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Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030
Address all correspondence and requests for reprints to: David R. Rowley, Ph.D., Department of Cell Biology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030.
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Multiple signal transduction pathways have been shown to influence steroid action, including tyrosine kinase receptors [i.e. epidermal growth factor (EGF) and insulin-like growth factor I] (10, 11), the protein kinase A pathway (12), and the protein kinase C pathway (12). Typically, these pathways lead to enhanced androgen responses and, in some instances, ligand-independent receptor activation. Moreover, certain growth factor pathways act to inhibit steroid pathways. Insulin-like growth factor II has been shown to decrease androgen-stimulated gene transcription 5-fold (11), and the addition of TGFß1 to a developing prostate organ culture model reportedly inhibited the androgen-dependent stromal induction of epithelial differentiation (8, 13). The potential linking of androgen and TGFß action have been shown in androgen deprivation studies in which TGFß1 message and protein are repressed by androgens in epithelial cells both in vitro and in vivo (14, 15, 16). The effects of TGFß1 on androgen action in prostate stromal cells may be particularly important in understanding the mechanisms of prostate carcinoma progression, where overexpression of TGFß1 by carcinoma cells (17, 18, 19) may affect tumor progression through action in the stromal compartment of cells (19). Accordingly, studies to specifically address the role of TGFß1 in androgen signaling in prostatic stromal cells are necessary to understand the complex interaction between this growth factor and androgen action pathways.
The PS-1 cell line was established and characterized to elucidate
mechanisms through which androgens regulate prostate stromal cell
biology. Rat ventral prostate-derived PS-1 stromal cells were positive
for smooth muscle 
-actin, desmin, AR (20), smooth muscle myosin
heavy chain, calponin (our unpublished observations), and ps20, a new
smooth muscle protein (21, 22). These data indicate that PS-1 cells are
derived from a smooth muscle lineage. Proliferation of PS-1 stromal
cells was growth stimulated by physiological concentrations of
androgens in chemically defined, serum-free medium conditions, and
several androgen-regulated marker messenger RNA (mRNA) transcripts were
identified through differential display (20). Accordingly, this cell
line model has the advantage of allowing examination of the expression
of endogenous mRNA transcripts and cell proliferation to assess
androgen action.
Our previous studies have shown that in serum-containing medium, PS-1 cells were essentially unresponsive to androgen action, and AR exhibited a cytoplasmic distribution pattern in the presence of ligand (testosterone or dihydrotestosterone) (20). In serum-free medium, however, AR was localized to the nucleus in a ligand-independent manner, and PS-1 cells were growth stimulated by physiological concentrations of androgens (10 nM) (20). These observations suggested that a serum component, possibly a peptide growth factor, was responsible for altered AR localization and activity. The data reported here show that of several growth factors tested in serum-free medium, only TGFß1 induced a nuclear to cytoplasmic shift in the distribution of AR in a specific manner coincident with an inhibition of androgen-induced proliferation and inhibited expression of transcript marker ddp17 in PS-1 cells. These studies suggest that cross-talk mechanisms between TGFß1 and AR signaling pathways may be important in directing androgen-regulated events in prostate gland stroma.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell culture
PS-1 prostate smooth muscle cell cultures were standardly
maintained in Bfs medium (90% DMEM, 5% FCS, 5% NuSerum, 5 µg/ml
insulin, 25 U/ml penicillin, and 25 µg/ml streptomycin) supplemented
with 0.5 µg/ml testosterone in 25-cm2 flasks and
incubated at 37 C with 5% CO2 as previously reported (20).
Medium was replaced every 48 h with fresh medium Bfs, and cultures
were passaged weekly. All experiments used PS-1 cell cultures at
passages 20–27. LNCaP cells were cultured in medium L (90% RPMI 1640,
10% FBS, 0.5 µg/ml testosterone, 25 U/ml penicillin, and 25 µg/ml
streptomycin). DDT1MF-2 smooth muscle cells (DDT-1/3B9 cell line
provided by Dr. Dorrie Lamb, Baylor College of Medicine, Houston, TX)
were cultured in medium D (low glucose 98% DMEM, 2% FBS, 25 U/ml
penicillin, and 25 µg/ml streptomycin). All cultures were routinely
assayed for mycoplasma contamination (MycoTect Kit, Life Technologies,
Gaithersburg, MD) every 3 months.
For growth factor and steroid hormone regulation studies, cells were seeded onto sterile glass coverslips (one coverslip per well) in six-well culture plates (no. 25810, Corning, Corning, NY) in medium Bfs (PS-1 cells), medium L (LNCaP cells), or medium D (DDT1MF-2 cells) and allowed to adhere for 24–48 h. Medium was then replaced with chemically defined medium M0, which consisted of MCDB110 basal medium, 5 µg/ml insulin, 5 µg/ml transferrin, and 5 ng/ml sodium selenite (ITS), as described previously (20), and supplemented with the indicated concentration(s) of androgen or growth factor for 24–96 h as described in the text.
For studies with charcoal-stripped serum (CSS), dextran-coated charcoal was prepared by incubating 25 g charcoal (Sigma) with 0.25 g dextran T-70 in 10 mM HEPES, 0.15 M NaCl, and 1.5 mM MgCl2, pH 7.4, overnight at 4 C with stirring. Charcoal was pelleted (2000 rpm, 10 min), and an equal volume of FCS was added, mixed, and incubated overnight at 4 C in the dark. Charcoal was then pelleted (7000 rpm, 1 h, 4 C), and serum was filter sterilized. Aliquots were stored at -20 C until use.
For protein synthesis inhibition and time-course studies, cyclohexamide (10 µg/ml, final concentration) was added for 1 h before the addition of TGFß1 or androgens, and cells were cultured for 30 min to 24 h before fixation. Control cultures did not receive cyclohexamide. Coverslips were removed from culture at the indicated time points and immediately fixed with 10% neutral buffered formalin for 20 min at room temperature, washed three times (5 min each time) in PBS, and stored in 70% ethanol at 4 C before immunohistochemistry. Additionally, replicate coverslips were fixed with 4% paraformaldehyde, which produced identical immunocytochemical results as with formalin fixation. The efficiency of cyclohexamide to inhibit protein synthesis was determined by [35S]methionine incorporation in cell lysates as follows. PS-1 cells were seeded as described above in triplicate in 24-well culture dishes in medium Bfs for 24 h. Medium was changed to defined medium M0 and 10 µg/ml cyclohexamide for 1 h before the addition of [35S]methionine (20 µCi/ml), dihydrotestosterone (DHT; 10 nM) and TGFß1 (25 pM). Cell lysates were harvested at 1 and 6 h and precipitated with equal volumes of 20% TCA as reported previously (23). Lysates were transferred to Whatman glass-fiber filters (Whatman, Clifton, NJ), washed three times in ice-cold ethanol, and quantitated by scintillation counting to allow determination of the percentage of protein synthesis inhibition (23). For studies using a pan-specific neutralizing antibody for TGFß1 (ND50 = 5 µg/ml), cells were cultured as described above, and 10–50 µg/ml of antibody were added at the time of TGFß1 addition.
Primary cultures of rat ventral prostate stromal cells were established
as we have reported previously for the initiation of the U4F and PS-1
cell lines (20, 24). Briefly, organ explant cultures were seeded
adjacent to coverslips in medium Bfs. Primary stromal cultures were
observed extending onto the coverslips after 1 week of culture. Medium
was changed to defined medium M0 for 24 h, followed by
supplementation with the indicated concentration(s) of androgen or
TGFß1 for 24 h as described in the text and Fig. 5
. Cultures
were fixed and processed for immunocytochemistry as described above for
the PS-1 cell line.
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Immunocytochemistry
Cells were seeded into six-well culture dishes containing
sterile glass coverslips at a density of 9.6 x 103
cells/cm2 and allowed to adhere for 48 h in fresh
serum-containing (Bfs, L, or D) medium. Medium was then changed to
basal M0 medium (serum free, steroid free) for 24 h,
followed by the experimental conditions described in the text and
figures. Coverslips were washed three times in PBS and fixed in 10%
neutral buffered formalin as described above. Cells were permeablized
by exposure to 0.1% Triton X-100 for 5 min in PBS at room temperature,
washed three times (5 min each time) in PBS, and incubated with anti-AR
antibody PG-21 (1:60 dilution) or anti-AR antibody U402 (1:100
dilution) in PBS containing 1% BSA for 24 h at 37 C. Coverslips
were then washed sequentially in PBS, followed by PBS-0.1% Triton
X-100, followed by PBS for 15 min/wash at room temperature on a rocking
platform. Secondary antibody staining consisted of fluorescein
isothiocyanate-conjugated goat antirabbit IgG (1:100 dilution of 1.5
mg/ml stock) in PBS-1% BSA for 45 min at 37 C. All coverslips for each
hormone/growth factor regimen were prepared in triplicate, and
replicate experiments were repeated on at least 2 separate days.
Coverslips were washed as described above and mounted onto glass slides
with antifade mounting solution [250 µg/ml
diazabicyclo-(2,2,2)octane in PBS-glycerol, 1:9]. Photomicroscopy was
performed on a Nikon Labophot-2 equipped for fluorescence (Nikon,
Melville, NY) using TMAX ASA 400 film or Ektachrome ASA 400 color slide
film (Eastman Kodak, Rochester, NY). To confirm immunocytochemistry
data, Western analysis of AR was performed using anti-AR antibody
PG-21, and total cell lysates of PS-1 cells were cultured as described
in the text following standard Western blot procedures that we have
published previously (22). A primary reactive species was observed at
111 kDa. The experiment was repeated in triplicate.
DNA probes
The ddp17 probe was prepared by digesting 10 µg plasmid
pCRII-ddp17 (20) with EcoRI (2 U/µg; Promega, Madison, WI)
at 37 C for 3 h. The 390-bp insert was purified by gel
electrophoresis, excised from a 1.2% agarose gel (Amresco), and
prepared for labeling with the QIAquick gel extraction kit (Qiagen,
Chatsworth, CA). Seventy nanograms of insert DNA were random prime
labeled following the manufacturer’s protocols (Random Prime labeling
kit, Boehringer Mannheim, Indianapolis, IN) with
[32P]deoxy-CTP (3000 Ci/mmol; New England Nuclear,
Boston, MA) at 37 C for 3 h and purified with the QIAquick
nucleotide removal kit according to the manufacturer’s protocols
(Qiagen).
RNA analysis
PS-1 cells were seeded into 75-cm2 flasks in medium
Bfs for 24 h, after which medium was changed to M0
(serum free, chemically defined) for 24 h, followed by the
experimental conditions described in the text. Total RNA was isolated
by extracting cells with RNA Stat-60 essentially according to
manufacturer’s suggested protocols. RNA was fractionated with a 1.2%
agarose gel containing 3.3 M formaldehyde and 20
mM NaPO4 (pH 7) at 30 V for 17 h in 20
mM NaPO4 running buffer (circulated throughout
run). Loading accuracy was determined by ethidium bromide staining of
gels. RNA was transferred to Nytran membranes by capillary transfer
with a SSC (standard saline citrate) gradient (Schleicher and Schuell,
Keene, NH). RNA was UV cross-linked to the membrane, prehybridized
(5 x SSC, 50% formamide, 0.1% SDS, 50 mM
NaPO4 (pH 7.0), and 1 µg/ml salmon sperm DNA) for 24
h, and then hybridized with 1 x 106 cpm/ml
[32P]deoxy-CTP random prime-labeled probe overnight at 42
C. Membranes were washed twice for 30 min each time in 2 x
SSC-0.1% SDS at room temperature with agitation, followed by two
20-min washes in 0.2 x SSC-0.1% SDS at 65 C. Membranes were
exposed to radiographic film for 24–48 h. Transfer efficiency was
determined by methylene blue staining of blots.
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Results |
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PS-1 control cultures in serum-containing medium Bfs (5% FCS and 5%
NuSerum) exhibited a cytoplasmic AR localization with a uniformly
diffuse perinuclear distribution, as shown in Fig. 1A. Testosterone concentrations ranging
from 10 nM to 1 µM did not affect the
cytoplasmic AR localization pattern in serum-containing medium (Fig. 1B). Further, as CSS has been frequently used as a steroid-free serum
source, both 10% and 1% CSS were tested with and without androgens.
In medium with 10% CSS, a cytoplasmic AR distribution pattern was
observed, identical to patterns in medium Bfs. Cultures in medium plus
1% CSS exhibited some nuclear accumulation of AR, however not to the
extent previously observed with chemically defined medium conditions.
These results are summarized in Table 1.
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, the 24-h period in unsupplemented medium
M0 induced a nuclear distribution of AR and thus allowed
for growth factors that might affect nuclear localization of AR to be
identified under these assay conditions. PS-1 cells cultured in either
vehicle control or 10 nM DHT or for 1–7 days in medium
M0 produced identical nuclear AR patterns (Fig. 1D).
Identical patterns were observed using either the PG-21 or U402 anti-AR
antibodies for immunocytochemistry as described in Materials and
Methods. These data indicate that androgens are not required in
defined M0 medium for AR to localize to the nucleus in PS-1
stromal cells. PS-1 cultures in M0 serum-free medium
supplemented with FGF-2 (5 ng/ml), EGF (10 ng/ml), or TGFß2 (10–200
pM) also exhibited nuclear AR staining patterns identical
to those of cultures in the unsupplemented basal medium alone (see
Table 1; localization pattern identical to that in Fig. 1C). Nuclear
localization patterns were also ligand independent for each growth
factor tested (Table 1). In contrast, PS-1 cells cultured in basal
M0 medium supplemented with TGFß1 (5–100 pM
for 24 h) exhibited a cytoplasmic distribution pattern of AR as
shown in Fig. 1E (25-pM TGFß1 concentration shown), also
independent of ligand concentration (0–1 µM DHT; Fig. 1F). Nuclear AR was not detectable in any cultures containing TGFß1.
TGFß1 at 5 pM produced a near-complete cytoplasmic
distribution of AR, and all higher concentrations up to 100
pM produced a complete redistribution of AR. The
cytoplasmic localization pattern of AR induced by TGFß1 was distinct
from that observed in serum-containing medium. The TGFß1-induced
pattern exhibited a considerably more filamentous pattern of
localization (Fig. 1, E compared with A and B).
To address the specificity of the TGFß1 response, a pan-specific
neutralizing antibody to TGFß1 (R&D Systems) was used. PS-1 cultures
that received both TGFß1 (25 pM) and neutralizing
antibody (10 µg/ml) exhibited a nuclear localization of AR, as shown
in Fig. 1G (compare with Fig. 1, E and F). Control cultures receiving
antibody alone exhibited no effect (data not shown). To assess whether
the cytoplasmic AR localization previously observed in serum-containing
medium might be attributed to TGFß1 activity in serum, TGFß1
neutralizing antibody was added to serum-containing cultures. Under
these conditions, a cytoplasmic localization pattern was retained
regardless of the antibody concentration (10–50 µg/ml; data not
shown). These results suggest that serum-induced cytoplasmic
localization of AR is not due to TGFß1 activity and that other serum
components, in addition to TGFß1, act to influence the cellular
distribution of AR in PS-1 cell cultures.
To determine whether the TGFß1-induced effects on AR distribution are
directly downstream of a signaling event or are mediated through newly
synthesized intermediary proteins, PS-1 cells were cultured with or
without TGFß1 in medium containing cyclohexamide. The addition of
cyclohexamide (10 µg/ml) in the absence of TGFß1 did not alter the
nuclear localization of AR observed in control cultures (data not
shown). The addition of cyclohexamide (10 µg/ml) and TGFß1 (25
pM) together in either the presence or absence of androgen
(DHT, 10 nM) resulted in the usual cytoplasmic localization
pattern, as shown in Fig. 1H. Cyclohexamide treatment under these
conditions produced 95.3% and 94.2% decreases in
[35S]methionine incorporation in cell lysate proteins in
PS-1 cultures (treated for 1 and 6 h, respectively), indicating a
nearly complete absence of new protein synthesis. These results
indicate that TGFß1-induced mechanisms for localizing AR from the
nucleus to cytoplasm in PS-1 cells is independent of new protein
synthesis.
To assess the time course of the TGFß1-induced shift of nuclear to
cytoplasmic AR, a series of time points was examined after the addition
of TGFß1 (25 pM). An increase in cytoplasmic AR staining
intensity was apparent after 1 h of incubation with TGFß1, as
shown in Fig. 2A. More extensive
filamentous cytoplasmic staining pattern and staining intensity were
apparent at 3 h (shown in Fig. 2B). The majority of AR was
cytoplasmic at the 6 h point (Fig. 2C), and the typical
cytoplasmic AR distribution pattern, with nuclei essentially void of
AR, was observed after 24 h (Fig. 2D). At 24 h, medium was
changed to basal conditions (medium M0) without TGFß1 to
determine reversibility. Within 1 h after the withdrawal of
TGFß1, a nuclear staining pattern of AR was again apparent (Fig. 2E),
and redistribution of AR to the nucleus was complete by 6 h (Fig. 2F). Identical time-course patterns and reversibility were observed in
medium without androgen or in medium containing cyclohexamide (10
µg/ml; data not shown).
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shows a
representative growth-response curve for PS-1 cells stimulated by 10
nM DHT compared with that for cells treated with a
steroid-free vehicle control, TGFß1, or TGFß1 plus DHT over a 6-day
course. DHT (10 nM) acted to significantly
(p < 0.01) stimulate the proliferation of PS-1
cells as reported previously (20). The addition of TGFß1 (25
pM) together with DHT (10 nM) produced a
consistent (n = 3 separate experiments) and significant inhibition
(p < 0.01) of cell proliferation relative to
that of DHT only (10 nM) cultures. No consistent
significant differences were noted between the control (minus androgen)
and TGFß1 plus DHT cultures. In addition, TGFß1 in the absence of
androgens did not alter the proliferation rate of PS-1 cells. These
data suggest that TGFß1-induced inhibition of PS-1 cells is mediated
through a block of androgen response in defined medium conditions.
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, and rogen-induced ddp17 expression (lane 2) was inhibited by TGFß1 (25
pM) to basal levels (lanes 1 and 4). TGFß1 alone did not
alter basal expression of ddp17 in androgen-free medium (lanes 1 and
3). To address the possibility that TGFß1 may induce a change in AR
protein levels concurrent with a redistribution of AR, Western analysis
of AR in total cell lysates from each of the experimental conditions
was examined. No changes in AR content, as determined by Western
analysis at two different protein concentrations (n = 3
experiments), was detectable under any of the experimental conditions
(data not shown). These data are consistent with the staining intensity
observed with immunocytochemistry, which suggested no overall
alteration in AR protein levels.
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, primary cultures of rat
ventral prostate stromal cells in defined medium M0 plus
DHT (10 nM) showed a TGFß1 (25 pM)-induced
cytoplasmic distribution of AR nearly identical to the altered
distribution observed in the PS-1 cell line. In contrast to either PS-1
cells or primary stromal cultures, LNCaP cells cultured with TGFß1
(25 pM) and androgen (10 nM or 1
µM) maintained a nuclear distribution of AR with no
apparent cytoplasmic redistribution of AR (data not shown). Also
different from PS-1 cells, LNCaP cells cultured in defined medium
(medium M0) without androgen exhibited a cytoplasmic
distribution of AR. Supplementation of medium with androgen (10
nM or 1 µM) resulted in a nuclear
localization of AR. Similarly, DDT1MF-2 cells exhibited a nuclear AR
distribution in medium (M0) supplemented with androgen (10
nM or 1 µM), which did not redistribute to
the cytoplasm in response to TGFß1 (25 pM). However, when
DDT1MF-2 cells were tested in medium containing cyclohexamide (10
µg/ml), a diminished nuclear staining was apparent, with an increase
in cytoplasmic staining in the presence of TGFß1 (data not shown).
This shift in AR distribution was not to the extent observed in PS-1
cells or primary stromal cell cultures. In contrast, no alterations in
AR distribution were noted when LNCaP cells were cultured with
cyclohexamide and TGFß1. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As only cell proliferation and the expression of ddp17 marker has been examined here, and little else is known about the androgen response in PS-1 cells, it is possible that not all effects of androgen are modulated by TGFß1. In addition, the observation that TGFß1 alters the distribution of AR in primary cultures of rat prostate stromal cells indicates that this response is not simply a unique feature of the PS-1 cell line. Rather, these results suggest that this response to TGFß1 is probably a central feature of prostate stromal cells in general. The cell type lineage of the PS-1 cell line or the primary stromal culture outgrowths is not known; however, all of the primary culture cells were AR positive. These data suggest the possibility of a selection pressure in the androgen-containing Bfs medium in selecting an AR-positive stromal cell outgrowth. Due to the lack of additional cell lines that are both androgen responsive and TGFß1 responsive, it is difficult to comment further on the cell type specificity of this response. The full extent to which TGFß1 regulates androgen action in vitro or in vivo is not yet known. Accordingly, the PS-1 stromal cell line will be beneficial to future studies to determine mechanisms of steroid receptor shuttling and translocation due to the regulatable nature of AR distribution within this cell. Additionally, this cell model system may aid in identifying specific cross-talk mechanisms between TGFß1 and androgen signal transduction.
Expression patterns and functional studies suggest that TGFß1 plays a major role in prostate gland development and cancer progression. However, the specific mechanisms of action, primary target cell types, and overall functional significance of TGFß1 action in the prostate are not known. In the rat coagulating gland (anterior prostate gland) morphogenesis model, TGFß1 reportedly inhibited the development of glandular differentiation (8, 13), a process fully dependent on androgen signaling in mesenchyme/stromal cells (8). Furthermore, TGFß1 acted to inhibit androgen-induced penile growth during development without reducing AR content in the tissue (25). The data presented here suggest that the development inhibitory action of TGFß1 in androgen-responsive tissues may be mediated through altered AR distribution and androgen response.
The role of elevated TGFß1 expression by prostate carcinoma cells (17, 18, 19) is also not known. In prostate cancer, TGFß receptor type II is maintained in stromal cells (19), whereas TGFß receptors are down-regulated in epithelial carcinoma cells (19, 26, 27, 28). Hence, TGFß1 action may be mediated through the stromal compartment of cells in prostate cancer progression. TGFß1 may regulate stromal cells to provide a stromal environment more supportive of carcinoma proliferation and invasion. A TGFß1-induced change in stromal cell response to androgen may be a central component of this process.
Specific cross-talk mechanisms of TGFß1-induced changes in AR distribution are not yet known. Cyclohexamide experiments indicate that new protein synthesis is not required, suggesting a direct effect on signal transduction events. Changes in AR phosphorylation status as a consequence of TGFß1 signaling may play a role. It has been well documented that steroid receptor phosphorylation alters transcriptional activity (29, 30, 31, 32, 33). Although no known phosphorylation sites in AR have been shown to influence nuclear localization or shuttling of receptor from nucleus to cytoplasm, several sites with unknown function have not yet been examined for directing receptor distribution (29, 32). It is possible that TGFß1 acts to alter the balance of constitutive shuttling of AR, where the nuclear export rate is more rapid than the import rate. Nuclear export of steroid hormone receptors is believed to be constitutive, although may vary between cell types (34, 35). TGFß1 may also regulate either a nuclear export factor or a cytoplasmic anchoring factor, such as heat shock protein 56 (hsp56). With the glucocorticoid receptor, cytoplasmic colocalization with microtubules is mediated through hsp56 (36, 37), and hsp56 is required for nuclear shuttling (38). The cytoplasmic pattern of AR staining induced by TGFß1 exhibited a filamentous network similar to a microtubule network observed with the glucocorticoid receptor. This concept is further supported by the fact that hsp56 has been reported in the AR-hsp complex (33, 39).
Of interest, the nuclear localization of AR in serum-free medium was not dependent on androgen with PS-1 cells. Nuclear localization of steroid receptors in the absence of hormone appears to be tissue and receptor type specific. Both estrogen and progesterone receptors typically show a nuclear distribution regardless of hormone (40, 41). In contrast, glucocorticoid receptors exhibit both nuclear and cytoplasmic distributions in the absence of hormone (42). The distribution pattern of AR in response to androgen is reported to be tissue and cell type specific in vivo. The lateral lobe of the rat prostate exhibited a nuclear AR pattern in both intact and castrate animals, whereas the ventral and dorsal lobes showed a gradual switch from nuclear to cytoplasmic with castration, the rate of which was region dependent, with the distal tips exhibiting a slower loss of nuclear AR (43, 44, 45). Regardless of the nuclear or cytoplasmic distribution pattern, all prostate tissue exhibited the typical castration-induced involution (43, 44). It has been suggested by Wilson and colleagues (43) that androgen serves to anchor or make more stable the interaction of AR with nuclear elements in some cell types and is permissive to nuclear transport, if not an absolute requirement. Together these studies indicate that the effect of androgen withdrawal on the distribution of AR in vivo is complex and probably cell type specific and tissue specific, with no clear-cut dogma on the effects of androgen on AR distribution being universally applicable to all androgen-responsive cell types.
Our data suggest that the possible anchoring of AR in the nucleus of PS-1 cells is destabilized by TGFß1 signaling as well as other unknown serum components. The effect of serum to induce a cytoplasmic distribution pattern of steroid receptors has been observed in other cell systems. Both human and rat vascular smooth muscle cultures exhibited a cytoplasmic localization of estrogen receptor when cultured in medium containing CSS (46, 47). Many model cell systems have reportedly required serum-free medium to exhibit a steroid response. Additionally, the recently reported DuK50 prostate stromal cell line was androgen insensitive in high serum-containing medium, yet exhibited androgen-stimulated proliferation in low serum medium (48). The data reported here may help to explain the lack of steroid hormone response generally observed with cells cultured in serum-containing medium, in contrast to specific responses generated in serum-free conditions.
TGFß1 did not affect AR distribution patterns in LNCaP carcinoma
cells. The proliferation of LNCaP human prostatic carcinoma cells is
stimulated by androgens; however, LNCaP cells express a mutated AR at
higher levels than normal prostate gland (49, 50). The responsivity of
LNCaP cells to TGFß1 and the role of androgens in a putative TGFß1
response are complex and not well understood. The majority of reports
have shown that TGFß1 does not affect LNCaP cells (51, 52, 53, 54) that lack
TGFß receptor type I (51) and lack high affinity binding sites to
TGFß1 (53). In contrast, two other reports have shown that LNCaP
cells are growth inhibited by TGFß1 only in the presence of specific
concentrations of androgen (55) and in a dose-dependent manner (56).
Moreover, TGFß1 has been shown to block EGF- and TGF-induced
proliferation of LNCaP cells (57, 58) and to increase prostatic acid
phophatase and AR levels (57). Hence, it is not clear whether the
inability of TGFß1 to affect AR distribution in LNCaP cells results
from altered TGFß1 signaling, mutations in AR, or other alterations
in AR shuttling mechanisms.
TGFß1 did stimulate DDT1MF-2 cells to redistribute some AR immunoreactivity from the nucleus to the cytoplasm, however only in the presence of cyclohexamide. These data suggest that maintained nuclear AR in TGFß1-treated DDT1MF-2 cells probably resulted from new AR synthesis. The DDT1MF-2 stromal cell line was derived from a hamster ductus deferens leiomyosarcoma, is AR positive, and exhibits androgen-stimulated proliferation as well as androgen-stimulated FGF-1 expression (59, 60, 61). In contrast to DDT1MF-2 cells, PS-1 cells exhibited a major loss of nuclear AR, and a cytoplasmic redistribution regardless of whether cyclohexamide was present. One possible explanation for these data is that PS-1 cells may have a lower AR degradation rate and/or a lower AR synthesis rate relative to DDT1MF-2 cells.
In evaluation of these data it should be kept in mind that of the three cell lines and primary cultures tested in the present study, only the PS-1 stromal cell line and primary stromal cell cultures were derived from normal (noncancerous) tissue (20). In addition, it is clear that TGFß1 generally induces apoptosis in epithelial cells, whereas TGFß1 is growth stimulatory to many stromal cell lines. Moreover, the differences in TGFß receptor types and content between these different cell lines are not known. How the differential TGFß1 responses in AR distribution relate to fundamental biological differences in cancer vs. noncancer cells or in epithelial cells vs. stromal cells is not yet clear.
The PS-1 cell culture model system will be useful for studies on TGFß1 and androgen mechanisms to better understand androgen action in the prostate smooth muscle cell type. Future studies can be directed to determine specific cross-talk mechanisms, cell type specificity, physiological relevance to prostate morphogenesis, and the stromal cell response in prostate cancer progression.
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Footnotes |
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Received December 29, 1997.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 S. C. Sharma, J. W. Clemens, M. D. Pisarska, and J. S. Richards Expression and Function of Estrogen Receptor Subtypes in Granulosa Cells: Regulation by Estradiol and Forskolin Endocrinology, September 1, 1999; 140(9): 4320 - 4334. [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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