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Endocrinology Vol. 144, No. 11 4841-4850
Copyright © 2003 by The Endocrine Society

Neonatal Estrogen Down-Regulates Prostatic Androgen Receptor through a Proteosome-Mediated Protein Degradation Pathway

Carl Woodham, Lynn Birch and Gail S. Prins

Department of Urology, College of Medicine, University of Illinois at Chicago, Chicago, Illinois 60612

Address all correspondence and requests for reprints to: Gail S. Prins, Ph.D., Department of Urology, M/C 955, University of Illinois at Chicago, 820 South Wood, Chicago, Illinois 60612. E-mail: gprins{at}uic.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Brief exposure of male rats to estrogens during the neonatal period interrupts normal prostate development, alters epithelial cell differentiation, and predisposes this gland to hyperplasia and severe dysplasia analogous to prostatic intraepithelial neoplasia (PIN) with aging. Previous work demonstrated that the reduced growth, secretory activity, and androgen sensitivity that are observed in the adult ventral lobe are a function of reduced androgen receptor (AR) levels. Down-regulation of AR protein was found to occur immediately following neonatal exposure to estradiol benzoate (EB) and persist through adulthood and aging, indicating a permanent imprint on the ability of the prostate to express normal AR levels. To determine the intracellular mechanism of AR down-regulation by estrogens, the present study examined the effect of neonatal EB on AR gene transcription, mRNA levels, protein translation, and protein degradation in the d 10 ventral prostate glands. Nuclear run-on assays showed no alteration in AR gene transcription following exposure to EB on d 1–5 compared with controls. In situ hybridization and quantitative (q) RT-PCR revealed no difference in mRNA levels in the stromal or epithelial cells in response to estrogen exposure which, taken together, indicate that estrogen down-regulation of AR is mediated at the posttranscriptional level. AR translation was assessed with an in vitro transcription-translation assay in the presence of prostatic lysates from oil and estrogen-exposed animals, and no treatment effect was noted. AR degradation was examined in an in vitro assay validated with adult intact and castrate prostates. Prostatic lysates from intact rats initiated AR degradation with a t1/2 of 2.31 h, whereas proteins from castrate rats accelerated AR degradation to a t1/2 of 1.34 h (P < 0.001). Prostatic lysates from control d 10 prostates induced AR degradation with a t1/2 of 1.49 h, whereas estrogenized prostates increased AR degradation to a t1/2 of 1.11 h (P < 0.001). Proteosome inhibitors MG132 and ALLnL were able to reverse AR degradation induced by prostatic lysates from adult intact and castrate rats as well as from developing and estrogenized prostates, indicating that AR degradation was mediated through the proteosome pathway. Furthermore, the proteosome-mediated AR degradation in the estrogenized d 10 prostate was associated with a marked suppression of Akt phosphorylation that has been linked to AR degradation in other systems. Taken together, the present data show that exposure to neonatal estrogens down-regulates AR protein levels in the ventral prostate gland by accelerating AR degradation, which is mediated through the proteosome pathway.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MORPHOGENESIS OF THE rodent prostate gland is initiated late in fetal life, whereas the majority of growth and differentiation occurs during the postnatal period. It is well established that this process is under the direct influence of several steroid hormones, most notably androgens and, to a lesser degree, estrogens and retinoids (1, 2, 3, 4, 5, 6, 7). Alterations in the balance of these steroid hormones leads to distinctive changes in prostate growth, differentiation, and function. Loss of androgens through castration or administration of antiandrogens at birth results in diminished prostate growth and secretory capacity (1, 8, 9, 10), whereas androgen replacement at various time points can largely reverse these deficits. Significantly, loss of androgens alone does not predispose the prostate to aging-associated diseases such as hyperplasia or cancer and, in fact, may be protective becausee eunuchs do not develop prostate cancer (11).

Brief exposure to high levels of estrogens during the critical developmental period (neonatal d 1–5) has also been shown to retard rat prostate growth, branching morphogenesis, and epithelial differentiation (7). However, unlike androgen ablation, these permanent effects have been associated with an increased incidence of prostatic intraepithelial neoplasia and hyperplasia with aging, suggesting that estrogen imprinting during the neonatal period is a predisposing factor for prostatic disease later in life. The mechanism whereby estrogen mediates these divergent effects is not completely understood. Initial work by Coffey and colleagues (12, 13) established that, in addition to reduced growth, the estrogenized prostate exhibited a reduced activational response to endogenous and exogenous androgens during adulthood. Because the amplitude of androgen sensitivity in the prostate is, in part, a function of androgen receptor (AR) content, we examined AR levels in the neonatally estrogen-exposed rat prostates and observed lobe-specific changes in AR levels. In the adult ventral lobe, AR expression was reduced to 6.4% of control levels, and androgen replacement in adulthood restored this to only 43% of control levels (14). This reduction in AR levels directly paralleled the growth reduction of the ventral lobe to 14% of control levels in estrogenized adult rats and 39% of control levels in those given testosterone. In addition, loss of AR in epithelial cells of estrogenized ventral lobes was directly linked to loss of prostate binding protein expression in those cells (15). Subsequent studies revealed that this permanent effect on AR expression was initiated immediately following estrogen exposure such that at d 10 of life, AR protein was barely detectable by Western analysis and immunocytochemistry in the ventral prostate (16). AR immunostain rapidly declined in epithelial cells and was retained in the cytoplasm of stromal cells at d 6 (17, 18), whereas by d 10, protein levels were very low in both cell types. It is intuitive that loss of prostatic AR during development will result in loss of androgen-driven growth and differentiation of the prostate gland, which is normally mediated through androgen dependent, stromal-derived paracrine factors (5). Thus, immediate and sustained down-regulation of AR protein can explain, in part, the prostatic growth retardation observed following neonatal estrogen exposure.

The present study was undertaken to delineate the intracellular mechanism of AR down-regulation in response to neonatal estrogen exposure. Past work in our laboratory has shown that autoregulation of AR expression in the rat prostate gland is mediated through transcriptional as well as posttranscriptional pathways (19). In the present study, we have evaluated the effects of estrogens on AR gene transcription rate, AR mRNA quantity and distribution, AR translation, and AR degradation to determine the site of estrogenic regulation. Our findings indicate that the primary pathway of AR down-regulation by estrogens as well as by androgen withdrawal in the prostate gland is through proteosome-mediated AR protein degradation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
All rats were handled in accordance with the principles and procedures of the National Institutes of Health Guiding Principles for the Care and Use of Animal Research, and experiments were approved by the Institutional Animal Care Committee. Timed pregnant female Sprague Dawley rats were purchased from Zivic-Miller (Pittsburgh, PA), housed individually in a temperature (21 C) and light (14-h light, 10-h dark) controlled room and fed standard Purina rat chow (Ralston-Purina, St. Louis, MO) ad libitum. They were monitored daily for delivery of pups, and the day of birth was designated as d 0. Pups were sexed according to ano-genital distance, and female pups were removed. All males from a single mother were assigned to one of two treatment groups given sc injections of either 25 µg estradiol benzoate (Sigma, St. Louis, MO) in 25 µl peanut oil or oil alone on neonatal d 1, 3, and 5. Pups from both treatment groups were killed by decapitation on d 10 of life and the accessory sex gland complexes were quickly removed and placed in ice-cold PBS. Ventral prostate lobes were microdissected at 4 C under a dissecting microscope and either snap frozen in liquid nitrogen (RT-PCR and translation studies), positioned longitudinally on a nylon square, covered with OCT compound, frozen in liquefied propane and subsequently stored in liquid nitrogen (in situ hybridization) or kept on ice and used fresh (nuclear run-on assays and degradation studies). For adult studies with testosterone withdrawal, d 90 rats were castrated via the scrotal route under ketamine/xylazine anesthesia [50 mg/kg Ketaset (Brisol Laboratories, Syracuse, NY); and 10 mg/kg Rompum (Mobay Corp., Shawnee, KS)]. Intact and 48-h castrate rats were killed by decapitation and ventral prostates lobes were collected and placed on ice.

Nuclear run-on assays
Nuclear run-on transcription assays were performed as previously described (19). Fresh ventral prostate tissue from d 10 oil and estrogen-treated rats were minced on ice and homogenized in 5.0 ml cell lysis buffer [0.25 M sucrose, 50 mM HEPES (pH 7.4), 5.0 mM MgCl2, 0.1% Triton X-100] at 4 C using a Dounce homogenizer (Baxtor Scientific, McGaw Park, IL). Nuclei were isolated by centrifugation through 1.0 M sucrose [with 50 mM HEPES (pH 7.4) and 5.0 mM MgCl2] at 1000 x g for 10 min. at 4 C. The pellet was resuspended in buffer [40% glycerol, 10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 5 mM MgCl2, and 1 mM dithiothreitol (DTT)] and nuclei were counted with a hemocytometer. Aliquots of an equal number of nuclei (1.5 x 106 to 3.0 x 107) from both groups were mixed in 2x transcription buffer [33% glycerol, 120 mM HEPES (pH 7.0), 4 mM DTT, 50 mM ammonium sulfate, 6 mM magnesium acetate, 6 mM MnCl2, 10 mM NaF, 18 µM creatine phosphate, and 32 µg/ml creatine phosphokinase]. An equal volume of reaction buffer (containing 0.5 mM each of rGTP and rATP, 150 µCi each of [{alpha}-32P]-rUTP and [{alpha}-32P]-rCTP and Inhibit Ace (5Prime->3Prime, Boulder, CO) at 1 U/30 µl) was added and the nuclei were incubated at 26 C for 45 min. The reaction was terminated by treatment with 20 U ribonuclease-free deoxyribonuclease (Promega Corp., Madison, WI) and 10 mM CaCl2 for 15 min at 37 C. The mix was treated with 10% (vol/vol) 10x SET buffer [5% sodium dodecyl sulfate (SDS), 50 mM EDTA, and 100 mM Tris-HCl (pH 7.4)], 10% (vol/vol) proteinase K buffer [2% SDS, 25 mM EDTA, 0.2 M Tris-HCl (pH 7.5), 0.4 M NaCl, 0.4 mg/ml proteinase K] and 100 µg Escherichia coli tRNA and further incubated at 37 C for 30 min. Nascent mRNA transcripts were extracted with 1.0 ml RNAzol B (Tel-Test, Inc., Friendswood, TX) and 10% chloroform, precipitated three times with isopropanol at -30 C and resuspended in 100 µl deionized H2O.

Two micrograms of denatured AR probe (714-bp HindIII/EcoRI fragment of rat AR cDNA), 1 µg pGEM-3Z (negative control) and 1 µg 28S cDNA (positive control) were immobilized on Zeta-Probe GT nylon membranes by slot blot. After incubation for 3 h at 42 C in prehybridization buffer (50% formamide, 5x saline sodium phosphate EDTA buffer, 5x Denhardt’s solution, 0.8% SDS, 100 µg/ml E. coli tRNA), the membranes were hybridized for 72 h at 42 C in 5 ml hybridization buffer (prehybridization buffer with 5% dextran sulfate) containing 0.5–1.5 x 107 cpm of run-on transcripts from either the oil or estrogen-exposed prostates. The membranes were washed three times for 15 min at room temperature, twice at 68 C in moderate salt buffer [2x SSC (single strength = 0.15 M sodium chloride, 15 mM sodium citrate), 0.1% SDS, 1x Denhardt’s without BSA], and three times at 60 C in low salt buffer (0.1x SSC-0.1% SDS). Run-on assays from both treatment groups were performed together and membranes were exposed on a single PhosphorImager plate. Signals were quantitated with a Molecular Dynamics (Sunnyvale, CA) PhosphorImager system, and the AR value was normalized to the 28S signal for each membrane. Six run-on assays were performed and mean values for treatment groups were compared by Student’s t test.

AR mRNA quantitation by RT-PCR
The quantitative (q) RT-PCR assay used to measure rat AR mRNA within small tissue samples involved coamplification of an exogenous standard under noncompetitive conditions and has been previously validated in our laboratory (19). A rat cDNA corresponding to the entire open reading frame (kindly supplied by Dr. S. Liao, University of Chicago, Chicago, IL) was inserted in pGEM-3Z plasmid and mutated by deletion of base pairs 1697–1862 following digestion with SacI. The product, designated as M-165, was linearized with AsuII and the sense strand was transcribed from the T7 promoter with a Riboprobe kit (Promega Corp). The resultant M-165 cRNA was quantitated by absorbance at 260 nm and the number of M-165 cRNA molecules calculated.

Total RNA from four pooled ventral lobes of d 10 oil or estrogen-treated rats was isolated with guanidinium thiocyanate/chloroform extraction (RNAzol B; Tel-Test, Inc.), and 4 µg were spiked with 9.1 x 106 molecules of M-165 cRNA. The mixture was reverse-transcribed in 100 µl PCR buffer (Perkin-Elmer, Norwalk, CT) with 10 mM deoxy-NTPs and 25 mM MgCl2 using the reverse primer and 400 U of murine leukemia virus reverse transcriptase (Promega). Six serial 1:1 dilutions of one eighth of the cDNA mixture were amplified in a Perkin-Elmer Cetus thermal cycler with 2.5 U Taq DNA polymerase (Perkin-Elmer) and 10 µCi [{alpha}-32P]-dCTP (Amersham Pharmacia Biotech, Inc., Piscataway, NJ). The forward primer; 5'-AGTGAAATGGGACCTTGGATGG-3' and reverse primer 5'-GGCTCAATGGCTTCCAGGACAT-3' amplified the AR cDNA between bp +1543 and 2038 and yield 486- and 321-bp products for the wild-type AR and M-165 sequences, respectively. Intact adult ventral prostate (AR positive) and spleen (AR negative) total RNA were included in the amplifications as controls. The radiolabeled DNAs were separated on a 4% NuSieve 3:1 agarose gel (Cambrex Life Sciences, Baltimore, MD) and bands were quantitated with a PhosphorImager system. Because the reaction rates of M-165 cRNA and prostate AR mRNA amplification are identical within the exponential phase of the PCR, this protocol allowed for the construction of a standard curve for M-165 cRNA and extrapolation of AR mRNA molecules in the prostate sample. Data were expressed as the number of AR mRNA molecules present in 100 ng of total RNA. A total of six RNA isolations and RT-PCR assays were performed with the oil and estrogen-treated prostates run in parallel for each assay and mean values for treatment groups were compared by Student’s t test.

In situ hybridization
In situ hybridization for AR mRNA was performed as previously described (19). Frozen 6-µm sections of d 6 and d 10 oil and estrogen-exposed prostates were thaw-mounted in parallel on coated slides (Superfrost Plus, Fisher Scientific, Itasca, IL) to allow for direct comparison of silver grain density between the two treatment groups. Prostates from three animals in each treatment group were analyzed. The slides were fixed in 4% formaldehyde (5 min at room temperature), acetylated for 10 min [0.25% acetic anhydride, 0.1 M triethanolamine, and 0.9% sodium chloride (pH 8.0)], rinsed in 2x SSC and dehydrated in ascending alcohol. Ninety microliters of heat-denatured hybridization solution (50% formamide, 0.25 M NaCl, 1x Denhardt’s solution, 10% dextran sulfate, 25 µg yeast tRNA, 500 µg total yeast RNA, 100 µg sheared salmon sperm DNA, 50 mM DTT, 0.05% sodium thiosulfate, and 0.25% SDS) containing 20 x 106 cpm/ml AR cRNA probe was applied to each slide. The 35S-labeled antisense cRNA probe was transcribed from an EcoRI/SmaI fragment (142 bp) of the rat cDNA subcloned into a pGEM-3Z plasmid. Following incubation for 16–20 h at 60 C in a humidified container, the slides were washed in 2x SSC, treated with ribonuclease for 30 min at 37 C, and rinsed in SSC under increasing stringency conditions with a final wash in 0.1x SSC at 60 C. After dehydration in alcohol, the slides were apposed to ß-max Hyperfilm (Amersham Pharmacia) for 7 d. The slides were then dipped in Kodak (Rochester, NY) NTB-3 emulsion and exposed for 3–5 wk at 4 C before developing. The slides were counterstained with hematoxylin, cleared with xylene and coverslipped with Permount (Fisher Scientific). No hybridization signal was detected on control slides incubated with radiolabeled sense strand RNA probes or in negative control tissue (spleen).

AR translation
To determine whether estrogenic exposure could influence the production of AR protein, an in vitro transcription-translation assay for full-length rat AR was performed in the presence of prostatic extracts from control and estrogen-treated d 10 prostates. Because data obtained from the above experiments had demonstrated that AR mRNA transcription in rat ventral prostates was not influenced by neonatal estrogen exposure, changes in the final AR product of a transcription-translation assay should reflect an estrogenic influence on AR translation. Rat AR (rAR) cDNA containing the entire 5' UTR (untranslated region) (kindly supplied by Dr. A. Roy, University of Texas, San Antonio, TX) was inserted into the KpnI/XbaI site in the multiple cloning region of pGEM-3Z and linearized using XbaI. The AR cDNA was in vitro transcribed and translated using a TNT Coupled Reticulocyte Lysate System (Promega) in the presence of [35S]-methionine (Amersham Pharmacia) and 1 µg cytoplasmic proteins from d 10 oil or estrogen-treated prostates. Cytoplasmic proteins were obtained from pooled d 10 ventral prostates homogenized in 10 mM HEPES (pH 7.5), 40 mM KCl, 3 mM MgCl2, 0.32 M sucrose, 1 mM DTT, 0.5 mM PMSF, 10 µg/µl leupeptin, and 2 µg/µl aprotinin on ice, using a Dounce homogenizer. The homogenate was centrifuged at 10,000 x g at 4 C for 10 min and the supernatant was centrifuged at 103,720 x g at 4 C for 1 h. The final supernatant was assayed for protein content using the Bio-Rad Protein Assay (Bio-Rad, Hercules, CA). For controls, the TNT reaction was run in the absence or presence of a similar amount of vehicle buffer. The TNT reaction product was separated on a 7% SDS-PAGE gel, the gel was dried under vacuum at 80 C, exposed to x-ray film and/or a Phosphor screen, and the resulting image quantitated using PhosphorImager software. AR protein levels in the treatment groups were normalized to the AR content in the buffer-TNT reaction. A total of six protein isolations and TNT assays were performed with the oil- and estrogen-treated prostates run in parallel for each assay. Mean values for treatment groups were compared by Student’s t test.

In vitro AR degradation assay
To determine the effect of prostatic proteins on the degradation of AR, an in vitro degradation assay was established. Prostatic lysates were prepared from fresh ventral prostates homogenized on ice in 20 mM Tris-HCl (pH 7.4), 300 mM NaCl, 0.2 mM DTT using a glass-glass homogenizer. For adult tissues, 100 mg tissue were homogenized in 1 ml buffer and for d 10 prostates, 25 mg pooled tissue from several rats were homogenized in 400 µl buffer. The homogenates were centrifuged at 10,000 x g for 30 min, the supernatants collected, and total protein content determined. Full-length radiolabeled rat AR protein was synthesized in vitro using the TNT assay described above. The purity of the AR protein was determined with a 7.0% SDS-PAGE gel that produced a single band at approximately 110 kDa. The specificity of this band as AR was confirmed by Western blot using PG-21 anti-AR antibody. Ten microliters of [35S]-rAR were incubated in the absence or presence of 2.5 µg prostatic protein at 37 C for 20, 40, 60, and 90 min in a 40-µl volume of 20 mM Tris-HCl, 50 mM NaCl, 0.2 mM DTT, and 2 mM ATP (pH 7.4). Control reactions consisted of [35S]-rAR incubated for 60 min (intact vs. castrate) or 90 min (oil vs. neoE2) in the presence of homogenization buffer without prostatic proteins. The reactions were terminated with the addition of Laemmli buffer and samples were separated by 7% SDS-PAGE. The gels were dried, autoradiography was performed and AR band intensity was calculated using a Molecular Dynamics densitometer. To determine whether proteosome activity was involved in AR degradation, [35S]-rAR was incubated for 90 min with protein from prostatic lysates in the presence of either 50 mM MG132 or 100 mM ALLnL (both from Sigma-Aldrich) in 0.5 µl dimethylsulfoxide (DMSO). Control incubations contained 0.5 µl DMSO buffer alone.

Treatment and control assays were performed in parallel and repeated three to six times. Degradation rates for AR were determined in each treatment group by classical first order kinetics. For determination of t1/2 for [35S]-rAR, the logarithm of the relative units was plotted against time of incubation, the slope (degradation rate constant or k) was determined and t1/2 was calculated from the formula t1/2 = 0.69/k. A t test applied between the regression lines for the control and treatment groups was used to determine significant differences in the degradation rate constants (slopes). For inhibitor assays, vehicle groups were compared with inhibitor values by one-way ANOVA followed by Student-Newman-Keuls multiple comparison test to establish significance. All data are expressed as the mean ± SEM.

Western blot analysis of Akt
To determine whether activation of the serine-threonine kinase Akt/protein kinase B was involved in targeted proteolysis of AR, total and phosphorylated Akt levels were measured in the d 10 control and estrogen-exposed ventral prostates using the PhosphoPlus Akt Antibody kit (Cell Signaling Technology, Beverly, MA). Prostates were homogenized in a glass-glass homogenizer at 4 C in 10 mM Tris-HCl (pH 7.4) containing 0.1 mM leupeptin, 1 IU/ml aprotinin, and 1 mM sodium orthovanadate. Following centrifugation at 3000 x g for 10 min, the supernatant was assayed for protein content and diluted 1:1 in sample buffer. Prostatic proteins (35 µg) and negative and positive control NIH-3 cells (nontreated and platelet-derived growth factor treated, respectively; supplied with kit) were electrophoresed through 10% SDS-PAGE and transferred to nitrocellulose membranes as previously described (20). Duplicate blots were exposed to Total Akt antibody and Phospho-Akt (Ser 473), respectively, overnight at 4 C followed by secondary antibody for 1 h at room temperature. Proteins were visualized by a chemiluminescent detection system (supplied with kit). Three separate Westerns were performed on tissues from different animals and comparable results were observed.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
AR transcription
Previous studies showed that AR protein levels were suppressed in the d 10 ventral prostate following estrogenic exposure of rats on neonatal d 1–5 (16, 18). To determine whether this effect was mediated through a decrease in the AR gene transcription rate, nuclear run-on assays were performed on d 10 control and estrogenized ventral prostates. As shown in Fig. 1Go, A and B, there was no change in the transcription rate of AR mRNA in the ventral prostate following neonatal estrogenic exposure. This indicates that estrogenic down-regulation of AR protein is mediated at a posttranscriptional level.



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  		FIG. 1. AR transcription rate and transcript levels in the d 10 oil and estrogen-exposed (EB) rat ventral prostate gland. A, Representative nuclear run-on assay where 32P-labeled transcripts from prostatic nuclei were hybridized to excess DNA fragments: 1 µg 714-bp HindIII/EcoR1 fragment of AR cDNA, 1 µg of pGEM-3Z for background control and 1 µg of rat 28S cDNA as an internal control. Tissues from control and estrogenized rats were analyzed together and exposed to the same piece of x-ray film. B, Bar graph representation of cumulative data from six separate nuclear run-on experiments shows no effect of EB treatment on AR gene transcription rate. C, Graphic representation of AR mRNA levels from six qRT-PCR assays shows no difference in AR mRNA content in prostates from oil- and estrogen-treated rats.

 
To determine whether estrogens affected the amount or stability of AR transcripts present within the ventral prostate, AR mRNA levels were quantified in the treatment groups by qRT-PCR. As shown in Fig. 1CGo, there was no difference in the number of total AR mRNA molecules in the d 10 ventral prostate lobes of rats treated neonatally with either oil or estrogen. Prostatic AR mRNA levels and cellular distribution were additionally assessed by in situ hybridization at d 6 and 10 of life immediately following estrogen exposure on d 1, 3, and 5. Silver grain density after 4 wk of slide exposure was modest, which correlates with what has been previously published for the adult rat prostate (19). In the control oil-treated prostates, silver grains were present at equivalent levels within the epithelial and stromal cells of developing prostates (Fig. 2AGo), which corresponds to the known presence of similar AR protein levels in those cell populations during development (18). In confirmation of the qRT-PCR findings, the in situ hybridization revealed equivalent levels of AR message in the prostatic lobes of oil and estrogenized animals (Fig. 2Go, A and B). Additionally, these results show that estrogen exposure did not alter the levels of AR mRNA in either the stromal or epithelial cell population.



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  		FIG. 2. In situ hybridization of AR mRNA in d 10 ventral prostate glands. Oil-treated control prostate (A) neonatally estrogen-exposed (EB) prostate (B) mounted on a single glass slide and hybridized with antisense strand of [35S]-rAR riboprobe. No difference in silver grain density was observed between the two treatment groups at any region of the gland. C, An adjacent section of estrogenized prostate shown in B hybridized with sense stand [35S]-rAR mRNA exhibits low background signal. e, Epithelium; s, stromal cells. Magnification, x80.

 
AR translation
The AR transcript contains a long 5' UTR that possesses multiple sites for specific protein binding. One of these sites, termed the AR suppressor has been linked to both transcriptional and translation regulation of the AR (21). Of interest, preliminary findings performed in collaboration with Dr. Tindall (Mayo Clinic, MN) revealed the loss of specific proteins that bound and supershifted rAR suppressor antisense single-stranded cDNA in the estrogenized prostatic extracts compared with the oil-control prostates (Tindall, D., M. Grossman, C. Woodham, and G. S. Prins, unpublished data). These data pointed to the possibility of specific proteins present in normal prostates that regulate AR translational efficiency which appear to be lost in the estrogenized prostates. To directly assess a potential estrogenic influence on the translational efficiency of AR, we attempted in vivo pulse-chase labeling of prostatic AR protein in oil- and estrogen-treated rats. Although we were able to effectively radiolabel AR protein in vivo, animal variability in labeling efficiency did not permit data normalization between animals killed at different time points following the [35S]-methionine pulse. Consequently, decay rates could not be reliably determined by this approach. As an alternative, we established an in vitro transcription-translation assay that was performed in the absence and presence of prostatic lysates from the treatment groups. Because the above data have clearly shown that AR transcription was not affected by estrogen treatment, we reasoned that a decrease in AR product in the presence of proteins from estrogenized prostates compared with control prostates would reflect interference in AR translation. Importantly, we used a full-length AR cDNA which contained the entire 5' UTR in our transcription-translation assay to allow for potential binding of prostatic proteins to these sites. A representative gel of the radiolabeled AR protein product from the transcription-translation assay in the absence and presence of prostatic proteins is shown in Fig. 3Go. The results of six separate experiments with increasing amounts of protein showed that prostatic proteins from control and estrogenized prostates had no influence on AR translation in vitro. However, it must be emphasized that the commercial extract may have been so optimized for AR translation that contributions from the estrogenized prostatic lysates may have been rendered ineffective despite increasing lystate concentrations. Nonetheless, although these findings lack definitive proof that estrogens are not altering AR translation, they shed doubt on this possibility.



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  		FIG. 3. Representative SDS-PAGE gel of [35S]-methionine-labeled AR from an in vitro transcription-translation reaction performed in the presence of 1 µg protein extract from d 10 oil- or estrogen-treated rat ventral prostates. + control, TNT reaction performed with rat AR cDNA in pGEM-3Z but no further additions; - control, TNT reaction performed without the rat AR cDNA; buffer, TNT reaction performed in the presence of the vehicle buffer used for prostate extraction. TNT reactions run in the presence of 1 µg prostatic extract from oil- and EB-treated rats were performed in duplicate for each experiment.

 
AR degradation
To determine whether estrogenic exposure lowered prostatic AR levels by influencing AR degradation, we developed an in vitro AR protein degradation assay performed in the absence and presence of lysates from control and estrogen-treated prostates. To first establish that this assay was effective in monitoring AR degradation, we performed the assay with lysates from adult ventral prostates of intact and 48 h castrate rats because previous studies have established that AR degradation is significantly increased in the prostate following androgen withdrawal (22, 23, 24). As shown in Fig. 4AGo, in vitro transcribed-translated full length AR protein slowly degraded over a 90-min period with a calculated half-life of 2.31 h when incubated in the presence of 2.5 µg prostatic protein from adult intact rat prostates. In contrast, AR protein incubated in buffer alone was stable and did not degrade over this time frame. Incubation of AR protein for 90 min in the presence of prostatic proteins from 48 h castrated rats significantly accelerated the AR degradation rate compared with the intact prostate (intact degradation slope = 0.0049 ± 0.00014 vs. castrate degradation slope = 0.0086 ± 0.00015, P < 0.001). This resulted in a mean AR half-life of 1.34 h following androgen withdrawal. Because these results confirm the previous findings from the Wilson laboratory (23, 24) that AR degradation is increased in the absence of ligand with a half-life of 1.5 h, these data indicate that the present assay is able to detect physiologic changes in the AR protein half-life.



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  		FIG. 4. In vitro AR degradation assay. A, Left, Representative SDS-PAGE gel of in vitro translated AR protein incubated for 20, 40, 60, or 90 min in the presence of 2.5 µg protein extracted from adult intact and 48-h castrate ventral prostates (VP). The "No protein" lane on the left shows the initial amount of AR protein at the start of the assay with no prostatic protein added. The "No protein - 90 min" lane on the right is AR incubated for 90 min in the absence of prostatic protein and documents that AR by itself did not degrade during the incubation period. Right, Degradation slopes of mean data from six separate assays plotted in a semilogarithmic manner show a significant increase in AR degradation rate in the presence of proteins from a castrate prostate vs. an intact prostate (*, P < 0.001). B, Left, Representative AR degradation assay performed in the presence of 2.5 µg prostatic proteins from d 10 oil- or estrogen-treated (neoEB) rat VP. Right, Degradation slopes of mean data from three separate assays plotted in a semilogarithmic manner show a significant increase in AR degradation rate in the presence of prostatic proteins from neoEB-treated rats vs. oil-treated d 10 rats (*, P < 0.001).

 
Figure 4BGo shows the effect of prostatic lysates from d 10 ventral prostates of oil-treated control and estrogenized rats. AR protein degraded at a faster rate in the presence of d 10 oil-treated prostate lysates compared with intact adult prostates (t1/2 = 1.49 vs. 2.31, respectively), which most likely reflects a lower circulating androgen level before puberty that would increase AR turnover. Importantly, exposure to neonatal estrogens significantly increased the AR degradation rate compared with d 10 control prostates (control degradation slope = 0.0077 ± 0.0003 vs. neonatal EB degradation slope = 0.0103 ± 0.0006, P < 0.001). The calculated half-life of AR was reduced to 1.11 h in the presence of prostatic proteins from the estrogenized rats.

To determine whether AR degradation in the presence of prostatic lysates was mediated through a proteosome pathway, AR degradation was assessed after a 60- or 90-min incubation in the absence and presence of specific proteosome inhibitors. Figure 5Go, A and B, shows representative and cumulative data from the adult intact and castrate prostates. At 60 min, there was a decrease in AR protein levels in the presence of intact prostate lysates compared with AR incubation without proteins and this was further decreased in the presence of 48-h castrate prostatic lysates. In contrast, the AR degradation induced by prostate proteins and accelerated by androgen withdrawal was effectively blocked by the proteosome inhibitors MG132 and ALLnL. Thus, we conclude that AR degradation in the prostate is mediated through a proteosome pathway. In Fig. 5Go, C and D, representative and cumulative data are shown for d 10 oil and estrogen-exposed ventral prostates. At 90 min of incubation, AR was degraded in the presence of control prostatic extracts compared with incubations without protein, and this degradation was accelerated in the presence of prostatic lysates from estrogenized prostates. The AR degradation in the control prostatic lysates was largely reversed by the addition of proteosome inhibitors, whereas the AR degradation in the presence of prostatic lysates from estrogenized prostates was completely blocked by addition of either ALLnL or MG132. These findings indicate that AR degradation induced by neonatal exposure to estrogens is mediated through a proteosome pathway.



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  		FIG. 5. In vitro AR degradation assay in the absence and presence of proteosome inhibitors. A, Representative SDS-PAGE gel of in vitro translated AR protein incubated for 60 min in the presence of 2.5 µg protein extracted from adult intact and 48-h castrate ventral prostates (VPs). The "- protein" lane shows AR protein incubated for 60 min with no prostatic proteins added, whereas the remaining lanes show AR protein incubated for 60 min with prostatic protein extracts in DMSO vehicle alone (60 min vehicle) or vehicle containing 50 mM MG132 or 100 mM ALLnL. B, Bar graph representation of cumulative data from four separate assays with intact and 48-h castrate adult VPs. No protein, In vitro translated AR levels after 60 min of incubation with no prostatic protein added; vehicle, in vitro translated AR protein incubated for 60 min in the presence of 2.5 µg protein extracted from adult intact and 48-h castrate VPs, and MG132 and ALLnL represent 60-min incubations of in vitro translated AR protein in the presence of prostatic protein extracts with the respective proteosome inhibitors. **, P < 0.05 vs. intact VP vehicle; +, P < 0.005 vs. no protein, {ddagger}, P < 0.02 vs. 48-h castrate VP vehicle. C, Representative SDS-PAGE gel of in vitro translated AR protein incubated for 90 min in the presence of 2.5 µg protein extracted from d 10 VPs treated neonatally with oil or estradiol. The "- protein" lane shows AR protein incubated for 90 min with no prostatic proteins added, whereas the remaining lanes show AR protein incubated for 90 min with prostatic protein extracts in DMSO vehicle alone (60 min vehicle) or vehicle containing 50 mM MG132 or 100 mM ALLnL. D, Bar graph representation of cumulative data from four separate assays with d 10 oil-treated VP and neonatally estradiol-treated VP (neoE2). - protein, In vitro translated AR levels with no prostatic protein added; 90 min - protein, in vitro translated AR levels with no prostatic protein added after 90 min of incubation; vehicle, in vitro translated AR protein incubated for 90 min in the presence of 2.5 µg protein extracted from d 10 oil and neoE2-treated VP, and MG132 and ALLnL represent 90-min incubations of in vitro translated AR protein in the presence of prostatic protein extracts with the respective proteosome inhibitors. **, P < 0.001 vs. no protein; +, P < 0.05 vs. oil vehicle; *, P < 0.01 vs. neoE2 vehicle, 2+ P < 0.001 vs. neoE2 vehicle.

 
A recent report using an in vitro system showed that activation of the serine-threonine kinase Akt/protein kinase B results in targeted proteolysis of AR (25). To investigate whether the Akt pathway might play a role in the increased AR degradation in the estrogenized ventral prostate, total and phosphorylated Akt levels were measured in d 10 control and estrogenized ventral prostate homogenates. Exposure to estrogens between d 1 and 5 of life did not change total Akt levels within the d 10 prostate but resulted in a significant decrease in phosphorylated Akt levels (Fig. 6Go).



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  		FIG. 6. Representative Western blot of total and phosphorylated-Akt levels in d 10 ventral prostates (VP) exposed to oil or estradiol (E-2) on d 1–5 of life. NIH-3 cells cultured in the absence (NT) and presence of platelet-derived growth factor treated serve as a negative and positive control, respectively, for hormone-induced Akt phosphorylation. Total Akt levels (top) were not changed in response to hormone treatment, whereas phosphorylation of Akt (middle) was markedly reduced in response to estrogen exposure. Bottom shows bar graph of scanned data for p-Akt levels normalized to total Akt for three separate experiments. *, P < 0.05 oil vs. E-2.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our previous studies demonstrated that neonatal estrogen exposure results in an immediate and sustained decrease in AR protein levels in the developing and adult rat ventral prostates that in turn leads to abnormal growth, decreased secretory capacity, and a dampened activational response to exogenous androgen replacement (14, 15, 16, 18). The present findings demonstrate that this AR down-regulation is not mediated at the transcriptional or translational level but rather through a posttranslational mechanism at the level of AR protein degradation. Furthermore, the present results indicate that AR protein is degraded by a ubiquitin-proteosome pathway in both the developing and adult rat ventral prostate glands. This targeted proteolysis is under hormonal regulation in vivo such that androgen withdrawal in the adult prostate and neonatal estrogen exposure in the developing prostate can accelerate the rate of AR degradation via the 26S proteosome pathway.

The process of ubiquitination and subsequent targeting of proteins for proteosome degradation consists of an enzymatic cascade involving a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin-protein ligase (E3), which directly links ubiquitin to the substrate protein (26). Following the covalent binding of polyubiquitin residues, the protein is recognized by the 26S proteosome complex that is responsible for protein proteolysis. Studies with multiple members of the steroid receptor gene superfamily including AR have shown that the ubiquitin-proteosome pathway is involved in steroid receptor degradation and is a point for hormonal regulation of steroid receptor levels (27, 28, 29, 30, 31, 32, 33, 34, 35). For AR specifically, treatment of human cell lines with proteosome inhibitors was capable of increasing AR levels (36), whereas a more recent study has shown that activation of Akt-mediated AR phosphorylation targets the AR for ubiquitination and degradation in several cells in vitro (25). Interestingly, in most instances, liganding of the steroid receptor is a critical step in targeting the protein for proteolysis and provides a negative feedback loop whereby ligand binding induces both transcriptional activation as well as degradation of the receptor. However, for both the AR (present results) and VDR (37), the opposite appears to occur where the cognate ligand is protective against proteosome-mediated degradation. This correlates with previous findings by Wilson (24) and others (22) that AR is more stable in the presence of androgens, whereas other steroid receptors are more labile in the presence of their cognate ligands and points to another site where AR regulation differs from that of the other members of this gene superfamily.

The intracellular site(s) for hormonal regulation of steroid receptors by targeted proteolysis remain to be elucidated. Many proteins are targeted for degradation by phosphorylation (26), which is of interest because steroid receptors, including AR, are phosphorylated by both ligand-dependent and ligand-independent mechanisms (38). However, which specific mechanism is involved in targeted proteolysis in vivo remains unclear. It has been shown that phosphorylation of progesterone receptor (PR) via the MAPKs is involved in targeting PR for ubiquitination-mediated proteolysis (32). More recently, Akt-dependent phosphorylation of the AR was shown to target AR for ubiquitination-proteosome degradation in an in vitro system in the presence of androgens (25). In those studies, dihydrotestosterone-treated COS cells transvected with constitutively active Akt showed increased ubiquitin binding to a transvected AR whose levels declined compared with those without cAkt. Further, IGF-1 induced activation of Akt in LNCaP cells resulted in reduced AR levels, suggesting that this system may be intact under more physiologic-type conditions. However, those findings contrast with other results where activation of the phosphatidylinositol 3-kinase-Akt pathway through various mechanisms including the IGF-1 pathway led to an increase in basal and dihydrotestosterone-induced levels of AR (39). In those studies using both vas deferens epithelial cells and LNCaP cells, blockade of Akt phosphorylation led to a loss of AR protein levels suggesting that activation of Akt was involved in stabilization and not degradation of AR. In the present study, neonatal estrogen exposure led to a decrease in levels of phosphorylated Akt in the ventral prostate glands which was associated with increased proteosome-mediated AR degradation. This would imply that if the Akt pathway is involved in in vivo AR degradation in response to estrogen exposure, it would be through decreased rather than increased Akt activation.

It is important to note that phosphorylation of other proteins such as c-fos and c-jun has been shown to actually prevent their degradation (26). Thus, it remains a possibility that ligand-induced or ligand-independent activation and phosphorylation of AR may be involved in suppressing its ubiquitinylation and degradation in vivo. It is recognized that PEST elements within proteins (regions enriched in Pro, Glu, Ser, and Thr) are also rich in S/TP sequences that are minimum consensus phosphorylation sites for protein kinases (26). In many, but not all instances, PEST elements contain phosphorylation sites necessary for regulation of protein degradation. In this regard, it is significant that AR contains a PEST sequence in its hinge region (36), which includes a proline-directed phosphorylation site at Ser-650 (numbering for human AR) known to be important in phosphorylation-regulated transactivation (40). It is possible that this sequence may function as an important site for phosphorylation-mediated protection from proteosome-mediated degradation of AR. This contrasts with the documented Akt-induced phosphorylation sites on human AR at Ser-213 and -791 (41) or Ser-210 and -790 (42), which were shown to be necessary in accelerating Akt-mediated AR degradation (25). It is possible that Akt-induced phosphorylation of AR may be involved in either degradation or stabilization of the AR in a context-dependent manner.

DNA binding is required for ligand-mediated targeted degradation of retinoic acid receptor (RAR) {alpha} (31), whereas for peroxisome proliferator-activated receptor {gamma}, recruitment to the transcription complex is not required for proteosome degradation but rather ligand-induced conformational changes are critical, which may involve recruitment of coactivators (35). Interestingly, several nuclear receptor-interacting proteins, some with coactivator/corepressor activity, have been identified that are also components of the ubiquitin system pointing to the complexity of this system (43, 44, 45, 46).

Cells typically contain a single species of E1, several species of E2, and multiple families of E3 ligase that appear to confer specificity and regulation to the process of ubiquitinylation (26). Several E2 and E3 enzymes have been shown to be associated with AR ubiquitination and subsequent degradation. Mdm2, a RING finger E3 ligase involved in the ubiquitinylation of p53, was recently shown to directly associate with AR following Akt-induced phosphorylation and this interaction was critical for Akt-induced ubiquitination of AR (25). Another E2-dependent E3, ARNIP, was recently isolated; it specifically interacts with the N-terminal region of AR and functions to ubiqitinate this molecule with a marked degree of specificity (47). Ubc9, a homolog of E2-conjugating enzymes, has been shown to interact with AR and covalently link it to a ubiquitin-like protein SUMO-1, a molecule involved in sumoylation and attenuation of AR transactivation (44, 48). Whether there is potential for hormonal regulation of these specific enzymes within prostate cells remains to be determined.

It is unclear whether the effects of estrogens on AR degradation in vivo are mediated directly through the estrogen receptor (ER) or indirectly through an intermediary signal. Studies with ER{alpha} and ERß knockout mice have previously determined that neonatal estrogenization is mediated through ER{alpha} (49). In the normal developing prostate, ER{alpha} is low and confined to periductal mesenchymal cells surrounding the proximal ducts. In response to neonatal estrogen exposure, there is an immediate up-regulation of ER{alpha} mRNA and protein within the periductal stromal cells along the entire length of the developing ducts, which allow for amplification of estrogen signals in these stromal cells (50). This in turn leads to transient expression of PR within these same cells (51); thus, it is possible that either ER{alpha}, PR, or other altered stromal signals may be involved in AR down-regulation within the prostatic stromal cells. However, ER{alpha} and PR are not expressed at any time in the prostatic epithelial cells, which implies that other intermediary signals may be involved in inducing AR degradation in the prostatic epithelium. Alterations in several signaling molecules have been identified by our laboratory in response to neonatal estrogens including TGFß (17), hox 13 genes, and Nkx 3.1 (7); however, other yet unidentified factors may also play a role. Additionally, we have demonstrated that immediately following neonatal estradiol exposure, intraprostatic levels of retinol, stromal cell expression of RAR{alpha}, and epithelial expression of RARß were markedly elevated and that these elevations persist through adulthood (6). This is of interest because it was reported that retinol administration was capable of decreasing AR protein levels in rat Sertoli cells through a ubiquitin-mediated process (52, 53). It is thus possible that sustained elevations in retinol and retinoic acid levels within the estrogenized prostate contribute to elevated AR degradation rates within the prostate gland throughout the life of the animal.

In closing, it is important to emphasize that although AR down-regulation via increased degradation contributes to the estrogen-induced aberrations in prostate growth and function, they alone do not fully explain the estrogen-induced lesions in the adult prostate gland. Other studies from our laboratory have pointed to a contribution from both transient and permanent alterations in the temporal and quantitative expression of several transcription factors and homeobox genes in determining the estrogenized phenotype of the aging prostate gland (6, 7, 50, 54, 55). Due to a loss in AR protein combined with an increase in ER{alpha}, PR, and RAR{alpha} and ß, we propose that the neonatally estrogenized prostate gland is shifted from a predominantly androgen-driven developmental pathway to one regulated predominantly by estrogens and retinoids during this critical period. It is hypothesized that this may set into motion a series of differentiation and growth defects that predispose this gland to neoplasia as the animal ages.


   Footnotes
 
This work was supported by NIH Grants NIDDK 40890 and NIDDK 09653.

Abbreviations: AR, Androgen receptor; DMSO, dimethylsulfoxide; DTT, dithiothreitol; E1, ubiquitin-activating enzmye; E2, ubiquitin-conjugating enzyme; E3, ubiquitin-protein ligase; EB, estradiol benzoate; ER, estrogen receptor; PEST, regions enriched in Pro, Glu, Ser, and Thr; PIN, prostatic intraepithelial neoplasia; PR, progesterone receptor; q, quantitative; rAR, rat AR; RAR, retinoic acid receptor; SDS, sodium dodecyl sulfate; UTR, untranslated region.

Received January 8, 2003.

Accepted for publication July 14, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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