Department of Biochemistry and Molecular Biology (J.C.-F., V.P., M.B.C., D.L.R., D.F.S.), Mayo Clinic College of Medicine, Scottsdale, Arizona 85259; and Department of Cell Biology (C.S.-Q.), Georgetown University Medical Center, Washington, D.C. 20057
Address all correspondence and requests for reprints to: David F. Smith, Department of Biochemistry and Molecular Biology, Johnson Research Building, Mayo Clinic Scottsdale, 13400 East Shea Boulevard, Scottsdale, Arizona 85259. E-mail: smith.david26{at}mayo.edu.
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ABSTRACT |
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INTRODUCTION |
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RESULTS |
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We performed a histological examination of selected male reproductive tissues (Fig. 3). Because FKBP52 is a known component of steroid receptor complexes and male reproductive organs are all androgen target tissues, expression patterns of AR and FKBP52 were compared by immunohistochemical staining of wild-type and 52KO tissues. Specific FKBP52 immunostaining was detected in most cell types of the wild-type testis (Fig. 3
, AC), yet testicular histology appears normal and all stages of spermatogenesis are observed in tissue from 52KO males (Fig. 3
, DF). The pattern of AR immunostaining is similar in testis slices from either wild-type (Fig. 3C
) or 52KO (Fig. 3F
) origin. In both backgrounds, AR immunostaining localizes to the nuclei of Sertoli, Leydig, and peritubular myoid cells, as previously described (18). Therefore, loss of FKBP52 does not grossly affect the pattern of AR expression or nuclear localization of AR. However, it does appear that the intensity of AR staining is somewhat reduced in 52KO testis, an issue that will be addressed further below.
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Chaperone Expression in Mouse Reproductive Tissues
More than a dozen molecular chaperones have been identified in complex with steroid receptors and many of these have been shown to influence different aspects of receptor assembly, functional maturation, or proteolytic degradation. Therefore, the balance of chaperones in a particular tissue could influence AR activity and the degree to which FKBP52 is important. To gain some insight into relative expression levels of multiple receptor-associated chaperones, extracts were prepared from male reproductive organs taken from wild-type mice and assayed by Western immunostaining for nine chaperones known to assemble with steroid receptors (Fig. 4A). Hsp90 is the central chaperone component in mature steroid receptor complexes, and Hsp70 is required at early stages of complex assembly. Hop is a cochaperone that binds both Hsp90 and Hsp70 and serves as an adaptor to recruit Hsp90 to preexisting Hsp70-receptor complexes. Hip is an Hsp70 cochaperone that participates with Hop at intermediate assembly stages. FKBP52 and FKBP51 are closely related but functionally distinct cochaperones that compete for binding a common site on Hsp90. PP5 is a protein phosphatase and CyP40 is a cyclophilin that also compete with FKBP52 for binding Hsp90. Finally, p23 is an Hsp90 cochaperone that stabilizes Hsp90 interactions with functionally mature receptor complexes.
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We further sought to determine whether disruption of the gene for FKBP52 results in compensatory changes in expression levels of other receptor-associated chaperones. Toward this end, extracts were prepared from testis and epididymis of age-matched wild-type or 52KO mice and Western blotted for nine receptor-associated chaperones, for AR, and for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a loading control (Fig. 4B). Despite the absence of FKBP52, there was no significant change in the protein level of other chaperones in either testis or epididymis. This observation lessens the likelihood that there are compensatory changes in chaperone expression resulting from FKBP52 ablation; furthermore, the unaltered level of Hsp90 and Hsp70 expression suggests that loss of FKBP52 does not induce a chronically stressful stimulus in tissues.
In contrast to the constant level of chaperone expression, the steady-state level of AR protein relative to GAPDH or multiple chaperones is reduced by two thirds in both testis and epididymis from 52KO mice. Clearly, reduced levels of AR could have some bearing on how well these tissues respond to hormone. We noted above that testis organogenesis and descent are abnormal in approximately 2025% of 52KO males, but further study is required to establish whether these defects relate to reduced AR levels. In fully descended testes of 52KO males, spermatogenesis appears normal, but there is one indication for an epididymal-related defect in sperm function. Multiple attempts to cross mildly defective 52KO males with wild-type females have failed to result in pregnancy. S. K. Dey, a collaborator examining aspects of 52KO reproductive physiology, notes that spermatozoa isolated from the epididymis of 52KO males are normal in number but display reduced motility and fertilization efficiency in vitro (Dey, S. K., and S. T. Kim, unpublished observations). Thus, defective sperm maturation in the epididymis, perhaps related to decreased AR levels, could contribute to 52KO infertility.
FKBP52 Expression in Human Tissues
To relate the mouse model more closely with potential human requirements for FKBP52, we immunostained for FKBP52 using extracts from multiple human reproductive tissues (Fig. 5A). FKBP52 was detected in all male and female reproductive tissues examined, although the relative level of FKBP52 protein in placenta appears to be much less than in other organs. We chose to look more carefully at FKBP52 expression in human prostate because this organ was affected in 52KO mice. Surgical waste samples were obtained from patients with prostatic hyperplasia, and tissue slices were immunostained for FKBP52 and AR (Fig. 5B
). Similar to the mouse, FKBP52 and AR are strongly coexpressed in luminal epithelial cells of human prostate. AR is also observed in some stromal cells, but not FKBP52, which suggests that prostate stromal and epithelial compartments are likely influenced differently by FKBP52.
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Knockdown of FKBP52 Expression in Human Cells
FKBP52 also supports AR activity in human cells (Fig. 7). HeLa cells stably expressing AR (22) were infected with virus expressing a short hairpin RNA (shRNA) that specifically targets FKBP52 mRNA (52KD) or a scrambled shRNA (Control). After viral infection, cells were transfected with either an empty plasmid (vect) or plasmid expressing human FKBP52 (p52). For the latter plasmid, silent mutations were introduced into the shRNA-targeted region to escape RNA inhibition. Cell extracts were prepared from each of the cell lines and Western immunostained for AR, FKBP52, or GAPDH as a loading control. GAPDH levels were constant in all cells examined. FKBP52 protein was reduced by over 70% in cells infected with 52KD vs. Control (Fig. 7A
), but FKBP52 levels were restored in cells transfected with plasmid p52. In parallel with changes in FKBP52 protein level, the steady-state level of AR protein was reduced by approximately 50% in 52KD cells. AR accumulation was partially restored in 52KD cells transfected with p52; presumably, the level of AR protein did not fully recover due to the limited efficiency of p52 transfection in the total 52KD population. Therefore, FKBP52 appears to influence the level of AR protein in HeLa cells, which is consistent with our observation in Fig. 4B
that AR levels are reduced in 52KO testis and epididymis.
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FKBP52 Rescues Activity of an AR Point Mutant
Because FKBP52 PPIase activity is critical for enhancing AR function, we speculated that one or more proline sites in AR, perhaps in the LBD where chaperone interactions are localized, might be relevant substrates for FKBP52. Detailed studies are underway to address this possibility. In the meantime, we accessed the Human AR Mutant Database (http://ww2.mcgill.ca/ androgendb/) to look for known mutations that might relate to FKBP52 interactions. One candidate of interest is a proline to serine substitution at position 723 of human AR (AR-P723S) that was identified in genital skin fibroblasts from a patient with complete androgen-insensitivity syndrome (23). We generated a cDNA encoding AR-P723S by site-directed mutagenesis and tested mutant function in yeast (Fig. 8A). When compared with the basal activity of wild-type AR (wtAR plus FKBP51), mutant AR alone (AR-P723S plus vector) or in the presence of FKBP51 exhibited very little ability to induce reporter expression. Remarkably, however, in the presence of FKBP52 (AR-P723S plus FKBP52), the activity of mutant AR matched the enhanced activity seen with wtAR in the presence of FKBP52. We previously showed that human Hip, a cochaperone that assembles with steroid receptor complexes and is absent in yeast, could enhance stability and function of misfolded GR in yeast (24); however, because neither Hip (AR-P723S plus Hip) nor FKBP51 has the ability to restore mutant AR function, it is clear that FKBP52 is functioning in a specific manner. As seen for FKBP52-dependent enhancement of wtAR function (Fig. 6C
), FKBP52 mutants deficient in Hsp90 binding (52-K354A) or PPIase (52-FD67DV) or a combination of both mutations (52-combo) had much reduced ability to rescue AR-P723S function. Rescue is not a result of proteolytic stabilization because the level of mutant AR protein is similar in yeast strains expressing various cochaperones (Fig. 8B
). Likewise, FKBP52, FKBP51, and mutant FKBP52 forms are expressed at similar levels in yeast, so the specific rescue of AR-P723S activity by FKBP52 must be due to native functions of wild-type FKBP52. DHT dose-response curves were generated for yeast reporter strains expressing AR-P723S plus either FKBP52 or, as a negative control, FKBP51 (Fig. 8C
). It is apparent that AR-P723S in the absence of FKBP52 has some residual ability to activate transcription, but only at supraphysiological levels of hormone. In the presence of FKBP52, AR-P723S responds robustly to hormone.
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DISCUSSION |
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Receptor Conformation and Chaperones
Because these are androgen-dependent tissues/processes and FKBP52 is a known component of steroid receptor complexes, we chose to focus on the functional interaction between FKBP52 and AR to begin to understand the molecular basis for the 52KO phenotype. These studies were informed by numerous preceding studies that have demonstrated the importance of Hsp90 and associated chaperones in the folding and functional maturation of steroid receptors. Relatively little study has been devoted to chaperone interactions with AR, but AR and steroid receptors other than estrogen receptor are known to require close association with Hsp90 to establish and maintain hormone binding ability at physiological temperatures (25, 26, 27, 28). A conformational change, sometimes referred to as refolding, is presumed to be stimulated by Hsp90 binding to the LBD, although the exact nature of chaperone-dependent conformational changes in the LBD that generate hormone binding ability has yet to be determined. An atomic structure for the unliganded LBD of a steroid receptor could be highly informative, but no such structure has been obtained, perhaps due to LBD conformational instabilities that also attract chaperones. In addition to Hsp90s central role in establishing basal hormone binding ability, it is becoming increasingly clear that cochaperones like FKBP52 can further influence receptor conformation and activity.
Mechanism of FKBP52 Action
In cellular models of AR function (Figs. 6 and 7
), hormone-dependent activation of reporter genes is clearly enhanced in the presence of FKBP52 (Fig. 6B
) and impaired by reduction of FKBP52 (Fig. 7B
). FKBP52 assembles with non-hormone-bound AR complexes in an Hsp90-dependent manner (Fig. 6
, A and C), similar to FKBP assembly with other steroid receptor complexes. Enhancement of AR function requires FKBP52 PPIase activity (Fig. 6
, C and D), and there is a specific requirement for FKBP52 because FKBP51, which has similar PPIase activity by in vitro assays (29), fails to alter AR function. Our working model is that FKBP52 piggybacks on Hsp90 into AR complexes where the FKBP52 PPIase domain is positioned to effectively interact with one or more prolines on the AR LBD. Unfortunately, direct demonstration of a potentially small FKBP52-dependent conformational change in AR LBD is technically prohibitive, especially because the noncovalent conformational change is probably transient in the absence of FKBP52 and measurements must be made in the context of large multichaperone complexes.
How Is AR Function Altered by FKBP52?
The answer to this question appears to depend on the cellular context of AR-FKBP52 interactions. In the yeast model, FKBP52 stimulates an increase in hormonal potency (Fig. 6B), as we previously observed with GR (20). By reducing the EC50 for hormone-dependent gene activation, the presence or absence of FKBP52 would have the greatest effect on AR activity at limiting concentrations of hormone that occur physiologically. Thus, failure of androgen target tissues to develop normally in the 52KO mouse could be due to desensitization of tissues to normal hormone levels.
In contrast to the yeast results, knockdown of FKBP52 expression in a human HeLa cell model reduced hormonal efficacy (Fig. 7D), which can be attributed to a decrease in AR protein (Fig. 7A
). A decrease in the steady-state level of AR was also observed in the testis and epididymis of 52KO mice (Fig. 4B
), but organogenesis of these tissues was not dramatically impacted in 52KO mice. As opposed to testis and epididymis, there does not appear to be a large decrease in AR levels in anterior prostate of 52KO mice (Fig. 3
) even though this organ was highly dysgenic.
The proteolytic stability of proteins can be greatly affected by the chaperone environment of cells. Certain cochaperones promote ubiquitination and proteasomal degradation of protein substrates. Two of these have been identified in steroid receptor complexes: BAG1 (BCL2-associated athanogen 1), which is an Hsp70 inhibitor and exists in multiple functionally distinct isoforms (30), and CHIP, which binds either Hsp70 or Hsp90 and contains ubiquitin ligase activity (31). In a dynamic manner that remains poorly understood, steroid receptors can proceed through chaperone assembly pathways to functionally mature complexes containing Hsp90 and FKBP, or the receptor can be side-tracked by BAG1 and CHIP (carboxyl terminus of the Hsc70-interacting protein) toward proteolytic degradation. The presence or absence of FKBP52 and the individual activities of BAG1, CHIP, and multiple other cochaperones in HeLa and other cells will influence whether AR is shunted toward maturation or degradation. Interestingly, yeast lack direct orthologs for either CHIP or BAG1, so the proteolytic triage decision for AR probably differs in this background.
Although CHIP and BAG1 isoforms were not assessed in mouse tissues, it is clear that the relative levels of other receptor-associated chaperones differ markedly among male reproductive organs of the mouse (Fig. 4A). Among these cochaperones are ones that promote or inhibit intermediate steps in assembly of receptor complexes, ones that influence the stability of Hsp90 interactions with receptor, and ones that compete with FKBP52 for assembly into receptor complexes. How AR function is impacted by FKBP52 may ultimately depend on a sizeable combination of chaperone and other cellular factors. Thus, androgen target organs and even individual cell types within these organs may need to be addressed individually to understand how AR is affected by FKBP52.
AR LBD Structure and Pro723
Aside from the indirect influences of other chaperones on FKBP52-AR interactions, we think it is likely that FKBP52 PPIase acts directly and specifically on one or more proline sites in the AR LBD. Directly proving which, if any, of 12 prolines in the human AR LBD is a relevant PPIase substrate and how isomerization structurally and functionally alters AR will be challenging propositions. As an initial analysis within a much larger study, we selected from the AR Mutant Database AR-P723S, which is a proline mutant associated with androgen insensitivity. Our initial hypothesis in testing AR-P723S was that the mutant receptor would retain basal activity while resisting enhancement by FKBP52. Instead, we find that basal activity is much below normal and that FKBP52 can dramatically restore function to this functionally defective mutant (Fig. 8). It would not appear that AR-P723S is grossly misfolded because its steady-state level in yeast is similar to wtAR and unaffected by FKBP52-mediated functional restoration.
One might exclude Pro723 as a relevant substrate for FKBP52 PPIase because this proline is absent from AR-P723S. However, PPIases can dock at sites lacking proline and potentially influence interactions independent of prolyl isomerization (32); residual interactions of the FKBP52 PPIase domain with AR-P723S could help overcome a folding barrier that deters functional maturation of mutant AR. It is perhaps instructive to note the position of Pro723 in the AR LBD and consider how a conformational change at this site, either through prolyl bond isomerization or mutation, might alter AR function beyond basic folding. To assist in this consideration, Fig. 8D depicts a cocrystal structure for an AR LBD bound to the synthetic androgenic agonist R1881 and a peptide from the transcriptional coactivator transcriptional intermediary factor 2/steroid receptor coactivator-2/GR-interacting protein 1 (33). Pro723 is in a solvent-accessible loop that connects helix 3, which directly participates in ligand binding and helix 4, which together with helices 3 and 12 forms the activation function (AF) 2 binding site for transcriptional coactivators. The AF2 pocket is also an interaction site for an N-terminal AR motif (33) and thus can contribute to interdomain binding between subunits in AR homodimers (34, 35, 36, 37). In addition to the AF2 interaction motif, the N-terminal domain also contains the AF1 transcriptional activation site, which is dominant in many AR transactivation events (33).
A change at Pro723 could potentially induce a twist in helix 3, reorient side chains known to be involved in ligand binding, and cause a change in hormone binding properties. Alternatively, Pro723 could potentially impact protein interactions involving helices 3 and 4 along the AF2 pocket. Interestingly, mutations in AF2 that disrupt N-terminal domain interactions, including a substitution of Phe725, have been correlated with partial androgen insensitivity syndrome (36, 38). Whether AF2 interactions are disrupted in AR-P723S and restored by FKBP52 has not been established. Finally, a conformational change through Pro723 or other proline sites could alter AR susceptibility to proteolysis.
Physiological Importance of FKBP52
We propose that the reproductive phenotype of male 52KO mice results from partial androgen insensitivity in anterior prostate, external genitalia, and perhaps other accessory sex organs due to loss of FKBP52-enhanced AR function. Thus, in these tissues FKBP52 serves a nonredundant, physiologically relevant role in up-regulating AR activity. In testis or other androgen-sensitive tissues that are less affected by loss of FKBP52, locally high hormone concentrations or alternative chaperone combinations could lessen the critical need for FKBP52 in AR complexes. As we demonstrated in Fig. 4, androgen target tissues in the mouse have highly distinctive chaperone repertoires, and the local concentration of androgens in the testis and nearby tissues is certain to be greater than in peripheral target tissues.
52KO male mice share some features with individuals harboring mutations in a gene for 5-reductase (16, 39), the enzyme responsible for converting testosterone to the more potent DHT, which is required for full development of prostate and virilization of external genitalia in humans. In mice, however, it is surprising that simultaneous disruption of both 5
-reductase genes is not a major detriment to virilization (17), presumably due to a compensating increase in tissue T levels of the urogenital sinus and urogenital tubercle. The 52KO mouse model suggests that FKBP52 is more critical than DHT for full virilization in mice. Whether FKBP52 is equally critical for complete human virilization, however, remains an open question.
Because FKBP52 assembles with multiple steroid receptor complexes, and we have previously shown that FKBPs can alter glucocorticoid receptor function, it is possible that FKBP52 is important for steroid response pathways other than androgen signaling. Preliminary evidence from the 52KO mouse model supports this possibility. 52KO mice have elevated circulating levels of corticosterone (results not shown) as would be consistent with resistance to glucocorticoids, a phenotype predicted from cellular studies of FKBP52-GR interactions (20). Additionally, 52KO female mice are infertile even though we observe no gross defects in female reproductive tissues or oogenesis. There do not appear to be deficits in estrogen signaling, which is consistent with the inability of FKBP52 to alter ER function (20). Further description and characterization of the female phenotype is the focus of ongoing investigations.
Clinical Implications for FKBP52
If FKBP52 acts by altering AR conformation, then it follows that mutant AR could either mimic the change induced by FKBP52, and thereby generate a constitutively high activity receptor, or block the change, and thus generate a receptor with constitutively lower activity. A high-activity receptor mutant could contribute to prostate hyperplasia or carcinogenesis, whereas a low-activity receptor could contribute to androgen insensitivity. Because there are more than 250 human AR mutations identified in androgen insensitive or prostate cancer patients (http://ww2.mcgill.ca/androgendb/), it will be interesting to further test whether any relate to FKBP52-directed changes in AR structure and function. Also, based on the 52KO mouse phenotype, mutation of FKBP52 rather than AR could underlie instances of androgen insensitivity that are not due to AR gene mutation (23, 40).
Hypospadias, a flaw in urethral tube formation, is an idiopathic birth defect that occurs in as many as 1 in 200 male infants (41). Because we consistently observe hypospadias in 52KO males, disruption of FKBP52 function at a critical time during embryonic development could contribute to this deformity. One could further speculate that FKBP52 is a toxicological target for environmental endocrine disruptors that dont act by directly binding steroid receptor (42, 43). Finally, targeting FKBP52 function might be a useful therapeutic means to alter AR activity in a tissue-restricted manner. For example, retarded prostate development in 52KO mice suggests that a specific FKBP52 blocker might inhibit androgen-dependent prostate proliferation without severely disrupting testis function. The 52KO mouse line will be highly valuable in experimentally addressing these issues and future studies into the physiological actions of FKBP52. At the same time, much remains to be learned about the molecular mechanisms by which FKBP52 alters steroid receptor function.
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MATERIALS AND METHODS |
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Antibodies
Mouse monoclonal antibodies 2G6 (Hip), F5 (Hop), Hi52A/Hi52C/Hi52D (each specific for FKPB52), and rabbit polyclonal antibodies anti-FKBP52 and anti-FKBP51 were prepared in the Smith lab. Monoclonal antibodies JJ3 (p23), BB70 (Hsp70), and H9010 (Hsp90) were provided by David Toft (Mayo Clinic, Rochester, MN), and anti-L3 (recognizing the yeast L3 ribosomal subunit) was provided by Jonathan Warner (Albert Einstein College of Medicine, Bronx, NY). Polyclonal anti-PP5 was provided by Michael Chinkers (University of South Alabama, Mobile, AL). Antibodies specific for CyP40 (PA3-022; Affinity Bioreagents, Golden, CO) and GAPDH (486504M; Biodesign International, Saco, MA) were obtained commercially.
Targeting Vector
Bacterial artificial chromosome (BAC) clones containing genomic regions for Fkbp4 were isolated by PCR screening the 129SvJ mouse BAC library (Genome Systems, St. Louis, MO). Restriction fragments were subcloned into pBluescript (pBS; Stratagene, La Jolla, CA) or pZero (Invitrogen, Carlsbad, CA) for further analysis and sequencing. PCR products amplified from the BAC clones were used to construct a targeting vector in pPGKneo (kindly provided by James Lee, Mayo Clinic Scottsdale). As illustrated in Fig. 1A, the targeting vector contained a neomycin resistance cassette flanked upstream by a 4-kb PCR product containing the 5' untranslated region of Fkbp4 up to exon 1 and downstream by a 1.5-kb PCR product containing a portion of exon 10 plus 3' sequences. Homologous recombination using the final construct resulted in removal of all coding exons (
9 kb) from Fkbp4. (The sequence for any PCR primer or hybridization probe used in these studies is available by request to the corresponding author.)
Generation of Targeted ES Cells and Knockout Mice
ES cells isolated from 129SvJ mouse were cultured in Knockout DMEM (Invitrogen) supplemented with 10% FBS, penicillin/streptomycin, essential amino acids, ESGRO (103 U/ml; Chemicon, Temecula, CA) with irradiated embryonic fibroblast feeder cells. ES cells were electroporated at 0.2 kV, 950 µF (Gene Pulser II; Bio-Rad, Hercules, CA) with linearized targeting vectors and selected with G418 (300 µg/ml). DNA from G418-resistant clones was isolated for Southern blot analysis. A DNA probe was used to distinguish EcoRI restriction fragments from wild-type (17 kb) and mutant (9 kb) alleles. Appropriate homologous recombination in ES cell clones was confirmed by PCR using primers complementary to sequences within the neomycin cassette and to 3' Fkbp4 sequences downstream from the recombination site. ES cell clones containing a mutant Fkbp4 allele were injected into C57BL/6 blastocysts and implanted into psuedopregnant 129SvJ females. Chimeric offspring were identified by coat patterns and mated to C57BL/6 mice to obtain germline transmission of the mutant allele.
Mouse Genotyping
To obtain DNA, tail pieces (58 mm) collected from weaned mice were digested in 400 µl proteinase K (1 µg/ml) at 55 C overnight; 200 µl saturated NaCl was added, and the mixture was centrifuged at 16,000 x g for 15 min at RT. Supernatant (500 µl) was mixed with 1 ml 100% ethanol, and the DNA precipitate was removed with a pipette tip and washed in 70% ethanol. The DNA pellet was allowed to dry briefly before resuspension in 100 µl TE buffer. Genotypes were determined by PCR using primers specific for the wild-type (2-kb product) or mutant (700-bp product) alleles.
Tissue Preparation and Histology
Testes, epididymides, and seminal vesicles were harvested from mice killed by CO2 asphyxiation. Human prostate samples were obtained as surgical waste after transurethral resection from patients with benign prostatic hyperplasia. Mouse tissues were fixed overnight in Bouins solution, and human prostate samples were fixed overnight in formalin. All tissues were processed for routine paraffin embedding by the Histology Core, Mayo Clinic Scottsdale. Paraffin sections (4 µm) were de-waxed by two 5-min rinses in xylene, and rehydrated in ascending grades of alcohol (5 min each) and a 20 min rinse in running dH2O. Tissue sections were stained with Harris hematoxylin (Sigma, St. Louis, MO) and Eosin-Y (Sigma). Slides were rinsed thoroughly in 100% ethanol (2 x 1 min), dipped in xylene, and mounted in Histomount (Zymed, South San Francisco, CA).
Immunohistochemistry
After dewaxing and rehydration, mounted paraffin sections (4 µm) were treated for antigen retrieval by heating in Antigen Unmasking Solution (Vector Laboratories, Burlingame, CA) using a 600-watt microwave oven (4 x 5 min). Slides were cooled to room temperature in a buffer bath. Tissue immunostaining was performed using a commercially available streptavidin-biotin-horse radish peroxidase kit (HistoPlus Kit; Zymed). Immunoreaction was visualized by applying aminoethyl carbozole substrate solution to the slide for 1530 min. In some cases, sections were counterstained with hematoxylin (Zymed) for 1 min before mounting (GVA Mount; Zymed). For AR immunostaining, endogenous peroxidase activity was blocked by treating sections with Peroxo Block (Zymed) for 45 sec, and endogenous avidin and biotin activities were blocked using avidin/biotin Blocking Kit (Zymed). Nonspecific antibody binding was blocked by incubating sections in 10% goat nonimmune serum for 10 min. Next, tissues were incubated in anti-AR PG-21 (10 ng/µl; Upstate, Lake Placid, NY) at 37 C for 1 h in a humid chamber, followed by a 10-min incubation in biotinlayted secondary antibody and a 10-min incubation in streptavidin-peroxidase. FKBP52 immunostaining was carried out essentially as described for AR with minor modification. To block nonspecific antibody binding, sections were preincubated in 10 mg/ml BSA (Sigma). Affinity-purified polyclonal anti-FKBP52 was applied to the sections at a 1:300 dilution and allowed to incubate at 37 C overnight in a humid chamber. Endogenous peroxidase activity was quenched by incubating sections in Peroxo Block for 2 min before secondary antibody/enzyme conjugate application and chromogen development. Images of immunostained tissues were captured using a Leica (Wetzler, Germany) DMRB microscope. Negative controls were performed similarly using a 1:1000 dilution of preimmune serum.
Western Blots
Mouse tissues were harvested from 10-wk-old animals and immediately Dounce-homogenized in ice-cold tissue homogenization buffer [10 mM Tris (pH 7.4), 2.5 mM EDTA, 10 mM monothioglycerol, 10% glycerol plus protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN)]. Human prostate samples were quick frozen in a liquid nitrogen bath and minced before Dounce homogenization. Tissue homogenates were centrifuged at 16,000 x g for 30 min, and supernatants were collected. Yeast lysates were prepared as described previously (20). HeLa-AR cell lysates were prepared with M-PER reagent (Pierce, Rockford, IL) according to suppliers directions. All tissue/cell extracts were mixed 1:1 with 2x sodium dodecyl sulfate (SDS) sample buffer, boiled, and subjected to SDS-PAGE. Proteins were transferred onto polyvinylidene difluoride membrane and immunostained with individual antibodies. In another experiment, a commercially available human multiple tissue blot (Human Normal Tissue Blot IV; ProSci, Poway, CA) was probed with antibodies recognizing FKBP52 or GAPDH.
AR Signaling Assays in Yeast
Yeast culture, transformation, and functional assays were performed essentially as described previously (20). Briefly, all yeast assays were performed in a common W303a parental strain (MATa leu2112 ura31 trp11 his311 15 ade21 can1100 GAL SUC2) transformed with a plasmid (p424GPD; Ref. 44) constitutively expressing human AR and a reporter plasmid (pUCS-26x) which carries the lacZ gene under control of hormone-responsive elements (45). For experimental purposes the common AR reporter strain was cotransformed with a plasmid constitutively expressing FKBP52 or other human chaperone proteins.
For hormone-dependent reporter assays, cells were grown at 28 C in liquid culture to an OD600 of 0.050.1. DHT (Sigma) was added to the cultures and cell density was regularly monitored by OD600 measurement. Beginning at 90 min after hormonal induction, 100 µl samples were withdrawn at 15-min intervals and added to an equal volume of ß-galactosidase assay reagent (Gal-Screen; Tropix, Bedford, MA) in 96-well microtiter plates at room temperature. The plate was read in a luminometer 2 h after the last sample was collected. The rate of increase in ß-galactosidase expression is the slope (R2 > 0.98) of light units plotted against increases in culture OD600 during the incubation period.
Assembly of FKBP52 with AR Complexes in Vitro
Radiolabeled human AR was generated by in vitro transcription/translation (TnT Kit, Promega, Madison, WI) of plasmid AR/pSPUTK in the presence of [35S]-methionine. The specific activity of labeled AR was determined by gel separation and autoradiography of aliquots of the synthesis mixture. For coimmunoprecipitation assays, anti-FKBP52 Hi52C (10 µg) or negative control antibody PR22 (10 µg) was bound to protein-G Sepharose (Amersham-Pharmacia Biotech, Piscataway, NJ) for 30 min at room temperature. Immune resins were washed (3 x 1 ml) with wash buffer (20 mM Tris, 50 nM NaCl, and 0.5% Tween 20) and added to 100 µl rabbit reticulocyte lysate (Green Hectares, Oregon, WI) supplemented with radiolabeled AR and an ATP regenerating system (10 mM phosphocreatine plus 50 µg/ml creatine phosphokinase). The reaction was incubated at 30 C for 30 min without addition or in the presence of DHT (100 nM), the Hsp90-inhibitor geldanamycin (36 mM; provided by Drug Synthesis & Chemistry Branch, Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute), or the peptidylprolyl isomerase inhibitor FK506 (2 mM; provided by Jon Clardy, Harvard Medical School, Boston, MA). Resin complexes were washed (3 x 1 ml) with ice-cold wash buffer, and bound proteins were extracted into SDS sample buffer and separated by SDS-PAGE. Gels were Coomassie-stained to visualize total proteins then dried and autoradiographed to visualize radiolabeled AR.
FKBP52 Knockdown
To test the functional requirement for FKBP52 in human cells, we used a HeLa-AR cell line (Ref. 22 ; provided by Michael Carey, UCLA, Los Angeles, CA) that stably expresses human AR. Cells were infected with a bicistronic viral vector (pQCXIX; CLONTECH, Palo Alto, CA) that expresses green fluorescent protein (GFP) and either an shRNA targeting FKBP52 or a scrambled shRNA. GP2293 packaging cells were grown in DMEM supplemented with 10% FBS (Hyclone, Logan, UT), and HeLa-AR were cultured in MEM supplemented with 10% FBS (Hyclone). A plasmid expressing the viral envelope protein (pVSV-G; CLONTECH) and the retroviral vector were transfected into GP2293 cells using Lipofectamine 2000 reagent (Invitrogen). Virus released from packaging cells were collected and concentrated by ultracentrifugation at 50,000 x g for 90 min. HeLa-AR were infected with concentrated virus and sorted for GFP expression by flow cytometry 48 h after infection. FKBP52 protein levels in GFP-positive HeLa-AR cells were compared by Western blots.
HeLa-AR expressing the control shRNA or FKBP52-specific shRNA were plated overnight in 12-well plates (1 x 105 cell/well). When cells had grown to approximately 6070% confluency, they were cotransfected with 20 ng pCMV-ß-galactosidase plasmid (CLONTECH), 0.7 µg probasin-luciferase reporter plasmid (244/96-pT81-Luc; provided by Robert Matusik, Vanderbilt, Nashville, TN) and 1 µg pCI-neo vector (Promega) lacking or containing a silently mutated FKBP52 cDNA that escapes shRNA knockdown. Twenty-four hours after transfection, 10 nM DHT was added and cells were incubated an additional 24 h. Treated cells were washed three times with PBS and lysed with 100 µl of M-PER reagent. For luciferase (Luciferase Assay System; Promega) and ß-galactosidase (Gal-Screen) assays, 30 µl and 20 µl, respectively, of cell lysate were added substrate mixtures and assayed according to suppliers instructions.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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First Published Online April 14, 2005
Abbreviations: AF, Activation function; AR, androgen receptor; BAC, bacterial artificial chromosome; BAG1, BCL2-associated athanogen 1; CHIP, carboxyl terminus of Hsc70-interacting protein; CyP40, 40-kDa cyclosporin A-binding cyclophilin; DHT, dihydrotestosterone; FKBP, FK506 binding protein; shRNA, short hairpin RNA; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; GR, glucocorticoid receptor; Hsp90, 90-kDa heat shock protein; 52KD, FKBP52 knockdown HeLa cells; 52KO, FKBP52 gene knockout mouse line; LBD, ligand binding domain; PP5, protein phosphatase 5; PPIase, peptidylprolyl isomerase; SDS, sodium dodecyl sulfate; TPR, tetratricopeptide repeat.
Received for publication February 2, 2005. Accepted for publication April 7, 2005.
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