal smooth muscle cells (8). We
propose that this thick fibroblast zone may act as a physical barrier
that constrains branching morphogenesis. As formation of this
fibroblast zone temporally coincides with the precocious increase in
TGFß1 levels in the periductal smooth muscle layer, it is possible
that the fibroblast proliferation is mediated by TGFß1, which is
known to stimulate fibroblast proliferation in several other systems
(54).
In conclusion, the present study has shown that the developing rat prostate gland has all the necessary signaling molecules for TGFß action, which implicates a specific role for this growth factor in prostate morphogenesis. Evidence has been presented here which suggests that TGFß is involved in the commitment of the prostatic epithelium to a differentiation pathway during prostatic development. Early exposure to estrogens, by interfering with TGFß isoform and receptor levels, may interrupt this delicate balance and ultimately predispose the prostate gland toward a dysplastic state as the animals age.
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Acknowledgments |
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Footnotes |
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Received October 1, 1998.
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 H. M. Theobald, B. L. Roman, T.-M. Lin, S. Ohtani, S.-W. Chen, and R. E. Peterson 2,3,7,8-Tetrachlorodibenzo-p-dioxin Inhibits Luminal Cell Differentiation and Androgen Responsiveness of the Ventral Prostate without Inhibiting Prostatic 5{alpha}-Dihydrotestosterone Formation or Testicular Androgen Production in Rat Offspring Toxicol. Sci., December 1, 2000; 58(2): 324 - 338. [Abstract] [Full Text] [PDF]
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I. Goddard, M. Bouras, M. Keramidas, J. C. Hendrick, J. J. Feige, and M. Benahmed Transforming Growth Factor-{beta} Receptor Types I and II in Cultured Porcine Leydig Cells: Expression and Hormonal Regulation Endocrinology, June 1, 2000; 141(6): 2068 - 2074. [Abstract] [Full Text] [PDF]
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