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Department of Urology (N.Z., E.B.P., X.C., E.B.C., S.L., R.C., Z.W.) and Department of Molecular Pharmacology and Biological Chemistry (Z.W.), Northwestern University Medical School, Chicago, Illinois 60611
Address all correspondence and requests for reprints to: Zhou Wang, Department of Urology, Tarry 11–715, Northwestern University Medical School, Chicago, Illinois 60611. E-mail: wangz{at}nwu.edu
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Abstract |
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Introduction |
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Recently, we have initiated a comprehensive search for
androgen-response genes (4) in the rat ventral prostate using a gene
expression screen, a highly sensitive PCR-based complementary DNA
(cDNA) subtraction method (5). A series of androgen-response genes were
identified on the basis of changes in gene expression induced by
androgen replacement in the prostate of a 7-day castrated rat. Sequence
analysis (4) has identified that one of the androgen-response genes
encodes calreticulin, a highly conserved intracellular
Ca++-binding protein in the lumen of endoplasmic reticulum
(6, 7, 8, 9). Homozygous knockout of calreticulin gene resulted in embryonic
lethality in mice (Coppolino, M. G., and S. Dedhar, manuscript
submitted), indicating that calreticulin plays an essential role in
animal development. Calreticulin seems to be involved in a wide variety
of cellular processes, including: 1) modulation of Ca++
signals; 2) storage and buffering of Ca++; 3) regulation of
steroid-dependent gene expression via direct interaction with steroid
receptors; 4) cell adhesion via direct binding to integrin 
; 5) as a
chaperone in protein folding; 6) autoimmune response; and 7) long-term
neuromodulations (7, 11). Calreticulin consists of 1 high-affinity and
approximately 25 low-affinity Ca++ binding sites and is a
major intracellular Ca++-binding protein in nonmuscle
cells. It has been demonstrated that calreticulin could inhibit
intracellular Ca++ oscillations (12). Down-regulation of
calreticulin by antisense oligo increases sensitivity of
neuroblastoma x glioma NG-108–15 cells to cytotoxic
Ca++ overload (13). Conversely, overexpression of
calreticulin has been shown to protect HeLa cells from apoptosis (14, 15). Recently, it is reported that the expression of calreticulin is
markedly decreased before the cell apoptosis in human leukemia HL-60
cells (16).
Because calreticulin is a protein with multiple functions, it is of great interest to study its role in androgen action in the prostate. Characterization of calreticulin expression during androgen manipulation is likely to provide insights into the function(s) of calreticulin in androgen action in the prostate. This paper describes the spatial and temporal expression of calreticulin in the prostate, tissue-specificity of androgen induction, and possible mechanisms by which androgen regulates calreticulin expression in the prostate.
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Materials and Methods |
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Animals
Young adult male Harlan Sprague-Dawley rats (250–300 g) and
BALB/c mice (35–40 g) were used in this research. The rats and mice
were castrated, in a room dedicated to animal surgery, according to a
protocol approved by the Northwestern University Animal Care and Use
Committee. Testes, fat pads, and epididymides were removed in the
castration. The castrated animals were maintained at Northwestern
University Animal Facility. Treatment of 7-day castrated rats with
exogenous androgen was carried out by daily sc injections of 0.2 ml
testosterone propionate dissolved in propylene glycol at 10 mg/ml for
up to 7 days. At various times after castration or androgen
replacement, at least three rats were killed by decapitation after
methoxyflurane anesthesia. The ventral prostate lobes were removed,
weighed, and frozen in liquid nitrogen before RNA or protein isolation.
The left lobes were used for isolation of RNA, and the right lobes were
used for preparation of protein extracts.
RNA isolation and Northern blot analysis
Total RNA was isolated using the guanidine thiocyanate/CsCl
gradient method (17). Purified RNA samples were fractionated in a 1%
agarose-formaldehyde gel. Ten micrograms of total RNA was loaded in
each lane. After electrophoresis, RNA was transferred to nylon membrane
by capillary blotting and then cross-linked to the membrane by UV
irradiation. Northern hybridization of the membrane was carried out at
42 C overnight in a buffer containing 5 x SSPE (0.9
M NaCl, 50 mM NaH2PO4,
50 mM EDTA, PH 7.4), 2 x Denhart’s solution, 0.1%
SDS, 0.1 mg/ml denatured salmon sperm DNA, and 50% formamide in the
presence of DNA probes labeled by random priming. The membrane was then
washed at room temperature with 1 x SSC (0.3 M
NaCl, 30 mM sodium citrate, pH 7.0) and 0.1% SDS for 20
min, followed by three 20-min washes at 65 C with 0.2 x SSC and
0.1% SDS. The autoradiogram for Northern blot was obtained by exposing
the film at -80 C with an intensifying screen.
In situ hybridization
DIG-labeled calreticulin sense and antisense RNA probes for
in situ hybridization were synthesized using linearized,
proteinase K-treated plasmid DNA templates. A full-length rat
calreticulin cDNA, inserted at the multiple cloning site of
pBluescript II SK plasmid, was used in template preparation.
Synthesis of sense and antisense RNA probes was carried out by in
vitro transcription using DIG RNA labeling mix of nucleotides
(Boehringer-Mannheim) with either T3 or T7 RNA polymerase
(Promega, Madison, WI).
In situ hybridization was carried out according to Furlow et al. (18), with small modifications. Rat ventral prostate tissue was isolated and fixed at 4 C overnight in 4% paraformaldehyde in SPB (3% sucrose, 0.15 mM CaCl2, 0.06 M phosphate buffer, pH 7.4) at 4 C. The tissue was then rinsed in 10% sucrose in SPB and incubated overnight at 4 C in 30% sucrose in SPB. The tissue was cryosectioned at 5-µm thickness and placed on ProbeOnR Plus microscope slides (Fisher Biotech). The slides were heated at 45 C for 3–5 h on a slide warmer. The sections were refixed with 4% paraformaldehyde and then digested with proteinase K at 20 µg/ml in PBS for 6 min. After refixing again in 4% paraformaldehyde, the sections were washed in PBS and acetylated in 0.25% acetic anhydride in triethanolamine (pH 8.0). The sections were washed again in PBS before hybridization. The probes for in situ hybridization were heated at 80 C for 5 min after being diluted 100-fold to 500-fold in hybridization buffer consisting of 1x Denhardt’s solution, 100 µg/ml DNA, 50% formamide, and 250 µg/ml yeast RNA in 5 x SSC buffer. Twenty microliters of the heated probe was put onto a coverslip. The slide was inverted, and the coverslip was allowed to attach by capillary action. The slides were incubated overnight at 67 C in a sealed chamber humidified with 5 x SSC buffer/50% formamide. After hybridization, the coverslips were removed by dipping the slides in 5 x SSC buffer at 72 C. The slides were washed in 0.2 x SSC buffer at 72 C for 1 h, followed by a 5-min room-temperature wash in 0.2 x SSC. The staining of the hybridized probes on the slides was the same as described by Furlow et al. (18).
Protein preparation and Western blot
Prostate tissue was homogenized in a lysis buffer consisting of
1 x PBS, 1% SDS, 10 mM EDTA, 100 µM
phenylmethylsulfonyl fluoride, 10 µM leupeptin, 0.2
mM 4-(2-aminoethyl)benzenesulfonyl fluoride, and 1
µM pepstatin. Insoluble materials were pelleted by
centrifugation at 10,000 x g for 10 min at 4 C. A very
small percentage of the protein in the prostate extracts was insoluble.
Protein concentration was determined using a BioRad DC protein assay
kit. Protein samples were separated by 10% SDS-PAGE and then
transferred onto Protran nitrocellular membranes (Schleicher &
Schuell, Keene, NH). Rabbit anticalreticulin primary antiserum
and a secondary antibody linked to horseradish peroxidase were used in
a standard Western blot procedure (19) for the detection of
calreticulin protein in the prostate. The polyclonal anticalreticulin
antiserum was generated using a GST
(glutathione-S-transferase)-calreticulin fusion protein in rabbits.
Anticalreticulin antibody is also available from StressGen
(Victoria, BC, Canada). The enhanced chemiluminescence detection method
(Amersham, Arlington Heights, IL) was used to detect secondary
antibody binding.
Immunohistochemistry
The ventral prostates from normal Sprague-Dawley rats were
collected, and fixed in 4% paraformaldehyde in PBS overnight at 4 C.
Tissues were dehydrated through an ethanol series, cleared in xylenes,
and embedded in paraffin. Eight-micrometer sections were cut on a
microtome, allowed to spread in water, placed on ProbeOn Plus
slides, and warmed on a slide warmer for 2 h at 55 C. The sections
were dewaxed three times, for 10 min each, in fresh xylenes, then twice
in 100% ethanol for 5 min each, twice in 95% ethanol for 5 min each,
and finally rinsed in distilled water twice for 5 min each. Endogenous
peroxidase activity was inactivated by incubating the sections in 3%
freshly-made hydrogen peroxide in methanol for 20 min. The nonspecific
sites were blocked with 2% normal goat serum in PBS for 1 h at
room temperature. The slides were incubated with 100 µl of the
primary antibody diluted 1500-fold in the blocking solution in a
humidified box overnight at 4 C. The control group was incubated in a
mixture of 100 µl of the same diluted primary antibody in the
presence of 10 µg GST-calreticulin fusion protein. The slides were
rinsed twice in PBS for 5 min each. Then the slides were processed as
described in the instructions in the Vectastain ABC Kit from Vector
Laboratories (Burlingame, CA) and were developed in DAB
substrate (Vector Laboratories) for 5 min. After stopping the reaction
in distilled water, the slides were dehydrated in an ethanol series,
cleared in xylenes, and dried overnight at room temperature. Finally,
the slides were mounted with Permont (Fisher). Pictures were taken
using an Olympus VANOX-S camera and an Olympus AH-2 microscope
(N. Nuhsbaum Inc., McHenry, IL).
Prostate organ culture
The prostate organ culture was essentially carried out, as
previously described, with some modification (20). Ventral prostates
were isolated from 7-day castrated rats and cut into pieces of about 1
mm3. Approximately 40 pieces were placed on a sheet of
sterile lens paper (4 cm x 6 cm) supported by a stainless steel
rack in a 100-mm culture dish. The prostate pieces were cultured 1 day
before various treatments in M199 organ culture medium containing 10%
charcoal-treated FBS and 1% penicillin-streptomycin in 95% air-5%
CO2 at 37 C. The cultured organs were treated with ethanol
(control), 10-6 M dihydrotestosterone (DHT),
cycloheximide at 50 µg/ml plus anisomycin at 80 µg/ml (CHX), or
both DHT and CHX. Ethanol was used as the vehicle for both DHT and CHX.
The CHX treatment condition inhibited protein synthesis 98% in this
organ-culture experiment, as assayed by 35S-methionine
incorporation (21) (data not shown). The protein synthesis inhibitors
were added in the culture media 2 h before the addition of DHT.
The prostate organ was cultured for 36 h after the addition of
DHT
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Results |
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shows down-regulation of calreticulin messenger RNA (mRNA) in the
prostate after androgen ablation by castration and up-regulation of
calreticulin mRNA in the 7-day castrated prostate by androgen
replacement. The time course of down-regulation shows that there is
over 90% decrease in calreticulin expression within 1 day after
castration. Androgen replacement induces up-regulation of calreticulin
mRNA within 14 h, reaching a peak level of expression in about
24 h (Fig. 1).
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shows the Western blot analysis of calreticulin expression in the rat
ventral prostate during hormonal manipulation. Calreticulin protein is
down-regulated by castration and up-regulated by androgen replacement
in the prostate. This result is consistent with the observation of
Bruchovsky et al. (23).
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) and Northern blot (Fig. 1)
results showed that up- or down-regulation of calreticulin mRNA occurs
1–2 days before the up- or down-regulation of calreticulin protein,
respectively, in the prostate during hormonal manipulation. The time
course of androgen regulation of calreticulin mRNA is directly
comparable with that of calreticulin protein, because the same batch of
animals was used in the Northern and Western experiments. The left
lobes of the ventral prostate were harvested for RNA isolation and the
right lobes for protein preparation. Calreticulin mRNA seems to be much
more labile than calreticulin protein, because the down-regulation of
the mRNA is much more rapid than the down-regulation of the protein in
the prostate after castration. The lag of calreticulin protein
up-regulation after its mRNA reflects the time required for
translation of calreticulin and the time needed to increase
translational efficiency in prostatic cells upon androgen
replacement.
Calreticulin is abundantly expressed in the prostate
The tissue specificity of calreticulin expression and androgen
induction was studied by Northern blot analysis, and the results are
shown in Fig. 3. Calreticulin is most
abundantly expressed in the prostate, among the tissues surveyed. In
the testis-intact rat, calreticulin mRNA is most abundant in the
ventral prostate. In comparison, calreticulin expression is much weaker
in the other tissues: heart, brain, muscle, kidney, liver, and seminal
vesicles.
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shows that androgen does not influence the expression
of calreticulin in liver, kidney, heart, brain, and muscle. The
expression of calreticulin is responsive to androgen in both seminal
vesicles and prostate. Castration leads to a very modest
down-regulation of calreticulin in the seminal vesicles, in contrast to
substantial down-regulation of calreticulin in the prostate after
castration. Although calreticulin down-regulation by castration in the
seminal vesicles is very limited, the up-regulation by androgen
replacement is marked. The level of calreticulin mRNA in the prostate
induced by androgen replacement is also higher than that in the
prostate of testis-intact animals.
Androgen induction of calreticulin expression partially resists
protein synthesis inhibition
One of the important questions concerning the regulation of
calreticulin expression by androgen is whether calreticulin is a
primary or a secondary response gene. To address this question, we
studied the effect of protein synthesis inhibition on the induction of
calreticulin by androgen in prostate organ culture. The induction of
calreticulin mRNA by 36 h of DHT treatment in the cultured
prostate organ is much weaker than the induction in vivo,
and the induction partially resists protein synthesis inhibition (Fig. 4). Calreticulin induction in prostate
organ culture could be enhanced by 48 h DHT treatment. However,
conducting 48 h CHX inhibition was not possible, because RNA in
prostate organ culture was almost completely degraded after 48 h
CHX treatment. The inhibition of protein synthesis by cycloheximide and
anisomycin in the prostate organ culture was over 98%, as assayed by
measuring 35S-methionine incorporation (data not shown).
This result suggests that calreticulin is a primary androgen-response
gene in the prostate.
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shows that
calreticulin mRNA is localized in the epithelial cells. The expression
of calreticulin mRNA in stromal cells was not detected, even with
intense staining in the epithelial cells. Because calreticulin mRNA is
dramatically regulated by androgen and localized in the epithelial
cells, calreticulin expression is responsive to androgen only in the
epithelial cells of the prostate.
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Calreticulin is an intracellular protein in prostatic epithelial
cells
Immunohistochemistry was employed to study whether calreticulin is
an intracellular protein or a secretory protein. The antibody staining
was localized in the epithelial cells, suggesting that calreticulin is
not a secreted protein in the prostate (Fig. 6). The staining to epithelial cells was
blocked by GST-calreticulin fusion protein, indicating that the
antibody staining was specific to calreticulin.
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shows the effect of
castration on the expression of calreticulin in the mouse prostate. At
day 3 after castration, the expression of calreticulin mRNA was
significantly down-regulated. Day 3 after castration was chosen because
calreticulin is significantly down-regulated in the rat model, yet the
change in epithelial-stromal ratio within 3 days after castration is
very limited. This experiment indicates that calreticulin expression is
regulated by androgen in the mouse prostate.
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). As a
control, the expression of cytokeratin 8 was also examined. Cytokeratin
8 is constitutively expressed in normal and abnormal prostatic
epithelial cells (26). Cytokeratin 8 expression in the cultured
prostate organ was virtually the same either in the presence or absence
of DHT, suggesting that the percentages of epithelial cells in the two
organ culture samples were very similar. The above observations suggest
that androgen also regulates calreticulin expression in the human
prostate. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The down-regulation of calreticulin by androgen ablation correlates
with the apoptosis of epithelial cells in the prostate. First, the time
course of calreticulin down-regulation correlates with the onset of
apoptosis in the prostate after castration (1). Castration-induced
apoptosis begins at day 2 and is most active between days 3 and 5. The
down-regulation of calreticulin mRNA occurs within 1 day after
castration, preceding the onset of apoptosis. However, the
down-regulation of calreticulin protein coincides with the onset of
apoptosis in the prostate after castration. Calreticulin protein is
modestly down-regulated within 1 day after castration and further
down-regulated several fold during the period of active apoptosis.
Second, the down-regulation occurs mainly, if not exclusively, in
prostatic epithelial cells, as demonstrated by in situ
hybridization (Fig. 5). Epithelial cells are the major type of the
cells undergoing apoptosis after castration (24, 25). Thus,
down-regulation of calreticulin has the potential to be involved in
prostatic epithelial cell death. It is important to point out that
down-regulation of calreticulin does not seem to be sufficient to cause
apoptosis, because epithelial cells in the castrated prostate, with
significantly low calreticulin expression (as detected by in
situ hybridization), are still alive. Therefore, in addition to
the down-regulation of calreticulin in the prostate, the expression
changes of other gene(s) are likely to be required for inducing
apoptosis in prostatic epithelial cells.
On the other hand, the up-regulation of calreticulin by androgen replacement in a 7-day castrated rat correlates with the proliferation and differentiation of prostatic epithelial cells. The induction of calreticulin mRNA is very rapid, occurring within 14 h after androgen replacement, preceding the onset of proliferation in the regrowth of the castrated prostate. The induction of calreticulin protein occurs most significantly around 2–3 days after the androgen replacement, at the onset of extensive cell proliferation and differentiation in the regrowth. The most extensive regrowth occurs between 3–5 days after the replacement. Although it is unlikely that calreticulin up-regulation alone could stimulate cell proliferation and/or differentiation, its accumulation may represent an important molecular event associated with cell proliferation and/or differentiation.
It is important to point out that the down-regulation of calreticulin expression, within 2–3 days after castration, in the prostate is not caused by the changes in the ratio of stromal-to-epithelial cells in the prostate. There is little or no change in the ratio between stromal and epithelial cells within 2–3 days after castration, because the nuclei number in the prostate remains virtually the same during this period (1). Similarly, the up-regulation of calreticulin expression, within 2–3 days after androgen replacement, in a 7-day castrated prostate is not caused by the changes in the ratio of stromal-to-epithelial cells, because significant cell proliferation is initiated 2–3 days after androgen replacement (1). Thus, dramatic up- or down-regulation of calreticulin, within 2–3 days after androgen manipulation, mainly reflects changes in calreticulin mRNA levels in epithelial cells.
One major function of calreticulin is involved in the modulation of intracellular Ca++ levels and/or signals (6, 7, 8, 9, 11). The abundant expression of calreticulin inside prostatic epithelial cells suggests that calreticulin could be an intracellular Ca++-buffering protein. Although the importance of intracellular Ca++ in androgen-induced prostate regrowth is not clear, evidence for the involvement of intracellular Ca++ in prostatic cell death exists. In the prostate, cell death induced by Ca++ ionophore is indistinguishable from the cell death induced by castration (27). Furthermore, Ca++ channel blockers can inhibit as much as 70% of the castration-induced increase in the rate of cell death (28) and suppress the induction of apoptosis-associated genes, including TRPM-2 and c-fos in the prostate (27, 28). Down-regulation of calreticulin, contributing to an elevation of intracellular Ca++, seems to be a potential mechanism for the initiation of apoptosis in the prostate after castration. The importance of calreticulin down-regulation in castration-induced apoptosis is supported by our observation that down-regulation of calreticulin correlates with apoptosis of prostatic epithelial cells.
As indicated in previous discussion, low calreticulin expression does not seem to be sufficient to cause apoptosis in the prostate. This is consistent with the observation that down-regulation of calreticulin does not trigger apoptosis in a number of cells, including PC-3 (29) and NG-108–15 cells (13). However, calreticulin down-regulation sensitizes the cells to cytotoxic intracellular Ca++ overload (13). Additional mechanisms are likely to be involved in triggering intracellular Ca++ influx.
Another possible function of calreticulin in the prostate is its effect
on cell adhesion via direct binding to integrin (7).
Down-regulation of calreticulin in several tumor cell lines, including
PC-3, an androgen-independent prostate cancer cell line, inhibits the
ability of these cells to attach and spread on extracellular matrix
(29). Embryonic stem cells, lacking both alleles of the calreticulin
gene, were impaired in their short-term attachment to fibronectin (7).
On the other hand, overexpression of calreticulin in mouse L
fibroblasts leads to a flattened shape, development of strong
cell-substratum adhesions, reorganization of actin into stress fibers,
and establishment of epithelial-like cell-cell junctions (30).
Expression of calreticulin could be important for cell-cell
interactions in the prostate. Given the importance of
stromal-epithelial interactions in the prostate (31, 32), calreticulin
may play an important role in the stromal-epithelial interactions.
Calreticulin could also be a chaperone in endoplasmic reticulum and a regulator of gene expression via interaction with steroid receptors (7). Although it is not clear, at all, whether calreticulin can function as a chaperone in the prostate, calreticulin does not seem to inhibit gene expression via direct interaction with the AR. Calreticulin is abundantly expressed in prostatic epithelial cells in the normal prostate, yet the prostate expresses all the genes that are up-regulated by androgen. Clearly, the AR is active in the presence of an abundance of calreticulin.
Androgen regulation of calreticulin seems to be conserved in different species, which is consistent with the observation that the promoters and genomic organization are highly conserved between human and mouse calreticulin genes (7). R. A. Clark has isolated and characterized more than 1900 bp of the 5' flanking sequence of the human calreticulin gene (7). It was reported that the transcription of calreticulin gene in HL-60 myeloid cell line is regulated by many transcription factors, possibly including CCAAT-binding factor, retinoblastoma protein, and some unidentified C-rich sequence binding factors. There is no report that an AR-binding site was identified from the calreticulin promoter. However, calreticulin seems to be a primary androgen-response gene, because its induction by androgen does not require protein synthesis. Two potential explanations exist. First, AR binding sites may be localized far away from the transcription initiation site of calreticulin promoter. Second, the AR may regulate gene expression without making direct contact with a DNA element(s), rather by interacting with another DNA-binding protein.
The up-regulation of calreticulin by androgen replacement is very rapid, which is likely to be controlled at the transcription level, because AR is a ligand-dependent transcription factor. The down-regulation of calreticulin by castration may be more complex and likely to involve inhibition of transcription, rapid degradation of mRNA, and then down-regulation of calreticulin protein. Within 1–2 days after castration, the calreticulin mRNA is down-regulated over 10-fold. Considering that the half-life of DHT in the prostate is longer than 6 h in the rat ventral prostate after castration (33), down-regulation of calreticulin mRNA by castration in the prostate is very efficient. After the mRNA down-regulation, calreticulin protein is gradually down-regulated. We do not know whether or not the stability of calreticulin mRNA and/or protein is also affected by hormonal manipulation in the prostate. It seems that androgen regulates calreticulin expression mainly at transcription.
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Acknowledgments |
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Footnotes |
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2 Recipient of the 1997 American Association for Cancer
Research-Glaxo Wellcome Oncology Clinical Research Scholar Travel Award
and the 1998 American Association for Cancer Research-AFLAC Scholars in
Cancer Research Travel Award.
3 Recipient of a Pfizer USPG Scholarship from American Foundation for
Urologic Disease.
4 Recipient of a Junior Faculty Research Award from the American
Cancer Society.
Received March 23, 1998.
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