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Center for Reproductive Biology, Department of Genetics and Cell Biology, Washington State University, Pullman, Washington 99164-4231
Address all correspondence and requests for reprints to: Michael K. Skinner, Ph.D., Center for Reproductive Biology, Department of Genetics and Cell Biology, Washington State University, Pullman, Washington 99164-4231. E-mail: skinner{at}mail.wsu.edu
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Abstract |
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These results indicate that the isoforms of TGFß2 and TGFß3 are differentially regulated during prostate development, suggesting distinct regulatory mechanisms. Testosterone did not affect TGFß expression in cultured prostatic cells. These observations suggest that the in vivo effects of castration on TGFßs are regulated indirectly through a complex network of growth factors, not simply by direct androgen depletion. The ability of EGF to inhibit prostatic ductal morphogenesis and growth in organ culture is postulated to be in part mediated by the increase in TGFß1 expression. In summary, a network of growth factor-mediated stromal-epithelial interactions is needed to maintain prostate growth and development. TGFß is postulated to have an important role in this process.
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Introduction |
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Three isoforms of TGFß (TGFß1, -2, and -3) have been identified in mammals, and their actions on cultured cells are similar in activity and potency (15). The biological actions of TGFß are numerous and include control of cell proliferation, adhesion, and differentiation (15). TGFßs generally have growth inhibitory activity on epithelial, neuronal, and lymphoid cells. TGFß activates the synthesis of extracellular matrix components (16). Recently, it was found that inhibition of cyclin-dependent kinases by TGFß results in growth arrest of cells in the G1 phase of the cell cycle (17, 18). TGFß has dramatic effects on the prostate (19, 20) through inhibiting the growth of both epithelial and stromal cells (13, 21, 22). These observations suggest that an alteration of TGFß expression may cause an imbalance in growth regulation of stromal and epithelial cells in the prostate. For example, TGFß may have a role in prostate cancer (23, 24). After ablation of androgen by castration, the expression of TGFß1 in the prostate increases and can be suppressed by in vivo treatment with androgen (9, 25). This observation suggests that the expression of TGFß may be regulated by androgen in normal prostate. TGFß has been identified in stromal cells in rat ventral prostate and in an epithelial cell line derived from rat dorsal prostate (26, 27). Cultured human prostatic stromal and epithelial cells have been shown to express TGFß1, -2, and -3 by RT-PCR (28). The developmental and hormonal regulation of TGFß1, -2, and -3 has not been thoroughly investigated.
The current study investigates the development and hormonal regulation of TGFß1, -2, and -3 expression. Isolated epithelial cells and stromal cells from rat ventral prostate were used in the in vitro experiments to investigate the actions of testosterone and growth factors.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Culture of ventral prostate cells
The isolated cell cultures used 20-day-old Sprague-Dawley rats;
these animals were killed, and ventral prostates were removed. Tissue
was incubated with Hanks’ Balanced Salt Solution (Life Technologies,
Gaithersburg, MD) containing 675 U collagenase activity/ml type II
collagenase (Sigma Chemical Co., St. Louis, MO) and 0.04%
deoxyribonuclease I (Sigma) at 37 C for 4 h. After incubation,
tissue was separated by centrifugation. The mixture was spun at 30
x g for 4 min to pellet the epithelial cells. The remaining
supernatant was centrifuged at 190 x g for 6 min to
pellet the stromal cells. Both pellets were resuspended and spun at
30 x g for 4 min. The supernatant from stromal cells
was removed and repelleted at 190 x g for 6 min. These
were then subsequently placed in 10% bovine calf serum F-12 medium in
six-well culture plates (Nunclon, Roskilde, Denmark) at 3 x
105 cells/well. After 24 h, the cells were washed in
serum-free F-12 medium and incubated for 24 h. On day 3 of
culture, the cells were maintained in serum-free conditions and
incubated with or without the following treatments: from
10-6-10-9 M testosterone (Sigma),
100 ng/ml EGF (Life Technologies), 25 ng/ml KGF (Life Technologies), 10
ng/ml human recombinant TGFß1 (Life Technologies), 10% bovine calf
serum (HyClone, Logan, UT), and a combination of TGFß1 and
testosterone (10-7 M), EGF, or 10% bovine
calf serum. The treatments were administered for a period of 72 h.
On the sixth day of culture, the medium was removed and stored for
measurement of acid phosphatase at -20 C, and cells were harvested for
RNA extraction.
Cell purity assay and immunocytochemistry
The purity of ventral prostate stromal and epithelial cells in
culture was analyzed by immunohistochemical methods. Isolated stromal
or epithelial cells were plated in four-well culture plates at 3
x 105 cells/well on Thermonox coverslips (Nunc, Inc.,
Naperville, IL). Two days postplating, cells were fixed in 100%
methanol for 5 min, then washed in decreasing ethanol conditions and
equilibrated in Tris-buffered saline (TBS; pH 7.4). Before antibody
staining, the cells were treated with H2O2 to
quench endogenous peroxidases and then incubated in TBS containing 1%
BSA and 0.5% normal serum for 1 h at 20 C. After rinsing three
times in TBS, cells were incubated for 2 h at 37 C with either
rabbit polyclonal antikeratin (Dako Corp., Carpenteria, CA) or a
monoclonal antivimentin (Sigma) antibody at dilutions of 1:250 and
1:300, respectively. Negative controls had no primary antiserum added,
and positive controls used epithelial (MCF-10A, human breast) and
stromal (SS-140, human fibroblast) cell lines. After primary antibody
incubations, the cells were rinsed three times in TBS and incubated
with either goat antirabbit or antimouse IgG horseradish
peroxidase-conjugated secondary antibody (Sigma). Visualization was
achieved using 50 mM Tris (pH 7.6) containing 0.6%
diaminobenzine and 0.03% H2O2 for 10 min at 20
C. The cells were then counterstained in hematoxylin and mounted on
slides using an aqueous mounting solution. Stained cells were counted
in four separate areas of the slide using cells from five different
experiments.
Ventral prostate sections were obtained from 20-day-old Sprague-Dawley rats. Tissue specimens were immediately fixed in Bouin’s solution for 3–6 h. Tissue was cut into 5-µm thick sections, deparaffinized, rehydrated, and treated with an aqueous solution of 3.0% H2O2 to quench endogenous peroxidase. After several washes in TBS buffer, sections were incubated in TBS containing 1% BSA for 2 h at 37 C to reduce nonspecific staining. After rinsing three times in TBS, a sheep antirat pan TGFß antibody (East Acres Biological, South Bridge, MA) was added at a 1:500 dilution and incubated overnight at 37 C followed by 2 h at 4 C. For controls, a nonimmune sheep IgG was used at a similar dilution. After three rinses in TBS, the primary antibody was detected with antisheep biotinylated secondary antibody, and the biotin was detected with an avidin-biotin-peroxidase kit (ABC-Elite, Vector Laboratories, Burlingame, CA). Diaminobenzidine tetrachloride was used as a chromagen, and serial sections were lightly counterstained with hematoxylin.
RNA preparation
Total RNA was obtained using Tri Reagent (Sigma). Briefly,
tissue or cells were lysed in Tri Reagent (1 ml/50–100 mg tissue or 1
ml/10 cm2 of culture plate). After adding 0.2 ml
chloroform/ml Tri Reagent, the mixture was centrifuged at 12,000
x g for 15 min at 4 C, the colorless upper aqueous phase
was transferred to a fresh tube, and 0.5 ml isopropanol/ml Tri Reagent
was added to pellet the RNA. The mixture was centrifuged at 12,000
x g for 10 min at 4 C. The RNA pellet was washed with 75%
ethanol and resuspended in diethylpyrocarbonate (DEPC)-treated
H2O. RNA was stored at -80 C until analysis.
RT
Complementary DNA (cDNA) was synthesized in a total 20-µl
volume containing 1 µg total RNA, 0.05 µM of specific
3'-primers (TGFß1, 5'-GGG GTG GCC ATG AGG AGC AGG-3'; TGFß2, 5'-GCG
CTG GGT GGG AGA TGT TAA-3'; TGFß3, 5'-CCT TTG AAT TTG ATC TCC A-3';
cyclophilin, 5'-ATT TGC CAT GGA CAA GAT GCC-3'), 4 µl 5 x first
strand PCR buffer (Life Technologies), 10 mM dithiothreitol
(Life Technologies), 0.125 mM deoxy-NTPs, 100 U Moloney
murine leukemia virus transcriptase (Life Technologies), 20 U
ribonuclease inhibitor (Promega, Madison, WI), and
DEPC-H2O. Initially, RNA was denatured and annealed in the
presence of each primer and DEPC-H2O at 65 C for 15 min.
The tube was placed on ice for 5 min. The other reagents were added to
the tube and incubated at 42 C for 2 h. To decrease the high
background signal, a higher RT reaction temperature (42 C) was used
(29). The mixture was incubated at 95 C for 5 min to inactivate Moloney
murine leukemia virus transcriptase. The mixture was diluted by
UV-treated H2O containing Bluescript plasmid DNA
(Stratagene, La Jolla, CA) as carrier DNA. The final concentrations of
cDNA and Bluescript plasmid were 1 ng/µl and 10 ng/µl,
respectively. This concentration of Bluescript plasmid was included in
all samples and standards.
Quantitative PCR assay
As a standard for the assay, PCR products of TGFß1, -2, and -3
and cyclophilin amplified by each specific primer were subcloned into
Bluescript plasmid (Stratagene). Each subclone was sequenced in both
directions and confirmed to be rat TGFß1, -2, and -3 and cyclophilin.
The size and base pair alignment of the PCR product generated were as
follows: 200 bp size from 1003–1203 bp alignment on the coding
sequence (accession no. 52498; TGFß1), 194 bp size from 355–549 bp
alignment on the coding sequence (TGFß2), 288 bp size from 865-1153
bp alignment on the coding sequence (accession no. 403491; TGFß3),
and 105 bp size from 244–348 bp alignment on the coding sequence
(accession no. M19533; cyclophilin). Plasmid DNA containing TGFß
subclones was used to generate standard curves ranging from 0.1 fg/µl
to 1.0 pg/µl, and for cyclophilin from 10 fg/µl to 100 pg/µl. PCR
was performed on a Perkin-Elmer GeneAmp PCR System 9600 (Perkin-Elmer,
Branchburg, NJ) and was carried out in a total 25-µl reaction volume
containing 5 µl plasmid DNA or RT reaction, 0.4 µM
3'-primer as shown above, 0.4 µM 5'-primer (TGFß1,
5'-TCG ATT TTG ACG TCA CTG GAG TTG T-3'; TGFß2, 5'-CCG CCC ACT TTC
TAC AGA CCC-3'; TGFß3, 5'-TGC CCA ACC CGA GCT CTA AGC G-3';
cyclophilin, 5'-ACA CGC CAT AAT GGC ACT GG-3'), 2.5 µl 10 x
GeneAmp PCR buffer (containing 1.5 mM MgCl2;
Perkin-Elmer), 25 µM deoxy-NTPs, 0.5 U Taq DNA
polymerase (Perkin-Elmer), and 1 µCi [
-32P]deoxy-CTP
(Amersham Life Science, Arlington Heights, IL).
The TGFß1 reaction cycle sequence comprised 5 min at 95 C, followed by 30 cycles of 1 min at 95 C, 1 min at 60 C, and 2 min at 72 C. The cyclophilin reaction cycle sequence comprised 5 min at 95%, followed by 25 cycles of 1 min at 95 C, 1 min at 60 C, and 2 min at 72 C. The TGFß2 reaction cycle sequence comprised 5 min at 95 C, followed by 33 cycles of 1 min at 95 C, 2 min at 60 C, and 3 min at 72 C. The TGFß3 reaction cycle sequence comprised 5 min at 95 C, followed by 35 cycles of 1 min at 95 C, 2 min at 55 C, and 3 min at 72 C. All reactions had a final extension for 10 min at 72 C.
After PCR, the products were electrophoretically separated on a 6% acrylamide gel. The gel was then dried and analyzed on a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) quantitatively. Each gene was assayed in separate PCR reactions from the same RT samples. Equivalent steady state messenger RNA (mRNA) levels for each gene were determined by comparing each sample to the appropriate standard curve. TGFß1, -2, and -3 were normalized for cyclophilin. For each assay, all samples were simultaneously measured in duplicate with intraassay variabilities of 7.5% (TGFß1), 9.4% (TGFß2), 9.9% (TGFß3), and 9.2% (cyclophilin).
Cell growth assay
Cell growth was analyzed by examining
[3H]thymidine incorporation into newly synthesized DNA.
Stromal and epithelial cells were placed at subconfluent densities (<1
million cells/cm2) in 0.5 ml DMEM containing 0.1% calf
serum. The low serum level does not stimulate growth and is required
for progression factors (i.e. insulin-like growth factor)
for the S phase of the cell cycle to detect the growth factor response.
After 48–72 h of culture, the cells were treated with various agents
for 24 h. After the 24-h treatment, 0.5 ml DMEM containing 2 µCi
[3H]thymidine was added to each well, and the cells were
incubated for 4 h at 37 C before sonication. The quantity of
[3H]thymidine incorporated into DNA was determined as
previously described (30). Data were normalized to total DNA per well
using an ethidium bromide procedure, described previously (31). Under
these subconfluent culture conditions, approximately 0.5–1.5 µg DNA
were detected per well. [3H]Thymidine incorporation was
generally greater than 2 x 103 cpm/µg DNA.
Organ culture of rat ventral prostate
Ventral prostates were removed from 0-day-old rats and cultured
in a drop of medium on a Millicell CM filter (Millipore, Bedford, MA)
floating on the surface of 0.5–1.0 ml CMRL 1066 medium (Life
Technologies, Gaithersburg, MD) supplemented with
penicillin-streptomycin, insulin (10 µg/ml), and transferrin (10
µg/ml) with or without treatments. The cultures were performed in a
four-well Nunclon surface dish (32). These plates were placed in the
incubator at 37 C in an atmosphere of 5% CO2 and 95% air.
The tissues were cultured for 6 days. Treatments were testosterone
(Sigma; 10-7 M), EGF (Life Technologies; 100
ng/ml), TGFß1 (Life Technologies; 10 ng/ml), and a combination of
testosterone and EGF or a combination of testosterone and TGFß1.
Images of the prostates were captured with an image analysis system
(Pixera, Pixera Corp., Los Gatos, CA). At the end of the culture, the
organs were fixed for histology. The analysis and quantitation of
ductal branching morphogenesis were previously described (32).
Statistical analysis
All data were analyzed by a JMP 3.1 statistical analysis program
(SAS Institute, Cary, NC). All values are expressed as the mean ±
SEM. Statistical analysis was performed using one-way
ANOVA. Significant differences were determined using the Dunnett’s
test for comparison to controls and using the Tukey-Kramer honestly
significant difference test for multiple comparisons. Statistical
difference was confirmed at P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The TGFß2 PCR
product continued to be amplified at 42 cycles. For TGFß2, 33 cycles
was midpoint on the linear amplification of the PCR product and was
selected for QRT-PCR (Fig. 1A). TGFß3 product started to increase
after 30 cycles and reached a plateau level at 40. Thirty-five cycles
was determined appropriate to use for TGFß3 (Fig. 1B). For
cyclophilin, 25 cycles was determined to be the appropriate cycle
number (Fig. 1B).
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, A–D).
The small error shown in each of the data points demonstrates minimal
variability in the assay (Figs. 1
and 2).
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).
Expression of TGFß1 and -2 was higher in stromal cells than in
epithelial cells. TGFß3 expression in epithelial cells was the same
as that in stromal cells. In cultured samples derived from 20-day-old
rat ventral prostate, TGFß1, -2, and -3 showed higher expression than
fresh samples and ranged from 7–700 x 10-3 fg
TGFß/fg cyclophilin. Contrary to that in fresh samples, expression in
epithelial cells in cultured samples was higher than that in stromal
cells. In both epithelial and stromal cells, TGFß1 had the lowest
level of expression, and TGFß3 had the highest level of expression
(Fig. 3).
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). After castration of 60-day-old rats,
TGFß1 significantly increased to 5-fold that in 60-day-old rats.
During the neonatal period, TGFß2 expression was elevated, then
decreased in pubertal and early adult periods (60 days of age) and was
elevated in adult rats (100 days old). After castration, TGFß2 was
increased 4-fold, but remained lower than that in 100-day-old rats. In
contrast to TGFß2, TGFß3 expression was very low in the 1- and
100-day-old prostates and was elevated during the pubertal and early
adult periods (60 days of age). After castration, TGFß3 expression
was significantly suppressed from that in 60-day-old rats.
Interestingly, TGFß3 expression was inversely related to TGFß2
expression (Fig. 4).
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Analysis of cell purity indicated that isolated epithelial cells showed 93.2 ± 1.2% (mean ± SEM; n = 5) keratin-positive cells and less than 3% vimentin-positive cells. Isolated stromal cells showed 86.0 ± 3.5% (mean ± SEM; n = 5) vimentin-positive and less than 5% keratin-positive cells. These results demonstrate that cell purity in both isolated stromal and epithelial cells were appropriate for data interpretation.
EGF significantly stimulated (6-fold) TGFß1 expression in stromal
cells (Fig. 5A) and significantly
stimulated the expression of TGFß1 in epithelial cells at a reduced
level (2.5-fold) compared to that in stromal cells. This effect was not
significantly different compared with that of other treatments, but was
different from the control. TGFß1 had no effect on the expression of
TGFß1 in either stromal or epithelial cells. The effects of EGF on
TGFß1 expression were not influenced by exogenous administration of
TGFß1 in either cell type. Neither testosterone nor KGF had any
effect on TGFß1 expression in either cell type (Fig. 5A).
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). In epithelial cells, only KGF
significantly stimulated (2.8-fold) TGFß2 expression. TGFß1 alone
did stimulate TGFß2 expression, but this stimulation was not
statistically different from that in control cultures (Fig. 5B).
Results indicate that prostate stromal cells were not influenced by any
treatment, and epithelial cells were only influenced by KGF with regard
to TGFß2 expression.
No treatment affected TGFß3 expression in either stromal or
epithelial cells (Fig. 5C). Although small effects were observed with
testosterone and TGFß1, these were not statistically different from
those in control cultures.
Combined observations indicate that EGF regulates TGFß1 expression by stromal and epithelial cells, KGF regulates TGFß2 expression by epithelial cells, and none of the treatments used in the current study influences TGFß3 expression. Testosterone had no effect on TGFß1, TGFß2, or TGFß3 in either cell type.
Regulation of prostate cell growth
Stromal and epithelial cells from rat ventral prostates were
cultured to examine the effects of testosterone, EGF, KGF, and TGFß1
on cell growth. A tritiated thymidine incorporation assay was performed
using 10% bovine calf serum as a positive control. Calf serum
significantly stimulated (
4-fold) DNA synthesis in both stromal and
epithelial cells (Fig. 6). Testosterone
and EGF stimulated (2.5- to 3-fold) DNA synthesis in stromal and
epithelial cells. KGF had a stimulatory effect on epithelial cells.
TGFß had no direct effect on DNA synthesis alone (Fig. 6). However,
TGFß significantly inhibited the growth effects of testosterone and
EGF on stromal cells as well as the growth effects of testosterone on
epithelial cells. These results indicate that testosterone and EGF can
stimulate the growth of both stromal cells and epithelial cells,
whereas TGFß can inhibit these actions and suppress growth. Although
the growth response for testosterone was observed in less than 24
h, potential indirect effects of testosterone mediated by other peptide
growth factors remain to be elucidated.
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).
TGFß1 alone caused a slight reduction in the number of branching
ducts, but the number was similar to that in control cultures. However,
TGFß1 completely inhibited the stimulatory effect of testosterone on
prostatic ductal branching morphogenesis (Figs. 7 and 8). EGF increased prostatic size;
however, ductal branching was significantly reduced (Figs. 7 and 8),
and ducts were swollen, with an increase in surrounding stromal layers
(Fig. 7). The histology of these organ cultures is shown in Fig. 9 and demonstrates that EGF does increase
the stromal/epithelial cell ratio as well as shows the epithelial cells
are less organized into ductal structures. Testosterone promotes a
polarized epithelial cell morphology, whereas EGF promotes a more
random proliferation of the epithelium (Fig. 9). A combination of
testosterone and EGF caused enlargement of the prostate; however, this
enlargement was not due to ductal formation and appears to be due to
stromal enlargement (Figs. 7 and 8). The degree of ductal branching
morphogenesis with testosterone and EGF was increased over that with
EGF alone (Fig. 8). The ratio of stroma/epithelium was higher in the
prostates treated with EGF than in control cultures. Combined results
indicate that TGFß can inhibit the stimulatory effects of
testosterone on prostate morphogenesis. The ability of EGF to stimulate
TGFß may have a role in the inhibitory effects of EGF on prostate
ductal branching morphogenesis.
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). TGFß staining was most intense
in the stromal cells adjacent to the epithelial cells. Although the
mRNA for TGFß was found in both cell types, the protein is primarily
localized in stromal cells. Whether this is due to localization in the
extracellular environment of the stromal cells or is a reflection of
production levels remains to be elucidated. Controls with nonimmune IgG
had no staining detected (Fig. 10).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All three isoforms of TGFß, TGFß1, -2, and -3, were expressed in both stromal cells and epithelial cells from freshly isolated cells and cultured cells. Freshly isolated prostatic stromal cells have a higher level of TGFß expression than epithelial cells. TGFß3 has the highest level of expression, whereas TGFß1 has the lowest. The high levels of TGFß mRNA in freshly isolated stromal cells correlated to the immunocytochemical data, with TGFß staining predominantly in the stromal cells. However, epithelial cells did express all isoforms of TGFß. During cell culture, the levels of TGFß1, -2, and -3 increased in both cell types. Interestingly, the epithelial cells developed a higher level of expression than stromal cells in culture. This increase in expression during culture may be due to the lack of regulatory agents that suppress expression in normal in vivo tissue. Previously, the type I and type II TGFß receptors (15) have been shown to be expressed in the prostate (35). These observations suggest that both prostate stromal and epithelial cells have the capacity to express TGFß1, -2, and -3, which may act as both paracrine and autocrine factors to influence prostate function.
The developmental regulation of TGFß1, -2, and -3 expression was investigated with neonatal day 0 rats, pubertal day 20 rats, early adult day 60 rats, and adult day 100 rats. TGFß1 expression (i.e. mRNA levels) remained constant during prostate development. TGFß2 expression was high during the neonatal and adult (100 day) periods and low during the pubertal and early adult (60 day) periods. In contrast, TGFß3 expression was low during the neonatal and adult (100 day) periods and high during the pubertal and early adult (60 day) periods. TGFß2 and TGFß3 expression appeared to be inversely related during prostate development. Recently, the ratio of stromal to epithelial cells was measured during prostate development and was constant throughout (36). Therefore, the changes in TGFß1, -2, and -3 do not appear to be due to alterations in the cell populations. Although the three isoforms of TGFß are highly homologous and have similar biological activities, the developmental expression during prostate development appears distinct. The inverse relation of TGFß2 and TGFß3 may reflect changes in the regulation of prostate function and may be required to coordinate local cell-cell interactions (37).
Androgens are postulated to be an essential factor to regulate TGFß expression in the prostate (9, 25). Both TGFß1 and TGFß2 expression increased after castration. This supports previous observations with TGFß1 (9, 10). Interestingly, TGFß3 expression decreased after castration. Previously, TGFß2 mRNA levels have been shown to be negatively correlated with androgen levels in both normal and malignant prostate (34). The changes in TGFß expression during development cannot be directly correlated with serum androgen levels. The relationship of serum androgen levels and TGFß expression remains to be elucidated. As discussed below, androgens have no effect on TGFß expression using cultured prostate cells. Another factor to explain the changes after castration is the alteration in the stromal/epithelial cell populations (36). These observations suggest that the relationship of TGFß expression and androgen levels remains to be elucidated and probably involves a combination of direct and indirect actions of androgens.
Several interesting observations came from the analysis of TGFß expression in cultured prostatic stromal and epithelial cells. Both isolated cell populations were approximately 90–95% pure by an immunocytochemical analysis of the cell populations. Androgens did not influence TGFß1, -2, or -3 in either stromal cell or epithelial cell cultures. The concentrations of androgen used ranged from 10-8-10-6 M, with no effect at any concentration (data not shown). This observation suggests that androgen may not have a dramatic direct action on TGFß expression. TGFß3 expression was not influenced by any of the treatments used in the current study. TGFß2 was also not affected in stromal cells by any treatment. KGF stimulated TGFß2 in epithelial cells. KGF has been shown to mediate stromal-epithelial cell interactions in the prostate and to influence prostate morphogenesis (3, 32, 36). The ability of KGF to influence TGFß2 expression in prostate epithelium will probably have a role in the actions of KGF on the prostate.
TGFß had no effect on TGFß1, -2, or -3 expression in either stromal cells or epithelial cells. Previously, TGFß1 was found to enhance bFGF expression in cultured human prostatic stromal cells (13, 14). In rat kidney fibroblasts, TGFß has been shown to stimulate TGFß expression (38). In the current study, TGFß did not have a major regulatory role in the control of TGFß expression.
Perhaps one of the more interesting observations made was the effect of EGF on TGFß1 expression. EGF stimulated the expression of TGFß1 by both prostatic stromal and epithelial cells. EGF has been shown to be a potent growth stimulator of prostatic epithelium (39, 40). The observations made in the current study suggest that EGF may also have an important regulatory role. The actions of EGF on prostate function and development discussed below will probably be mediated in part by these effects on TGFß1 expression.
The effects of TGFß on cell growth were examined to investigate the functional significance of TGFß in the prostate. Both EGF and testosterone dramatically stimulated prostate stromal and epithelial cell growth. The potential indirect actions of testosterone via peptide growth factors need to be considered. TGFß inhibited the actions of testosterone and EGF on both cell types. This correlates with the previously identified function of TGFß as a growth inhibitory substance. Previously, TGFß has been shown to inhibit the growth of human prostate epithelial cells (21). An important function for TGFß will probably be as a growth inhibitor in the prostate.
Another functional parameter of the prostate examined was ductal branching morphogenesis in prostate organ cultures. This organ culture system has been developed to investigate morphogenesis of the prostate and actions of agents such as androgens and KGF (36, 32). In the current study, androgen was found to promote ductal branching morphogenesis, and TGFß was found to suppress the actions of androgens. This suggests that TGFß may have a role in regulating prostate morphogenesis. Interestingly, EGF also dramatically suppressed ductal branching morphogenesis. This was in part due to a proliferation of stromal cells and a disorganization of the epithelium. The ability of TGFß to suppress ductal branching morphogenesis and inhibit EGF-stimulated growth suggests that TGFß may have a role in this process. In support of this proposal is the ability of EGF to stimulate TGFß1 expression. These observations suggest that TGFß will have a role in prostate growth and morphogenesis. The actions of regulatory agents (e.g. EGF) on the prostate may be mediated indirectly through the production and action of TGFß.
The combined observations suggest that a network of locally produced growth factors, such as KGF, EGF, and TGFß, will coordinate prostate development and morphogenesis. This is supported by observations in the current study regarding the developmental and hormonal regulation of TGFß1, -2, and -3 expression in prostate stromal and epithelial cells. Regulatory agents such as androgen probably involved both direct and indirect actions mediated through this network of growth factors to control prostate function. The abnormal phenotypes in prostate cancer and BPH will probably in part be due to the inability of this network of regulatory agents and growth factors to maintain normal prostate function. It is speculated that TGFß will have a role in this process.
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Urology, Sapporo Medical University,
Sapporo, Japan.
Received September 24, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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EGF on cell proliferation and cell death in rat
ventral prostatic epithelial cells in culture. J Androl 16:482–490,25-dihydroxyvitamin
D3 in NRP-152 rat prostatic epithelial cells. J Cell
Physiol 166:231–239[CrossRef][Medline]
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D. C. Tomlinson, J. C. Grindley, and A. A. Thomson Regulation of Fgf10 Gene Expression in the Prostate: Identification of Transforming Growth Factor-{beta}1 and Promoter Elements Endocrinology, April 1, 2004; 145(4): 1988 - 1995. [Abstract] [Full Text] [PDF]
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W. Zhou, I. Park, M. Pins, J. M. Kozlowski, B. Jovanovic, J. Zhang, C. Lee, and K. Ilio Dual Regulation of Proliferation and Growth Arrest in Prostatic Stromal Cells by Transforming Growth Factor-{beta}1 Endocrinology, October 1, 2003; 144(10): 4280 - 4284. [Abstract] [Full Text] [PDF]
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 A. S. Cupp, M. Uzumcu, H. Suzuki, K. Dirks, B. Phillips, and M. K. Skinner Effect of Transient Embryonic In Vivo Exposure to the Endocrine Disruptor Methoxychlor on Embryonic and Postnatal Testis Development J Androl, September 1, 2003; 24(5): 736 - 745. [Abstract] [Full Text] [PDF]
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M. Uzumcu, K. A. Dirks, and M. K. Skinner Inhibition of Platelet-Derived Growth Factor Actions in the Embryonic Testis Influences Normal Cord Development and Morphology Biol Reprod, March 1, 2002; 66(3): 745 - 753. [Abstract] [Full Text] [PDF]
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A. S. Cupp, G. H. Kim, and M. K. Skinner Expression and Action of Neurotropin-3 and Nerve Growth Factor in Embryonic and Early Postnatal Rat Testis Development Biol Reprod, December 1, 2000; 63(6): 1617 - 1628. [Abstract] [Full Text]
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 C. Gupta Reproductive Malformation of the Male Offspring Following Maternal Exposure to Estrogenic Chemicals Experimental Biology and Medicine, June 1, 2000; 224(2): 61 - 68. [Abstract] [Full Text]
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E. Levine, A. S. Cupp, and M. K. Skinner Role of Neurotropins in Rat Embryonic Testis Morphogenesis (Cord Formation) Biol Reprod, January 1, 2000; 62(1): 132 - 142. [Abstract] [Full Text]
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W. Y. Chang, L. Birch, C. Woodham, L. I. Gold, and G. S. Prins Neonatal Estrogen Exposure Alters the Transforming Growth Factor-{beta} Signaling System in the Developing Rat Prostate and Blocks the Transient p21cip1/waf1 Expression Associated with Epithelial Differentiation Endocrinology, June 1, 1999; 140(6): 2801 - 2813. [Abstract] [Full Text]
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A. S. Cupp, G. Kim, and M. K. Skinner Expression and Action of Transforming Growth Factor Beta (TGFß1, TGFß2, and TGFß3) during Embryonic Rat Testis Development Biol Reprod, June 1, 1999; 60(6): 1304 - 1313. [Abstract] [Full Text]
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A. S. Cupp, J. M. Dufour, G. Kim, M. K. Skinner, and K. H. Kim Action of Retinoids on Embryonic and Early Postnatal Testis Development Endocrinology, May 1, 1999; 140(5): 2343 - 2352. [Abstract] [Full Text]
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N. Itoh, U. Patel, and M. K. Skinner Developmental and Hormonal Regulation of Transforming Growth Factor-{alpha} and Epidermal Growth Factor Receptor Gene Expression in Isolated Prostatic Epithelial and Stromal Cells Endocrinology, March 1, 1998; 139(3): 1369 - 1377. [Abstract] [Full Text] [PDF]
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