The Atypical Orphan Nuclear Receptor DAX-1 Interacts with Orphan ...

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Molecular Endocrinology 18(8):1929–1940 Copyright © 2004 by The Endocrine Society doi: 10.1210/me.2004-0043

The Atypical Orphan Nuclear Receptor DAX-1 Interacts with Orphan Nuclear Receptor Nur77 and Represses Its Transactivation KWANG-HOON SONG, YUN-YOUNG PARK, KI CHEOL PARK, CHEOL YI HONG, JIN HEE PARK, MINHO SHONG, KEESOOK LEE, AND HUENG-SIK CHOI Hormone Research Center, School of Biological Sciences and Technology (K.-H.S., Y.-Y.P., C.Y.H., J.H.P., K.L., H.-S.C.), Chonnam National University, Gwangju 500-757, Korea; and Laboratory of Endocrine Cell Biology (K.C.P., M.S.), National Research Laboratory Program, Department of Internal Medicine, Chungnam National University School of Medicine, Daejeon 301-721, Korea DAX-1 (dosage-sensitive sex reversal adrenal hypoplasia congenital critical region on the X chromosome, gene 1) (NROB1) is an atypical member of the nuclear receptor family, which lacks the classical zinc finger DNA binding domain and acts as a coregulator of a number of nuclear receptors. In this study, we have found that DAX-1 is a novel coregulator of the orphan nuclear receptor Nur77 (NR4A1). We demonstrate that DAX-1 represses the Nur77 transactivation by transient transfection assays. Specific interaction between Nur77 and DAX-1 was detected by coimmunoprecipitation, yeast two-hybrid, and glutathione-S-transferase pull-down assays. The ligand binding domain of DAX-1 and the activation function-2 domain of Nur77 were determined as the direct interaction domains between DAX-1 and Nur77. In vitro competition binding assay showed that DAX-1 repressed Nur77 transactivation through the compe-

tition with steroid receptor coactivator-1 for the binding of Nur77. Moreover, DAX-1 repressed Nur77- and LH-dependent increase of cytochrome P450 protein 17 promoter activity in transient transfection assays. Furthermore, Nur77-mediated transactivation was significantly increased by downregulation of DAX-1 expression with DAX-1 small interfering RNA in testicular Leydig cell line, K28. LH treatment induced a transient increase in Nur77 mRNA, whereas LH repressed DAX-1 expression in a time- and dose-dependent manner in K28 cells. In addition, immunohistochemical analysis showed the expression of Nur77 in mouse testicular Leydig cells. These results suggest that DAX-1 acts as a novel coregulator of the orphan nuclear receptor Nur77, and that the DAX-1 may play a key role in the regulation of Nur77-mediated steroidogenesis in testicular Leydig cells. (Molecular Endocrinology 18: 1929–1940, 2004)

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clear receptors are composed of three distinct domains: an N-terminal domain containing a transactivation function (AF-1), a DNA binding domain (DBD) consisting of two zinc finger motifs, and the C-terminal ligand binding domain (LBD), containing the dimerization interface and a second activation domain, AF-2, responsible for ligand-induced activation (3–6). Transcriptional activation of nuclear receptors involves binding of the cognate ligand, dimerization, binding to target DNA sequences, and modulation of gene transcription via interactions with chromatin components and with the basal transcriptional machinery (4). Broad ranges of coregulatory factors, which associate with DNA-bound nuclear receptors, have been isolated, acting either as coactivators or corepressors of gene expression (7, 8). Transcriptional coregulators either bridge transcription factors and the components of the basal transcriptional machinery or remodel the chromatin structures (9–11). Among the nuclear receptors, three members (NR4A), Nur77, Nurr-1, and NOR1 have similar structural features of conserved DBD and LBD but retain variable sequence in the N-terminal AF-1 domain (3, 12). Specific ligands for these molecules have not yet

UCLEAR RECEPTORS ARE grouped into a large superfamily and are thought to be evolutionarily conserved protein consists of structurally and functionally related transcription factors (1, 2). Nuclear receptor superfamily regulates pivotal gene networks important for eukaryotic cell growth, development, and homeostasis, and also includes orphan nuclear receptors that do not have known ligands (3–6). NuAbbreviations: aa, Amino acids; AF-1, activation function-1; AF-2, activation function-2; AHC, adrenal hypoplasia congenital; AR, androgen receptor; DAX-1, dosage-sensitive sex reversal AHC critical region on the X chromosome, gene 1; CYP, cytochrome P450 protein; DBD, DNA binding domain; ER, estrogen receptor; FSK, forskolin; GST, glutathione-Stransferase; HA, hemagglutin; HDAC, histone deacetylase; K28, mouse testicular Leydig cell line; LBD-1, ligand binding domain; LRH-1, liver receptor homolog-1; luc, luciferase; NBRE, Nur77 response element; N-CoR, nuclear receptor corepressor; NT, N-terminal repeat region; PKA, protein kinase A; PR, progesterone receptor; SF-1, steroidogenic factor 1; siRNA, small interfering RNA; SRC-1, steroid receptor coactivator-1; tk, thymidine kinase; TSA, trichostatin A. Molecular Endocrinology is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving the endocrine community.

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been identified (3), and NR4A subfamily members are classified as immediate-early genes that are induced rapidly but transiently by a variety of stimuli (13–15). Evidence accumulated over the past decade suggests that this subfamily is involved in important signaling functions in the hypothalamo-pituitary-adrenal axis (16–18), apoptotic functions in T cells (19–23), lung cancer cells (24), and prostate cancer cells (25, 26). Recently, it has been demonstrated that LH treatment induces Nur77 gene expression in ovarian granulosa cells (27) and testicular Leydig cells (15), and that Nur77 is involved in the regulation of cytochrome P450 protein (CYP) 17 gene expression (28). Moreover, Nur77 also regulates steroid 21-hydroxylase and 20␣-hydroxysteroid dehydrogenase gene expression (16, 29), suggesting that Nur77 may play an important role in steroidogenesis. Furthermore, it has been reported that the Nur77 family behaves as endpoint effectors of the protein kinase A (PKA) signaling pathway acting through dimmers, and that the AF-1 domain of Nur77 plays a major role in transcriptional activation, cofactor recruitment, and intra- and intermolecular interactions (30–32). In addition, 6-mercaptopurine, a purine antimetabolite, regulates Nurr-1 and NOR1 transactivation through the AF-1 domain of NOR1 and Nurr-1 (32, 33). Although the Nur77 functions as an immediately early response gene (13–15, 27, 34) and its posttranslational modifications (35–39) have been well characterized, coregulators involved in Nur77 transactivation are not fully characterized. Recently, steroid receptor coactivator (SRC)-1 and silencing mediator for retinoid and thyroid hormone receptors (SMRT) have been shown to regulate the transactivation of Nur77 through direct protein-protein interactions (30, 40). The DAX-1 [dosage-sensitive sex reversal, adrenal hypoplasia congenital (AHC) critical region on the X chromosome, gene 1; NR0B1] gene was identified through a search for gene linked to AHC, a disease affecting the normal development of the adrenal cortex and often associated with hypogonadotropic hypogonadism (41). DAX-1 lacks the zinc finger DBD, a typical structure of most nuclear receptors, but its C terminus consists of a putative LBD, although no ligand has been identified yet (3). Instead, its N terminus consists of a unique repeat domain implicated in single-stranded DNA and RNA binding (42, 43) as well as in protein-protein interactions (44, 45). Previous works have established that DAX-1 functions as a coregulatory protein rather than a typical transcription factor because it inhibits the transcriptional activity of the other nuclear receptor such as steroidogenic factor 1 (SF-1) (44), liver receptor homolog-1 (LRH-1) (46), estrogen receptor (ER) (45), androgen receptor (AR) (47, 48), and progesterone receptor (PR) (48). Furthermore, DAX-1 inhibits gene expression through the recruitment of the corepressors N-CoR (nuclear receptor corepressor) (44) and Alien (49) to a potent silencing domain localized in the C terminus of DAX-1. Interestingly, a large number of DAX-1 mutations found in

Song et al. • DAX-1 Regulates Nur77 Transactivation

patients with adrenal hypoplasia congenita have the common feature of an altered C terminus (50, 51), which abolish a potent silencing function within the LBD and have lost the ability to recruit corepressors such as N-CoR and Alien (49, 51). DAX-1 shows restricted gene expression pattern in tissues directly involved in steroidogenesis and reproductive function, such as adrenal cortex, testis, ovary, and pituitary (51–53). Interestingly, this pattern of DAX-1 gene expression is overlapped with that of Nur77, suggesting that both receptors may cooperate in these tissues. In this study, we show that DAX-1 is a novel transcriptional coregulator of Nur77. We further present evidences that DAX-1 physically interacts with Nur77 and represses the Nur77 transactivation through interfering binding of coactivator SRC-1. Moreover, LH represses DAX-1 gene expression but increases Nur77 gene expression in testicular Leydig cells. We propose that DAX-1 is a novel coregulator of Nur77 transactivation and may play an important role by modulating Nur77-mediated cellular responses.

RESULTS DAX-1 Inhibits Nur77 Transactivation Previous studies have demonstrated that DAX-1 physically interacts with SF-1, LRH-1, ERs, AR, and PR, and acts as a potent corepressor (44–48). Moreover, DAX-1 and Nur77 are expressed in similarly restricted tissues (15, 27, 29, 51–53). To determine whether DAX-1 plays any regulatory role in the transcriptional activity of Nur77, we performed transient transfection with the reporter construct containing the Nur77 response element (NBRE), Nur77, and DAX-1 expression vectors in CV-1 cells. Coexpression of increasing amounts of DAX-1 with a constant amount of Nur77 expression vector caused progressive repression of Nur77-mediated transactivation in a dose-dependent manner, and 4-fold excess of DAX-1 resulted in up to 80% inhibition of the Nur77 transactivation (Fig. 1, upper). Western blot analysis showed that DAX-1 protein level was increased as increasing amounts of the DAX-1 expression vector, whereas Nur77 protein level was not significantly changed (Fig. 1, lower). These results suggest that DAX-1 is a novel coregulator of Nur77 transactivation. Nur77 Interacts with DAX-1 in Vivo and in Vitro To determine whether the repressive function of DAX-1 on Nur77 transactivation is due to the direct physical interaction of DAX-1 with Nur77, we performed coimmunoprecipitation assay using cells from a mouse testicular Leydig cell line (K28) treated with LH for1 h. As shown in Fig. 2A, DAX-1 was specifically coimmunoprecipitated with Nur77 in LH-treated cells but not in untreated cells. Expression level of DAX-1 proteins was also confirmed by Western blot analysis


Fig. 1. Inhibition of Nur77 Transactivation by DAX-1 Upper, CV-1 cells cultured on 24-well plates were transfected with 200 ng of NBRE-Luc reporter, 50 ng of pH␤Nur77 (Nur77), 100 ng of pCMV-␤-gal, and increasing amounts (50, 100, and 200 ng) of pcDNA-HA-DAX-1 (DAX-1). The total amount of DNA was kept constant by adding empty pcDNA vector. After 36 h, the cells were harvested, and luciferase activities were determined. The transfection efficiency was normalized using ␤-galactosidase activity. Lower, Fifty micrograms of cellular extracts from the transient transfection assay were resolved on SDS-PAGE, transferred to a nitrocellulose membrane and detected the expression of Nur77 and DAX-1 as described in Materials and Methods.

using crude cell lysates with the antibody against DAX-1. These results suggest that DAX-1 physically interacts with Nur77 in vivo. To further investigate which region of DAX-1 and Nur77 is involved in the physical interaction of both proteins, we performed yeast two-hybrid assay and glutathione-S-transferase (GST) pull-down assay. Because it has been reported that N-terminal repeat region of DAX-1 (DAX-1-NT), which contains LXXLL motif and is involved in the interaction with SF-1 (44) and ER (45), we divided DAX-1 into two parts, N-terminal repeat domain (DAX-1-NT) and LBD (DAX-1-LBD) (Fig. 2B). Interestingly, yeast two-hybrid assay revealed that not only the full-length DAX-1 but also N-terminal deletion construct DAX-1-LBD retained the interaction ability to Nur77 (Fig. 2B), suggesting that C-terminal LBD domain of DAX-1 is necessary for Nur77 interaction. It has previously shown that DAX-1 specifically requires the carboxy-terminal domain of nuclear receptors for both interaction and repression (44–46). To test whether repressive function of DAX-1 also requires the carboxy-terminal region of Nur77, a series of deletion constructs of Nur77 were used to map the Nur77 interaction domain (Fig. 2C). Yeast two-hybrid assay showed that Nur77-dAF-2, the deletion construct of Nur77 AF-2 domain, could not interact with DAX-1, suggesting that the AF-2 domain of Nur77 is required for the interaction (Fig. 2C). AF-1 domain of Nur77 (Nur77-AB; amino acids 1–254) or DBD domain (Nur77-CD; amino acid 255–354) alone did not interact with DAX-1, whereas Nur77-CDE, which contains the

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AF-2 domain of Nur77 interacted with DAX-1, suggesting that the AF-1 and DBD domains of Nur77 are not essential for the interaction between DAX-1 and Nur77. Interaction between DAX-1 and SF-1 was used as a positive control. To confirm the domain mediating DAX-1 and Nur77 interaction in vitro, two deletion constructs of DAX-1 were made and fused to hemagglutinin (HA) (DAX1-NT and DAX-1-LBD), and evaluated their binding to Nur77 in GST pull-down assay. As shown in Fig. 2D, wild-type Nur77 bound specifically to not only wildtype DAX-1 but also DAX-1-LBD. However, DAX-1-NT did not bind to Nur77. Furthermore, DAX-1 binding was lost with GST alone and GST-Nur77-dAF-2. Taken together, these results demonstrate that that DAX-1 requires the AF-2 domain of Nur77 for the direct physical interaction. Functional Consequences of the Interaction between Nur77 and DAX-1 To confirm whether the AF-2 domain of Nur77 is involved in DAX-1-mediated repression, we cotransfected GAL4-Nur77 and GAL4-Nur77-dAF-2 with or without DAX-1 expression vector. Transfection of GAL4-Nur77 and GAL4-Nur77-dAF-2 efficiently induced the expression of reporter GAL4-tk-Luc relative to the GAL4 DBD, and the transcriptional activity of GAL4-Nur77 was repressed significantly by the addition of DAX-1, whereas the activity of GAL4-Nur77dAF-2 was not affected by DAX-1 (Fig. 3A). Moreover, it has been reported that deletion of the putative AF-2 domain of Nur77 does not affect Nur77 transactivation (40). Thus, we examined the effect of DAX-1 on Nur77dAF-2 transactivation. Consistent with the interaction of DAX-1 with the AF-2 domain of Nur77 (Fig. 2, C and D), DAX-1 could not repress Nur77-dAF-2 transactivation, suggesting that AF-2 domain of Nur77 is critical for DAX-1-mediated repression of Nur77 transactivation. Because DAX-1 LBD domain is required for interaction with Nur77, we investigated whether DAX-1 LBD alone could repress Nur77-mediated transactivation. Transient transfection assay was performed with Nur77 in the presence of HA-DAX-1-NT or HA-DAX-1-LBD (Fig. 3C). As expected from interaction assays, cotransfection with DAX-1-NT failed to reduce Nur77-mediated transactivation, whereas DAX-1-LBD caused progressive suppression of Nur77-mediated transactivation, suggesting that DAX-1 LBD domain is involved in the interaction and repression of Nur77 transactivation. It has been reported that Nur77 subfamily members, Nurr-1 and NOR1, share extensive homology with Nur77 in their AF-2 domain (3, 12). To explore the possibility that DAX-1 performs a similar function to Nurr-1 or NOR1, we cotransfected these Nur77 subfamily members with or without DAX-1. Although the degree of repression by DAX-1 was variable among Nur77 family members, DAX-1 also significantly inhibited the transactivation of Nurr-1 and NOR1 (Fig. 3D),

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Fig. 2. Determination of Interaction Domain between Nur77 and DAX-1 A, Physical interaction of Nur77 with DAX-1 in coimmunoprecipitation assay. Protein extracts were prepared after treatment with or without LH for 1 h in K28 cells, and immunoprecipitated with anti-Nur77 antibody (IP-Nur77). Immunoprecipitated proteins were resolved on SDS-PAGE and analyzed by Western blotting with anti-DAX-1 and anti-Nur77 antibodies. B, DAX-1 LBD is required for Nur77 interaction. LexA-DAX-1 wild-type and deletion constructs, as indicated in the upper panel, were cotransformed with B42-Nur77 into EGY48 yeast cells. Transformants were selected on plates with appropriate selection marker, and the ␤-galactosidase activity was measured. The results shown are the mean of ␤-galactosidase value from five independent transformant colonies. NT, N-terminal aa 1–200; LBD, C-terminal aa 201–470. C, Analysis of DAX-1 interaction domain of Nur77. LexA-DAX-1 and B42-Nur77 wild-type and deletion constructs, as indicated upper panel, or B42-SF-1 were cotransformed into EGY48 yeast cells. Yeast two-hybrid assay was done as described above in B. D, GST pull-down assay. Purified GST-Nur77 or GST-Nur77-dAF-2 or GST alone bound to glutathione-Sepharose beads were incubated with 35S-labeled a series of DAX-1. After extensive washing, the reactions were analyzed by SDS-PAGE and bound DAX-1 was visualized by autoradiography. The input represents 10% of the labeled DAX-1 used for the pull-down assay.

suggesting that DAX-1 represses the transactivation of Nur77 family. Taken together, these results suggest that interaction region of Nur77 and DAX-1 is critical for the repressive function. DAX-1 Competes for and Represses Binding of SRC-1 to Nur77 To investigate the functional mechanism of Nur77 repression by DAX-1, we compared wild-type DAX-1 with naturally occurring DAX-1 mutant (DAX-1-R267P) which is lost the intrinsic repression potential possibly caused by its inability to bind the corepressors N-CoR and Alien (49, 51). By cotransfecting the same amounts of expression vectors for wild-type and mu-

tant DAX-1, we observed that both wild-type DAX-1 and DAX-1-R267P significantly repressed Nur77mediated transactivation in a dose-dependent manner. However, DAX-1-R267P showed reduced ability to repress Nur77-mediated transactivation compared with the wild-type DAX-1 (Fig. 4A). DAX-1 has neither histone deacetylase (HDAC) catalytic activity nor a putative HDAC binding site, but it interacts with N-CoR, which recruits HDACs (44, 45). Thus, we investigated whether trichostatin A (TSA), a HDAC inhibitor, could release the repressive action of DAX-1. As shown in Fig. 4B, the repressive effect of DAX-1 on Nur77 remained unchanged in the presence of TSA, although TSA increased overall activity of



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Fig. 3. Domains of Nur77 and DAX-1 Necessary for Functional Interaction A, The AF-2 domain of Nur77 is essential for DAX-1-mediated Nur77 repression. CV-1 cells were transfected with 250 ng of Gal4-tk-Luc reporter, 100 ng of pCMV-GAL4 DBD-Nur77 (Gal4-Nur77 WT) or pCMV-GAL4 DBD-Nur77-dAF-2 (Gal4-Nur77dAF-2) and increasing concentrations (100 and 200 ng) of pcDNA-HA-DAX-1 (DAX-1). The transfection efficiency was normalized using ␤-galactosidase activity. B, CV-1 cells were cotransfected with 200 ng of NBRE-Luc reporter, 50 ng of pcDNA3-Nur77 (Nur77) or pcDNA3-Nur77-dAF- (Nur77-dAF-2), 100 ng of pCMV-␤-galactosidase, and increasing amounts (100 and 300 ng) of pcDNA-HA-DAX-1 (DAX-1). The transfection efficiency was normalized using ␤-galactosidase activity. C, DAX-1 LBD is required for Nur77 repression. CV-1 cells cultured on 24-well plates were transfected with 200 ng of NBRE-Luc reporter, 50 ng of pH␤-Nur77 (Nur77), 100 ng of pCMV-␤-gal, and increasing amounts (50, 100, and 200 ng) of pcDNA-HA-DAX-1 (DAX-1-WT) or pcDNA-HA-DAX-1-NT (DAX-1-NT) or pcDNA-HA-DAX-1-LBD (DAX-1-LBD). D, Expression of DAX-1 leads to inhibition of transactivation of all Nur77 family members. CV-1 cells were cotransfected with 200 ng of NBRE-Luc reporter along with Nur77 family member, 50 ng of pCMX-Nur77 (Nur77), 50 ng of pCMX-Nurr1 (Nurr-1), and 50 ng of pCMX-NOR1 (NOR1) and increasing amounts (100 and 200 ng) of pcDNA-HA-DAX-1 (DAX-1). The total amount of DNA was kept constant by adding empty pcDNA vector. The transfection efficiency was normalized using ␤-galactosidase activity.

Nur77. These results suggest that TSA-sensitive HDACs may not be involved in DAX-1-mediated repression of Nur77 transactivation. It has been reported that coactivators p300/cAMP response element binding protein-binding protein and glucocorticoid receptor-interacting protein-1 directly interact with Nur77 and increase Nur77 transcriptional activity (30, 31). Furthermore, the fact that both SRC-1 (30) and DAX-1 interaction with Nur77 require the AF-2 domain led us to hypothesize that the DAX-1-mediated repression of Nur77 transactivation might be through competition for its AF-2 domain. We transfected NBRE-Luc reporter together with expression vectors for Nur77, Nur77-dAF-2, SRC-1, and DAX-1 into CV-1 cells. Cotransfection of SRC-1 enhanced Nur77- and Nur77-dAF-2-mediated transactivation

and increasing amount of DAX-1 repressed the positive effect of SRC-1 on Nur77-mediated transactivation in a dose-dependent manner, whereas no significant repression by DAX-1 was observed on SRC-1-mediated enhancement of Nur77-dAF-2 transactivation (Fig. 4C). To confirm the direct competition of DAX-1 and SRC-1 on Nur77 binding, we performed in vitro competition binding assays using [35S]methionine-labeled SRC-1 and DAX-1 with GST-fused Nur77. As shown in Fig. 4D, upper panel, both SRC-1 and DAX-1 interacted with Nur77 and the increasing amounts of DAX-1 protein caused a dose-dependent decrease in the binding of SRC-1 and a corresponding increase in the binding of DAX-1. However, GSTNur77-dAF-2 interacted with SRC-1 but not with DAX-1, and even increasing amounts of DAX-1 could



Fig. 4. DAX-1 Competes for Binding of SRC-1 to the AF-2 Domain of Nur77 A, CV-1 cells cultured on 24-well plates were transfected with 200 ng of NBRE-Luc reporter, 50 ng of pH␤-Nur77 (Nur77), 100 ng of pCMV-␤-gal, and increasing amounts (100 and 200 ng) of pcDNA-HA-DAX-1 (DAX-1-WT) or pcDNA-HA-DAX-1-R267P (DAX-1-R267P). B, CV-1 cells cultured on 24-well plates were transfected with 200 ng of NBRE-Luc reporter, 50 ng of pH␤-Nur77 (Nur77), 100 ng of pCMV-␤-gal, and increasing amounts (50, 100, and 200 ng) of pcDNA-HA-DAX-1 (DAX-1). Cells were grown for 24 h before harvest either in the absence or presence of 100 nM TSA. C, CV-1 cells were cotransfected with 200 ng of NBRE-Luc reporter, 50 ng of pH␤-Nur77 (Nur77), 50 ng of pcDNA-HA-Nur77-dAF-2 (Nur77-dAF-2), 200 ng of pcDNA-SRC-1 (SRC-1), 100 ng of pCMV-␤-gal, and increasing amounts (100 and 300 ng) of pcDNA-HA-DAX-1 (DAX-1). The total amount of DNA was kept constant by adding empty pcDNA vector. After 36 h, the cells were harvested, and luciferase activities were determined. The transfection efficiency was normalized using ␤-galactosidase activity. D, lsqb]35S]Methionine-labeled in vitro-translated SRC-1 and increasing amounts of DAX-1 (1, 2, 5, or 10 ␮l) were incubated with a GST-Nur77 and GST-Nur77-dAF-2 fusion protein. The protein mixtures or [35S]methionine-labeled SRC-1 or DAX-1 alone were incubated with glutathione-Sepharose 4B beads and analyzed by SDS-PAGE (8%). The experiment was repeated three times.

not decrease SRC-1 binding to Nur77 (Fig. 4D, lower panel). Taken together, these results suggest that DAX-1 represses Nur77-mediated transactivation probably by the binding of SRC-1 to the AF-1 of Nur77. Effect of DAX-1 on CYP17 Promoter Activity To confirm the significance of repressive function of DAX-1 on Nur77 transactivation, the inhibitory effect of DAX-1 on Nur77- and LH-mediated CYP17 promoter activity was examined in K28. Consistent with a previous report (28), cotransfection with CYP17 promoter and Nur77 expression vector showed that the CYP17 promoter activity was dramatically increased by Nur77 (Fig. 5A). Significant inhibition of both Nur77- and LHmediated CYP17 promoter activities were observed after cotransfection with DAX-1 (Fig. 5, A and B).

These results suggest that DAX-1 represses Nur77and LH-dependent CYP17 gene transcription in testicular Leydig cells. To investigate the role of endogenous DAX-1 in regulating the Nur77 transactivation, we tested the NBRE-Luc reporter expression with or without transfection of DAX-1 small interfering RNA (siRNA) duplex. As shown Fig. 5C, siRNA#2, which is targeted from approximately 1288–1309 bp of the mouse DAX-1 cDNA, was effective in the inhibition of endogenous DAX-1 expression. However siRNA#1, which is targeted from approximately 907–92 bp, did not show significant inhibition of the endogenous DAX-1 gene expression. As a result, siRNA#1 was used as the control siRNA for siRNA#2. Transfection of DAX1siRNA#2 increased the Nur77-mediated transactivation about 2-fold in the cells with knock-down of en-



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dogenous DAX-1 (Fig. 5C), whereas the control siRNA#1 did not affect the Nur77 transactivation. These observations suggest that regulation of the intracellular DAX-1 gene expression may determine the Nur77-mediated target gene transcription. LH Regulates DAX-1 Gene Expression in Testicular Leydig Cells

Fig. 5. Effects of DAX-1 on the CYP17 Promoter Activity in K28 A, DAX-1 inhibited Nur77-induced CYP17 promoter activity in K28 cells. K28 cells were cotransfected with 200 ng of CYP17 promoter-Luc reporter, 100 ng of pH␤-Nur77 (Nur77), 100 ng of pCMV-␤-galactosidase, and increasing amounts (100 and 300 ng) of pcDNA3-HA-DAX-1 (DAX-1). The total amount of DNA was kept constant by adding empty pcDNA3 vector. The transfection efficiency was normalized using ␤-galactosidase activity. The mean ⫾ SD values from at least three independent experiments are shown. B, DAX-1 inhibited LH-induced CYP17 promoter activity in K28 cells. K28 cells were transfected with 200 ng of indicated Luciferase reporter and increasing amounts (100 and 300 ng) of pcDNA3-HA-DAX-1 (DAX-1). Cells were treated with LH (200 ng/ml), and assayed for luciferase activity 36 h after transfection. The transfection efficiency was normalized using ␤-galactosidase activity. Data represent the means of five independent experiments. C, Knockdown of the DAX-1 protein induces transactivation of Nur77. The effects of siRNAs on the DAX-1 expression were measured by the Western blot analysis in K28 cells as described in Materials and Methods (upper). K28 cells were transfected with DAX-1 siRNA#1 or #2. Twenty-four hours after transfection, the cells were cotransfected with NBRE-Luc and Nur77 expression vector together with pCMV-␤-gal vector (lower). After a 36-h transfection, cells were lysed, and luciferase activity was measured.

A large number of studies have shown that the secretion of LH is increased during the puberty in the testis, and LH is a major stimulus for the biosynthesis of testosterone in the Leydig cells (54, 55). Moreover, our previous study has demonstrated that LH rapidly increases the expression of Nur77 mRNA in testicular Leydig cells (15). Although DAX-1 expression is well documented in the testis (51–53), the hormonal regulation of DAX-1 in Leydig cell has not been fully characterized. Therefore, we analyzed the regulation of DAX-1 expression by LH in K28 cells. Northern blot analysis showed that DAX-1 expression was significantly decreased in a time- and dose-dependent manner (Fig. 6, A and B), whereas Nur77 expression was dramatically increased by LH treatment (Fig. 6B). Moreover, adenylate cyclase activator, forskolin (FSK, 10 ␮M) also represses DAX-1 expression in a timedependent manner (Fig. 6C). This result suggests that LH-mediated repression of DAX-1 expression might be due to the increase of intracellular cAMP level. We further studied Nur77 expression during the postnatal development of the testis. In the interstitial region where Leydig cells are located, immunoreactive cells against Nur77 antibody were rarely detected at postnatal d 14 (data not shown), but easily detected at postnatal d 38 (Fig. 6D). The antibody raised against Nur77 exclusively stained the nuclei of Leydig cells in the testis (indicated by arrows), in which Nur77 expression is known to be regulated by LH (15). No signal was detected in control sections that were incubated with preimmune serum (data not shown). This result demonstrates that Nur77 is localized exclusively in Leydig cells where LH-mediated steroidogenesis occurs.

DISCUSSION It has been reported that DAX-1 regulates the transactivation of classical nuclear receptors AR (47, 48), ER (45), PR (48), and the previously described orphan nuclear receptor SF-1 (44) and LRH-1 (46). Previous studies have shown that DAX-1 uses its N-terminal repeating region, which is defined as the LXXLLcontaining interaction domain, for the interaction with nuclear receptors (44, 45). However, in this study we demonstrate that DAX-1 interacts with Nur77 using its C-terminal LBD domain. In parallel with the mapping of interaction domain, we assessed the functional effects of DAX-1 on Nur77-mediated transactivation. Consis-



Fig. 6. LH Represses DAX-1 Gene Expression in K28 Proliferating K28 cells were cultured in serum-free condition for 24 h. These quiescent cells were then treated with 200 ng/ml LH for up to 24 h (A) or with different doses of LH for 1 h (B). The quiescent cells were treated with FSK (C; 10 ␮M) for up to 24 h. Total RNA (20 ␮g) was analyzed by Northern blotting using a cDNA probe for DAX-1 and Nur77. DAX-1 and Nur77 transcripts are indicated by arrow on right. The migration distance of 28S and 18S ribosomal RNA is indicated at left. The expression of GAPDH was used as an internal control. D, Immunohistochemical detection of Nur77 expression in testicular Leydig cells. Wild-type mouse testis at postnatal d 38 was immunostained with antimouse Nur77 antibody as described in Materials and Methods. Data are representative of at least three independent experiments. Magnification, ⫻400.

tent with the involvement of the DAX-1 LBD domain in interaction with Nur77, DAX-1-LBD represses Nur77meidated transactivation, whereas DAX-1-NT shows no significant effect on the repression of Nur77, suggesting that DAX-1 LBD domain is sufficient for the interaction and repression of Nur77. Recent reports demonstrate that coactivators p300/ cAMP response element binding protein-binding protein and glucocorticoid receptor-interacting protein-1 directly interact with Nur77 through its AF-1 domain and increase Nur77 transcriptional activity (20, 21), and DAX-1 inhibits the activity of the N terminus of AR (48). However, cotransfection experiments with GAL4fused Nur77-AB and DAX-1 shows that the AF-1 domain of Nur77 is not involved in the DAX-1-mediated repression (data not shown). Moreover, cotransfection with Nur77-dAF-2, the deletion construct of the AF-2 core domain of Nur77, does not affect the Nur77 transactivation (40), and the fact that DAX-1 could not repress Nur77-dAF-2 transactivation (Fig. 3B), suggest the requirement of the AF-2 domain of Nur77 for DAX1-mediated Nur77 repression. Nurr-1 and NOR1, the members of Nur77 superfamily, are structurally related

to Nur77 (3, 22), and their AF-2 sequences (I/V V/I D K I/L F M/L) are highly conserved. The result, that DAX-1 represses both Nurr-1 and NOR1 transactivation (Fig. 3D), strongly suggests the significance of the AF-2 domain of Nur77 family in DAX-1-mediated repression. Previous studies have suggested that DAX-1 inhibits nuclear receptor signaling by various mechanisms: 1) DAX-1 competes for the DNA binding of the retinoic acid receptor (41); 2) DAX-1 directly interacts with SF-1 and recruits corepressors N-CoR and Alien, which can make complex with HDACs (44). In the case of Nur77, EMSA demonstrated that DAX-1 was unable to bind directly to the NBRE that was used in transfection assays, and the DAX-1 could not interfere with the binding of Nur77 on NBRE (data not shown). Moreover, DAX-1 represses GAL4-fused Nur77 transactivation, suggesting that DAX-1 does not require direct contact with DNA to modulate Nur77 transactivation. Although DAX-1 represses transcription by recruiting N-CoR (44) and Alien (49), which interact with HDACs, the HDAC inhibitor, TSA, does not block DAX-1mediated repression of Nur77 (Fig. 4B). Furthermore,


DAX-1 mutant R269P, which is unable to bind the corepressors N-CoR and Alien, represses Nur77 transactivation (Fig. 4A), suggesting that the DAX-1mediated inhibitory mechanism for Nur77 is quite different from SF-1. The results presented in Fig. 4, C and D, indicate that DAX-1 counteracts the positive effect of SRC-1 on Nur77 transactivation. Moreover, DAX-1 competes the interaction between SRC-1 and Nur77, indicating that DAX-1 represses Nur77-mediated transactivation via competing the binding of coactivators such as SRC-1. It has been well documented that both Nur77 (15) and DAX-1 (56) are expressed in the testicular Leydig cells. However, hormonal regulation of DAX-1 expression in Leydig cells, a major place of steroidogenesis, is not fully characterized yet. Here, we demonstrate that DAX-1 expression is down-regulated by LH or FSK treatment, whereas LH increases Nur77 gene expression in cultured Leydig cells (15), suggesting that induction of Nur77 and repression of DAX-1 expression are critical for steroidogenesis in Leydig cells. This result is reminiscent of the previous report that activation of cAMP pathway by FSH or dibutyryl cAMP leads to the potent down-regulation of DAX-1 expression in cultured Sertoli cells (53). In addition, a recent report that activation of PKA is able to disrupt or weaken the interaction between DAX-1 and SF-1, and therefore rescues the SF-1 transactivation capability (57), suggesting that activation of PKA by LH potentially weakens the interaction between Nur77 and DAX-1. More intriguingly, during the testis development, significant increase of Nur77 expression in Leydig cell after puberty (Fig. 6D; Ref. 15) is relevant to the increase of steroidogenesis. In addition to the fact that expressions of Nur77 and DAX-1 is regulated by LH in testicular Leydig cells, the ability of DAX-1 to inhibit Nur77- and LH-induced activation of the CYP17 promoter supports a physiological role of DAX-1 in LH signaling in Leydig cells. Furthermore, we showed that the treatment of DAX-1 siRNA significantly increased the Nur77 transactivation, suggesting that DAX-1 plays an important role in Nur77-mediated CYP17 gene regulation. The ability of DAX-1 to repress CYP17 gene transcription suggests that the increased expression of DAX-1 effectively inhibits steroidogenesis by repressing CYP17 gene expression. In addition, recent reports that Nur77 levels are significantly elevated in SF ⫾ adrenal (58) and that Nur77 regulates CYP11B2 gene expression (59), suggesting that Nur77 serves a critical role in steroidogenesis. Furthermore, Nur77 and DAX-1 are coexpressed in many tissues, including the testis, ovary, pituitary, and adrenal gland. In summary, here we report that Nur77 and DAX-1 and the AF-2 domain of Nur77 and LBD domain of DAX-1 are involved in this interaction. DAX-1 inhibits Nur77 transactivation via competing with coactivator SRC-1 for the binding of Nur77 and represses LHmediated CYP17 expression. Our current study sug-


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gests that DAX-1 acts as a novel coregulator of Nur77 and represses Nur77 transactivation.

MATERIALS AND METHODS Hormone and Reagents Ovine LH (LH-s-26; 2300 IU/mg) was obtained from the National Hormone and Pituitary Distribution Program, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health (Baltimore, MD). TSA and FSK were purchased from Sigma Chemical Co. (St. Louis, MO). Plasmids The mammalian Nur77 expression vector, NBRE-tk-Luc (NBRE-Luc), and Gal4-tk-Luc reporter construct were as described previously (15, 60). The pCMX-Nurr1 and pCMXNOR1 were obtained from Dr. Thomas Perlmann (The Ludwig Institute for Cancer Research, Stockholm, Sweden). The mouse pCMV-DAX-1 and DAX-1 R267P mutants were obtained from Dr. Jeffrey Milbrandt (Washington University School of Medicine, St. Louis, MO). Mouse P450 17␣-hydroxylase/C17–20-lyase-luciferase reporter construct (CYP17-Luc: ⫺1021) was kindly provided by Dr. Anita Payne (Stanford University School of Medicine, Stanford, CA). The various deletion constructs of DAX-1 [DAX-1-NT, amino acids (aa) 1–200; DAX-1-LBD, aa 201–470] and Nur77 (Nur77-dAF-2, aa 1–590; Nur77-AB, aa 1–254; Nur77-CD, aa 254–335; Nur77-CDE, aa 254–601) were made by PCR with suitable restriction endonucleases and inserted into the pcDNA3-HA or pCMX-GAL4 DBD or the yeast LexA or B42 expression vector (CLONTECH Laboratories, Inc., Palo Alto, CA). For bacterial expression, GST-fused full-length of Nur77 and Nur77-dAF-2 were constructed by inserting EcoRI-XhoI fragments of Nur77 from B42-Nur77 and B42-Nur77dAF-2 into pGEX4T-1 vector (Amersham Biosciences, Piscataway, NJ) and pCMX vector. GST-SF-1 was as described previously (61). All the clones were confirmed by sequencing analysis. Coimmunoprecipitation K28 cells were treated with LH for 1 h and nuclear extract was preincubated with Nur77 antibody for 4 h at 4 C. Protein-A/G agarose beads were added, and the mixture was incubated for 4 h at 4 C. Antibody complexes were pelleted and washed three times with radioimmunoprecipitation assay buffer containing protease inhibitors (20 ␮g/ml leupeptin, 10 ␮g/ml pepstatin A, 2 ␮g/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride). Pellets were resuspended in loading buffer and analyzed by SDS-PAGE gel and immunoblotting using DAX-1 and Nur77 antibodies. Yeast Two-Hybrid Assay For the yeast two-hybrid system, full-length or deletion constructs of LexA-DAX-1 and B42-Nur77 were cotransformed into Saccharomyces cerevisiae EGY48 strain containing the LacZ reporter plasmid, p80p-Lac Z. Characterization of LacZ expression on plates was carried out as described elsewhere (61). Similar results were obtained in at least three independent experiments. GST Pull-Down Assay [35S]Methionine-labeled proteins were prepared using pcDNA3-HA vectors containing cDNAs encoding for full-


length and deletion constructs of DAX-1 and the TNTcoupled transcriptional translation system with conditions as described by the manufacturer (Promega, Madison, WI). GST-fused wild-type Nur77 (GST-Nur77) and Nur77 (GSTNur77-dAF-2) were expressed in Escherichia coli BL21 (DE3) strain and purified using glutathione-Sepharose 4B beads (Amersham Biosciences). In vitro protein-protein interaction assays were carried out as described previously (61). Transient Transfection and ␤-Galactosidase Assay CV-1 and K28 cells were maintained in DMEM (Invitrogen Life Technologies, Carlsbad, CA) in the presence of 10% and 15% fetal bovine serum (Invitrogen Life Technologies), respectively. For luciferase assays, cells were plated in 24-well plates 24 h before transfection and transfections were carried out with Superfect reagent (QIAGEN, Valencia, CA) or LipofectAMINE Plus reagent (Life Technologies, Inc., Gaithersburg, MD) according to the manufacturer’s instructions. Total DNA used in each transfection was adjusted by adding appropriate amount of pcDNA3 vector. Approximately 48 h post transfection, cells were harvested, and the luciferase activity was measured as described previously (15) and normalized against ␤-galactosidase activity as an internal control. Western Blot Analysis CV-1 cells were transfected with 50 ng of Nur77, and 50, 100, and 200 ng of DAX-1 using the Superfect reagent (QIAGEN), respectively. Forty-eight hours after transfection, culture plates were rinsed twice with cold PBS (pH 7.4), and cells were harvested and resuspended in lysis buffer [50 mM TrisHCl (pH 7.4), 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 1 ␮g/ml aprotinin, 1 ␮g/ml leupeptin, 1 mM HaF]. Proteins were fractionated by SDS-PAGE and electrophoretically transferred to Hybond nitrocellulose (Amersham Biosciences) as described previously (15). Enhanced chemiluminescence Western blotting (Amersham Biosciences) was performed according to the manufacturer’s instructions. Nur77 and DAX-1 proteins were detected by incubation of blots with an anti-Nur77 antibody (1:1000 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and anti-DAX-1 antibody (1:1000 dilution; Santa Cruz Biotechnology, Inc.), respectively. siRNA Experiments The siRNAs for DAX-1 were chemically synthesized (Dharmacon Research, Lafayette, CO), deprotected, annealed, and transfected according to the manufacturer’s instructions. The siRNA sequences are as follows: siRNA#1, GAUCACCUGCACUUCGAGdTdT; siRNA#2, CUGAACAGUGCCCUUUUCCdTdT. For the experiments in Fig. 5C, K28 cells were transfected with siRNA using Oligofectamine reagent (QIAGEN). Forty-eight hours after transfection, cells were extracted for Western blot analysis for DAX-1 and for ␤-actin as a control Northern Blot Analysis Northern blot analysis was performed using DAX-1 and Nur77 cDNA as a probe and after procedures are described previously (15). Immunohistochemistry For immunostaining of Nur77, paraffin-embedded sections of wild-type testes (38 d) were deparaffinized in Histoclear (Amresco, Solon, OH) and rehydrated in an ethanol series, fol-


lowed by blocking endogenous peroxidases with 10% hydrogen peroxide in 1⫻ PBS for 10 min. After washing in 1⫻ PBS, the section was processed by blocking for 10 min in blocking solution of Histostain-Plus kit (Zymed Laboratories, Inc., South San Francisco, CA) and incubated for overnight in Nur77 antibody with 1:100 dilution in blocking solution. After three rinses in PBS, the slide was treated for 10 min with biotinylated universal secondary antibody from HistostainPlus kit. Next, the slide was rinsed again in PBS and then incubated for 10 min in horseradish peroxidase-streptavidin conjugate reagent. Finally, the section was rinsed again in PBS and developed using the aminoethyl carbazole substrate kit for approximately 5 min or until adequate signal was seen. Slides were then washed in distilled water, mounted with glycerol vinyl alcohol mounting solution (Zymed), and observed under light microscope with bright-field illumination. Where indicated, the section was counterstained slightly with Mayer’s hematoxylin.

Acknowledgments We thank Dr. Thomas Perlmann for kind gifts of pCMXNurr1 and pCMX-NOR1, Dr. Anita Payne for the mouse P450 17␣-hydroxylase/C17–20-lyase-luciferase reporter construct, (CYP17-Luc, -1021), Dr. Jeffrey Milbrandt for the pCMVDAX-1 and DAX-1 R267P mutant constructs, and Dr. Sean Lee for the critical reading of the manuscript. We also thank Dr. Kyung-Tae Kim for technical assistance on siRNA experiment.

Received February 2, 2004. Accepted May 11, 2004. Address all correspondence and requests for reprints to: Hueng-Sik Choi, Ph.D., Hormone Research Center, Chungnam National University, Gwangju 500-757, Republic of Korea. E-mail: hsc@chonnam.ac.kr. This work was supported by KOSEF R02-2003-00010064-0 and Hormone Research Center (to H.S.C.). K.H.S. was a recipient of a Brain Korea 21 program in 2003.

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