The orphan nuclear receptor LRH-1 (liver receptor homologue-1; NR5A2) plays a critical role in development, bile acid synthesis and cholesterol metabolism. LRH-1 is also expressed in the ovary where it is implicated in the regulation of steroidogenic genes for steroid hormone synthesis. In the present study, we investigated the molecular mechanisms of the transcriptional regulation of CYP11A1 by LRH-1 and found that LRH-1-mediated transactivation was markedly repressed by PIASy [protein inhibitor of activated STAT (signal transducer and activator of transcription) y], the shortest member of the PIAS family. The suppression of LRH-1 activity requires the N-terminal repression domain. Although PIAS proteins also function as E3 SUMO (small ubiquitin-related modifier) ligases and enhance SUMO conjugation, PIASy-mediated repression was independent of LRH-1 SUMOylation status. In addition, histone deacetylase activity was not involved in the inhibition of LRH-1 by PIASy. Immunoprecipitation and mammalian two-hybrid analyses indicated that PIASy interacted with LRH-1 through the C-terminal region, including the AF-2 (activation function-2) motif, which was also involved in the interaction between LRH-1 and the co-activator SRC-1 (steroid receptor co-activator-1). PIASy inhibited the binding of SRC-1 to LRH-1, although overexpression of SRC-1 partially overcame the PIASy inhibition of LRH-1 induction of the CYP11A1 promoter. The results of the present study suggest that competition with co-activators may be an important mechanism underlying the PIASy repression of LRH-1-mediated transactivation.
- cholesterol side-chain cleavage cytochrome P450 (CYP11A1)
- liver receptor homologue-1 (LRH-1)
- protein inhibitor of activated STAT (PIAS)
- steroid receptor co-activator-1 (SRC-1)
LRH-1 (liver receptor homologue-1; NR5A2), related to the Drosophila Ftz-F1 (Fushi tarazu factor 1), belongs to the nuclear hormone receptor NR5A subfamily . LRH-1-null mice die at embryonic day 7.5, indicating that it is essential for embryo development . LRH-1 is predominantly expressed in liver, pancreas, intestine, ovary and preadipocytes [3–7]. In enterohepatic tissues, LRH-1 is associated with liver and pancreas differentiation, bile acid synthesis and cholesterol homoeostasis [6,8–11]. LRH-1 is also known to play a role in steroidogenesis: it is abundantly expressed in ovarian granulosa cells and corpus luteum [3,12], and regulates the transcription of several steroidogenic genes including CYP11A1 [13,14]. In addition, LRH-1 controls the expression of aromatase in breast adipose tissue .
LRH-1 has a typical nuclear hormone receptor structure, including an N-terminal A/B domain, a zinc-finger-containing DBD (DNA-binding domain), a hinge region and a C-terminal LBD (ligand-binding domain) that usually contains a ligand-dependent AD (activation domain), in this case AF-2 (activation function-2) [7,15]. LRH-1 is an orphan receptor, for which the natural ligands have yet to be identified. However, the AF-2 domain is functional and essential for the transactivity of LRH-1 because AF-2-truncated LRH-1 acts as a dominant-negative mutant . Like many nuclear receptor, specific co-regulators such as MBF-1 (multiprotein bridging factor-1) , SHP (short heterodimer partner) [10,17] and SRC-1 (steroid receptor co-activator-1)  have been identified to associate with LRH-1 and modulate the LRH-1-mediated transcriptional activation. In addition, several post-translational modifications are known to affect the functional activity of LRH-1. Phosphorylation of the hinge domain enhances transcriptional activity , whereas SUMOylation (where SUMO is small ubiquitin-related modifier) of LRH-1 is associated with transcriptional repression and subnuclear localization .
The PIAS [protein inhibitor of activated STAT (signal transducer and activator of transcription)] family of proteins was initially identified as inhibitors of STAT [21,22]. Subsequently, PIAS proteins have been shown to interact with many other proteins and regulate the activity of transcription factors . The PIAS family is generally associated with transcriptional repression through different mechanisms. For example, PIAS1 blocks DNA-binding by NF-κB (nuclear factor κB) p65 and inhibits NF-κB-mediated gene activation . PIASy interacts with HDACs (histone deacetylases) and represses the transcriptional activity of AR (androgen receptor) and Smad3 by recruiting HDAC [25,26]. Previous studies have indicated that PIAS proteins have SUMO E3 ligase activity to enhance the transfer of SUMO from Ubc9 to substrate proteins [27,28]. PIAS proteins may affect transcriptional activity by promoting the SUMOylation of transcription factors. For example, PIAS1 stimulated the SUMO conjugation of Sp3 and mutation of the SUMO acceptor sites increased the transcriptional activity of Sp3 .
LRH-1 has been shown to regulate steroidogenic gene expression, yet the molecular mechanisms of LRH-1 function in steroidogenesis are not clear. In the present study, we have demonstrated that the LRH-1-dependent stimulation of CYP11A1 promoter activity is impaired by PIASy. We show that PIASy interacted with the C-terminal domain of LRH-1 and could interfere with the binding of the co-activator SRC-1 to the same region. Our results suggest that PIASy may compete with co-activators that interact with LRH-1 to repress gene transcription.
MATERIALS AND METHODS
Plasmid pCMX-mLRH-1 was generously provided by Dr D. J. Mangelsdorf (Howard Hughes Medical Institute, Chevy Chase, MD, U.S.A.) . The full coding sequence of LRH-1 was digested with EcoRI and BamHI from pCMX-mLRH-1 and then subcloned into the pFlag-CMV2 vector (Sigma). Arginine-substitution LRH-1 mutants (K173R, K213R, K289R, K329R and K389R) were generated by PCR-based site-directed mutagenesis and verified by DNA sequencing. The luciferase reporter CYP11A1–Luc was produced by cloning upstream regions (−4400 to +55) of the human CYP11A1 into pGL3-Basic vector (Promega). The plasmid pFLAG–PIASy, which encodes FLAG-tagged human full-length PIASy, was kindly provided by Dr K. Shuai (University of California, Los Angeles, CA, U.S.A.) . pFLAG–PIASy was digested with EcoRI and ScaI, the ends were filled with Klenow and then self-ligated to generate pFLAG–PIASyΔRD1 (amino acids 115–510). The C-terminus of PIASy (amino acids 332–510) was amplified by PCR (forward primer, 5′-TGGTGAAGCTTCGGCTCTCCGTG-3′; reverse primer, 5′-ACTGGAGTGGCAACTTCCAG-3′), digested with HindIII and subsequently ligated in-frame into the HindIII site (amino acids 159) of pFLAG–PIASy to generate pFLAG–PIASyΔRD2. Expression plasmids for HDAC1, 2 and 3 were gifts from Dr W. M. Yang (Institute of Molecular Biology, National Chung Hsin University, Taichung, Taiwan). The plasmid pGal4 was made by cloning the Gal4 DBD into the pcDNA3 vector (Invitrogen). Gal4 and LRH-1 fusion constructs were generated by insertion of the corresponding cDNA fragments of LRH-1 in-frame after the Gal DBD in pGal4. The VP16 AD cloned into pGal4 (pGal4–VP16) and the Gal4-dependent reporter construct p5xGAL4-E1B-Luc were kindly provided by Dr B. C. Chung (Institute of Molecular Biology, Academia Sinica, Taiwan). The Gal4 DBD was deleted from pGal4–VP16 to generate plasmid pVP16. PIASy and VP16 fusion constructs were produced by cloning the desired coding sequence of PIASy upstream of the VP16 AD in pVP16. The plasmid pCR3.1-SRC-1 encoding full-length SRC-1 was a gift from Dr M. J. Tsai (Baylor College of Medicine, Houston, TX, U.S.A.) . The VP16 AD was amplified by PCR (forward primer, 5′-CTCGAGCCGGACCGGAACC-3′; reverse primer, 5′-CAGTGGGATCCAATACCCACC-3′) and cloned into the TA vector. The VP16 AD fragment was then released by digestion with KpnI and BamHI and subcloned upstream of the SRC-1 coding sequence in pCR3.1-SRC-1.
The coding sequence corresponding to residues 1–115 of mouse LRH-1 was cloned into the SalI/HindIII sites of pQE-32 (Qiagen) to produce pQE-mLRH1-N. Plasmid pQE-mLRH1-N was transformed into bacteria M15 (Qiagen) and induced by 0.8 mM IPTG (isopropyl β-D-thiogalactoside) for 5 h at 37 °C. Cells were pelleted, then lysed with 2 mg/ml lysozyme in phosphate buffer [10 mM Tris/HCl (pH 8.0) and 100 mM NaH2PO4] containing 0.1 mg/ml DNase. After sonication and centrifugation (13000 g for 10 min at 4 °C), the cell pellet was dissolved in 6 M guanidine hydrochloride in phosphate buffer. Following centrifugation (13000 g for 10 min at 4 °C), the recombinant protein was electrophoresed in SDS/PAGE and electroeluted. Antiserum produced by inoculating a rabbit (250 μg of gel-purified recombinant protein, four doses) was collected, and the specificity of the polyclonal anti-LRH-1 antibody was tested in HEK (human embryonic kidney)-293T cells transiently transfected with LRH-1. The procedures were approved by the National Taiwan University College of Medicine Public Health Institutional Animal Care and Use Committee.
Cell transfection and luciferase assays
HEK-293T cells were grown in high-glucose (3%) DMEM (Dulbecco's modified Eagle's medium) supplemented with 10% (v/v) FBS (foetal bovine serum) and transfected with Lipofectamine™ (Invitrogen). Y1 cells were grown in DMEM-F12 supplemented with 10% (v/v) FBS and transfected with TurboFect (Fermentas). HEK-293T cells were subcultured on to 24-well or 12-well plates as indicated and transfected the following day with equal amounts of total plasmid DNA. After 24 h, cells were harvested and the luciferase activities were determined using the Dual-Glo Luciferase Assay System (Promega). For reagent treatments, after the transfection, medium was replaced with fresh medium containing 100 nM TSA (trichostatin A), 1 mM NaB (sodium butyrate) or 1 mM VPA (valproic acid) (all reagents were purchased from Sigma–Aldrich), and incubated for a further 24 h. The results were normalized to internal Renilla luciferase activities. Data were obtained from at least three independent experiments and are presented as the mean±S.E.M. The significance of differences between group means was analysed using one-way ANOVA.
Western blotting and immunoprecipitation
Cells were lysed in buffer [20 mM Tris/HCl (pH 7.9), 137 mM NaCl, 10 mM NaF, 5 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 10% (w/v) glycerol, 1% (v/v) Triton X-100, 1 mM sodium pyrophosphate, 0.1 mM β-glycerophosphate, 5 mM DTT (dithiothreitol), 2 mM PMSF and 10 μg/ml leupeptin] and incubated on ice for 1 h. After centrifugation (13000 g for 30 min at 4 °C), the supernatant fraction was collected and either analysed directly by Western blotting or subjected to immunoprecipitation. The protein concentration of each sample was determined using the Bradford method (Bio-Rad). Western blot analysis was carried out using anti-FLAG (M2, Sigma), anti-acetyl histone H3 (Upstate Biotechnology), anti-VP16 (sc-7545; Santa Cruz Biotechnology) and anti-β-actin (Sigma) antibodies. For immunoprecipitation assays, the anti-VP16 antibody was incubated with 200 μl of rProtein G agarose beads (Invitrogen) at 4 °C for 1 h and the beads were collected by centrifugation (190 g for 2 min at 4 °C). Next, the cell extracts were precleaned with 50 μl of rProtein G agarose beads and then incubated with antibody-bound beads at 4 °C for 2 h with gentle agitation. After washing with lysis buffer (as above), the beads were collected, resuspended in protein sample buffer and subjected to SDS/PAGE (8% gels). Western blots were carried out using anti-VP16 or anti-LRH-1 antibodies, with the signal detected using SuperSignal West Femto (Thermo Fisher Scientific).
PIASy inhibits the LRH-1-induced CYP11A1 promoter
We studied the effect of various PIAS proteins on LRH-1-regulated gene activation. To understand the effect of PIAS proteins on LRH-1 activity, we initially examined five members of the PIAS family and found that PIASy was most effective in inhibiting LRH-1 activity. The LRH-1-dependent reporter CYP11A1–Luc contains a 4.4 kb CYP11A1 promoter linked to a luciferase gene. LRH-1-induced CYP11A1 activity was dramatically inhibited by PIASy. As shown in Figure 1(A), LRH-1 increased the activity of the 4.4 kb CYP11A1 promoter by approx. 25-fold in HEK-293T cells. Co-expression of PIASy inhibited up to 90% of the LRH-1-enhanced CYP11A1 promoter activity. In the absence of LRH-1, PIASy alone had no effect on CYP11A1 reporter activity compared with the vehicle control. The PIASy inhibition of LRH-1-stimulated CYP11A1 activity was concentration-dependent (Figure 1B).
PIASy has two intrinsic repression domains, RD1 and RD2 . To examine the part played by the individual domains in the repression of LRH-1 transactivity, two PIASy mutants, pFLAG–PIASyΔRD1 (N-terminal deletion of amino acids 1–114) and pFLAG–PIASyΔRD2 (internal deletion of amino acids 161–331), were constructed (Figure 2A). As shown in Figure 2(B), the PIASy repression of LRH-1-mediated transcription was almost entirely blocked by removal of RD1, whereas the RD2 deletion was nearly as effective as the wild-type. Western blot analysis demonstrated that mutant and wild-type proteins were expressed at similar levels (Figure 2C). These results strongly suggest that the RD1 region of PIASy is responsible for the inhibition of LRH-1 transcriptional activity.
PIASy-mediated LRH-1 repression is independent of LRH-1 SUMOylation
LRH-1 has been shown to be a substrate for SUMO conjugation, and SUMOylation represses its transactivation [20,32]. We had previously mutated five lysine residues in consensus SUMO sites and found that Lys289 was most efficiently conjugated by SUMO-1 In vitro . PIAS proteins act as E3 ligases to enhance the SUMO conjugation of substrate proteins. To examine whether the inhibition of LRH-1 transcriptional activity by PIASy correlates with SUMOylation, five lysine-to-arginine mutants of LRH-1 were used in a transfection assay with or without co-expression of PIASy (Figure 3). While the SUMOylation-deficient mutant K289R had more than twice as much transcriptional activity as wild-type LRH-1, PIASy also repressed the transcriptional activity of K289R to the same level as the wild-type and other lysine mutants. This suggests that the inhibition of LRH-1-mediated transcription by PIASy is not associated with LRH-1 SUMOylation.
PIASy interacts with LRH-1
To test whether PIASy interacts with LRH-1 in vivo, a mammalian two-hybrid assay was performed to identify the interaction domain. PIASy fragments of various lengths fused to the VP16 AD were co-transfected into HEK-293T cells with the Gal4-dependent reporter p5xGAL4-E1B-Luc and expression plasmids for LRH-1 fused to the Gal4 DBD or the empty pGal4 vector. As shown in Figure 4(A), co-expression of VP16-PIASy181-510 with Gal4–LRH-1 induced more reporter activity than co-expression with control Gal4. Even greater induction was observed when Gal4–LRH-1 was co-expressed with VP16–PIASy331-510, but not with VP16–PIASy160-334. Immunoblotting analysis showed that the plasmids VP16–PIASy1-159, VP16–PIASy160-334 and VP16–PIASy331-510 were expressed at comparable levels, whereas VP16–PIASy181-510 was expressed at a lower level (Figure 4B). This might explain why VP16–PIASy181-510 showed less induction of reporter with Gal4–LRH-1 than VP16–PIASy331-510. These results demonstrate that the C-terminal residues 331–510 are responsible for the interaction of PIASy with LRH-1.
To define the domains of LRH-1 involved in the interaction with PIASy, various fusion proteins of Gal4–LRH-1 were constructed. As shown in Figure 4(C), reporter activity was enhanced by VP16–PIASy331-510 in the presence of Gal4–LRH-1193-560, but not Gal4–LRH-11-191, Gal4–LRH-1193-487 or Gal4–LRH-1450-560. These results indicate that LRH-1 interacts with PIASy through the C-terminal region and the AF-2 motif is essential for the interaction.
Association of PIASy with LRH-1 in vivo was further assessed by co-immunoprecipitation analysis. The LRH-1 expression plasmid pFLAG–LRH-1 was co-transfected with pVP16–PIASy331-510 or empty vector pVP16 into HEK-293T cells. Cell lysates were immunoblotted with anti-VP16 or anti-LRH-1 antibodies to confirm the expression of VP16-fused PIASy331-510 and LRH-1 proteins respectively (Figures 5A and 5B). Cell extracts were immunoprecipitated with an anti-VP16 antibody. Immunoblots with anti-LRH-1 antibody revealed that LRH-1 was co-immunoprecipitated with VP16–PIASy331-510, but not the empty VP16 alone (Figures 5C and 5D). These results indicate that PIASy331-510 can form a complex with LRH-1.
PIASy repression of LRH-1 transactivation does not involve the HDAC pathway
Recruitment of HDACs is an important mechanism whereby some transcriptional repressors control gene transcription [34,35]. To examine whether HDAC is involved in PIASy repression of LRH-1-dependent transcription, three different HDAC inhibitors, TSA, NaB and VPA, were used to inhibit HDAC activity. Treatment with these HDAC inhibitors had no effect on the repression of the LRH-1-stimulated CYP11A1 activity by PIASy (Figure 6A). Western blot analysis showed that cells treated with HDAC inhibitors had increased accumulation of acetylated histone H3, demonstrating that these inhibitors did indeed inhibit HDAC (Figure 6B). These results suggest that inhibition of HDAC activity could not overcome the PIASy repression of the LRH-1 transactivity. We also tested whether overexpression of HDAC could further enhance this repression. In order to clearly observe the action of HDAC, cells were transfected with minimal amounts of PIASy in this assay to reduce the PIASy-mediated repression to a much smaller extent. As shown in Figure 6(C), co-transfection of different HDAC expression vectors, 1, 2 or 3, in HEK-293T cells had little effect on the PIASy-mediated repression of LRH-1 transactivity. Western blot analysis was performed to verify the expression of HDAC proteins (Figure 6D). Taken together, these results indicate that HDAC may not be involved in the PIASy-mediated repression of LRH-1 transcriptional activity.
PIASy interferes with the interaction of SRC-1 with LRH-1
We next determined whether PIASy inhibits LRH-1-mediated gene expression by influencing the function of the co-activators of LRH-1. SRC-1 has been demonstrated to induce the transactivity of human LRH-1 through an interaction with its LBD . Because this interaction site overlaps with the PIASy–LRH-1 interaction site, we explored whether SRC-1 is associated with the PIASy-mediated repression of LRH-1. As shown in Figure 7, co-expression with SRC-1 modestly enhanced the LRH-1-stimulated CYP11A1 promoter activity. An increase in the amount of SRC-1 did not further increase the promoter activity. However, co-transfection with SRC-1 partly eliminated the inhibitory effect of PIASy on LRH-1-mediated transactivation in a dosage-dependent manner (Figure 7). The interaction of LRH-1 and SRC-1 was then assessed using a mammalian two-hybrid assay. Consistent with a previous report , a significant transactivation was observed when VP16–SRC-1 was co-expressed with Gal4–LRH-1193-560 (Figure 8A), indicating a strong interaction of SRC-1 with the C-terminus of LRH-1. Co-transfection with PIASy abolished the interaction between VP16–SRC-1 and Gal4–LRH-1193-560 (Figure 8B), suggesting that PIASy competes for LRH-1 binding with SRC-1.
PIASy represses Cyp11a1 expression in Y1 cells
Because our results implied that PIASy could participate in the control of CYP11A1 transcription, we investigated whether the CYP11A1 expression was sensitive to PIASy in cells. As shown in Figure 9, when PIASy was overexpressed in the mouse adrenocortical Y1 cells, there was a marked decrease by approx. 50% in the protein levels of endogenous Cyp11a1. This result suggests that PIASy may be involved in the regulation of CYP11A1 expression in steroidogenic cells.
In general, a number of co-regulatory molecules are recruited to enhance or repress the expression of genes regulated by nuclear receptors. Several cofactors have been implicated to modulate the transcriptional activity of LRH-1. The p160 co-activator member SRC-1  and co-regulator MBF-1  can interact with LRH-1 and enhance its activity in lipid metabolism. SHP [10,17] and Dax-1 (dosage-sensitive sex reversal-adrenal hypoplasia gene on the X chromosome gene 1)  have been shown to function as co-repressors to inhibit LRH-1-mediated transcription. In the present study, we found that PIASy can interact with LRH-1 through its C-terminus and repress the LRH-1-stimulated transcriptional activity of the CYP11A1 promoter. The inhibitory effect of PIASy on LRH-1-mediated transcription can be partly relieved by overexpression of SRC-1.
PIASy has been reported to interact with numerous transcription factors, including the ligand-dependent nuclear hormone receptor, and is able to alter their transcriptional activity . Similar to most of the cases, we found that PIASy negatively regulates LRH-1-mediated transcription. PIAS proteins possess SUMO E3 ligase activity that stimulates SUMO conjugation of protein targets. SUMOylation of LRH-1 is known to be associated with transcriptional repression and subnuclear targeting [20,32]; however, our results show that mutation of the main SUMO attachment site did not prevent the transcriptional repression of LRH-1 by PIASy. In addition, co-expression with PIASy had no obvious effect on the subnuclear localization of LRH-1 (results not shown). This suggests that PIASy negatively regulates the transcriptional activity of LRH-1 in a manner independent of LRH-1 SUMOylation. Similar results have also been described with several transcription factors such as Ets-1 (E twenty-six-1)  and a nuclear receptor member, AR .
Gross et al.  identified two transcriptional repression domains of PIASy by mammalian one-hybrid assay and demonstrated that the N-terminal repression domain 1 (termed RD1) is required for PIASy-mediated repression of AR. Similarly, our results show that deletion of RD1, but not RD2, results in the loss of inhibition of LRH-1-dependent activation, suggesting that the RD1 domain is necessary for the PIASy repression of LRH-1. The RD1 domain contains an LXXLL motif which occurs in various co-activators and co-repressors and appears to be important for the interaction with nuclear receptors or between co-regulators [38,39]. This LXXLL sequence in PIASy has been shown elsewhere to be essential for the repression of AR and STAT1 without influencing their DNA-binding activity [25,40]. PIASy can interact with HDAC1 and 2 through the LXXLL-containing RD1 region, and HDAC inhibitors block PIASy from inhibiting AR- and Smad-dependent transcription [25,26]. It was suggested that PIASy could recruit HDACs to suppress the activity of transcription factors. However, we show in the present study that neither HDAC inhibitors nor overexpression of HDAC proteins have any apparent effect on transcriptional repression of LRH-1 by PIASy, suggesting that PIASy represses the activity of LRH-1 by an HDAC-independent pathway. It would be interesting to know whether PIASy has the ability to recruit other co-repressors, such as CtBP (C-terminal binding protein) , through HDAC-independent mechanisms to inhibit LRH-1 transactivation. In addition, a putative nuclear matrix association SAP domain is present in the N-terminal RD1 region of PIASy . It has been shown that PIASy recruits LEF1 (lymphoid enhancer factor 1) and Ets-1 to the nuclear matrix [37,42]; however, it is still unknown whether targeting proteins to the nuclear matrix is important for PIASy-mediated transrepression.
Negative regulators are able to interact with components of the basal transcription machinery to influence gene transcription. For example, human ZNF76 (zinc finger protein 76) targets TBP (TATA-binding protein), an essential component of transcription initiation, to negatively regulate gene expression . Previously, PIAS proteins were also identified as the interaction proteins of TBP by a yeast two-hybrid system . To examine whether the interaction with TBP was involved in the PIASy-mediated repression, we tested whether transfection of TBP expression plasmid could relieve this repression; however, we could not demonstrate any effect of TBP on PIASy repression because overexpression of TBP strongly inhibited LRH-1-mediated CYP11A1 promoter activity (results not shown).
The two-hybrid results in the present study indicated that the AF-2 transactivation motif in the LBD is crucial for the interaction of LRH-1 with PIASy. This domain is also the target for the interaction between LRH-1 with the co-activator SRC-1 and other repressors, SHP and Dax-1 [18,36,45]. In the present study we show that PIASy could interrupt the interaction between LRH-1 and SRC-1, and overexpression of SRC-1 partly reversed the transcriptional repression of PIASy. This suggests one possible mechanism by which PIASy may limit the recruitment of co-activators through competition for association with LRH-1. A similar situation has also been observed with SHP, which is able to inhibit LRH-1-mediated transactivation through competition with the p160 co-activator SRC-1 . However, SRC-1 can only partially reverse the repression by PIASy, which is probably due to the fact that SRC-1 has only a minor effect on transcriptional activation of LRH-1-mediated CYP11A1 promoter. Little is known regarding the co-activators of LRH-1. Whether more co-activators of LRH-1 are involved in the PIASy-regulated repression awaits further investigation.
PIAS proteins have been implicated in diverse biological processes such as the immune system, cell senescence and apoptosis [23,46]. LRH-1 is abundantly expressed in the ovary and has been shown to activate the transcription of steroidogenic genes including CYP11A1 in granulosa cells . The results of the present study demonstrate a significant inhibition of LRH-1-stimulated CYP11A1 activity by PIASy. In addition, PIASy overexpression reduces the expression levels of endogenous Cyp11a1 in Y1 cells. We therefore suggest that PIASy may play an important role in the regulation of steroidogenic function when its level was increased.
This work was supported by the National Science Council, Taiwan [grant number NSC95-2320-B-002-063].
We are grateful to Dr K. Shuai, Dr David J. Mangelsdorf, Dr B. C. Chung, Dr M. J. Tsai and Dr W. M. Yang for providing various expression plasmids.
Abbreviations: AD, activation domain; AF-2, activation function-2; AR, androgen receptor; Dax-1, dosage-sensitive sex reversal-adrenal hypoplasia gene on the X chromosome gene 1; DBD, DNA-binding domain; DMEM, Dulbecco's modified Eagle's medium; Ets-1, E twenty-six-1; FBS, foetal bovine serum; HDAC, histone deacetylase; HEK, human embryonic kidney; LBD, ligand-binding domain; LRH-1, liver receptor homologue-1; MBF-1, multiprotein bridging factor-1; NaB, sodium butyrate; NF-κB, nuclear factor κB; PIAS, protein inhibitor of activated STAT (signal transducer and activator of transcription); RD, repression domain; SHP, short heterodimer partner; SRC-1, steroid receptor co-activator-1; STAT, signal transducer and activator of transcription; SUMO, small ubiquitin-related modifier; TBP, TATA-binding protein; TSA, trichostatin A; VPA, valproic acid
- © The Authors Journal compilation © 2009 Biochemical Society