Death-associated Protein 6 (Daxx) Mediates cAMP-dependent Stimulation of Cyp11a1 (P450scc) Transcription*

Background: HIPK3 mediates cAMP signaling and stimulates SF-1 activity to activate Cyp11a1 transcription. Results: In response to cAMP, HIPK3 phosphorylates Daxx, leading to JNK/c-Jun activation and the enhancement of SF-1-dependent Cyp11a1 expression. Conclusion: Daxx mediates HIPK3 effect to activate JNK/c-Jun for SF-1-dependent Cyp11a1 expression. Significance: The cAMP signaling pathway for the activation of the steroidogenic gene Cyp11a1 uses the apoptotic pathway components HIPK3/Daxx/JNK/c-Jun. SF-1 is a key transcription factor for all steroidogenic genes. It up-regulates the expression of the steroidogenic Cyp11a1 gene in the adrenal in a pathway stimulated by cAMP through HIPK3-mediated JNK/c-Jun phosphorylation. In the present study, we have investigated the factors mediating cAMP-dependent HIPK3 action to potentiate the activity of SF-1 for Cyp11a1 transcription in mouse adrenocortical Y1 cells. We found Daxx, a HIPK kinase substrate in the apoptosis pathway, was phosphorylated by HIPK3 at Ser-669 in response to cAMP stimulation. Daxx participated in SF-1-dependent Cyp11a1 expression as shown by experiments involving both overexpression and down-regulation via a dominant negative Daxx mutant. The S669A mutant of Daxx, which could not be phosphorylated by HIPK3, lost the ability to potentiate SF-1 activity for Cyp11a1 expression. The enhancement of SF-1 activity by Daxx required JNK and c-Jun phosphorylation. Thus, Daxx functioned as a signal transducer linking cAMP-stimulated HIPK3 activity with JNK/c-Jun phosphorylation and SF-1-dependent Cyp11a1 transcription for steroid synthesis.

SF-1 (Ad4BP, NR5A1) (1) is a key regulator for steroid biosynthesis, metabolism, and reproduction in the adrenals and gonads (2). It activates the transcription of many steroidogenic genes like Cyp11a1, StAR, Cyp21, etc (3). One SF-1 target gene, Cyp11a1, encodes P450scc that catalyzes the first and rate-limiting step of steroidogenesis. Cyp11a1 is expressed in a tightly regulated manner in the adrenals and gonads in response to the stimulation of adrenocorticotropin and gonadotropin, respectively. Upon stimulation by these tropic hormones, the intracellular cAMP level is increased to trigger a downstream signal-ing cascade that leads to increased Cyp11a1 expression. Although proteins like CREB and c-Jun potentiate SF-1 activity for Cyp11a1 expression (4), the components in the signaling pathway that lead to the enhancement of SF-1 activity are not well characterized. SF-1 activity is modulated by post-translational modifications (5)(6)(7) and interactions with other protein partners (8,9). One SF-1-interacting protein, homeodomaininteracting protein kinase 3 (HIPK3), 2 increases the ability of SF-1 to stimulate Cyp11a1 transcription in response to cAMP (10).
The roles of Daxx were initially established in apoptosis. Daxx mediates apoptosis stimulated by the death receptor Fas (16), UV irradiation (17), or TGF-␤ signaling (18). However, Daxx also possesses anti-apoptotic functions (19 -21), and Daxx is required for Mdm2 stability in the degradation of the pro-apoptotic protein p53 (22). Thus, Daxx plays dual functions in cell death.
Daxx serves as a scaffold protein and signal transducer. It up-regulates ASK-1 kinase activity (23) and the subsequent MKK/JNK signaling pathway (18,24), mediates the HIPK2 signal regulating JNK activity in TGF-␤-induced apoptosis (13), and mediates the activation of ASK-1/JNK/c-Jun and GLUT4 in response to serum deprivation (25). * This work was supported by grants from Academia Sinica, NHRI In addition to the roles in apoptosis and signal transduction, Daxx is a transcription regulator. Daxx represses c-Met transcription by recruiting HDAC2 to the gene (26). It also represses the activities of androgen receptor (27), CCAAT/enhancer-binding protein ␤ (28), AIRE (29), and Tcf4 proteins (30).
Despite numerous studies on Daxx, its role in steroidogenesis has never been reported. Here we show that Daxx participates in cAMP-stimulated steroidogenic Cyp11a1 transcription by mediating the effect of HIPK3. We found HIPK3 phosphorylated Daxx at Ser-669 resulting in the transactivation of SF-1 in mouse adrenal Y1 cells. Mutation of Ser-669 or depletion of Daxx resulted in Cyp11a1 down-regulation. Therefore, we uncovered a novel function of Daxx in steroidogenesis and the signal transduction pathway of HIPK3/Daxx/c-Jun in the regulation of SF-1 activity.

EXPERIMENTAL PROCEDURES
Cell Culture and Reagents-Y1 mouse adrenocortical tumor cells were maintained in Dulbecco's modified Eagle's medium/ F12 supplemented with 10% fetal calf serum. The human lung adenocarcinoma H1299 cell was maintained in RPMI 1640 medium supplemented with 10% fetal calf serum. 8-Br-cAMP (1 mM) was added to Y1 cells for 1 to 24 h for stimulation experiments. The Jun N-terminal kinase inhibitor SP600125 (Calbiochem) was added to cells for 6 h at a final concentration of 25 M. To remove phosphate groups from proteins, cell extract was incubated with 5 mM lambda protein phosphatase (New England Biolabs, Ipswich, MA) in reaction buffer supplemented with 1 mM MnCl 2 for 30 min at 37°C.
For luciferase assays, cells in 24-well plates were harvested in lysis buffer (100 mM potassium phosphate, pH 7.8, 0.2% Triton X-100, 0.5 mM DTT, 0.2 mM PMSF) 48 h after transfection and subjected to luciferase or ␤-galactosidase assays. Reporter activities were normalized against the internal control RSV-␤gal. At least three independent experiments were performed, and the standard deviations from the means are presented by error bars.
Western Blotting-Cells were harvested and lysed 48 h after DNA transfection or 3 days after siRNA transfection. Equal amounts of total protein mixture were separated by gel electrophoresis, transferred to Immobilon TM -P membrane (Millipore, MA), and incubated with specific antibodies overnight at 4°C followed by horseradish peroxidase-conjugated secondary antibody for 45 min. Signals were detected by chemiluminescence assays.
Co-immunoprecipitation (Co-IP) and in Vitro Kinase Assay-H1299 cells were lysed in IPH buffer (150 mM NaCl, 50 mM Tris-HCl, pH 8.0, 5 mM EDTA, 0.5% Nonidet P-40) plus 1X complete protease inhibitor mixture (Roche) 48 h post-transfection with FLAG-sHIPK3 and HA-Daxx. The cell lysate was then incubated with 1 g of mouse anti-FLAG antibody or normal mouse IgG and 30 l of 50% protein A-Sepharose beads for 2 h. Proteins bound to Sepharose beads were precipitated, dissolved, and denatured in sample buffer for gel electrophoresis and immunoblotting. For the in vitro kinase assay, protein substrates purified from Escherichia coli or from mammalian cells were incubated with active HIPK3 protein (Millipore) in buffer (20 mM Hepes, 1 mM EGTA, 0.4 mM EDTA, 5 mM MgCl 2 , 0.1 mM CaCl 2 , 0.05 mM dithiothreitol, 0.2 mg/ml phosphatidylinositol, 2.5 mM ␤-glycerophosphate, 1 M PMA, and 10 Ci [␥-32 P]ATP) at 30°C for 20 min before the reaction was stopped by heating for 5 min at 100°C in 4ϫ sample buffer (0.2 M Tris-HCl, 0.4 M DTT, 8% SDS, 0.4% bromphenol blue, 40% glycerol). The samples were separated by gel electrophoresis, stained with Coomassie Blue, and dried prior to exposure to x-ray film.

RESULTS
Cyclic AMP Stimulates HIPK3 Activity, c-Jun Phosphorylation, and CYP11A1 Expression-We have previously shown that HIPK3 mediates the cAMP signal and stimulates SF-1 activity to activate the transcription of CYP11A1 (10), which encodes an enzyme that controls the first and rate-limiting step of steroidogenesis. To investigate the mechanism by which HIPK3 mediates the effect of cAMP to stimulate gene expression, we examined whether HIPK3 kinase activity was regulated by 8-Br-cAMP using an in vitro kinase assay. FLAG-sHIPK3 was isolated from adrenocortical Y1 cells by immunoprecipitation, and FLAG-sHIPK3 auto-phosphorylation was increased with time after 8-Br-cAMP treatment (Fig. 1A). The kinasedead mutant of HIPK3, FLAG-K226R, had no activity (Fig. 1A). The phosphorylation level of c-Jun and the amount of CYP11A1 were also elevated after stimulation (Fig. 1B). Thus, HIPK3 activity, c-Jun phosphorylation, and CYP11A1 expression were all enhanced upon the increase of intracellular cAMP levels.
HIPK3 Interacts with and Phosphorylates Daxx-We next searched for HIPK3 substrates that participate in HIPK3-dependent CYP11A1 gene activation. Daxx is a direct substrate for HIPK1 and HIPK2 (13,15) and is therefore a likely candidate as a HIPK3 substrate. We first examined whether HIPK3 and Daxx interacted with each other by co-immunoprecipitation. The N-terminal (aa 1-500), but not the C-terminal (aa 501-740), part of Daxx immunoprecipitated together with FLAG-HIPK3, while the full-length Daxx displayed a weak interaction with HIPK3 ( Fig. 2A).
HIPK3 kinase activity was then examined in vitro using recombinant substrates fusing GST to Daxx, c-Jun, or different fragments of SF-1 (Fig. 2B). The bottom gel indicates the amounts of GST fusion proteins used in the experiment. The top gel of 32 P incorporation detected the phosphorylation signal only when Daxx or the positive control MBP was used as a substrate (Fig. 2B). SF-1, c-Jun, JNK1, and JNK2 were not efficient HIPK3 substrates ( Fig. 2B and supplemental Fig. S1). Phosphorylated Daxx was not detected when no kinase was added (supplemental Fig. S1), indicating the specificity of the assay.
HIPK3 Directly Phosphorylates C-terminal Daxx-To identify the domain of Daxx phosphorylated by HIPK3, we performed an in vitro kinase assay by incubating active-HIPK3 with different regions of HA-Daxx proteins that were isolated by immunoprecipitation. The full-length and C-terminal Daxx were phosphorylated by HIPK3, but N-terminal Daxx was not (Fig. 2C). The right panel is a control showing that equal amounts of Daxx proteins were present in this kinase assay. Thus, this experiment indicates that HIPK3 phosphorylates Daxx in the C-terminal region.
In vivo, two Daxx-myc bands were detected in H1299 cells transfected with 0.5 g of Daxx-myc (Fig. 2D). After co-expression of WT-FLAG-HIPK3, the intensity of the lower band was decreased while that of the upper band was enhanced (Fig. 2D). The kinase-deficient K226R mutant of HIPK3 could not induce Daxx mobility. In Y1 cells, Daxx mobility shift was increased with time after 8-Br-cAMP treatment. This gel shift was abolished following phosphatase treatment (Fig. 2E), indicating the band shift was due to phosphorylation. Thus, Daxx is a phosphorylation substrate of HIPK3 in vitro and in vivo, and Daxx is phosphorylated in response to cAMP stimulation.
Daxx Mediates HIPK3 Effect in SF-1-dependent CYP11A1 Expression-Because HIPK3 participates in the stimulation of CYP11A1 transcription (10), we examined whether Daxx was  also involved. CYP11A1 transcription was measured by examining the expression of luciferase driven by the 2.3 kb human CYP11A1 promoter. While having no function by itself, Daxx increased the ability of SF-1 to activate the CYP11A1 promoter (Fig. 3A). It also led to an increase of endogenous CYP11A1 in Y1 cells (Fig. 3B). Thus, Daxx functions as an SF-1 co-activator to increase CYP11A1 expression.
In the loss-of-function experiment, Daxx was depleted from H1299 cells by the introduction of shRNA driven by a pSuper-Daxx plasmid. In these cells, HIPK3 and SF-1 activate the CYP11A1 promoter synergistically (10). This activity, however, was diminished when the amounts of pSuper-Daxx were increased (Fig. 3C). Endogenous CYP11A1 levels in the presence or the absence of 8-Br-cAMP were also reduced following pSuper-Daxx transfection in Y1 cells (Fig. 3D). In human adrenocortical H295 cells, depletion of Daxx by pSuper-Daxx also led to a decrease of 8-Br-cAMP-dependent CYP11A1 and StAR expression (supplemental Fig. S2). However, when CYP11A1 levels were already enhanced by cAMP, more Daxx did not activate it further (supplemental Fig. S3). This indicates that Daxx mediates cAMP stimulation and HIPK3 activity to enhance SF-1 activity resulting in CYP11A1 expression.
JNK/c-Jun Activities Are Required for Daxx-dependent CYP11A1 Expression-To understand how Daxx activates CYP11A1 expression, we examined JNK/c-Jun since HIPKs/ Daxx signaling utilizes the JNK/c-Jun pathway in cell death regulation (13). With increasing amounts of Daxx in Y1 cells, we detected proportionally elevated c-Jun phosphorylation levels (Fig. 4A). Furthermore, over-expression of the dominant negative Ala-c-Jun resulted in a decrease of Daxx/SF-1-dependent CYP11A1 reporter activity; this activity was not affected by WT-c-Jun (Fig. 4B). This indicates that c-Jun participates in the synergistic effects of Daxx and SF-1. Furthermore, phosphorylation of c-Jun, which was induced by FLAG-HIPK3, was also abolished by pSuper-Daxx (Fig. 4C). Thus, c-Jun phosphorylation caused by HIPK3/Daxx signaling is important for SF-1 activation.
In addition to c-Jun, the role of JNK was also investigated by treating cells with the JNK inhibitor SP600125 (Fig. 5A) or by depleting endogenous JNK with a dominant negative DN-JNK mutant (Fig. 5B). While Daxx overexpression enhanced the level of CYP11A1 detected by immunobloting (Fig. 5A) and reporter assay (Fig. 5B), down-regulation of JNK activity by SP600125 reduced CYP11A1 protein levels. Moreover, the reduction of CYP11A1 promoter activity was rescued by the co-transfection of constitutively active Asp-c-Jun even though DN-JNK was present (Fig. 5B, last bar). These results indicate that the Daxx signal is transduced first to JNK and then to c-Jun in the regulation of CYP11A1 expression.
Ser-669 of Daxx Is Important for HIPK3 Phosphorylation and cAMP Stimulation-To further narrow down the residue of Daxx targeted by HIPK3 for phosphorylation, we focused on two residues, Ser-502 and Ser-669, because they were found   FEBRUARY 17, 2012 • VOLUME 287 • NUMBER 8 phosphorylated when co-expressed with HIPK1 (15). Both WT and S502A-Daxx migrated more slowly on the gel when they were co-expressed with HIPK3, but not with the K226R mutant (Fig. 6A), indicating they were phosphorylated by HIPK3. However, neither the S669A nor S502A/S669A mutant bands were shifted (Fig. 6A), indicating that Ser-669 but not Ser-502 was the residue of Daxx targeted for phosphorylation by HIPK3. In Y1 cells, while the WT Daxx was phosphorylated with time after 8-Br-cAMP treatment, the S669A mutant was not phosphorylated (Fig. 6B). This indicates that Daxx is phosphorylated at Ser-669 upon the increase of cAMP stimulation in Y1 cells.

Daxx Mediates HIPK3 Action to Enhance SF-1 Activity
S669A Mutant of Daxx Fails to Activate c-Jun and CYP11A1 but Still Interacts with HIPK3-The effect of Daxx phosphorylation on CYP11A1 promoter activity was further tested. WT-Daxx synergized with SF-1 to upregulate the expression of CYP11A1 as determined by reporter assay (Fig. 7A) and immunoblotting (Fig. 7B), while the S669A mutant had no effect. Furthermore, the phosphorylation level of c-Jun was also increased when wild type but not S669A Daxx was expressed in Y1 cells (Fig. 7B). Although having no function, the S669A mutant was still physically associated with FLAG-HIPK3 in the co-immunoprecipitation experiment (Fig. 7C). This result shows that the Ser-669 residue of Daxx is important for signal transduction and functional activation but not in physical association with HIPK3.

DISCUSSION
In this study, we have investigated the cAMP signaling pathway leading to increased steroidogenic Cyp11a1 expression. The novelty of our finding is the discovery that an established pathway in apoptosis, HIPK/Daxx/JNK/c-Jun (16,18), can transduce the cAMP signal to stimulate Cyp11a1 transcription. In adrenal cells an increased cAMP level activates HIPK3, which phosphorylates Daxx at Ser-669 leading to JNK/c-Jun phosphorylation, thus potentiating SF-1 activity to up-regulate Cyp11a1. This is the first report to point out the function of Daxx in steroidogenesis. We have also elucidated the mechanism of Daxx modification and activation following cAMP stimulation.   Daxx Function in Steroidogenesis versus Apoptosis-We find that the stimulation of Cyp11a1 transcription goes through a pathway known to trigger apoptosis (16,18). Although this pathway participates in both apoptosis and steroidogenesis, the cell type and target genes involved in these two events are different (Fig. 8). Daxx activates JNK/c-Jun to induce p53-independent apoptosis in response to TGF-␤ in liver cancer Hep3B cells (13), whereas Daxx activates SF-1-mediated Cyp11a1 expression for steroid synthesis in response to cAMP. This implies that different upstream stimuli could use the same signaling molecules to regulate distinct transcription factors for different outcomes. This observation indicates that the same signaling pathway can be used in different types of cell events. Furthermore, members of this pathway might be modified at different sites when the stimuli are different. For example, in addition to phosphorylation at Ser-669 by HIPK proteins, phosphorylation by CK2 kinase at Ser-737 and Ser-739 renders Daxx a better substrate for SUMO-1 conjugation and cell death sensitization (42).
Effects of Daxx on Nuclear Receptors-In this report, we document that Daxx enhances the transcriptional activity of the nuclear receptor SF-1. However, in a previous study, Daxx was shown to suppress the activity of another nuclear receptor family member, androgen receptor (AR) (27). This indicates that Daxx may have multiple functions. We show here that Daxx potentiates SF-1 activity indirectly by activating a signaling cascade involving JNK/c-Jun, while Daxx interferes with the DNA binding ability of AR via direct binding to SUMO-modified AR (27). SF-1 was found in the same complex as Daxx by co-immunoprecipitation assay (supplemental Fig. S4). We suggest that Daxx, HIPK3, c-Jun, and SF-1 may form a complex to upregulate the activity of SF-1, because HIPK3 fails to phosphorylate SF-1 directly (Fig. 2B). Thus, Daxx functions as both a positive and negative regulator of transcription depending on the cellular context and the molecules with which it interacts.
The Identity of Daxx Signal Transducer-Daxx participates in the stimulation of steroidogenic gene expression by transferring the phosphorylation signal from HIPK3 to JNK/c-Jun. There are probably a few steps between Daxx and JNK, and the direct downstream effector of Daxx is not yet clear. For apoptosis, Daxx activates signal-regulating kinase 1 (ASK-1) for the signal flow to JNK (23). It is unclear whether ASK-1 is also involved in steroidogenesis since we could not detect ASK-1 in Y1 cells (data not shown). Overexpression of exogenous ASK-1 in Y1 cells could stimulate the JNK/c-Jun signaling cascade leading to increased Cyp11a1 promoter activity (data not shown). Thus, Daxx might regulate some protein kinase like ASK-1 in a signaling pathway leading to c-Jun phosphorylation and SF-1 activation.
Physical Interaction between HIPKs and Daxx-Here we show that the N-terminal, but not the C-terminal, part of Daxx physically interacts with HIPK3. Furthermore, N-terminal Daxx interacted with HIPK3 better than the full-length Daxx, implying that the C terminus of Daxx probably exerts conformational constraint that inhibits its N-terminal domain from interacting within HIPKs. This C-terminal region of Daxx, in contrast, was important for HIPK3 phosphorylation. It seems only very weak binding is essential for HIPK3 to phosphorylate the Daxx C-terminal region, while the phosphorylation status of Daxx does not affect its own binding to HIPK3. Daxx appears to bind to HIPK3 in both basal and stimulated conditions.
Daxx Phosphorylation Sites-Daxx is a phosphoprotein with at least seven residues capable of being phosphorylated by different kinases (15). In the present report, we found Ser669 was phosphorylated by HIPK3 in response to cAMP stimulation. However, Daxx appears to be heavily phosphorylated at multiple sites in addition to Ser-669 upon 8-Br-cAMP treatment, as exemplified by the dramatic gel mobility shift. S669A-Daxx was hypophosphorylated, indicating that the mobility shift of Daxx may rely on the phosphorylation of Ser-669. Thus, Ser-669 may be a major HIPK3 modification site although not the only one, and the phosphorylation of Ser-669 may be required to trigger the phosphorylation of other residues.
Daxx represses transcription in many cases (26 -30). Yet phosphorylation of Daxx at Ser-669 by HIPK1 abrogates its repressive function (15). We also show here that Ser-669 phosphorylation by HIPK3 is required for increased Cyp11a1 transcription. Thus, Ser-669 phosphorylation is often associated with the activation function of Daxx.