Autoregulation of androgen receptor in rat ventral prostate: involvement of c-fos as a negative regulator

bhcularand eel Mar Emlocrinokw

ELSEVIER

Molecular and Cellular Endocrinology 124 (1996) 11I ~ 120

Autoregulation of androgen receptor in rat ventral prostate: involvement of c-fus as a negative regulator Gloria R. Mora, Virendra B. Mahesh”

Abstract

Recent work from our laboratory has focused on elucidating the mechanism of androgen regulation of the androgen receptor (AR). We have demonstrated that testosterone increases AR protein and binding within 1 h in the ventral prostate of adult rats castrated for 24 h. Cycloheximide administered with testosterone reduces AR and AR mRNA levels. AP-l/c-f& transcription factor has been shown to function as a negative repressor in many systems. c-fos mRNA levels were decreased 1 h after testosterone treatment in the ventral prostate, whereas they were increased in cycloheximide alone or cycloheximide-testosterone treated groups as compared to vehicle control. clfos protein was also increased in the testosterone-cycloheximide treated group as compared to testosterone alone or cycloheximide alone groups at 1 h. By 3 h, the tissue recovers from the inhibitory effect of cycloheximide as evidenced by restoration of AR and an increase in AR mRNA levels. At this time c-j&s protein levels were reduced after treatment with cycloheximide and testosterone and c-fos mRNA levels were comparable to the controls. These results suggest that elevation of cTfos expression is associated with a decrease in AR and AR mRNA and provide correlative data supporting negative repression by c-fos on androgen receptor levels. Between the age of 2.5-85 days, serum testosterone levels reached adult levels by the age of 55 days. Steady-state AR mRNA levels increased significantly by the age of 85 days while c+s mRNA levels remained at low baseline levels at all ages. Thus, in addition to the circulating levels of serum testosterone, other age related factors are also involved in the regulation of AR mRNA levels. Furthermore, androgens appear to maintain androgen receptor levels and androgen sensitivity by continuous suppression of the repressor, c-fos. Copyright 0 1996 Elsevier Science Ireland

Ltd.

Keywords:

Age; Androgen

receptor; Androgen

receptor mRNA; C.-$X: c:fos mRNA; Cycloheximide;

1. Introduction The androgen receptor (AR) acts as a ligand-activated transcription factor in the nuclei of androgen-sensitive cells [l]. Therefore, factors that regulate AR levels

available for androgen binding dictate the expression of specific target genes during androgen action. Although there is ample evidence of androgen controlling the expression of its own receptor, the chain of events involved are not yet known. Using both in vivo and in vitro models, a discrepancy between the regulation of AR mRNA and AR protein levels has been shown. * Corresponding 72 17299.

author. Tel.: + I 706 7212781: fax: + 1 706

Rat ventral prostate

While AR mRNA is negatively regulated after androgen administration [2-41, AR protein levels appear to increase [3], decrease [4] or do not change [5] depending upon the conditions used. Cis-acting elements at the S-untranslated region of the human and mouse gene involved in the regulation of AR gene transcription have been identified [6,7]. However, there is only limited information regarding the trans-acting factors that bind to such elements [8]. Less documented are the regulation of AR mRNA translation [9] and control of AR stability. Recently, using recombinant hAR it was revealed that sequences mediating androgen downregulation of AR mRNA are located within the AR cDNA [lo]. Autoregulatory sequences have also been identified

0303.7207/96/$1.5.00 Copyright 8 1996 Elsevier Science Ireland Ltd. All rights reserved PI1 SO303-7207(96)03939-I

112

G.R. Moru, V.B. Mahesh 1 Molecular und Crllulur Endocrinology 124 (1996) 1 I l-120

within the coding region for the human estrogen receptor and the human glucocorticoid receptor [11,12]. Numerous studies have demonstrated steroid regulation in the expression of the immediate early response genes such as c-fos, c-jun and c-myc [13]. Androgens down-regulate c-myc transcripts in the human prostate carcinoma cell line LNCaP and antiandrogens block this response, indicating that the regulation of c-myc gene expression is AR mediated [14]. In addition, c-fos mRNA is primarily expressed in the rat prostate after 36 h of castration [ 151 and transiently expressed as early as 1 h during androgen stimulated prostate regrowth in a 7 day castrated rat [16]. In a previous study, we demonstrated that the effect of testosterone treatment for 1 h on the increased nuclear accumulation of AR in the rat ventral prostate after 24 h of castration was prevented by cycloheximide [17]. The cycloheximide dependent decline in AR protein was accompanied by a remarkable decrease in AR mRNA levels which could not be explained on the basis of a decrease in transcription during this period. Thus, it was postulated that cycloheximide inhibited a protein(s) that regulated AR mRNA stability and possibly its translation as well. The purpose of this study was to examine whether AP-1 type transcription factor (c-fos was chosen as an example) was involved in AR mRNA stability and/or its translation. To interpret changes in c-fos mRNA and c-fos expression with respect to AR expression, it was considered desirable to examine c-fos changes during the period when cycloheximide decreases testosterone regulated AR mRNA and AR levels in the prostate as well as the period when the prostate recovers from the effects of cycloheximide. Finally, because of the dependence of AR mRNA on testosterone levels, it was of interest to determine the expression of steady-state AR mRNA in the ventral prostate with age and to determine if they were related to c-fos mRNA levels and circulating testosterone levels. This study was also of interest because of reports in the literature that the expression of steady-state levels of AR mRNA in the rat liver is age dependent [l&19].

2. Materials and methods 2.1. Animals Experiments were performed on adult male Virusfree Sprague Dawley rats obtained from Harlan (Madison, WI). Adult male rats (80-90 days old) were fed Purina chow and tap water, ad libitum, and were allowed 2 days of acclimatization period in an air-conditioned, light controlled room with a 12 h light-dark cycle. Bilateral orchidectomy, when indicated, was carried out via the scrotal route under ether anesthesia 24 h prior to each experiment. Groups of 4-6 animals

were treated 1 h or 3 h before sacrifice intraperitoneally (i.p.) with vehicle (l:l, ethanol:saline) testosterone (400 pg/lOO g body wt.), cycloheximide (400 pg/lOO g body wt.), or testosterone in conjunction with cycloheximide. Injection volume containing the doses required per rat were adjusted at 1:lOOOof rat’s body weight to avoid volume changes and/or excess of ethanol. Control rats were either intact, or vehicle-treated 1 day castrated rats. The cycloheximide dose is an accepted dose in the literature for blocking translation and is non-toxic [20]. For age dependent studies, intact rats were killed in groups of four each at ages 25 to 85 days for the measurement of serum testosterone, AR and c-fos mRNA levels. All animal studies carried out were approved by the Medical College of Georgia Institutional Committee for the Care and Use of Animals in Research and Education in accordance with the guidelines of the NIH and USDA. 2.2. lmmunocytochemistr~ Ventral prostates were rapidly excised and placed in ice-cold phosphate-buffer saline (PBS). The individual full-length ductal system arrays were microdissected as described by Sugimura et al. [21]. Tissues were frozen and sectioned longitudinally at 8 iurn, keeping the proximal-distal orientation [22]. In each experimental group, three animals were used and at least six individual ductal systems were dissected and sectioned for each ventral lobe. Tissue sections were fixed with 2% paraformaldehyde and picric acid, blocked with 2% goat serum and incubated overnight at 4°C with a polyclonal peptide antiserum raised against the first 21 aminoacids of the rat AR, PG-21 (2 pug/ml) [22]. Control experiments were performed using antibody preadsorbed with 50 x M excess of the antigenic peptide. AR immunocomplexes were detected using a biotinylated antirabbit secondary antibody, with subsequent amplification using an avidin-biotin peroxidase kit (ABC-Elite, Vector Laboratories, Burlingame, CA) and diaminobenzidine tetrahydrochloride as a chromagen. Sections were sequentially dehydrated with alcohol, cleared with xylene, and mounted with histological mounting medium. Representative sections were photographed with a Zeiss microscopic system. For c-fos immunocytochemistry, tissue was embedded in OCT and frozen in liquid nitrogen. Random sections (12 pm) of the ventral prostate mounted on gelatin coated slides were pretreated with 10% H,02 and 10% methanol in PBS. After washing with PBS, sections were blocked for 1 h in 5% goat serum, 0.005% TX-100 and then were incubated in a humidified chamber overnight at 4°C with a polyclonal c-fos antibody (0.2 @g/ml, Santa Cruz Biotechnology). Control sections received preadsorbed antibody using 2 pg/ml of the antigenic peptide. After overnight incubation, sections were processed as described above.

G.R. Mora, V.B. Mahesh / Molecular and Cellular Endocrinology 124 (1996) I I I ~-120

2.3. RNA isolution and Northern blot analysis Total prostatic RNA was isolated with RNAzol (Biotex Laboratories, Houston, TX) from individual ventral prostate tissue, using 2-4 rats per group. Polyadenylated RNA (Poly A+ mRNA) was isolated from the total RNA by chromatography on oligo(dT) cellulose (Oncor, Gaithersburg, MD), following a standard technique [23]. From each tissue sample, 3 pg of poly A+ mRNA were denatured with glyoxal and dimethyl sulphoxide for 1 h at 50°C [24], electrophoresed in a 1% agarose gel in 10 mM Na,PO, (pH 6.8) and transferred by capillary action to a nylon membrane (Biotrans, ICN, Irvine, CA) with 20 x SSC (1 x SSC = 0.15 M NACl, 15 mM sodium citrate). Membranes were crosslinked by brief exposure to UV light and baked for 1 h at 80°C. A 1 kb EcoRI fragment of the rat AR cDNR, kindly provided by Drs E. Wilson and F. French (University of North Carolina, Chapel Hill, NC) and a commercially available c-fos cDNA probe (ATCC, Rockville, MD) were used for AR and c-fos hybridization, respectively. Reactions were carried out overnight at 42°C in 50% formamide, 5 x SSC, 1 x Denhart’s, I50 gg denatured salmon sperm DNA, 0.5% SDS, 10% dextran sulfate and 1.5-2.0 x lo6 cpm/ng of 32P random prime labeled probe ([a3’P]dCTP, 3000 Ci/mmoi; Amersham, Arlington Heights, IL. DNA labeling kit, Promega, Madison, WI). Subsequently, membranes were washed at room temperature twice with 2 x SSC, 0.1% SDS for 5 min and then twice at 65°C with 0.1% SSC, 0.1% SDS for 20 min, before being subjected to autoradiography. The blots were stripped in 50% formamide, 10 mM Na,PO, (pH 6.8) at 65°C and rehybridized with a chicken /3-actin cDNA or a human 28s rRNA oligoprobe to control for the qualities and quantities of the mRNA loads on the gel. Densitometric analysis of the autoradiograms was performed with a Shimadzu CS900 densitometer (Shimadzu Instruments, Columbia, MD) or in a digital imaging system IS-1000 (Alpha lnnotech, San Leandro, CA). Levels of AR mRNA were expressed in arbitrary densitometric units and, when indicated, the data was normalized to the level of p-actin mRNA or 28s rRNA.

113

transferred to a PVDF microporous membrane (Immobilon-P, Millipore, Bedford, MA) using an electroblotting apparatus (BioRad, Richmond, CA). Membranes were blocked for 1 h in 5% non-fat dried milk in Tris buffer saline (TBS) plus 0.1% Tween 20 (TTBS). After blocking, membranes were incubated in TTBS containing the c-fos antibody (Polyclonal (0.2 pg/ml), Santa Cruz Biotechnology and Monoclonal (0.5 pug/ml), Oncogene Science). The unbound primary antibody was washed out with TTBS and the binding of the primary antibody was detected using enhanced chemiluminescence according to the manufacturer’s instructions (ECL, RPN 2106 Amersham). In order to confirm that equal transfer and loading had occurred, the blots were reacted with an antitubulin monoclonal antibody, following the same procedure described above. 2.5. Rndioinmrunoassuy of testosterone After decapitation, trunk blood was collected and serum testosterone levels were measured with Coat-ACount solid phase radioimmunoassay kit (Diagnostic Products, Los Angeles, CA), according to manufacturer’s protocol. This method is based on a specific antibody to testosterone being immobilized to the wall of a polypropylene tube. ‘Z51-labeled testosterone competes for antibody binding with testosterone in the serum sample. The assay can detect as little as 0.04 ngjml (0.14 nmol/l), and its cross reactivity with Sa-DHT is less than 5%. The intraassay and interassay coefficients of variation were 5 and 6.4% respectively. 2.6. Statistical unalysis The data was analyzed by one-way analysis of variance with post-hoc comparison by Tukey’s test. A comparison between two groups was performed by pairwise analysis using Student’s t-test.

3. Results 3.1. c-fos immunocytochernistry and undrogen regulation of c-jtis le/lels in the presence or in the absence of cycloheximide after 1 h qf’ treatment

2.4. Western blot analysis Prostatic tissue was homogenized at 4°C with a Polytron using ice cold detergent buffer (10 mM Hepes, 15 mM NaCl, 1 mM EDTA, 1% NP-40, 1 PM leupeptin, 1 PM aprotinin, and 1 mM PMSF). Homogenates were quickly centrifuged to discard cell debris ( 10 000 x g, 5-7 min). 30 pug (for minigels) or 100 pug (for standard gels) of total protein measured by the Bradford method [25] were loaded in Laemmli SDS denaturing polyacrylamide gels [26]. After electrophoresis, proteins were

To determine if androgens regulated c-fbs expression in the prostate, it was necessary to show the occurrence of c-fos in the target cell of androgen action namely the epithelial cells of the ventral prostate. As shown in Fig. lB, c-j&r protein is primarily localized in the nuclei of the epithelial cells. The lack of nuclear staining in the preadsorbed sections (Fig. 1A) indicates the specificity of c-fos staining. Since AR expression changes dramatically after 1 h of a combination of testosterone and cycloheximide

G.R. Aloru, C’.B. Muhesh 1 Molecular and Cellular Endocrinology 124 (1996) Ill-120

114

treatment [17], we decided to investigate the changes in c-fos at this time point. Fig. 2 shows the evaluation by Western blot analysis of total c-fos protein levels in prostate lysates from vehicle control, testosterone, cycloheximide and testosterone-cycloheximide treated groups, 1 h after their administration. Using a polyclonal antibody, higher levels of c-fos (immunoreactive protein band of 55 kDa) were found in the combined treatment after 1 h, when compared to androgen treatment alone. The c-fos band was eliminated by preincubation of the primary antibody with an excess of antigenic peptide, confirming the specificity of the immunoreaction. The results in Fig. 2A normalized with tubulin show an increase of 120% in c-fos in the presence of cycloheximide and testosterone at 1 h, as compared to the testosterone group. In spite of an indication of increased c-fos levels in the testosterone and cycloheximide treatment at 1 h, the use of only one pooled sample for analysis and the small percentage increase at 1 h over testosterone required confirmation. Therefore, the experiment was

A FOS

Fos preadsorbed Tu bulin

c-fos/Tubulin

1.35 2.70 1.27 3.25

B c-fos

Tubulin A

c-fos/Tubulin

0.87

0.95 0.95 1.62

Fig. 2. (A) Western blot analysis of c-fos in ventral prostate of 1 day castrated rats that received various treatments for 1 h. 30 pg of total protein from tissue lysates were electrophoresed in a 4420% gradient minigel and transferred to a nylon membrane. The blot was incubated with a c-fos polyclonal antibody and with a monoclonal antibody against tubulin to check for consistency in loading and transfer. Preadsorption of the c-jos antiserum checked for the specificity of the immunoreaction. The reactive bands were visualized using enhanced chemiluminescence (ECL). (B) Western blot analysis of c-fos in ventral prostate of 1 day castrated rats that received various treatments for 1 h. 100 pg of total protein from tissue lysates were electrophoresed in a 10% standard gel and transferred to a nylon membrane. The membrane was incubated with a monoclonal c-jbs antibody and with a monoclonal antibody against tubulin to check for consistency in loading and transfer. lmmunodetection mS performed with ECL.

repeated and a c-fos monoclonal antibody was used to detect c-fos (Fig. 2B). In this experiment the testosterone cycloheximide treated group showed a 170% increase in c-fos as compared to the testosterone alone treated group.

Fig. 1. lmmunocytochemical localization of c:fos in the prostatic ducts of 24 h castrated rats. (A) The control shows the staining specificity when the c-fos polyclonal antibody was adsorbed with the antigenic peptide. (B) Random sections were incubated with c-j&r antiserum and stained with the avidin-biotin peroxidase method. Magnification 40 x

3.2. Androgen regulation of c-fos mRNA levels in the presence or in the absence of cycloheximide after I h of treatment Cycloheximide is well known to increase the stability of c-fos mRNA transcripts [27]. Fig. 3 shows the results

G.R. Mora, V.B. Mahesh / Molecular and Cellular Endocrinology 124 (1996) Ill-

of Northern blot analysis of steady-state c-fos mRNA levels in the rat ventral prostate of a 24 h castrated rat after various treatments for 1 h. Testosterone decreased c-fos mRNA levels (Fig. 3, lanes 5-8) as compared to vehicle treated controls (lanes l-4). Cycloheximide when administered alone (Fig. 3, lanes 9-12) or together with testosterone (Fig. 6, lanes 13-16) significantly increased c-fos mRNA levels (P < 0.001) Furthermore, when c-fos mRNA densitometric units obtained from cycloheximide alone were compared to the c-fos mRNA densitometric units obtained from testosterone in combination with cycloheximide, the combined treatment caused a further increase of 63% in c--&s mRNA levels. Undetectable levels of c-fos mRNA were obtained in intact or castrated untreated rats (Fig. 3, lanes 17-24). 3.3. Androgen regulation of AR protein in the presence or in the absence of cycloheximide after 3 h of treatment In order to further examine the role of c-fos in the regulation of AR and AR mRNA levels, it was of interest to study changes in rats castrated for 24 h and treated with testosterone and cycloheximide at a time period when the animals recover from the effects of cycloheximide. A previous study with a limited data based on cytosolic AR had suggested that 3 h may be an appropriate time of study [20]. Therefore, the effects of testosterone in the presence of cycloheximide were investigated 3 h after their administration by immunocytochemical analyses.

IIIII

Vehicle

1 2

3

Testosterone 4

5

6

7

Cycloheximide 8

Cycloheximide Castrated t Testosterone (no treatment) I-w 13 14 15 16 17 18 19 20

9

10 11 12

Intact

I20

115

Control studies showed absence of nuclear staining in prostatic ductal sections from intact rats when the antigenic peptide (Fig. 4A) was used to preadsorb the PG-21 peptide antiserum. The two control groups, vehicle (ethanol:saline) and cycloheximide alone (400 ,ug/ 100 g body wt.) show low AR levels. (Fig. 4B and Fig. 4C). However, we found that 3 h of testosterone treatment (400 pg/lOO g body wt.) resulted in significantly elevated AR levels (Fig. 4D), similar to what it was found previously after 1 h of treatment [ 171. However, in contrast to the previous 1 h study, cycloheximide did not block the androgen-induced upregulation of AR, indicating that the tissue had recovered from the effects of cycloheximide with respect to AR by 3 h (Fig. 4E). 3.4. Androgen regulation of AR mRNA and c-fos mRNA levels in the presence or in the absence of cycloheximide after 3 h of treatment To determine whether an increase on AR mRNA levels accounted for the AR protein levels observed when testosterone was administered in the presence or in the absence of cycloheximide. AR mRNA levels were determined 3 h after treatment by Northern blot analysis. As shown in Fig. 5, AR mRNA steady-state levels were higher in the ventral prostate of animals treated with testosterone in the presence of cycloheximide (as percentage of B-actin 208.6 L- 69.5), as compared to intact controls (41.0 + 5.7), vehicle (11.6 f 8.8) and testosterone treated (50.8 +_1.5) groups. Even though the sample number were two in each treatment, the 4-fold increase in the testosterone plus cycioheximide treated group as compared to testosterone treatment is a sharp contrast from the 5-fold decrease after 1 h of the combined treatment reported earlier [17]. The superinduction of c--&s mRNA levels by cycloheximide alone or in the presence of testosterone was maintained after 3 h, as shown in Fig. 5. However, in contrast to the 1 h values (Fig. 3), the c-j& mRNA levels in the cycloheximide-testosterone group were comparable to those with cycloheximide alone. 3.5. Androgen regulation of c-fos levels in the presence or absence of cycloheximide after 3 h of treatment

21 22 23 24

c-fos mRNA p-actin mRNA Fig. 3. Northern blot analysis of c-fos mRNA levels in ventral prostate of 1 day castrated rats that received various treatments for 1 h. 3 pg of poly A+ RNA loaded in each lane were transferred to a nylon membrane and sequentially hybridized with 3ZP-labeled c-fos and /I-actin cDNA probes. RNA was isolated from four individual animals for each treatment group.

The results of c-fos levels after 3 h of testosterone or cycloheximide treatment or a combination of the two using a polyclonal antibody are shown in Fig. 6. The vehicle treated control values are comparable after 1 h (Fig. 2A) or 3 h (Fig. 6) of treatment. However, the c-fos levels in the testosterone-cycloheximide treated group at 3 h (Fig. 6) are 51.7% of those found at the 1 h time period (Fig. 2A). This is the time when both AR levels (Fig. 4) and AR mRNA levels (Fig. 5) have recovered from the effects of cycloheximide.

116

G.R. Mom, V.B. Mahesh / Molecular and Cellular Endocrinology 1.24 (1996) Ill-120

A: Control

B: Vehicle

C: Cycloheximide

D: Testosterone

E: Testosterone

+ Cycloheximide

Fig. 4. lmmunocytochemical detection ‘of AR in the ventral prostate of 24 h castrated rats that received various treatments for 3 h. The control shows the staining specificity when the PG-21 antibody (2 fig/ml) was adsorbed with antigenic peptide at 50 x M excess (A). AR was detected in the epithelial cells of prostatic ducts of 24 h castrated rats treated with vehicle (B), cycloheximide (C), testosterone (D) and testosterone together with cycloheximide (E) using the avidin-biotin peroxidase method. Sections were lightly counterstained with matoxylin. Magnification, 40 x

3.6. Age dependency and c-fos mRNA

on the androgen

regulation

of AR

Because of the relationship between androgens and AR and C-SOSlevels, it was of considerable interest to study AR mRNA and c-fos mRNA levels during development when testosterone levels change for the low pubertal levels to the adult levels. Furthermore, it has been shown in a previous study that AR mRNA expression in the liver is regulated by age-dependent factors [l&19]. Thus whether this phenomenon occurred in the ventral prostate was of considerable interest and was investigated. Serum testosterone increased in an age dependent manner reaching adult levels at day 55 (Fig. 7A). AR mRNA was found to be present

at low basal levels from days 25 to 55 of age and showed a dramatic increase on day 85 of age (Fig. 7B). c-fos mRNA levels were found to be at low basal levels at all ages studied (Fig. 7B).

4. Discussion Circulating androgens regulate the growth and secretory activity of the prostate gland. A correlation has been shown to exist between AR levels and sensitivity of the gland to androgens. Therefore, a fine regulatory mechanism of the AR should be operational to prevent overgrowth or oversecretion. Evidence obtained during recent years has demonstrated that steady-state AR

G.R. Mora. V.B. Mahesh / Molecular and Cellular Endocrinology

124 (1996) I I I - 120

mRNA AR

P-actin 25

35

55

45

85

Days after birth

B

he

Fig. 5. Northern blot analysis of AR mRNA and c-fos mRNA levels in ventral prostate of 1 day castrated rats that received various treatments for 3 h. 3 pg of poly A+ RNA loaded in each lane were transferred to a nylon membrane and sequentially hybridized with 3’P-labeled AR, c-fos and /I-actin cDNA probes. This experiment was performed in duplicate.

mRNA levels are elevated after androgen withdrawal, [3,4,27,28]. However, in spite of this increase in AR mRNA levels with androgen ablation, AR protein levels are decreased which result in a decrease in tissue sensitivity [29,30]. This decline in AR protein and tissue responsiveness can be reversed by androgen administration. With this treatment the increased post-castration

c-fos/Tubulin

1.271.360.52 1.68

Fig. 6. Western blot analysis for c-Jbs in ventral prostate of 1 day castrated rats that received various treatments for 3 h. See Fig. 2A for details.

c-fos mRNA

Fig. 7. Serum testosterone levels and Northern blot analysis of AR and c-fos mRNA in ventral prostate of intact animals of varying ages. (A) Each bar represents serum levels (n = 4) of samples in duplicate + SE. * Adult serum testosterone levels are reached 55 days after birth. (B) 3 ,ug of poly A + RNA loaded in each lane were transferred to a nylon membrane and sequentially hybridized with 22P-labeled c-fos and AR cDNA probes. and with a 28s rRNA oligoprobe.

AR mRNA levels return to intact levels [31]. Previously we have shown that a dramatic decrease in AR protein occurs 1 day after castration in the epithelial cells of the young adult rat prostatic ducts. Testosterone administered to castrated rats restored ventral prostate AR protein after 1 h. However, this restoration was blocked by cycloheximide [I 71 suggesting that AR autoregulation involved a protein synthesis-mediated mechanism. Cycloheximide blocks protein synthesis by inhibiting the peptidyl-transferase activity of the 60s ribosomal subunit. The effect of cycloheximide on blocking protein synthesis does not appear to be present at 3 h after its administration because castrated rats treated with a combination of cycloheximide and testosterone show the same degree of AR staining of epithelial cells as those treated with testosterone

118

G.R. More,

V.B. Muhesh 1 Molecular ond Cellular Endocrinology 124 (1996) II I- I20

alone. This increase in AR staining as compared to 1 h of treatment is accompanied by a significant increase in steady-state AR mRNA levels suggesting that AR replenishment occurs at least in part by de novo AR synthesis. In our previous work it was demonstrated that testosterone maintained the steady-state levels of AR mRNA after 1 h of treatment in the castrated rat. However, in the ‘presence of cycloheximide there was a dramatic reduction of steady-state AR mRNA levels. Since there appeared to be no change in the transcription rate during this period as shown by the use of actinomycin D [17], it was postulated that testosterone induced a short half-life protein that prevented the degradation of AR mRNA. In the absence of such an induction by the use of the protein synthesis inhibitor cycloheximide, AR mRNA was rapidly degraded. An increase in AR mRNA by 3 h of the combined testosterone-cycloheximide treatment suggests that in addition to increased transcription, the protein that stabilized AR mRNA had reappeared. The half life of this protein could be as short as I-2 h because effects observed at 1 h were no longer present at 3 h. c-fos protein binds to the consensus sequence for HeLa cell activator protein 1 [32] and directly participates in the transcriptional activation or transcriptional repression of different genes [33,34]. c-fos mRNA belongs to a group of messages termed as immediate early gene (IEG) mRNAs. After acute stimulation, the transcription of the c-fos gene is very rapid and maximum message levels are found within 15 min [35]. Several theories have been proposed for the superinduction of c-jbs and c-&n mRNAs in the presence of cycloheximide. The theory of existence of labile repressors has been challenged with experiments showing that cycloheximide induces a delay in shutting off transcription and increases message stability [36]. Experiments presented herein have also shown the induction of c-fos in the presence of cycloheximide. However, testosterone further superinduce c-jos expression in the presence of cycloheximide. The specific colocalization of AR and c-fos in the nuclei of the epithelial cells of the prostate suggests that this two proteins could directly or indirectly interact during testosterone action. Along these lines, our results show that protein synthesis regulation of the expression of these two transcription factors is oppositely modulated by androgen. The low AR protein levels, previously shown at 1 h. when testosterone is administered together with cycloheximide, are accompanied by higher levels of c-fos protein. These findings suggest that androgens downregulation of c-jos stability is protein synthesis dependent. At the same time, given our findings that androgen-increased AR protein is lowered by cycloheximide, it can be hypothesized that c-j&s could then be participating in down-regulating AR expression. Negative regulation with other

members of the steroid receptor superfamily has been reported, such as the mutual inhibition between the action of GR and API [37,38] or retinoic acid and APl [39]. This negative regulation between GR and AP-1 transcription factors appears to be mediated by direct protein-protein interaction [40]. However, it should be pointed out that this attractive hypothesis, which has the potential of explaining the role of c-jos in androgen action requires further confirmation. In fact, a rapid androgen regulation of calcium influx has been observed in LNCaP cells [41] and recently it has been shown that calcium downregulates AR expression in this system [42]. Decreased c-j& expression with calcium channel blocking agents in the prostate gland of castrated rats has been noted [15]. Therefore, these effects may also be a consequence of decreased synthesis of specific androgen induced calcium binding/effecter proteins that oppositely modulate c-fos and AR expression. A study of the relationship between changes in androgen levels and changes in AR mRNA and c-fos mRNA level between the ages of 25 to 85 days led to the interesting finding was that levels of AR mRNA in the ventral prostate change dramatically by 85 days of age even though adult levels of serum testosterone are reached by day 55 of age. These results indicate that shift in greater amounts of AR mRNA observed by day 85 of age is not triggered by testosterone but by other age-dependent factor. In the liver, the rat AR gene promoter contains two adjacent regulatory sites involved in the age dependent, expression of the AR gene [l&19]. These cis elements bind the ubiquitous age dependent factor (ADF) and another liver restricted associated factor (AF). Binding of these proteins to their elements correlate with the expression of AR mRNA in the liver. Interestingly, as in our studies, these investigators did not find a correlation between ADF-AF binding and androgen levels, suggesting as well that a signal other than circulating androgens is regulating ADF-AF activity and in turn AR gene expression. Since during the period of sexual development the c-fos mRNA levels are present only at basal levels, it appears that androgens may maintain androgen receptor levels and androgen sensitivity by continuous suppression of the repressor c-fos. In conclusion, the findings that c-fos and AR are oppositely regulated by testosterone and cycloheximide treatments suggest that androgens regulate an effector protein(s) involved in increasing the stability of AR and/or the translation of AR mRNA while on the contrary decreasing the stability of c-j&~. Therefore, the androgen regulation of AR and c-j& expression could be interdependent and may involve c-fos as a negative effector of AR autoregulation in ventral prostate. Finally, in addition to testosterone, the prostate gland seems to have an age dependent factor involved in enhancing the expression of the AR gene.

References [II Rundlett.

S.E., Wu. X.-P. and Miesfeld, R.L. (1990) Functional characterizations of the androgen receptor confirm that the molecular basis of androgen action is transcriptional regulation. Mol. Endocrinol. 4, 708-714. W.G.. Lubahn. D.B.. French, F.S. PI Quarmby. V.E.. Jarbrough. and Wilson, E.M. (1990) Autologous down-regulation of androgen receptor messenger ribonucleic acid. Mol. Endocrinol. 4, 22-28. A.. Wilson, C.M.. Wilson. J.D., Allman. D.R. and PI Krongrad. McPhaul. M.J. (1991) Androgen increases androgen receptor protein while decreasing receptor mRNA in LNCaP cells. Mol. Cell. Endocrinol.

76. 79 -88. M.C. and Janne. 0. (1990) Regulation of androgen receptor protein and mRNA concentrations by androgens in rat ventral prostate and seminal vesicles and in human

[41 Shan, L.X., Rodriguez.

hepatoma cells. Mol. Endocrinol. 4. 1636 - 1646. D.A., Herzinger. T.. Hermeking. H.. Blaschke,

[51 Wolf,

D. and

Horz. W. (1993) Transcriptional and posttranscriptional regulation of human androgen receptor expression by androgens. Mol. Endocrinol. 7, 924 936. 161 Mizokami, A., Yeh, S.-Y. and Chang, C. ( 1994) Identification of 3’.5’-cyclic adenosine monophosphate response element and other ciA-acting elements in the human androgen receptor gene promoter. Mol. Endocrinol. 8. 77-88. M.E.. Lindzey. J., Kumar, M.V. and Tindall, D.J. [71 Grossmann. ( 1994) The mouse androgen receptor is suppressed by the 5’-untranslated

region

of the gene. Mol. Endocrinol.

8, 448-455.

PI Grossmann.

M.E. and Tindall, D.J. (1995) The androgen receptor is transcriptionally suppressed by proteins that bind singlestranded DNA. J. Biol. Chem. 270, 10968-10975. [91 Mizokami. A. and Chang, C. (1994) Induction of translation by the 5’.untranslated region of human androgen receptor mRNA. J. Biol. Chem. 269, 25655-25659. K.L., Maiorino. C.A.. Dai, J.L., Cameron. D.J. ( 1995) Androgen and glucocorticoid regulation of androgen receptor cDNA expression. Mol. Cell. Endocrinol. 115.177- 186. t111 Kaneko. K.J., Furlow, J.D. and Gorski, J. (1993) Involvement of the coding sequence for the estrogen receptor gene in autologous l&and-dependent down-regulation. Mol. Endocrinol. 7, 879 888. K.L.. Jewell. C.M.. Sar. M. and Cidlowski, J.A. [I21 Burnstein.

[lOI Burnstein.

( 1994) lntragenic sequences of the human glucocorticoid receptor complementary DNA mediate hormone-inducible receptor messenger RNA down-regulation through multiple mechanisms. Mol. Endocrinol. 8. I764 1773. (131 Schuchard.

M., Landers.

( 1993) Steroid hormone Endocr.

J.P., Sandhu. N.P. and Spelsberg, T.C. regulation of nuclear proto-oncogenes,

Rev. 14, 659-669.

iI41 Wolf, D., Kohlhuber.

F.. Schulz. P.. Fittler, F. and Eick, D. (1992) Transcriptional down-regulation of c-myc in human prostate carcinoma cells by the synthetic androgen mibolerone. Br. J. Cancer 65. 376-382. [15] Buttyan. R.. Zakeri. Z., Lockshin, R. and Wolgemuth. D. (1988) Cascade induction of c_fOs, c-nzyc and heat shock 70 K transcripts during regression of the rat ventral prostate gland. Mol. Endocrinol. 2, 650-657. [16] Katz, A.E.. Benson, M.C., Wise. G.J.. Olsson. C.A., Bandyk. M.G., Sawczuk. I-S.. Tomashefsky. P. and Buttyan, R. (1989) Gene activity during the early phase of androgen-stimulated rat prostate regrowth. Cancer Res. 49, 5889-5894. [17] Mord. G., Prins, G. and Mahesh, V.B. (1996) Testosterone regulation of the androgen receptor is protein synthesis mediated. J. Steroid Biochem. Mol. Biol. (in Press).

[I 81 Song. C.S., Rao. T.R.. Demyan. W.F., Mancini. M.A.. Chatterjee. B. and Roy. A.K. (1991) Androgen receptor messenger ribonucleic acid (mRNA) in the rat liver: changes in mRNA levels during maturation, aging, and calorie restriction. Endocrinol. 128. 349-356. [19] Supakar. P.C., Song, C.S., Jung, M.H.. Slomczynska. M.A.. Kim. J.-M., Vellanoweth, R.L., Chatterjee, B. and Roy. A.K. (1993) A novel regulatory element associated with age-dependent expression of the rat androgen receptor gene. J. Biol. Chem. 268. 26400 26408. [20] Steinsapir, J.. Evans. A.C.. Bryhan. M. and Muldoon. T.G. (1985) Androge receptors dynamics in the rat ventral prostate. Biochem. Biophys. Acta 842. I II. [21] Sugimura, Y.. Cunha. G.R. and Donjacour. A.A. (1986) Morphogenesis of ductal networks in the mouse prostate. Biol. Reprod. 34, 961 97 I. [22] Prins, J.. Birch. L. and Greene. Cr. (1991) Androgen receptor localization in different ceil types of the adult rat prostate. Endocrinol. 129, 3187.-3199. [23] Davis. L.C.. Dibner. M.D. and Battey. J.F. (eds.) (1986) Selection of poly(A + )RNA on oligo(dT) cellulose In: Basic Methods in Molecular Biology Section I l-3. pp. 139- 142. [24] Carmichael, G.G. and McMaster. G.K. (1980) The analysis of nucleic acids in gels using glyoxal and acridine orange. Methods Enzymol. 65. 380 -391. [25] Bradford. M. (1976) A rapid and sensitive method for quantitation of microgram quantities of protein utilizing Ihe principle of protein-dye binding. Anal. Biochem. 72, 248 754. [26] Laemmli. U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680 -685. [27] Blok. L.J.. Bartlett, J.M.S.. Bolt-de Vries. J., Themmen, A.P.N.. Brinkmann. A.O., Weinbauer. G.F.. Nieschlag. E. and Grootegoed. J.A. (1992) Effect of testosterone deprivation on expression of the androgen receptor in rat prostate. epididymis and testis. Int. J. Androl. 15, 182- 198. [28] Burguess. L.H. and Handa. R.J. (1993) Hormonal regulation of androgen receptor mRNA in the brain and anterior pituitary gland of the male rat. Mol. Brain Res. 19. 31-38. [29] Greenstein, B.D. (1979) Androgen receptors in the rat brain. anterior pituitary gland and ventral prostate gland: effects of orchidectomy and aging. J. Endocrinol. 81. 75 81. [30] Prins. G. and Birch, L. (1993) lmmunocytochemical analysis of androgen receptor along the ducts of the separate rat prostate lobes after androgen withdrawal and replacement. Endocrinol. 132, 169-178. [31] Prins, G.S. and Woodham. C. (lY95) Autologoua regulation ol androgen receptor mRNA in the separate lobes of the rat prostate gland. Biol. Reprod. 53. 609- 619. [33] Franza. Jr. B.R.. Rauscher. III F.J.. Josephs. S.F. and Curran. T. (1988) The fos complex and fos-related antigens recognize sequence elements that contain AP-I binding sites. Science 239. 1150~1153. [33] Distel. R.J.. Ro. H.-S.. Rosen. B.S.. Groves. D.L. and Spiegelman, B.M. (1987) Nucleoprotein complexes that regulate gene expression in adipocyte differentiation: direct participation of c-fos. Cell 49. 835. 844. [341 Diamond, M.I.. Miner. J.N.. Yoshinaga. S.K. and Yamamoto. K.R. (1990) Transcription factor interactions: Selectors of positive or negative regulation from a single DNA element. Science 249. 1266&127’. [351 Greenberg. M.E. and Ziff, E.B. (1984) Stimulation of 3T3 cells induces transcription of the c,-fo.\ proto-oncogene. Nature 311. 433 -438. L.C. (1992) Protein synthesis [361 Edwards. D.R. and Mahadevan. inhibitors differentially superinduce c-fbs and c-jlaz by three distinct mechanisms: lack of evidence for labile repressors. EMBO J. I I. 2415.-2424.

120

G.R. Mora, V.B. Mahesh / Molecular and Cellular Endocrinology

[37] Lucibello, F.C., Slater, E.P., Jooss, K.U., Beato, M. and Miiller,

R. (1990) Mutual transrepression of Fos and the glucocorticoid receptor: involvement of a functional domain in Fos which is absent in FosB. EMBO J. 9, 2827-2837. [38] Jonat, C., Rahmsdorf, H.J., Park, K.-K., Cato, A.C.B., Gebel, S., Ponta, H. and Herrlich, P. (1990) Antitumor promotion and antiinflammation: down-modulation of AP-1 (Fos/Jun) activity by glucocorticoid hormone. Cell 62, 1189- 1204. [39] Yang-Yen, H.-F., Zhang, X.-K, Graupner, G., Tzukerman, M., Sakamoto, B., Karin, M. and Pfahl, M. (1991) Antagonism between retinoic acid and AP-1: implications for tumor promotion and inflammation. New Biol. 3, 1206-1219.

124 (1996) 11 l-120

[40] Yang-Yen, H.-F., Chambard, J.-C., Sun, Y.-L., Smeal, T., Schmidt, T.J., Drouin, J. and Karin, M. (1990) Transcriptional interference between c-jun and the glucocorticoid receptor: mutual inhibition of DNA binding due to direct protein-protein interaction. Cell 62, 1205-1215. [41] Steinsapir, J., Socci, R. and Reinach, P. (1991) Effects of androgen on intracellular calcium of LNCaP cells. Biochem. Biophys. Res. Commun. 179, 90-96. [42] Gong, Y., Blok, L.J., Perry, J.E.. Lindzey, J.K. and Tindall, D.J. (1995) Calcium regulation of androgen receptor expression in the human prostate cancer cell line LNCaP. Endocrinol. 136, 21722178.