Endocrinology Vol. 139, No. 4 2111-2119
Copyright © 1998 by The Endocrine Society
Clathrin Gene Expression Is Androgen Regulated in the Prostate1
James L. Prescott2 and
Donald J. Tindall
Departments of Biochemistry and Molecular Biology (J.L.P., D.L.T.)
and Urology Research (D.L.T.), Mayo Foundation, Rochester, Minnesota
55905
Address all correspondence and requests for reprints to: Dr. D. J. Tindall, Department of Urology Research, Mayo Foundation, 200 First Street SW, Rochester, Minnesota 55905. E-mail: tindall{at}mayo.edu
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Abstract
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Androgens are required for the development and function of the
prostate. In a normal human prostate, androgens control the synthesis
of proteins such as prostate-specific antigen and human glandular
kallikrein. The prostate secretes these proteins as well as a number of
other compounds to form the prostatic fluid. Using differential display
PCR to detect novel androgen-regulated genes, clathrin heavy chain
expression was identified as potentially being up-regulated by
androgens in the prostate cancer cell line LNCaP. We report here that
the clathrin heavy chain and light chain genes are regulated by
androgens. Clathrin heavy chain messenger RNA was up-regulated by
androgens in a concentration- and time-specific manner in the LNCaP
cell line. Translation of clathrin heavy chain messenger RNA was
stimulated by androgens. Steady state levels of clathrin light chains a
and b were up-regulated in the presence of androgen in LNCaP cells.
Clathrin gene expression was examined in normal rat prostates, and
similar results were found. Clathrin heavy chain protein levels in the
rat prostate are also affected by the androgen status of the animal. We
hypothesize that clathrin may be involved in the exocytosis of
androgen-regulated secretory proteins such as prostate-specific antigen
and human glandular kallikrein.
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Introduction
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THE PROSTATE is an exocrine sex gland found
in all mammals. In humans, the prostate is located at the base of the
bladder and surrounds the urethra. The function of the prostate is to
secrete chemicals and compounds that constitute approximately 15% of
the volume of the semen. The development and function of the prostate
are strictly dependent on androgens. A lack of androgens or defects in
androgen metabolism during development will result in a lack of
prostatic development. In the adult, surgical or chemical castration
will result in a loss of secretory activity and eventually an
involution of the prostate and loss of prostatic mass. Addition of
supplemental androgens will recover prostatic size and function.
The functional activities of the prostate include secretion, transport,
and reabsorption. Proteins such as prostate-specific antigen (PSA)
and human glandular kallikrein (hK2) are synthesized and secreted in
the prostatic epithelium. PSA and hK2 expression are both under the
control of androgens. A number of small molecules and ions are
transported across the epithelial acinar cell and concentrated in the
prostatic fluid (for review, see Ref.1). Sodium and chloride are able
to be reabsorbed by the prostatic epithelium under basal
(i.e. nonejaculating) conditions (1). During ejaculation,
there is a rapid increase in the volume of prostatic fluid as well as
an increase in the protein content of the fluid, indicating that there
is an increased rate of secretion.
One of the most important proteins known to be involved in the
secretion and transport of vesicles is clathrin heavy chain. Clathrin
heavy chain forms the backbone of the protein basket surrounding the
formation of some types of secretory vesicles in the cell (2). Clathrin
heavy chain has also been reported to be involved in the transport of
vesicles from the rough endoplasmic reticulum to the Golgi network (3)
and from the Golgi network to the plasma membrane (2). Associated with
clathrin heavy chain during vesicle formation are the clathrin light
chains a and b (4).
We report here that the clathrin heavy chain and the clathrin light
chain a and b genes are regulated by androgens. This regulation is seen
in both in vivo and in vitro models. We
hypothesize that the androgen regulation of clathrin gene expression is
part of the androgen control of secretion in the prostate.
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Materials and Methods
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Cell culture and steroid treatment
The prostate cancer cell line LNCaP was acquired from the
American Type Culture Collection (Rockville, MD). Cells were grown in
RPMI 1640 medium (Celox Corp., Hopkins, MN) supplemented with 9% FBS,
10 U/ml penicillin, 10 µg/ml streptomycin, and 0.25 µg/ml
fungizone. Cells were split 1:5 every 7 days. Experiments were
performed on cells between passages 25–40. Cells were placed in medium
containing 9% charcoal-stripped FBS for 72 h before treatment
with steroids. Steroids were dissolved in 100% ethanol at a 1000-fold
final experimental concentration. The final ethanol concentration added
to the cells (both experimental and controls) was 0.1%. After
treatment, LNCaP cells were quick-frozen at -70 C until
processing.
Animals
Rats were purchased from Harlan Sprague-Dawley at approximately
10 weeks of age (200–225 g). Animals were housed and treated in
accordance with all applicable institutional and national regulations.
Animals were divided into four groups: sham operated, castrated,
castrated and supplemented with 0.5 mg/kg testosterone propionate, and
castrated and supplemented with 0.5 mg/kg testosterone propionate and
20 mg/kg flutamide. Animals were treated for 48 h. Subcutaneous
injections were made immediately after surgery and 24 and 48 h
later with 1 ml corn oil with or without the above reagents.
Dorsolateral and ventral prostates were harvested and frozen in liquid
nitrogen or fixed in neutral buffered formalin (for
immunohistochemistry), and the RNA was prepared as described below.
RNA isolation
RNA was isolated by Trizol reagent (Life Technologies, Grand
Island, NY) according to the manufacturer’s directions. Briefly, 0.75
ml Trizol was added to a 60-mm dish of LNCaP cells, and the dish was
scraped to release the cells. The mixture was pipetted to mix,
transferred to a 1.5-ml microfuge tube, and allowed to sit for 5 min at
room temperature. Chloroform (0.25 ml) was added, mixed, and allowed to
incubate at room temperature for 2 min. The mixture was centrifuged at
12,000 x g for 15 min, and the supernatant was
transferred to a fresh tube. One milliliter of isopropanol was added
and mixed, and the solution was centrifuged at 12,000 x
g for 15 min. For the rat prostates, one prostatic lobe was
homogenized in 3 ml Trizol using a Dounce homogenizer (Kontes Co.,
Vineland, NJ). The remaining volumes were adjusted accordingly, and the
RNA was purified as above. The pellet was washed once with 70%
ethanol, resuspended in H2O, and quantitated. The RNA was
ethanol precipitated, pelleted, resuspended in H2O, and
stored at -100 C.
Differential display PCR (DD-PCR)
LNCaP cells were treated with 1 nM mibolerone for 0,
4, 8, or 16 h, and the RNA was isolated. DD-PCR was performed as
described previously (5). Briefly, equal amounts of RNA were reverse
transcribed using Moloney murine leukemia virus reverse transcriptase
(Life Technologies) and deoxythymidine11 (dT11)
MN oligos (where M is A, C, or G, and N is A, C, G, or T). The
complementary DNA (cDNA) was used immediately as template for the PCR.
The PCR mix included the identical poly(dT) primer, a random 10-mer,
and [35S]deoxy-ATP. Clathrin heavy chain was isolated
using the dT primer T11 MC and random primer A
(5'-CTGATCCATG-3'). The reaction was cycled 40 times at 94 C for 30
sec, at 40 C for 2 min, and at 72 C for 30 sec (5). After PCR, the
samples were diluted in stop solution and loaded on a 6% acrylamide
sequencing gel. The gel was electrophoresed for 4 h at 80 watts.
The gel was fixed in 5% methanol-5% acetic acid for 30 min and vacuum
dried. The gel was exposed to Kodak X-AR film (Eastman Kodak,
Rochester, NY) overnight at room temperature. The developed film was
aligned with the dried gel, and the band of interest was excised. The
DNA was isolated by electroelution in a Hoefer Six-Pac electroeluter
(Hoefer, San Francisco, CA) and concentrated by ethanol precipitation.
The pelleted cDNA was used as template in the PCR as described above.
The DNA was amplified twice, and the reaction was electrophoresed on a
1% agarose gel. The DNA was isolated from the gel by GeneClean (BIO
101, Vista, CA) and cloned using the T/A Cloning Kit (Invitrogen, San
Diego, CA). Cloned DNA was sequenced using the fmol cycle sequencing
kit (Promega, Madison, WI) and fluorescent automated sequencing
(Applied Biosystems, Foster City, CA).
DNA isolation and quantitation
DNA isolation was performed using the Trizol reagent and the
manufacturer’s recommended procedure. Briefly, after RNA extraction,
the phenol phase was precipitated with an equal volume of 100%
isopropanol and incubated, and the DNA was pelleted by centrifugation.
The pelleted was washed with 75% ethanol twice and resuspended in 300
µl H2O. The DNA was quantitated using Hoescht 33258
fluorescent dye (Sigma Chemical Co., St. Louis, MO) under the
conditions outlined in Current Protocols in Molecular
Biology (5a).
Northern blotting
The RNA was fractionated by electrophoresis in a 1% agarose-2%
formaldehyde denaturing gel in MOPS buffer (20 mM MOPS, pH
7.0; 5 mM sodium acetate; and 1 mM EDTA) for
1.5 h at a constant 100 V. The RNA was transferred to a Hybond
N+ membrane (Amersham, Arlington Heights, IL) by capillary
transfer overnight, fixed to the membrane by UV cross-linking, and
prehybridized at 42 C for at least 1 h in hybridization solution
[10 mM phosphate, 45% formamide, 5 x Denhardt’s
solution, 5 x SSC (standard saline citrate), 0.5% SDS, 50 mg/ml
dextran sulfate, and 100 µg/ml herring sperm DNA]. A clathrin heavy
chain cDNA was provided by Dr. R. Iozzo, Thomas Jefferson University
(Philadelphia, PA) (7). Probes for clathrin light chains a and b were
provided by Dr. A. Jackson, Cambridge University (Cambridge, MA) (6).
DNA probes were labeled by the random primed Klenow technique and
purified over a 1-ml Sephadex G-50 column. Purified probes were
denatured by heating in a boiling water bath for 3 min and added to the
membrane. Hybridization was allowed to proceed overnight at 42 C in a
shaking water bath. Membranes were washed to a final stringency of
1 x SSC-0.1% SDS at 42 C. Membranes were air-dried and exposed
to a PhosphorImager cassette (Molecular Dynamics, Sunnyvale, CA) for
quantitation.
Cell labeling
LNCaP cells were treated as described above. For the last 6
h of androgen treatment, 0.5 mCi [35S]methionine was
added to the culture medium (4 ml in a 60-mm dish). After treatment,
the cells were quick-frozen and stored at -70 C. Total cellular
protein was extracted by resuspending the cells in 0.8 ml RIPA buffer
(PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 0.1 mg/ml
phenylmethylsulfonylfluoride, 1 mM sodium orthovanadate,
and 2 µg/ml aprotinin), aspirating the cells through a 21-gauge
needle six times, and transferring them to a 1.5-ml microfuge tube. The
plate was rinsed with 0.4 ml RIPA; the solution was aspirated and added
to the previous volume. The solution was incubated for 60 min on ice
and microfuged at 15,000 x g for 20 min at 4 C. The
supernatant was transferred to a fresh tube, and the pellet was
discarded. The protein concentration was determined using the DC
Protein Assay Kit (Bio-Rad, Richmond, CA) and BSA for the standard
curve.
Immunoprecipitation
Fifteen micrograms of total LNCaP protein extract were incubated
with goat anticlathrin heavy chain antiserum (Sigma Chemical Co., St.
Louis, MO) at a 1:1000 final dilution in a final volume of 100 µl for
2 h at room temperature on a rotator platform. Fifteen microliters
of protein G-agarose (Boehringer Mannheim, Indianapolis, IN) were added
and allowed to incubate for 2 h at room temperature on a rotator
platform. The agarose was pelleted by brief centrifugation and washed
with 200 µl RIPA buffer, followed by three washes with 200 µl TBST
(10 mM Tris-HCl, pH 8; 150 mM NaCl; and 0.5%
Tween-20). The remaining supernatant was removed, and the pellets were
vacuum-dried. The pellets were resuspended in 15 µl SDS sample buffer
(250 mM Tris HCl, pH 6.8; 20% glycerol; 2.5% SDS; and 5%
2-mercaptoethanol), denatured by heating at 100 C for 5 min, and loaded
on a precast 4–20% Tris-glycine acrylamide minigels (Novex, San
Diego, CA). The gels were run for 2 h at 110 V and then
vacuum-dried to 3MM chromatography paper (Whatman, Clifton, NJ). The
dried gels were exposed to a PhosphorImager cassette for
quantitation.
Quantitation
Blots were exposed to a PhosphorImager model 425 (Molecular
Dynamics) overnight and scanned at a resolution of 88 µm (288 dpi),
and the band intensity was determined using ImageQuant software
(Molecular Dynamics). Gene expression was standardized to that of
glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Fold changes were
calculated relative to ethanol-treated control samples. In the rat
prostate experiments, gene expression was standardized relative to DNA,
and fold changes were calculated relative to the sham-operated animals.
All calculations were performed on a Power Macintosh 8100/80 or 7100/80
(Apple Computer, Cupertino, CA) using Excel 5.0 (Microsoft, Redmond,
WA) and graphed using C/A Cricket Graph III (Computer Associates
International, Malvern, PA). Each experiment was performed using a
minimum of four separate samples. Statistical analysis was performed
using the Analysis Toolpak in Excel 5.0 (Microsoft). Single factor
ANOVA was used to determine the statistical significance of the
results.
Immunohistochemistry
Formalin-fixed rat prostates were paraffin embedded, and 5-µm
sections were taken. Slides were deparaffinized in 2 5-min zylene
incubations, followed by ten dips in absolute ethanol and 10 dips in
95% ethanol, repeated once. Any endogenous peroxidase activity was
blocked with a 10-min room temperature incubation in 1.5%
H2O2-50% methanol. Slides were pretreated with
citrate buffer (10 mM sodium citrate, pH 6, for 30 min in
steam). Nonspecific binding was blocked using 5% normal horse serum in
PBS-0.05% Tween-20 for 10 min at room temperature. Representative
slides were removed for illustration of nonspecific staining.
Anticlathrin heavy chain antiserum (Sigma Immunochemical) was diluted
1:750 in PBS-0.05% Tween-20–1% normal horse serum and incubated on
the slides for 30 min at room temperature. Slides were rinsed twice
with water. Biotinylated secondary antibody was diluted 1:300 in
PBS-0.05% Tween-20–1% normal horse serum and incubated on all slides
for 30 min at room temperature. Slides were rinsed in water followed by
PBS-0.05% Tween-20. Slides were then incubated with
peroxidase-conjugated strepavidin in PBS-0.05% Tween-20–1% normal
horse serum for 30 min at room temperature, followed by a water rinse.
Color was developed by incubating in 3-amino-9-ethylcarbazole (0.2
mg/ml) in 5% N,N'-dimethylformamide-0.03%
H2O2 in 10 mM sodium acetate, pH
5.2, for 15 min at room temperature. A hematoxylin counterstain was
applied.
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Results
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Clathrin heavy chain was initially detected as an up-regulated
cDNA during a screen for androgen-regulated genes in LNCaP cells. This
DD-PCR fragment was 127 bp in length and showed homology to the rat
clathrin heavy chain. This fragment was used as a probe to screen a
prostate cDNA library (5'-STRETCH, Clontech, Palo Alto, CA) and yielded
a 1.1-kilobase clone with greater than 95% homology to the rat
clathrin heavy chain messenger RNA (mRNA). The high A+T content of this
clone made it impractical for use as a probe in examining the androgen
regulation of clathrin heavy chain; the human clathrin cDNA used here
was provided by Dr. R. Iozzo, Thomas Jefferson University (7).
The regulation of clathrin heavy chain expression by androgens was
examined by Northern blot of RNA isolated from LNCaP (8) cells treated
with 1 nM of the synthetic androgen mibolerone (9) for
various times (Fig. 1
). There was a
brief, rapid, 2-fold up-regulation within 8 h, followed by a more
gradual increase for the remaining period examined. In 72 h, there
was a greater than 5-fold increase in clathrin heavy chain steady state
mRNA levels. This biphasic response was reproducible over several
experiments. Clathrin heavy chain mRNA levels were also sensitive to
the concentration of mibolerone (Fig. 2).
LNCaP cells were treated with varying concentrations of mibolerone for
48 h, and the RNA was analyzed by Northern blot. A very low
concentration of mibolerone (0.01 nM) showed almost no
induction of clathrin heavy chain mRNA. This concentration is below the
Kd of mibolerone for the androgen receptor (
0.1
nM) (9). However, 0.1 nM mibolerone induced a
significant (P < 0.01) increase in clathrin heavy
chain mRNA. Saturating amounts of mibolerone (1 nM or more)
also produced significant (P < 0.01) inductions of
expression. We conclude from these experiments that the steady state
level of clathrin heavy chain mRNA expression is induced by androgens
in a time- and concentration-dependent manner.
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Figure 1. Time course of androgen induction of clathrin
heavy chain mRNA in LNCaP cells. Representative samples of androgen
induction of clathrin heavy chain mRNA for the indicated times (A) and
the quantitation of that data (B). LNCaP cells were treated with 1
nM mibolerone for the indicated times, and total RNA was
isolated, Northern blotted, and probed with clathrin heavy chain and
GAPDH. Clathrin heavy chain expression was standardized to GAPDH
expression, and the fold induction relative to the ethanol-treated
control value was calculated. Each point is the
mean ± SE of five plates. *, P <
0.01, by ANOVA (one-tailed).
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Figure 2. Dose response of androgen induction of clathrin
heavy chain mRNA in LNCaP cells. Representative samples of androgen
induction of clathrin heavy chain mRNA for the indicated mibolerone
concentrations (A) and the quantitation of that data (B). LNCaP cells
were treated with the indicated concentrations of mibolerone for
48 h, and total RNA was isolated, Northern blotted, and probed
with clathrin heavy chain and GAPDH. Clathrin heavy chain expression
was standardized to GAPDH expression, and the fold induction relative
to the ethanol-treated control value was calculated. Each
point is the mean ± SE of five plates.
*, P < 0.001, by ANOVA (one-tailed).
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We also examined the androgen regulation of clathrin light chains a and
b. The probes for clathrin light chains a and b were provided by Dr. A.
Jackson, Cambridge University (6). Clathrin light chain a and b mRNAs
also responded to androgens (Fig. 3 and 4), although the time course differed
from that seen with clathrin heavy chain. The androgen induction of
both genes was very similar. Neither light chains had a statistically
significant increase until at least 24 h after mibolerone
treatment. The magnitude of the induction was also lower than that seen
with the heavy chain, only about 2-fold as opposed to the 5-fold heavy
chain induction.
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Figure 3. Time course of androgen induction of clathrin
light chain a mRNA in LNCaP cells. Representative samples of androgen
induction of clathrin light chain a mRNA for the indicated times (A)
and the quantitation of that data (B). LNCaP cells were treated with 1
nM mibolerone for the indicated times, and total RNA was
isolated, Northern blotted, and probed with clathrin light chain a and
GAPDH. Clathrin light chain a expression was standardized to GAPDH
expression, and the fold induction relative to the ethanol-treated
control value was calculated. Each point is the
mean ± SE of five plates. *, P <
0.01, by ANOVA (one-tailed).
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Figure 4. Time course of androgen induction of clathrin
light chain b mRNA in LNCaP cells. Representative samples of androgen
induction of clathrin light chain b mRNA for the indicated times (A)
and the quantitation of that data (B). LNCaP cells were treated with 1
nM mibolerone for the indicated times, and total RNA was
isolated, Northern blotted, and probed with clathrin light chain b and
GAPDH. Clathrin light chain b expression was standardized to GAPDH
expression, and the fold induction relative to the ethanol-treated
control value was calculated. Each point is the
mean ± SE of five plates. *, P <
0.01, by ANOVA (one-tailed).
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The sensitivity of the clathrin light chain genes to varying mibolerone
concentrations was also examined. Both light chains had similar
responses to varying concentrations of mibolerone (Figs. 5 and 6).
The light chains required a slightly higher mibolerone concentration
than the clathrin heavy chain. Subsaturating concentrations (<1
nM) of mibolerone did not elicit any detectable change in
the light chain mRNAs. Concentrations of 1 nM or more
produced similar and significant increases in mRNA levels. We conclude
from these experiments that clathrin light chain a and b mRNAs are also
regulated by androgens in a time- and concentration-specific
manner.
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Figure 5. Dose response of androgen induction of clathrin
light chain a mRNA in LNCaP cells. Representative samples of androgen
induction of clathrin light chain a mRNA for the indicated mibolerone
concentrations (A) and the quantitation of that data (B). LNCaP cells
were treated with the indicated concentrations of mibolerone for
48 h, and total RNA was isolated, Northern blotted, and probed
with clathrin light chain a and GAPDH. Clathrin light chain a
expression was standardized to GAPDH expression, and the fold induction
relative to the ethanol-treated control value was calculated. Each
point is the mean ± SE of five plates.
*, P < 0.001, by ANOVA (one-tailed).
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Figure 6. Dose response of androgen induction of clathrin
light chain b mRNA in LNCaP cells. Representative samples of androgen
induction of clathrin light chain b mRNA for the indicated mibolerone
concentrations (A) and the quantitation of that data (B). LNCaP cells
were treated with the indicated concentrations of mibolerone for
48 h, and total RNA was isolated, Northern blotted, and probed
with clathrin light chain b and GAPDH. Clathrin light chain b
expression was standardized to GAPDH expression, and the fold induction
relative to the ethanol-treated control value was calculated. Each
point is the mean ± SE of five plates.
*, P < 0.001, by ANOVA (one-tailed).
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We next examined whether the mibolerone-induced increase in clathrin
heavy chain mRNA could be detected at the translational level. LNCaP
cells were treated with 1 nM mibolerone for varying times.
For the last 6 h of treatment, the cells were incubated with
[35S]methionine. Total protein was extracted from the
cells, and clathrin heavy chain was immunoprecipitated using a
commercially available goat anticlathrin heavy chain antiserum (Sigma
Chemical Co.). After SDS-PAGE, the radiolabeled clathrin heavy chain
band was quantitated. There was a time-dependent increase in the amount
of new clathrin heavy chain being translated (Fig. 7). This change in the rate of
translation was similar to the increase in clathrin heavy chain mRNA
seen with similar mibolerone treatment (Fig. 1
). We have concluded from
this experiment that androgens also increase the amount of new clathrin
heavy chain being synthesized.
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Figure 7. Effects of androgens on the translation of
clathrin heavy chain. LNCaP cells were treated with 1 nM
mibolerone for the indicated times; the cells were labeled for the last
6 h with [35S]methionine. Total protein was purified
as described (Materials and Methods),
immunoprecipitated, and subjected to denaturing SDS-PAGE. The labeled
clathrin heavy chain protein was quantitated by PhosphorImager, and the
fold increase was calculated. Each point is the
mean ± SE of nine (three separate experiments, each
assayed three times). *, P < 0.02.
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To determine whether androgen regulation of clathrin gene expression is
physiologically significant, we used the rat prostate as an in
vivo model. Ten- to 12-week-old male rats were subjected to
castration, castration and immediate supplementation with 0.5 mg/rat
testosterone propionate, castration and immediate supplementation with
0.5 mg/rat testosterone propionate and 20 mg/kg flutamide, or sham
operation. Rats were injected daily with 1 ml corn oil containing the
above compounds for 3 days. At the time of death, blood was taken, and
serum testosterone levels were determined using a clinical serum
testosterone assay. The intact animals had a mean level of 108 ng/dl.
The castrated animals, as expected, had serum levels below the
detectability of the assay. The testosterone propionate-supplemented
and the testosterone propionate- plus flutamide-supplemented animals
had almost identical serum levels: 557 and 546 ng/dl, respectively. The
dorsolateral and ventral prostatic lobes were removed, and the RNA was
purified, Northern blotted, and probed with clathrin heavy chain, light
chain a, and light chain b. DNA was also isolated from each sample, and
gene expression relative to DNA was calculated. DNA was used as a
control for expression instead of GAPDH, because it is known that the
prostate will begin to undergo apoptosis, and the levels of GAPDH may
vary under these conditions. This is in contrast to the LNCaP cell
line, which does not undergo apoptosis upon removal of androgens. In
the dorsolateral prostate (Fig. 8A),
castration caused a 50% decrease in the expression of clathrin heavy
chain mRNA (P < 0.02; n = 5). Supplementation
with testosterone propionate blocked this decrease (P
< 0.02; n = 5). The addition of flutamide with testosterone
propionate produced a significant reduction in clathrin heavy chain
mRNA compared with the effect of supplementation with only testosterone
propionate (P < 0.05; n = 5). Clathrin light
chain b also showed a 50% decrease in mRNA level with castration
(P < 0.02; n = 5), and testosterone
supplementation recovered the mRNA to near-intact levels
(P < 0.05; n = 5). Flutamide treatment induced no
change in the clathrin light chain b mRNA levels. Clathrin light chain
a showed no significant change in mRNA levels with the varying
treatments. Clathrin heavy chain was the most responsive to castration
and testosterone treatment, similar to that in LNCaP cells. The
clathrin light chains were less affected by the conditions, again
similar to the in vitro data. In the ventral prostate,
similar results were found (Fig. 8B). There were no significant changes
in the expression of light chain a. Clathrin heavy chain expression was
reduced 50% by castration (P < 0.05). Testosterone
treatment increased the expression of clathrin heavy chain by 60% over
that in the intact animals, but was only statistically significant
relative to expression in the castrated group (P <
0.01). Flutamide was able to antagonize the testosterone-induced
expression of clathrin heavy chain to a significant degree
(P < 0.05). Light chain b expression was also reduced
by approximately 50% in the castrated group (P <
0.02), and testosterone supplementation reversed this action
(P < 0.01). No significant change in the clathrin
light chain b mRNA level was detected in the flutamide-treated animals.
Although there are some noticeable differences between the androgen
induction of clathrin genes in the LNCaP cells vs. the rat
prostates, we conclude from these data that the androgen induction of
clathrin gene mRNAs seen in the in vitro model is
reproducible in an in vivo model.
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Figure 8. Clathrin gene expression is androgen responsive in
the rat dorsolateral and ventral prostates. Rats were treated as
described in Materials and Methods, and total RNA and
DNA were isolated from the dorsolateral prostate (A) or the ventral
prostate (B), Northern blotted, and probed for the expression of
clathrin heavy chain and light chains a and b. Gene expression was
standardized to the DNA content of the tissue, and the fold change was
calculated. Each bar is the mean ± SE
of data from five separate animals. Significance was calculated by
ANOVA. CHC, Clathrin heavy chain mRNA; Lca, clathrin light chain a;
Lcb, clathrin light chain b; Intact, sham-operated prostate; Cast, 72-h
castrated prostate; TP, testosterone propionate-supplemented castrated
prostate; TP+F, testosterone propionate- plus flutamide-supplemented
castrated prostate.
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To examine the protein levels of clathrin heavy chain in the
in vitro model, randomly selected rat prostates were
subjected to immunohistochemistry using the anticlathrin heavy chain
antiserum. As shown in Fig. 9, the intact
dorsolateral (Fig. 9B) and ventral (Fig. 9G) prostatic lobes showed
high levels of anticlathrin heavy chain immunoreactivity in the
secretory epithelial cells. In Fig. 9, C (dorsolateral) and H
(ventral), the castrated prostatic lobes had lost this staining,
indicating that in only 72 h the clathrin heavy chain proteins
were either degraded or lost through apoptosis of the cells. Figure 9, D (dorsolateral) and I (ventral), shows data from animals that were
supplemented with testosterone propionate, and the levels of clathrin
heavy chain remained similar to those in the intact animals. Animals
treated with testosterone propionate and flutamide showed lower levels
of clathrin heavy chain protein levels (Fig. 9E for dorsolateral and
Fig. 9J for ventral prostate). We conclude from these data that
androgens regulate the levels of clathrin heavy chain protein in the
rat prostate and, as the antiandrogen flutamide can inhibit this
regulation, that this effect is mediated through the androgen
receptor.
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Figure 9. Immunohistochemistry of clathrin heavy chains in
the rat dorsolateral and ventral prostate. Rats were treated as
described in Materials and Methods, and the dorsolateral
prostate and ventral prostate lobes were fixed, sectioned, and treated
for immunohistochemistry. A–E, Dorsolateral prostate. F–J, Ventral
prostate. Treatments are as follows: A and F, nonimmune staining of
intact prostate; B and G, anticlathrin heavy chain staining of intact
prostate; C and H, anticlathrin heavy chain staining of 72-h castrated
prostate; D and I, anticlathrin heavy chain staining of 72-h castrated
prostate supplemented with testosterone; E and J, anticlathrin heavy
chain staining of 72-h castrated prostate supplemented with
testosterone and flutamide. Magnification, x400.
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Discussion
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We describe in this manuscript the androgen regulation of clathrin
gene expression. Clathrin heavy chain and clathrin light chain a and b
mRNAs were up-regulated by androgens in a time- and
concentration-specific manner in the prostate cancer cell line LNCaP.
Using the rat prostate as an in vitro model, androgens were
able to maintain the expression of the clathrin genes compared with
that in the castrated state. The antiandrogen flutamide was able to
partially block the androgen maintenance of clathrin expression,
implying that the effect is mediated by the androgen receptor.
The mechanism of androgen action has been extensively studied (for a
review, see Ref.10). In the human prostate, the circulating androgen
testosterone is metabolized to the more active form,
dihydrotestosterone. Dihydrotestosterone binds to the androgen
receptor, a member of the nuclear hormone receptor superfamily, and
allows the androgen receptor to modulate the expression of the target
gene. There is an increasing number of genes that are reported as
androgen regulated. In the human prostate, two of the best
characterized genes are PSA and hK2. Both of these genes have been
shown to be androgen regulated at the transcriptional level (11). The
secretion of PSA glycoprotein from LNCaP cells into the culture media
is also androgen dependent (12). The time course of PSA secretion is
similar to the production of clathrin heavy chain mRNA, with both
events experiencing the largest increases after 24 h of androgen
stimulation.
As mentioned previously, the development and function of the prostate
require the presence of androgens, specifically dihydrotestosterone.
Defects in the enzyme 5
-reductase, which reduces testosterone to
dihydrotestosterone, will result in an individual with an
underdeveloped prostate. Removal of androgens by surgical or chemical
castration will result in a loss of prostatic function (13). This is
followed by a loss of prostatic mass through an apoptotic involution of
the prostate (14). Thus, as the function of the prostate is under
androgen control, and one of the primary functions of the prostate is
to secrete proteins that are androgen regulated, it follows that there
should be points in the secretion pathway that are under androgen
control. We have shown here that the expression of the clathrin genes
is under androgen control.
It is interesting to note that there are differences in the regulation
of the clathrin genes between the in vitro (LNCaP) model and
the in vivo (rat prostate) model. In neither the
dorsolateral nor the ventral prostatic lobes did the expression of
light chain a change to a significant degree. This could be due to the
fact that the LNCaP cell line has undergone changes during the
development of the metastatic lesion giving rise to the cell line or
during the propagation of the cells in culture. This difference may
also reflect a subtle difference between rat and human prostates.
However, the expression of heavy chain and that of light chain b were
both affected by the androgen state of the animals. The magnitude of
these changes was not as dramatic as that seen in vitro, but
this could be due to the more complicated state of the prostate, with
extensive paracrine and endocrine interactions available that are not
seen in the relatively more defined in vitro cell culture
model. With regard to the lack of changes in expression of light chain
a in the rat prostate, Acton and Brodsky have shown that the ratio of
light chain a to light chain b varies depending upon the type of
secretory activity in which the cell engages (15). This implies that
there are differing roles for the two light chains. As the LNCaP cell
line is by definition mutant, and the prostate does have basal as well
as regulated secretory activities, it may be that light chain a is, in
fact, not regulated by androgens in vivo, and the LNCaP
results are artifactual. This certainly bears further investigation and
may position the rat prostate as a viable model for dissection of the
putatively differing roles of the light chains.
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Acknowledgments
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The authors acknowledge Dr. R. Iozzo, Thomas Jefferson
University, for the clathrin heavy chain cDNA probe, and Dr. A.
Jackson, Cambridge University, for the clathrin light chains a and b
cDNA probes. The authors thank Lucy Schmidt for technical assistance,
and Mary Craddock and Denise Lecy for secretarial support.
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Footnotes
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1 This work was supported by NIH Grants DK-47592 and HD-09140 (to
D.J.T.). 
2 Current address: UroSciences Group, UroCor, Inc., 800 Research
Parkway, Oklahoma City, Oklahoma 73104.
Received October 24, 1997.
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Abstract
Introduction
