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Department of Biosciences at Novum (E.H., S.M., S.K., J.Å.G., E.T.), Department of Medical Nutrition (J.Å.G.), Karolinska Institute, S-14157 Huddinge, Sweden; Department of Anatomy (S.M., L.S.), University of Turku, FIN-20520 Turku, Finland, Department of Clinical Chemistry (O.A.J.); Institute of Biotechnology (J.J.P.); Institute of Biomedicine and Biomedicum Helsinki (N.K., O.A.J., J.J.P.), University of Helsinki, FIN-00014 Helsinki, Finland
Address all correspondence and requests for reprints to: Dr. Eckardt Treuter, Department of Biosciences at Novum, Karolinska Institute, S-14157 Huddinge, Sweden. E-mail: eckardt.treuter{at}cbt.ki.se.
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INTRODUCTION |
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Previous work has established a coregulatory role of DAX-1 because it inhibits the activity of the orphan receptor steroidogenic factor 1 (SF-1), a critical regulator of gonadal and adrenal differentiation (8, 11, 12, 13), as well as of the two ERs, ER
and ERß (9). Presumably, DAX-1 acts as corepressor for these receptors via direct occupation of the coactivator-binding surface AF-2 and subsequent recruitment of other corepressors. A distinct cytoplasmic and nongenomic function has recently been attributed to DAX-1, i.e. the association of DAX-1 with polyribosomes, which is mediated by binding of the unique N-terminal repeat domain to RNA (7). Although the in vivo relevance of this finding is currently unclear, it indicates the existence of novel mechanisms whereby DAX-1 may interfere with fundamental processes in the cytoplasm.
DAX-1 is predominantly expressed in both male and female reproductive tissues of mammals, e.g. in testis and ovary, as well as in adrenal, hypothalamus and pituitary (14, 15, 16, 17). Multiple mutations have been detected in DAX-1 that cause an X-linked form of adrenal hypoplasia congenita (AHC) (12, 18, 19). The disorder is limited to males and is characterized by neonatal adrenal insufficiency and failure to undergo puberty because of hypogonadotropic hypogonadism. AHC mutations in DAX-1 (e.g. R267P) eliminate its repression function (12). While the function of DAX-1 in females is largely unknown, recent in vivo evidence from genetic knock-out mice points at a particular importance of DAX-1 for the development and maintenance of male reproductive functions (20, 21, 22). In addition to defects in spermatogenesis, DAX-1 (-/-) mice display significantly increased expression of the aromatase gene in the Leydig cells (23).
In light of these cumulative data on the function and expression of DAX-1, we became interested in studying possible connections between DAX-1 and another crucial regulator of the male reproductive system, the AR (also NR3C4). AR is a member of the steroid hormone receptor branch of the nuclear receptor superfamily (24) and, as mediator of androgen signaling, it plays important roles for the coordinated gene expression in male reproductive tissues (25, 26, 27, 28, 29). Previous functional and the structural studies have provided detailed insights into the mechanisms of AR activation (24, 30 and the references therein). Briefly, ligand binding to the cytoplasmic receptor induces conformational changes that, in turn, cause dissociation of the receptor from heat shock proteins, nuclear localization, binding to hormone response elements and association with coregulatory factors (31). A number of distinct coregulatory factors may play particular roles in AR signaling (32). While many of these factors function as bona fide coactivators or corepressors by directly communicating with chromatin and the transcription machinery, additional coregulators may exist that function in an antagonistic manner by preventing, disrupting or redirecting interactions with bona fide coactivators and corepressors.
In this study, we identify DAX-1 as an inhibitory coregulator for AR. We provide evidence for previously uncovered aspects of DAX-1 mechanisms of action in the nucleus and in the cytoplasm. These data strongly suggest that DAX-1 antagonism could play a physiological role in modulating AR-dependent gene regulation in reproductive tissues.
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, A and C) and pPB(-285/+32)-LUC containing the natural probasin promoter (Fig. 1B). In the absence of DAX-1, AR activated both reporters in an agonist-dependent fashion. Coexpression of increasing amounts of DAX-1 decreased AR activity in a dose-dependent manner, and 200 ng of DAX-1/well typically resulted in up to 80% inhibition of the AR activity (Fig. 1, A and B). DAX-1 inhibition was similarly observed in human HeLa cells and resulted in approximately 50% inhibition of the AR activity (data not shown). To get insight into mechanisms of AR inhibition by DAX-1, we studied the effect of the natural DAX-1 R267P mutant that has lost the intrinsic repression potential (12). By cotransfecting the same amounts of expression plasmids coding for wt and mutant DAX-1 proteins, we observed that the inhibitory potential of DAX-1 R267P on AR was significantly reduced, particularly at lower DAX-1 concentrations, but not abolished (compare Fig. 1, A and C). The idea that cellular cofactors affect DAX-1 inhibition is further consistent with the observation, that both DAX-1 wt and the R267P mutant were less inhibitory in HeLa cells than in Cos-7 cells (data not shown). As a control for the specificity of the AR inhibition by DAX-1, we coexpressed the TR with AR. Although unliganded TR is a potent transcriptional repressor and possibly shares cellular corepressors such as N-CoR and Alien with DAX-1 (8, 33), coexpression of TR did not inhibit AR activity in Cos-7 or in HeLa cells (data not shown). This suggests that coexpression of a transcriptional repressor, i.e. unliganded TR, that presumably does not physically interact with AR, is not sufficient to antagonize AR activation. Finally, previous studies have indicated that proper AR function requires interdomain communication between the AF-1 and AF-2 activation domains (34, 35). To investigate whether DAX-1 affects this interaction, we performed a mammalian two-hybrid assay with GAL4-AR LBD and VP16-AR-N-terminus (Fig. 1D). In the presence of 100 nM T, we observed an approximately 90-fold activation of the GAL4-responsive reporter gene (pG5-LUC). In contrast, cotransfecting with increasing amount of pSG5-DAX-1 resulted in a reduction of the reporter gene activity to one-fifth of control with 100 ng pSG5-DAX-1. This indicates that DAX-1 is indeed able to interfere with the interdomain communication of the AR.
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, left panel). In the remaining approximately 20% of the cells, DAX-1 was both nuclear and cytoplasmic (Fig. 2B, middle panel) or entirely nuclear (Fig. 2B, right panel). Apparently, nuclear DAX-1 displayed a rather structured pattern that is distinct from the more uniform AR pattern (compare Fig. 2, B and D). Further, DAX-1 but not AR displays a specific granulae-like pattern in the cytoplasm (compare Fig. 2, B and C), which appears very similar to that previously reported for the localization of both endogenous and overexpressed DAX-1 (7).
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, C and D) or wt AR that was detected by indirect immunofluorescence (data not shown). In both cases, we found AR to reside mainly in the cytoplasm in the absence of ligand (Fig. 2C), but after addition of 10 nM 5-dihydrotestosterone (DHT) to the cell culture medium, AR translocated into the nucleus (Fig. 2D). This is in agreement with previous studies utilizing these GFP-AR fusions (36). In contrast, when GFP-AR was coexpressed with DAX-1 (Fig. 3), we found in approximately 80% of the cells GFP-AR localized to the cytoplasm in an unique pattern, identical to that seen for DAX-1, both in the absence (Fig. 3A, upper panel) or presence of DHT (Fig. 3C, upper panel). This indicates that cytoplasmic DAX-1 either prevents AR from ligand-induced import into the nucleus or probably mediates AR export out of the nucleus. Additionally, activation of AR by ligand appears not required for colocalization with DAX-1 in the cytoplasm. In contrast, in approximately 17% of the coexpressing cells where DAX-1 was nuclear, we could not detect any colocalization with AR in the absence of ligand (Fig. 3A, lower panel). This is not unexpected because unliganded AR is entirely cytoplasmic and therefore cannot interact with nuclear localized DAX-1. Interestingly, this also indicates that DAX-1, which most likely shuttles between cytoplasm and nucleus, is unable to import unliganded AR to the nucleus. However, in the presence of ligand (Fig. 3C, lower panel) AR and DAX-1 colocalized in the nucleus in approximately 29% of the coexpressing cells because liganded AR is entirely nuclear and therefore can interact with nuclear localized DAX-1. For clarity, we estimated the average percentages of cells displaying a distinct localization or colocalization pattern of AR and DAX-1 in the absence (Fig. 3B) or presence (Fig. 3D) of ligand. This estimation shows that a significant number of cells does not coexpress the two receptors, which may in part explain why DAX-1 inhibition of AR activity in the transfection studies is not complete, as it would have been expected from the colocalization studies in the presence of AR ligand. In summary, these results demonstrate colocalization of DAX-1 with AR in intact cells both in cytoplasm and in nucleus and suggest that intracellular tethering of the AR represents a novel feature of DAX-1. Notably, it appears that in all cases of colocalization AR was tethered by DAX-1 in a dominant fashion, i.e. AR adopted the DAX-1 typical intracellular pattern.
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LBD coexpressed with DAX-1 (Fig. 4, A and B). The subcellular distribution of GFP-AR-LBD and GFP-AR-LBD in the absence of DAX-1 (Fig. 4, A and B, left panel) was as previously demonstrated (36). However, while coexpression of DAX-1 both in the absence and presence (data not shown) of ligand clearly had an effect on the subcellular distribution of GFP-AR-LBD (Fig. 4A, right panel), the localization pattern of GFP-AR-LBD was unaffected (Fig. 4B, right panel). These results indicate that the AR LBD is responsible for the cytoplasmic tethering of AR. Moreover, in the approximately 20% of the cells where DAX-1 was nuclear, GFP-AR-LBD and DAX-1 colocalized in the nucleus in the presence of ligand, whereas cells containing GFP-AR-LBD showed no similarities with the DAX-1 pattern in the nucleus (data not shown).
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). DAX-1 was expressed in the absence or presence of different AR domains (ARwt, -AR-LBD, -AR-LBD), proteins were extracted and subjected to immunoprecipitation using a mouse monoclonal AR antibody. Precipitated AR and DAX-1 proteins were visualized by immunoblotting using specific antibodies. We found that DAX-1 was only detected in association with ARwt or LBD (Fig. 4C, lanes 2 and 3), whereas no DAX-1 was precipitated in the absence of AR or with AR-LBD (Fig. 4C, lanes 1 and 4). This indicates that the two receptors interact in cells and that this interaction requires the LBD of AR, consistent with the colocalization data described above. Similar results were obtained in immunoprecipitations using FLAG-tagged AR, and the direct character of these interactions was supported by the results of GST pull-down assays (see below and Fig. 5C).
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) irrespective of the ligand status (data not shown), indicating that the N-terminal part of DAX-1 is sufficient for the tethering of AR in the cytoplasm. Unfortunately, when analyzing the GFP-DAX-1-LBD, the interpretation of the microscopic images appeared difficult because the distribution of GFP-DAX-1-LBD equals GFP alone and the number of cytoplasmic dots indicating colocalization was significantly lower when compared with GFP-DAX-1-NT (data not shown). Therefore, we investigated in the next experiment whether DAX-1 N- or C-terminal domains would interfere with the interdomain communication of AR and performed a mammalian two-hybrid assay with VP16-AR-NT and GAL4-AR-LBD (as described in Fig. 1D) in the presence of GFP, GFP-DAX-1-NT or GFP-DAX-1-LBD (Fig. 5B). Intriguingly, the DAX-1 N-terminal repeat domain and to a lesser extend also the putative LBD, but not GFP alone, inhibited the AR domain interaction, most likely due to competitive binding of DAX-1 to the AR C-terminus.
To obtain additional evidence for presumably direct interactions of AR with DAX-1, we performed GST pull-down assays with partially purified GST-DAX-1 fusion proteins and radiolabeled AR synthesized by in vitro translation. As seen in Fig. 5C, all three GST-DAX-1 fusion proteins, but not GST alone, were able to adsorb AR. Apparent quantitative differences may not necessarily be relevant for the in vivo situation because the GST-DAX-1 proteins are highly insoluble proteins whose quality and functionality might be not equal. However, binding of AR to GST-DAX-1 was considerably stronger than to a GST-TIF2 LXXLL- domain protein (data not shown), whereas the ERs bound well to both GST-TIF2 and GST-DAX-1 (9, 38). Furthermore, the lack of ligand effects on the AR interactions with DAX-1 is in agreement with the ligand-independence seen in the colocalization experiments and contrast the agonist-dependence seen with ERs (Ref. 9 and data not shown). Taken together, these results suggest that DAX-1 binds to the LBD of AR irrespective of its ligand status and that this binding is mediated by the N-terminal repeat domain, possibly with contribution of the putative LBD, of DAX-1.
DAX-1 Expression in the Prostate, a Major Target Tissue of AR Action
While DAX-1 expression is well documented in the testis (14, 15, 16, 17, 39), it was unknown whether DAX-1 is also expressed in the prostate, an important site of AR expression and function in the male (29). Therefore, we analyzed the expression of DAX-1 in human prostate using immunohistochemistry (Fig. 6). Interestingly, DAX-1 was mainly detected in epithelial cells, which are known to express high levels of both AR and ERß (40, 41, 42). DAX-1 was not detected in the stroma cells, sites of ER expression (42), and apparently weakly expressed in the basal cell layer adjacent to the epithelial cell layer. DAX-1 expression was similarly observed in the rat prostate but interestingly seems to be lost in the AR-positive human prostate cancer LNCaP cell line (data not shown).
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A model aiming at integrating these alternative mechanisms is presented in Fig. 7. Androgen signaling via the AR can be envisaged as a multistep cascade involving the dissociation of cytoplasmic chaperone/heat shock protein complexes upon ligand-binding, nuclear localization, DNA binding, and the association of AR with various bona fide coactivators (reviewed in Ref. 31) such as histone acetyltransferases [p160s, cAMP response element binding protein (CREB) binding protein (CBP), p300, p300/CBP-associated factor (PCAF), Tat-interacting protein 60 TIP60)] and a number of unrelated proteins, such as PIAS (protein inhibitor of activated STAT) proteins (43), AR-interacting protein (ARIP)/small nuclear ring finger protein (SNURF)/RNF4s, and AR-interacting nuclear protein kinase (ANPK) (reviewed in Ref. 32). Considerably less is known about the mechanisms by which androgen-dependent transcription is inhibited, and candidate corepressors have only recently been identified. They include the amino- terminal enhancer of split, a member of the groucho/transducin-like enhancer of split family of corepressors, that is not associated with histone deacetylases but instead functions through direct contacts to the basal transcription factor TFIIE (44). Other repressors of androgen action include cyclin D1, which directly antagonizes the acetyltransferase PCAF (45); SMAD3, an intracellular mediator of the TGFß pathway (46); and the protein kinases PAK6 and Akt, which presumably repress AR activity via direct phosphorylation (47, 48). Moreover, a novel covalent modification of AR by attachment of small ubiquitin-related modifier 1 in certain contexts inhibits the transcriptional activity of AR (49).
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Modulation of AR Signaling by DAX-1 at Multiple Levels
The results of our study imply intracellular (i.e. cytoplasmic or nuclear) tethering of AR by DAX-1 as a novel strategy to modulate hormone signaling, which may possibly apply to other DAX-1 targets as well. Unfortunately, structural and functional parameters regulating the intracellular localization of DAX-1 are largely unknown and remain to be elucidated to verify the physiological significance of this strategy. Regarding nuclear import, atypical NLS sequences seem to reside within the N-terminal repeat domain of DAX-1 (13). Regarding nuclear export, there is evidence showing that both AR and DAX-1 are actively exported into the cytoplasm, and that this export is unaffected by leptomycin B, a specific inhibitor of the exportin CRM1 (7, 37). Similarly, the signals regulating the intracellular localization of DAX-1 are unknown, but as yet unidentified ligands or protein modifications are good candidates (see below).
We have demonstrated here that DAX-1 can sequester AR in the cytoplasm, indicating a possible function of DAX-1 as a cytoplasmic retention factor. While the precise mechanisms behind these phenomena remain unclear, DAX-1 most likely interferes with events required for AR activation in the cytoplasm. This may include interference with the association and dissociation of chaperones or interference with nuclear import by masking nuclear localization signals of AR (see model Fig. 7). In support of the generality of this mechanism, we have obtained preliminary evidence that DAX-1 also is able to sequester GFP-ERß in the cytoplasm in an agonist-dependent manner (Holter, E., and E. Treuter, unpublished data). We further believe, that the cytoplasmic structures of the AR associated with DAX-1 described here seem substantially distinct from the previously described cytoplasmic aggregates formed by overexpressed polyglutamine-expanded GFP-AR (51). Although our results are based on ectopic expression of DAX-1, results from a previous study argue that the cytoplasmic pattern of endogenous DAX-1 in an adrenal cell line resembles that of DAX-1 expressed in Cos-7 cells (7). In support of the in vivo relevance is the fact that endogenous DAX-1 can be detected by immunohistochemistry, using different antibodies, in the cytoplasmic compartments of adrenal, testis, and prostate cells (7, 16, 52). Numerous previous studies have addressed the subcellular distribution and dynamics of AR and nuclear cofactors in cells (36, 37, 51, 53), but only a few cytoplasmic target proteins have been identified so far. These include, in addition to the well-studied heat shock protein chaperone complexes, the actin-binding protein filamin (53), and the membrane protein caveolin-1 (54). Interestingly, AR shuttling was defective in filamin-deficient cell lines, implying that disruption of AR interactions with cytoplasmic structural components provides yet another possibility for DAX-1 to interfere with the translocation of AR to the nucleus. Moreover, recent work that has indicated distinct nongenomic effects for AR and ERs (55, 56), suggesting a novel paradigm for steroid hormone action that involves the activation of a cytoplasmic kinase signaling pathway and attenuation of apoptosis. However, a direct visualization of the postulated endogenous AR complexes in the cytoplasm remains to be accomplished in future studies.
In view of previous evidence suggesting nuclear mechanisms of DAX-1 inhibition of SF-1 and ER- mediated transcriptional activation (8, 9, 11, 33), our study does not exclude the possibility that DAX-1 also inhibits AR function as a corepressor in the nucleus. This would be consistent with our observation that in a significant percentage of transfected cells liganded AR colocalized with DAX-1 in the nucleus (Fig. 3C). Moreover, we have demonstrated that DAX-1 can inhibit the AR-specific interdomain communication between AF-1 and AF-2 (Fig. 1D), suggesting that DAX-1 is able to interfere with a crucial event for AR activation in the nucleus (34, 35, 57). Interestingly, DNA-dependent protein-protein-interaction assays suggest the existence of DAX-1 containing ternary complexes with DNA-bound AR (Holter, E., and E. Treuter, unpublished data), which would be consistent with our previous demonstration of DNA-bound ER-DAX-1 complexes (9).
Conceivably, complexity for the in vivo situation certainly arises from the fact that DAX-1 is an orphan receptor that could have ligands. Because ligand binding is expected to change the structure and subsequently features of the DAX-1 LBD, caution must be exercised when speculating about the relative importance of transcriptional vs. nontranscriptional pathways. Regulation of DAX-1 localization by yet unidentified ligands or protein modifications may increase the amount of nuclear DAX-1 in a cell-type specific manner. Indeed, immunohistochemistry revealed that DAX-1 in the adrenal cortex is localized in both cytoplasmic and nuclear compartments, whereas entirely nuclear localization was seen in Sertoli cells of the testes (52). Ligand binding or modifications could also alter the interaction characteristics of DAX-1 with associated proteins, which include target nuclear receptors and coregulatory proteins.
Implications for AR Regulation in Normal and Malignant Reproductive Tissues
AR function is often changed in humans with reproductive abnormalities as well as in prostate cancer due to mutations within the LBD (26, 29). While some of these mutations have been demonstrated to affect the ligand-binding capacity and specificity, others are proposed to influence interdomain communication or direct interactions with coactivators (35, 58, 59, 60, 61). Similarly, multiple DAX-1 mutations have been detected that primarily target the putative LBD of DAX-1 (12, 18, 19). One of these mutations (DAX-1 R267P) was used in this study and found to be less potent in AR inhibition, consistent with previous results on ER inhibition (9). However, as this mutation presumably affects several features of the DAX-1 LBD, further investigations have to determine whether mutated DAX-1 displays changes with respect to intracellular tethering, coactivator competition or corepressor recruitment (see model Fig. 7). AR and DAX-1 are the only two reproductive nuclear receptors in which high numbers of natural mutations have been detected in male human beings (20). Notably, the genes for both AR and DAX-1 are on the X-chromosome; therefore, all mutations affecting the function yield a phenotype. Interactions between DAX-1 and AR may be important for the proper development of the male reproductive system, and it remains to be seen whether inappropriate interactions due to mutations in AR or DAX-1 may play roles in cancer development as well.
In addition to AR mutations, AR-associated coregulators have been considered as possible factors contributing to the development and maintenance of prostate cancer by altering the normal function of AR (29, 32). However, more direct evidence came only recently when two coactivators of the p160 family, namely TIF2 and SRC-1, were found to be overexpressed in AR-positive recurrent prostate cancer (62). These findings suggested that the association of overexpressed coactivators with AR provides a mechanism for AR-mediated transactivation in the absence of circulating androgens, which could account for the growth of recurrent prostate cancer after androgen deprivation. Here we demonstrate that DAX-1 is expressed in the epithelial cells of the prostate, which express high levels of the DAX-1 target receptors AR and ERß (41). Intriguingly, ERß appears to be a candidate regulator of AR expression in prostate and in ovary, and it has been suggested that loss of ERß could be associated with prostate cancer progression (63). Indeed, similar observations have been made for other putative corepressors such as SMAD3 (46) and the p21-activated kinase PAK6 (48). Conceivably, the lack of association of AR with inhibitory coregulators such as DAX-1 might contribute to the increased AR-transactivation potency in prostate cancer, and coactivator antagonism could play a physiological role in balancing transcriptional androgen responses in the normal prostate. Important physiological implications further arise from the possibility that DAX-1 expression could be regulated by sex hormones. Indeed, DAX-1 expression in testis has been found to peak during the androgen-sensitive phase of spermatogenesis (39). It remains to be seen whether male estrogens, which in the prostate may include the ERß agonist 5-androstane-3ß,17ß-diol rather than 17ß-E2 (41), also affect the expression of DAX-1. Finally, the possibility that either expression or function of DAX-1 could be regulated by ligands bears an exciting therapeutic potential in the treatment of reproductive organ cancers.
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aa 149–295), pSG5-rAR-LBD (aa 641–902), pcDNA3-Flag-hARwt, pEGFPC1-hARwt, pEGFPC1-hAR-LBD (aa 658–919), pEGFPC1-hAR-LBD (aa 1–657), pVP16-hAR-NT (aa 5–538), pGAL4-hAR-LBD (aa 624–919) (36, 57, 64, 65); reporter plasmids pPB (-285/+32)-LUC, pARE2-TATA-LUC, pG5-LUC (35, 36, 65) and DAX-1 plasmids pSG5-hDAX-1, pSG5-hDAX-1 R267P, pGEX4T1-hDAX-1wt, pGEX4T1-hDAX1-NT (aa 1–253) (9).
Mammalian Cell Transfections
Cos-7 monkey kidney cells were maintained in DMEM supplemented with 10% FBS, penicillin (100 µl/ml), and streptomycin (10 µl/ml) (Life Technologies, Inc., Gaithersburg, MD) For assaying the transactivation by AR, Cos-7 cells on 12-well plates received fresh medium containing 10% charcoal-stripped (CS) FBS 4 h before transfection. The cells were transfected with 20 ng pSG5-ARwt, 50–200 ng pSG5-DAX-1 or pSG5-DAX-1 R267P and 200 ng pARE2-TATA-LUC or pPB(-285/+32) with 20 ng ß-galactosidase internal control plasmid (pCMVß) using FuGene (Roche Molecular Biochemicals, Indianapolis, IN). Eighteen hours after transfection, the cells received fresh medium containing 2% CS FBS and 100 nM T or vehicle. Thirty hours after transfection, the cells were harvested and luciferase activities were measured (43). For the mammalian two-hybrid assay, the transfections were performed with 100 ng pGAL4-AR-LBD, 100 ng pVP16-AR-NT, 50–100 ng pSG5-DAX-1, or pEGFPC2-DAX-1-NT or pEGFPC2-DAX-1-LBD and 200 ng pG5-LUC.
Coimmunoprecipitations
For preparation of whole cell extracts, Cos-7 cells were plated on 150-mm diameter plates and transfected with total 10 µg expression plasmid (pSG5-ARwt/LBD/LBD, pSG5-DAX-1) using DEAE dextran (Amersham Pharmacia Biotech). After 24 h, the cells were collected in PBS and the extracts were prepared in 10 mM HEPES-KOH buffer (pH 7.9) containing 400 mM NaCl, 0.1 mM EDTA, 5% glycerol and protease inhibitor cocktail (Roche Molecular Biochemicals). Protein extract was incubated with protein A/G agarose beads (sc-2002, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 15 min at 4 C to minimize unspecific binding. After washing, the extract was further incubated with mouse monoclonal AR antibody Ab-1 (NeoMarkers, MS-443-P1ABX, raised against aa299–315 of hAR) at 4 C in IP-T150 buffer containing 50 mM Tris HCl (pH 7.4), 150 mM NaCl, 0.2% NP40, 1 mM EDTA, and protease inhibitors in the presence of 100 nM DHT. After 1 h Protein A/G Plus-Agarose beads were added and the incubation continued over night at 4 C. After three washes in IP-T150 buffer, the pellets were resuspended in electrophoresis sample buffer, boiled for 5 min, and analyzed on 12% SDS polyacrylamide gel. Proteins were transferred to a nitrocellulose membrane and visualized using the mouse monoclonal anti-AR antibody Ab-1 (dilution 1:1000) or a polyclonal rabbit anti-DAX-1 antibody (K-17, Santa Cruz Biotechnology, Inc.) (dilution 1:4000), respectively.
GST Pull-Down Assays
Interaction studies were performed essentially as described previously (9). Briefly, partially purified GST-DAX-1wt, -NT, or -LBD and in vitro translated [35S]methionine-labeled Flag-AR were incubated in pull-down buffer containing 50 mM KPi (pH 7.4), 100 mM NaCl, 1 mM MgCl2, 10% glycerol, 0.1% Tween 20, and 1.5% BSA for 2 h at room temperature in the absence or presence of 1 µM DHT. After extensive washing with pull-down buffer lacking BSA, bound AR protein was analyzed by SDS-PAGE and visualized by autoradiography.
Analysis of Intracellular Localization Using Confocal Microscopy
Cos-7 cells were plated on glass coverslips in six-well cell culture plates and grown in DMEM supplemented with 10% CS FBS, penicillin and streptomycin for 12 h. The cells were transfected for 8 h with 0.5 µg of each plasmid (total amount 1 µg) using Lipofectin (Life Technologies, Inc.). The cells were grown for 24 h after transfection before fixing with 3% paraformaldehyde in 5% sucrose/PBS for 20 min at room temperature. Four hours before fixing, 10 nM DHT was added. For indirect immunofluorescence, fixed cells were rinsed three times with PBS before being permeabilized with PBS/Tween (0.1%) three times for 5 min at room temperature. The cells were blocked with 5% goat serum (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) in PBS/Tween (1 h at room temperature) before incubating with polyclonal rabbit anti-DAX-1 antibody (K-17, Santa Cruz Biotechnology, Inc.) for 1 h (diluted 1:200 in PBS/Tween). After removal of the DAX-1 antibody by washing three times 5 min with PBS/Tween, cells were treated with Lissamine Rhodamine-conjugated AffiniPure goat antirabbit IgG (H + L) (Jackson ImmunoResearch Laboratories, Inc.) for 1 h at room temperature. Cells were washed five times for 5 min in PBS/Tween before being fixed to slides using antiphotobleaching fluorosave (Calbiochem, La Jolla, CA). Subcellular localization was determined using a TCS SP Multiband Confocal Imaging System (Leica Corp., Deerfield, IL). AR was detected in similar manner using monoclonal mouse anti-AR antibody Ab-1 (NeoMarkers, MS-443-P1ABX) (diluted 1:200 in PBS/Tween) followed by tetramethyl rhodamine isothiocyanate-conjugated goat antimouse IgG (H + L) (Jackson ImmunoResearch) when used together with GFP-DAX-1-NT.
Immunohistochemistry
The immunohistochemical staining was performed as described previously (66) with the following modifications. Frozen tissues from human prostate were thawed and then fixed in 4% paraformaldehyde. Blocking with 1% hydrogen peroxidase in 50% methanol/50%PBS and incubation with 10% rabbit or goat serum were performed to block the endogenous peroxidase and to reduce nonspecific staining of the secondary antibody. Between each step, washing with PBS was performed. The sections were treated with 0.5% Triton X-100 in PBS before they were incubated overnight at 4 C with rabbit polyclonal anti-DAX-1 (K-17, Santa Cruz Biotechnology, Inc.) diluted in PBS with 3% BSA (1:1000). The sections were washed with PBS and incubated for 1 h at room temperature with the secondary peroxidase-conjugated goat antirabbit antibody (Sigma) diluted in PBS with 3% human serum albumin (1:400), followed by washing. 3,3'-diaminobenzidine tetrahydrochloride liquid (DAKO Corp. A/S, Glostrup, Denmark) was used for color development. After coloring and rinsing with distilled water, the sections were counterstained slightly with Mayer’s hematoxylin, dehydrated in graded alcohols, cleared in xylene and mounted using Pertex (Histolab).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: aa, Amino acids; AHC, adrenal hypoplasia congenita; DAX-1, dosage-sensitive sex reversal, AHC critical region on the X chromosome, gene 1; DHT, 5-dihydrotestosterone; GFP, green fluorescent protein; LBD, ligand-binding domain; SF-1, steroidogenic factor 1.
Received for publication August 13, 2001. Accepted for publication December 6, 2001.
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REFERENCES |
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and ß in human testis. Mol Hum Reprod 7:497–503This article has been cited by other articles:
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  B. Nedumaran, G. S. Kim, S. Hong, Y.-S. Yoon, Y.-H. Kim, C.-H. Lee, Y. C. Lee, S.-H. Koo, and H.-S. Choi Orphan Nuclear Receptor DAX-1 Acts as a Novel Corepressor of Liver X Receptor {alpha} and Inhibits Hepatic Lipogenesis J. Biol. Chem., March 19, 2010; 285(12): 9221 - 9232. [Abstract] [Full Text] [PDF]
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B. Nedumaran, S. Hong, Y.-B. Xie, Y.-H. Kim, W.-Y. Seo, M.-W. Lee, C. H. Lee, S.-H. Koo, and H.-S. Choi DAX-1 Acts as a Novel Corepressor of Orphan Nuclear Receptor HNF4{alpha} and Negatively Regulates Gluconeogenic Enzyme Gene Expression J. Biol. Chem., October 2, 2009; 284(40): 27511 - 27523. [Abstract] [Full Text] [PDF]
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 A. Ehrlund, E. H. Anthonisen, N. Gustafsson, N. Venteclef, K. Robertson Remen, A. E. Damdimopoulos, A. Galeeva, M. Pelto-Huikko, E. Lalli, K. R. Steffensen, et al. E3 Ubiquitin Ligase RNF31 Cooperates with DAX-1 in Transcriptional Repression of Steroidogenesis Mol. Cell. Biol., April 15, 2009; 29(8): 2230 - 2242. [Abstract] [Full Text] [PDF]
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B. Xu, W.-H. Yang, I. Gerin, C.-D. Hu, G. D. Hammer, and R. J. Koenig Dax-1 and Steroid Receptor RNA Activator (SRA) Function as Transcriptional Coactivators for Steroidogenic Factor 1 in Steroidogenesis Mol. Cell. Biol., April 1, 2009; 29(7): 1719 - 1734. [Abstract] [Full Text] [PDF]
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 E. P. Sablin, A. Woods, I. N. Krylova, P. Hwang, H. A. Ingraham, and R. J. Fletterick The structure of corepressor Dax-1 bound to its target nuclear receptor LRH-1 PNAS, November 25, 2008; 105(47): 18390 - 18395. [Abstract] [Full Text] [PDF]
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 J. Zhou, R. H. Oakley, and J. A. Cidlowski DAX-1 (Dosage-Sensitive Sex Reversal-Adrenal Hypoplasia Congenita Critical Region on the X-Chromosome, Gene 1) Selectively Inhibits Transactivation But Not Transrepression Mediated by the Glucocorticoid Receptor in a LXXLL-Dependent Manner Mol. Endocrinol., July 1, 2008; 22(7): 1521 - 1534. [Abstract] [Full Text] [PDF]
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 H. V. Heemers and D. J. Tindall Androgen Receptor (AR) Coregulators: A Diversity of Functions Converging on and Regulating the AR Transcriptional Complex Endocr. Rev., December 1, 2007; 28(7): 778 - 808. [Abstract] [Full Text] [PDF]
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 D. Saxena, R. Escamilla-Hernandez, L. Little-Ihrig, and A. J. Zeleznik Liver Receptor Homolog-1 and Steroidogenic Factor-1 Have Similar Actions on Rat Granulosa Cell Steroidogenesis Endocrinology, February 1, 2007; 148(2): 726 - 734. [Abstract] [Full Text] [PDF]
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 S. Harlap, O. Paltiel, Y. Friedlander, R. Calderon-Margalit, L. Deutsch, K. R. Kleinhaus, O. Manor, A. I. Neugut, M. Opler, M. C. Perrin, et al. Prostate Cancer in Fathers With Fewer Male Offspring: the Jerusalem Perinatal Study Cohort J Natl Cancer Inst, January 3, 2007; 99(1): 77 - 81. [Abstract] [Full Text] [PDF]
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 D. Wilhelm, S. Palmer, and P. Koopman Sex Determination and Gonadal Development in Mammals Physiol Rev, January 1, 2007; 87(1): 1 - 28. [Abstract] [Full Text] [PDF]
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 C. J Burd, L. M Morey, and K. E Knudsen Androgen receptor corepressors and prostate cancer Endocr. Relat. Cancer, December 1, 2006; 13(4): 979 - 994. [Abstract] [Full Text] [PDF]
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 G. Benoit, A. Cooney, V. Giguere, H. Ingraham, M. Lazar, G. Muscat, T. Perlmann, J.-P. Renaud, J. Schwabe, F. Sladek, et al. International Union of Pharmacology. LXVI. Orphan Nuclear Receptors Pharmacol. Rev., December 1, 2006; 58(4): 798 - 836. [Abstract] [Full Text] [PDF]
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A. K. Iyer, Y.-H. Zhang, and E. R. B. McCabe Dosage-Sensitive Sex Reversal Adrenal Hypoplasia Congenita Critical Region on the X Chromosome, Gene 1 (DAX1) (NR0B1) and Small Heterodimer Partner (SHP) (NR0B2) Form Homodimers Individually, as Well as DAX1-SHP Heterodimers Mol. Endocrinol., October 1, 2006; 20(10): 2326 - 2342. [Abstract] [Full Text] [PDF]
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S. Carascossa, J. Gobinet, V. Georget, A. Lucas, E. Badia, A. Castet, R. White, J.-C. Nicolas, V. Cavailles, and S. Jalaguier Receptor-Interacting Protein 140 Is a Repressor of the Androgen Receptor Activity Mol. Endocrinol., July 1, 2006; 20(7): 1506 - 1518. [Abstract] [Full Text] [PDF]
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L. A. Helguero, M. Hedengran Faulds, C. Forster, J.-A. Gustafsson, and L.-A. Haldosen DAX-1 Expression Is Regulated during Mammary Epithelial Cell Differentiation Endocrinology, July 1, 2006; 147(7): 3249 - 3259. [Abstract] [Full Text] [PDF]
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S. Chattopadhyay, E.-Y. Gong, M. Hwang, E. Park, H. J. Lee, C. Y. Hong, H.-S. Choi, J.-H. Cheong, H. B. Kwon, and K. Lee The CCAAT Enhancer-Binding Protein-{alpha} Negatively Regulates the Transactivation of Androgen Receptor in Prostate Cancer Cells Mol. Endocrinol., May 1, 2006; 20(5): 984 - 995. [Abstract] [Full Text] [PDF]
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 Y.-Y. Park, S.-W. Ahn, H.-J. Kim, J.-M. Kim, I.-K. Lee, H. Kang, and H.-S. Choi An autoregulatory loop controlling orphan nuclear receptor DAX-1 gene expression by orphan nuclear receptor ERR{gamma} Nucleic Acids Res., November 28, 2005; 33(21): 6756 - 6768. [Abstract] [Full Text] [PDF]
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 J. M. Naciff, K. A. Hess, G. J. Overmann, S. M. Torontali, G. J. Carr, J. P. Tiesman, L. M. Foertsch, B. D. Richardson, J. E. Martinez, and G. P. Daston Gene Expression Changes Induced in the Testis by Transplacental Exposure to High and Low Doses of 17{alpha}-Ethynyl Estradiol, Genistein, or Bisphenol A Toxicol. Sci., August 1, 2005; 86(2): 396 - 416. [Abstract] [Full Text] [PDF]
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 S. Y. Park, J. J. Meeks, G. Raverot, L. E. Pfaff, J. Weiss, G. D. Hammer, and J. L. Jameson Nuclear receptors Sf1 and Dax1 function cooperatively to mediate somatic cell differentiation during testis development Development, May 15, 2005; 132(10): 2415 - 2423. [Abstract] [Full Text] [PDF]
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 R. Kumar, A. E. Gururaj, R. K. Vadlamudi, and S. K. Rayala The Clinical Relevance of Steroid Hormone Receptor Corepressors Clin. Cancer Res., April 15, 2005; 11(8): 2822 - 2831. [Abstract] [Full Text] [PDF]
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Y. Jo and D. M. Stocco Regulation of Steroidogenesis and Steroidogenic Acute Regulatory Protein in R2C Cells by DAX-1 (Dosage-Sensitive Sex Reversal, Adrenal Hypoplasia Congenita, Critical Region on the X Chromosome, Gene-1) Endocrinology, December 1, 2004; 145(12): 5629 - 5637. [Abstract] [Full Text] [PDF]
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K.-H. Song, Y.-Y. Park, K. C. Park, C. Y. Hong, J. H. Park, M. Shong, K. Lee, and H.-S. Choi The Atypical Orphan Nuclear Receptor DAX-1 Interacts with Orphan Nuclear Receptor Nur77 and Represses Its Transactivation Mol. Endocrinol., August 1, 2004; 18(8): 1929 - 1940. [Abstract] [Full Text] [PDF]
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Y.-Y. Park, H.-J. Kim, J.-Y. Kim, M.-Y. Kim, K.-H. Song, K. Cheol Park, K.-Y. Yu, M. Shong, K.-H. Kim, and H.-S. Choi Differential Role of the Loop Region between Helices H6 and H7 within the Orphan Nuclear Receptors Small Heterodimer Partner and DAX-1 Mol. Endocrinol., May 1, 2004; 18(5): 1082 - 1095. [Abstract] [Full Text] [PDF]
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 C. E. Petre-Draviam, S. L. Cook, C. J. Burd, T. W. Marshall, Y. B. Wetherill, and K. E. Knudsen Specificity of Cyclin D1 for Androgen Receptor Regulation Cancer Res., August 15, 2003; 63(16): 4903 - 4913. [Abstract] [Full Text] [PDF]
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I. U. Agoulnik, W. C. Krause, W. E. Bingman III, H. T. Rahman, M. Amrikachi, G. E. Ayala, and N. L. Weigel Repressors of Androgen and Progesterone Receptor Action J. Biol. Chem., August 15, 2003; 278(33): 31136 - 31148. [Abstract] [Full Text] [PDF]
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E. Lalli and P. Sassone-Corsi DAX-1, an Unusual Orphan Receptor at the Crossroads of Steroidogenic Function and Sexual Differentiation Mol. Endocrinol., August 1, 2003; 17(8): 1445 - 1453. [Abstract] [Full Text] [PDF]
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K. Kawajiri, T. Ikuta, T. Suzuki, M. Kusaka, M. Muramatsu, K. Fujieda, M. Tachibana, and K.-i. Morohashi Role of the LXXLL-Motif and Activation Function 2 Domain in Subcellular Localization of Dax-1 (Dosage-Sensitive Sex Reversal-Adrenal Hypoplasia Congenita Critical Region on the X Chromosome, Gene 1) Mol. Endocrinol., June 1, 2003; 17(6): 994 - 1004. [Abstract] [Full Text] [PDF]
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H. Mizusaki, K. Kawabe, T. Mukai, E. Ariyoshi, M. Kasahara, H. Yoshioka, A. Swain, and K.-i. Morohashi Dax-1 (Dosage-Sensitive Sex Reversal-Adrenal Hypoplasia Congenita Critical Region on the X Chromosome, Gene 1) Gene Transcription Is Regulated by Wnt4 in the Female Developing Gonad Mol. Endocrinol., April 1, 2003; 17(4): 507 - 519. [Abstract] [Full Text] [PDF]
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 P. Brown, G. A. Scobie, J. Townsend, R. A. L. Bayne, J. R. Seckl, P. T. K. Saunders, and R. A. Anderson Identification of a Novel Missense Mutation That Is as Damaging to DAX-1 Repressor Function as a Nonsense Mutation J. Clin. Endocrinol. Metab., March 1, 2003; 88(3): 1341 - 1349. [Abstract] [Full Text] [PDF]
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J. J. Meeks, S. E. Crawford, T. A. Russell, K.-i. Morohashi, J. Weiss, and J. L. Jameson Dax1 regulates testis cord organization during gonadal differentiation Development, March 1, 2003; 130(5): 1029 - 1036. [Abstract] [Full Text] [PDF]
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C. Brendel, K. Schoonjans, O. A. Botrugno, E. Treuter, and J. Auwerx The Small Heterodimer Partner Interacts with the Liver X Receptor {alpha} and Represses Its Transcriptional Activity Mol. Endocrinol., September 1, 2002; 16(9): 2065 - 2076. [Abstract] [Full Text] [PDF]
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55 kDa) cross-reacting with the secondary antibodies.
