Autoregulation of the androgen receptor at the translational level:

Steroids 64 (1999) 587–591

Autoregulation of the androgen receptor at the translational level: Testosterone induces accumulation of androgen receptor mRNA in the rat ventral prostate polyribosomes Gloria R. Moraa,*, Virendra B. Maheshb a

b

Department of Urology Research, Mayo Clinic, 200 First St. SW, Rochester, Minnesota 55905, USA Department of Physiology and Endocrinology, Medical College of Georgia, Augusta, Georgia 30901-3000, USA

Abstract Several studies have documented that androgens have the ability to autoregulate their own receptor levels; however, the mechanism of such autoregulation remains poorly understood. Along these lines, our laboratory has shown that testosterone increased androgen receptor (AR) protein levels and binding in the castrated rat ventral prostate within 1 h. Ongoing protein synthesis was required for the testosterone effect, as the protein synthesis inhibitor cycloheximide blocked this effect. Testosterone and/or actinomycin D, an mRNA synthesis inhibitor, did not affect the steady-state AR mRNA levels. Therefore, we suggest that the early events induced by testosterone are posttranscriptional and that protein synthesis is required for the maintenance of AR protein and AR mRNA levels. In addition, we hypothesize that the testosterone posttranscriptional effect is primarily through the sequestering of AR mRNA in the prostate polyribosomes. To test this hypothesis, total RNA was isolated from prostate polyribosomes of controls and testosterone-treated rats and AR mRNA levels were quantitated by competitive reverse transcription–polymerase chain reaction. Polyribosomes profiles on linear sucrose gradients showed no difference in the sedimentation characteristics of ribosomal particles from the vehicle-treated control or testosterone-treated animals. Furthermore, because both polyribosomal preparations can direct protein synthesis to the same extent in a cell-free system, testosterone does not increase the efficiency of translation. However, competitive reverse transcription–polymerase chain reaction revealed that testosterone increases AR mRNA associated with polyribosomes by threefold after 1 h of treatment compared with control. These data suggest a rapid testosterone-mediated posttranscriptional mechanism, in which testosterone regulates the stability of the AR mRNA by sequestering it in polyribosomes, and consequently increasing its translation. © 1999 Elsevier Science Inc. All rights reserved. Keywords: Androgen receptor mRNA; Rat ventral prostate; Polyribosomes; Quantitative reverse transcription–polymerase chain reaction

1. Introduction Androgens regulate the growth and secretory activity of the prostate gland by binding to high-affinity intracellular androgen receptors (AR), which in turn activate gene transcription [1]. Previous data from several laboratories have indicated that androgens are capable of modulating their own receptor levels; however, the overall mechanism for this action is unclear [2– 8]. For instance, androgen readministration to castrated male rats, or human androgendepleted AR-positive prostate cells in culture, causes downregulation of AR mRNA levels. In contrast, in most cases the AR mRNA changes observed in androgen-sensitive tis* Corresponding author. Tel.: ⫹1-507-284-8138; fax: ⫹1-507-2842384. E-mail address: [email protected] (G.R. Mora)

sues or human cell lines do not parallel changes in AR protein [2– 8]. Along these lines, we have shown that 24 h after castration, a significant decrease in the AR protein level occurs in the rat ventral prostate; conversely, the AR mRNA levels were significantly elevated [8]. When testosterone was administered intraperitoneally to the castrated rats, it rapidly restored the AR protein in the epithelial cells of the ventral prostate, without altering the cellular AR mRNA steady-state levels. This AR protein accumulation required de novo protein synthesis without transcriptional activation of the AR gene [8]. Based on these findings, because testosterone induced a net increase in AR protein without changing the AR mRNA levels, we hypothesize that testosterone induces AR synthesis by stabilizing the AR mRNA during active protein synthesis and/or that testosterone may induce AR mRNA accumulation in the polyribosomes, the site of protein

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synthesis. This may increase the total efficiency of translation and/or preferentially increase the AR mRNA translation resulting in augmented AR protein in the rat ventral prostate. In the present study, we set out to determine whether total efficiency of translation is increased after testosterone treatment and whether levels of AR mRNA in prostatic polyribosomes of 24-h castrated rats differ from those that received a testosterone replacement. A competitive quantitative reverse transcription–polymerase chain reaction (RT-PCR) was designed specifically to compare AR mRNA levels in isolated polyribosomes. Our results demonstrate that total efficiency of translation does not change after testosterone treatment and that the posttranscriptional regulation of AR by testosterone involves sequestering AR mRNA into the prostatic polyribosomes. This preferential testosterone-induced accumulation of AR mRNA in polyribosomes might explain the findings in our experimental model, e.g. increased AR synthesis but AR mRNA steadystate levels remain unaltered.

2. Materials and methods 2.1. Animals Experiments were performed on adult male, virus-free, 80- to 90-day-old, Sprague–Dawley rats obtained from Harlan (Madison, WI, USA). Animals were fed with Purina chow and tap water ad libitum and were allowed a 2-day acclimatization period in an air-conditioned, light-controlled room with a 12-h light/dark cycle. Bilateral orchidectomy was performed via the scrotal route under ether anesthesia 24 h before each experiment. Groups of six animals were treated intraperitoneally with vehicle (1:1, ethanol/saline) or testosterone (400 ␮g/100 g body weight) 1 h before they were killed. Injection volumes containing the doses required per rat were adjusted to 1:1000 of the rat’s body weight to avoid volume changes and/or excess of ethanol. All animal studies performed 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 National Institutes of Health and the US Department of Agriculture. 2.2. Isolation and characterization of polyribosomes Ventral prostate polyribosomes from castrated rat controls (vehicle) or rats treated with testosterone were isolated following conventional cell fractionation techniques previously described [9,10]. These techniques were adapted to the prostatic tissue as follows. All procedures were performed at 4°C in a cold room. Tissue from individual rats was homogenized with a Polytron PT-10 homogenizer in isotonic Tris buffer (0.25 M sucrose, 5 mM MgCl2, 100 mM KCl, 1 mM dithiothreitol, 1 ␮M leupeptin, 1 ␮M aprotinin, and 500 ␮g/ml sodium heparin in 50 mM Tris buffer, pH

7.4, at 4°C, Reagents from: Sigma, St. Louis, MO, USA; Fisher Scientific, Pittsburgh, PA, USA), at a buffer/tissue ratio of 0.5 to 1 ml/prostate. The homogenate was centrifuged at 14 600 ⫻ g for 10 min. The resultant supernatant was adjusted to 1% Nonidet P-40 and 1% sodium deoxycholate with fresh detergent mixture. Aliquots of the detergent-treated supernatant were overlayed on a discontinuous sucrose gradient (2.5 M sucrose and 1.0 M sucrose). The layered supernatant was centrifuged at 201 000 ⫻ g for 90 min at 4°C, and the polyribosomes showed as an opalescent band at the 2.5 M sucrose interface. After dilution with isotonic Tris buffer, polyribosomes were centrifuged at 325 000 ⫻ g for 5 min at 4°C, resuspended in homogenization buffer, and the ratio A260/A280 determined (which, under these conditions, is ⱖ1.95). Sedimentation of total polyribosomes was performed on 15 to 55% linear sucrose gradients centrifuged for 6 to 8.5 h at 4°C at 27 000 rpm in a Beckman SW 28 rotor. The polyribosome profile was determined by absorbance at 254 nm, using a density gradient fractionator (ISCO-640) attached to an absorbance monitor (ISCO-UA-5). To assess for the functional activity of the isolated polyribosomes, the kinetics of incorporation of [35S]methionine were measured by using a cell-free system (wheat germ and rabbit reticulocyte lysate system), according to the manufacturer’s instructions (Promega, Madison, WI, USA). Ten A260 units of polyribosomes were used in each translation reaction in the presence of 7-methylguanosine-5⬘monophosphate, an inhibitor of translational initiation [11]. The reaction mixture was supplemented with 40 units of rRNasin ribonuclease inhibitor, 1 mM amino acid mixture (without methionine), and [35S]methionine at 0.8 mCi/ml. At various times aliquots were taken and labeled amino acid incorporation was measured in hot trichloroacetic acid– insoluble material in the presence of 0.1 M unlabeled methionine [12]. Measurements of each time point were performed in duplicate, using two different polysomal preparations for each group. 2.3. RNA isolation from polyribosomes Total polyribosomal RNA was isolated with RNAzol (Biotex Laboratories, Houston, TX, USA), using 30 to 60 A260 units of polyribosomal ventral prostate RNA preparations from the sucrose gradients. A minimum of six rats per group was required to collect the polyribosomal fractions. 2.4. Quantitative RT-PCR The apolipoprotein B (ApoB) gene is not naturally expressed in the prostate; thus, it was used as internal standard to correct for technique variations in polyribosomal AR mRNA measurements. ␤-Actin was used as a housekeeping gene to control for loading variations; previously we have shown that its expression does not change after 1 h of testosterone treatment [8]. Internal standards for competi-

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Fig. 1. Sedimentation of rat ventral prostate polyribosomes in a continuous sucrose gradient. Fractions were collected from the top, sedimentation is left to right. Twenty-five A260 units were layered over a linear sucrose gradient (15–55%) and centrifuged for 6 to 8.5 h at 27 000 rpm in a Beckman SW 28 rotor. After centrifugation, the absorbancy at 254 nm was monitored by an ISCO fractionator coupled to an ultraviolet monitor.

tive RT-PCR for AR and ␤-actin were designed as previously suggested [13,14]. AR and ␤-actin–attached ApoB cDNA fragments flanking T7 sequences were synthesized by PCR by using rat liver cDNA and specific composite primers (AR composite forward primer: 5⬘-GAGGATCCTAATACGACTCACTATAGGGGTTGGCGGTCCTTCACTAATGTTCTCGACTTCCAACGATCAT-3⬘; AR composite reverse primer: 5⬘-TTGATTTTTCAGCCCATCCACTGACCTTGGAGACACGATCTG-3⬘; ␤-actin composite forward primer: 5⬘-GGGAGGAGGGCGGACAACCTCCTTGCAGCTCCTCTCGACTTCCAACG-3⬘; ␤-actin composite reverse primer: 5⬘-ACAATGCCGTGTTCAATGGAAAGCCTTGTTGACACTG-3⬘). The amplified cDNA fragments were gel-purified and in vitro transcribed to generate cRNA internal standards, as per the manufacturer’s protocol (Ambion, Maxiscript kit). Five to 1000 ng of polyribosomal RNA together with 2 ng AR cRNA and 25 ng ␤-actin cRNA internal standards were reverse-transcribed by using Moloney murine leukemia virus reverse transcriptase and random hexamers at 42°C for 1 h. A primer pair specific for AR cDNA (forward primer: 5⬘-GGTTGGCGGTCCTTCACTAATGT; reverse primer: 5⬘-TTGATTTTTCAGCCCATCCACTG) was used to amplify a 225-bp AR cDNA fragment and a 381-bp of ApoB–AR cDNA fragment, at 63°C for 30 cycles. A primer pair specific for ␤-actin cDNA (forward primer: 5⬘-TACAACCTCCTTGCAGCTCC-3⬘; reverse primer: 5⬘ACAATGCCGTGTTCAATGG-3⬘) was used to amplify a 280-bp ␤-actin cDNA fragment and a 334-bp of ApoB–␤actin cDNA fragment, at 58°C for 30 cycles. The PCR profile used was as follows: 3 min at 94°C for 1 cycle, 1 min at 94°C, 1 min at 58°C, 1 min at 72°C, followed by 10 min at 72°C. Fragments were separated on a 3% metaphor/ ethidium bromide gel in single-strength Tris/borate/EDTA

buffer. Ethidium bromide intensities in each band were quantitated with a digital imaging system, IS-1000 (Alpha Innotech Corp., San Leandro, CA, USA). Calibration curves were created by using logarithmic values of the target versus the cRNA standard for AR and ␤-actin. By using the curve parameters, the ratio of AR mRNA versus ␤-actin mRNA was calculated as previously described [13,14].

3. Results 3.1. Characterization of intact polyribosomes To ensure the integrity of the isolated polyribosomes, their absorbancy profiles were followed at 254 nm on 15 to 55% linear sucrose gradients. Fig. 1 shows that identical sedimentation profiles are found in the ventral prostate of control rats (vehicle) and rats treated with testosterone. The largest peak corresponds to 80 S monosomes, and polyribosomes of up to 9 monosomes can be distinguished. 3.2. Translation efficiency of isolated polyribosomes The capability of isolated polyribosomes to perform protein synthesis was tested by measuring the kinetics of incorporation of [35S]methionine into the trichloroacetic acid– insoluble material in a cell-free system (Fig. 2). Similar results were obtained by using a rabbit reticulocyte lysate or a wheat germ cell-free system. The results shown in Fig. 2 are representative of results using the wheat germ system. Fig. 2 shows that polyribosomes isolated from both groups, control as well as testosterone treated, can direct protein synthesis in vitro to the same extent. Therefore, testosterone does not induce a general increase in the efficiency of

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Fig. 2. Kinetics of incorporation of [35S]methionine into newly synthesized proteins. Ten A260 units of polyribosomes were used in each translation reaction, and at various times, aliquots were taken and labeled amino acid incorporation was measured into the trichloroacetic acid–insoluble material. Each time point represents the mean of measurements in duplicate in two different polysomal preparations for each group.

translation. Differences in AR synthesis between control and testosterone-treated polyribosomes could only be demonstrated by further purification of the newly synthesized AR protein by immunoprecipitation. However, numerous attempts to detect the newly synthesized AR protein were unsuccessful presumably because of the small amount of protein being synthesized. We, therefore, took an alternative approach, reasoning that the net increase in AR protein in ventral prostate after testosterone treatment [8], without an increase in the efficiency of translation (Fig. 2), should be proportional to AR mRNA sequestered in polyribosomes, the site of translation. Thus, AR mRNA levels were next examined in ventral prostate polyribosomes from control and testosterone-treated rats. 3.3. Quantification of AR mRNA in polyribosomes of control and testosterone-treated rats by quantitative competitive RT-PCR Initially, we designed a quantitative RT-PCR by using 28 S rRNA as an internal standard. However, we encountered numerous problems with the calibration curve because of the relatively low levels of AR mRNA and the extremely high levels of 28 S rRNA in polyribosomes. Nevertheless, AR mRNA levels in the ventral prostate polysomes of testosterone-treated rats were found to be 50% higher compared with controls (data not shown). To further quantify AR mRNA levels in the polyribosomes, a quantitative RTPCR with an appropriate internal standard (␤-actin) was used. As shown in Fig. 3, 5 to 1000 ng of polyribosomal RNA from control rat ventral prostate was used to create a calibration curve for AR mRNA and ␤-actin mRNA, using a constant amount of their respective cRNA internal standards. Applying the calibration curve parameters, the relative amount of AR mRNA and ␤-actin mRNA levels were calculated in control and testosterone-treated animals, and

Fig. 3. Quantification of androgen receptor (AR) mRNA in ventral prostate polyribosomes by competitive reverse transcription–polymerase chain reaction analysis. Total RNA was extracted from prostatic polyribosomes from groups of six rats each; 100 ng of polyribosomal RNA from control and testosterone groups was used for quantification. The calibration curve was prepared as described in Methods, and the relative amount of androgen receptor mRNA in each group is expressed as a percentage of ␤-actin.

the AR mRNA levels in polyribosomes were expressed as a relative amount of ␤-actin mRNA. The ratio of AR mRNA/ ␤-actin mRNA in testosterone-treated rat ventral prostate polysomes was threefold higher compared with vehicletreated controls. The same experiment was performed with three independent polysomal preparations and the threefold increase after testosterone treatment was confirmed.

4. Discussion A great deal of evidence supports the process of autoregulation of ARs as an active control mechanism during androgen action. The results in this study indicate that the testosterone regulation of AR in rat ventral prostate 24 h after castration involves regulation of AR mRNA at the translational level. Previously, we have shown that in male rats castrated for 24 h and treated with testosterone for 1 h, steady-state AR mRNA levels were unchanged. Indirect evidence showed an increase in AR protein synthesis after androgen treatment during this time. Evidence from other investigators has shown that in rat and mouse prostate 3 days after castration

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the reduced AR protein and mRNA levels are restored to normal intact levels after testosterone treatment [15]. This effect required 5␣-dihydrotestosterone replacement for 2 to 3 days. In our experimental model, however, a rapid effect of testosterone occurred within 1 h. To understand how androgens increase the synthesis of AR protein without changing AR mRNA levels, we theorized that androgens may increase the amount and/or the efficiency of AR mRNA translation in polyribosomes. The possibility that androgens can regulate the synthesis of a particular protein only at the translational level has been proposed before by other investigators [5,16]. The results obtained in this study provide evidence that this may be applicable to the AR as well. The localization of specific mRNA in the cell is a highly regulated biological process. There is evidence that supports that after a specific signal, mRNAs interact with cytoskeletal elements and can be directed to the cell’s storage sites or to the site of protein synthesis, the polyribosomes [17]. Therefore, we optimized techniques to isolate intact and functional total polyribosomes from the prostate of control and testosterone-treated rats. Testosterone did not increase the kinetics of peptide elongation in prostatic polyribosomes during the first hour after administration. These findings are in sharp contrast to other steroid effects, such as the estrogen effect in polyribosomes from rat uterus, where estradiol increases significantly the kinetics of protein synthesis after 1 h [18]. Nevertheless, the use of competitive quantitative RT-PCR demonstrated that AR mRNA levels accumulate in the prostatic polyribosomes after testosterone treatment. Technical difficulties were encountered in the quantification of AR mRNA levels in polyribosomes by RT-PCR using 28 S rRNA for standardization, and therefore, ␤-actin was used for standardizing ribosomal RNA to mRNA. Overall, these results show that testosterone induces a rapid accumulation of AR mRNA in polyribosomes, the site of protein synthesis. The effect of testosterone results in a net increase of AR protein synthesis. In addition, by sequestering the AR mRNA in polyribosomes testosterone may also increase the AR mRNA stability. These two factors may contribute to the posttranscriptional regulation of AR synthesis by testosterone.

Acknowledgments We thank Ms Lynn Chorich for excellent technical assistance.

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