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Molecular and Cellular Biology, November 2007, p. 7486-7496, Vol. 27, No. 21
0270-7306/07/$08.00+0     doi:10.1128/MCB.00409-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

A Regulatory Circuit Mediating Convergence between Nurr1 Transcriptional Regulation and Wnt Signaling{triangledown}

Hirochika Kitagawa ,1,2,{dagger} William J. Ray ,1,{dagger},{ddagger} Helmut Glantschnig,1 Pascale V. Nantermet,1 Yuanjiang Yu,1,{ddagger} Chih-Tai Leu,1 Shun-ichi Harada,1 Shigeaki Kato,2,3 and Leonard P. Freedman1*

Department of Molecular Endocrinology, Merck Research Laboratories, West Point, Pennsylvania 19486,1 Institute of Molecular and Cellular Biosciences, University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-0032, Japan,2 ERATO, Japan Science and Technology Agency, Honcho 4-1-8, Kawaguchi, Saitama 332-0012, Japan3

Received 8 March 2007/ Returned for modification 10 April 2007/ Accepted 3 August 2007


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ABSTRACT
 
The orphan nuclear receptor Nurr1 is essential for the development and maintenance of midbrain dopaminergic neurons, the cells that degenerate during Parkinson's disease, by promoting the transcription of genes involved in dopaminergic neurotransmission. Since Nurr1 lacks a classical ligand-binding pocket, it is not clear which factors regulate its activity and how these factors are affected during disease pathogenesis. Since Wnt signaling via ß-catenin promotes the differentiation of Nurr1+ dopaminergic precursors in vitro, we tested for functional interactions between these systems. We found that ß-catenin and Nurr1 functionally interact at multiple levels. In the absence of ß-catenin, Nurr1 is associated with Lef-1 in corepressor complexes. ß-Catenin binds Nurr1 and disrupts these corepressor complexes, leading to coactivator recruitment and induction of Wnt- and Nurr1-responsive genes. We then identified KCNIP4/calsenilin-like protein as being responsive to concurrent activation by Nurr1 and ß-catenin. Since KCNIP4 interacts with presenilins, the Alzheimer's disease-associated proteins that promote ß-catenin degradation, we tested the possibility that KCNIP4 induction regulates ß-catenin signaling. KCNIP4 induction limited ß-catenin activity in a presenilin-dependent manner, thereby serving as a negative feedback loop; furthermore, Nurr1 inhibition of ß-catenin activity was absent in PS1–/– cells or in the presence of small interfering RNAs specific to KCNIP4. These data describe regulatory convergence between Nurr1 and ß-catenin, providing a mechanism by which Nurr1 could be regulated by Wnt signaling.


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INTRODUCTION
 
Expression and maintenance of the dopaminergic phenotype in the ventral midbrain (VM) require the orphan nuclear receptor (NR) Nurr1 (NR4A2) (48, 59). Genetic ablation of Nurr1 produces embryonic lethality due to a nearly complete absence specifically of mesencephalic dopaminergic neurons, which are critical for motor function. Nurr1 regulates both the differentiation and the maintenance of these dopaminergic cells, as Nurr1+/– mice appear normal at birth but develop motor deficits resulting from reduced numbers of dopaminergic neurons and lower dopamine levels in the striatum (22). At the molecular level, Nurr1 binds specific response elements in the promoters of genes involved in dopaminergic neurotransmission, such as the genes encoding tyrosine hydroxylase (TH), L-aromatic amino acid decarboxylase, and the dopamine transporter (20, 26, 44, 45). In Parkinson's disease, mesencephalic dopaminergic neurons degenerate, ultimately leading to severe motor deficits; correspondingly, Nurr1 levels appear to be reduced (10, 11). Therapeutic strategies that promote Nurr1 function in Parkinson's disease might therefore restore dopaminergic function or even increase the number of dopaminergic neurons. However, Nurr1 has a closed ligand-binding pocket and thus appears to be regulated by ligand-independent mechanisms (55). These mechanisms include changes in the expression of its RNA and protein (36, 47, 54) and changes by second messenger signaling systems and coactivators that modify its transcriptional activity (15, 21, 23, 27, 43). Identifying factors that govern Nurr1 activity is thus important for understanding the development and pathophysiology of VM dopaminergic neurons.

Among the potential factors regulating Nurr1 function, a strong candidate is signaling downstream of the Wnt family of secreted glycoproteins. Wnt signaling is required for the establishment of the midbrain/hindbrain region of the developing nervous system, including VM dopaminergic neurons (reviewed in reference 5), in part by promoting the expression of transcription factors that specify regional identity, such as engrailed (13). In the mouse VM region, the Wnt signaling molecule ß-catenin is highly expressed and active in Nurr1+ precursor cells, as evidenced by local expression of TOPGAL, a ß-catenin-responsive reporter (7). Specific Wnt molecules also promote the proliferation and differentiation of Nurr1+ dopaminergic precursor cells cultured from the VM (7), and Wnt-5a is expressed in VM glial cells, potentially explaining their ability to induce the dopaminergic phenotype in vivo (6). Thus, there is clear evidence that Wnt signaling via ß-catenin regulates the development of Nurr1+ precursors in vivo, but the molecular mechanisms underlying this effect are unknown.

Canonical Wnt signaling is triggered by binding of a secreted Wnt family member to the membrane receptor complex of low-density lipoprotein-related protein 5 (LRP-5) or LRP-6 and frizzled, leading to the accumulation of cellular ß-catenin protein through inhibition of protein degradation of ß-catenin (reviewed in reference 4). ß-Catenin is constitutively degraded through sequential phosphorylation events promoted within at least two distinct molecular complexes. The major complex consists of axin and its associated proteins, and the second complex involves presenilin-1 (PS1) and PS2, the membrane proteins responsible for the intramembranous processing of the ß-amyloid precursor protein in Alzheimer's disease (24, 25, 39, 51, 57). ß-Catenin phosphorylation is inhibited by Wnt activation of frizzled/LRP receptors, leading to protein accumulation and translocation to the nucleus, where it activates TCF/LEF transcriptional complexes in the promoters of target genes. Additionally, ß-catenin can act as a coactivator for several NRs and modify transcription in that manner (29, 32, 50, 52; reviewed in reference 35).

In the present study, we examined the potential relationship between Nurr1 and Wnt signaling. We found that Nurr1 is present in corepressor complexes on TCF/LEF elements prior to Wnt signaling, after which ß-catenin binds to Nurr1 and acts as a transcriptional cofactor. Furthermore, we identified KCNIP4 as a ß-catenin/Nurr1 target that modulates Wnt signaling by interacting with the PS complex.


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MATERIALS AND METHODS
 
Reagents. Full-length human Nurr1, human ß-catenin, and rat LEF-1 cDNAs were cloned into pcDNA3.1 (Invitrogen) and, for Nurr1, also inserted into FLAG- and hemagglutinin (HA)-tagged pIRES2-EGFP Q-vector (Clontech). ß-Catenin S33A and mutant promoter vectors were made with a site-directed mutagenesis kit (Stratagene). For glutathione S-transferase (GST) pull-down assays, each deletion mutant was inserted into pGEX4-1 5-2 vector (Amersham Biosciences). TOPFLASH was purchased from Upstate Biotechnology. The 2x-NBRE (AAAGGTCA) and promoter regions of cyclin D1 (positions –872 to +8), TH (positions –1110 to 0), and KCNIP4 (positions –1043 to –2) were cloned from a human genomic library (Clontech) and inserted into the PGL3-basic vector containing thymidine kinase to generate luciferase reporters.

The following commercially available antibodies were used: anti-Nurr1, anti-C-terminal binding protein (anti-CtBP), anti-transducin-like enhancer (anti-TLE), anti-protein inhibitor of activated STATy (anti-PIASy), and anti-axin from Santa Cruz Biotechnology; anti-PS1 from Chemicon; anti-CREB-binding protein (anti-CBP) and anti-LEF-1 from Upstate Biotechnology; anti-histone deacetylase 1 (anti-HDAC-1) and anti-HDAC-3 from Affinity Bioreagents; and anti-ß-catenin, anti-p45 ß-catenin, and anti-p33/37/41 ß-catenin from Cell Signaling. Anti-KCNIP4 antibody was obtained from Takeshi Iwatsubo (University of Tokyo).

The following small interfering RNA (siRNA) pools (SMARTpool) were purchased from Dharmacon: Nurr1, M-003427-00; CtBP1, M-008609-01; TLE1, M-015528-00; HDAC-1, M-003493-02; HDAC-3, M-003496-00; PIASy, M-006445-00; ß-catenin, M-003482-00; CBP, M-003477-01; KCNIP4, M-021472-00; PS1, M-004998-01; and nonspecific control, D-001210-02-05.

Cell culture. PS1–/– murine embryonic fibroblasts were kindly given by Bart de Strooper (Leuven, Belgium). All cells were routinely maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. To establish stable transformants, 293F cells were infected with retrovirus made with Amphopac 293F cells as the packaging cell line. The packaging cell line was made by transfection of pQCXIN retroviral vectors and was cultured for 2 weeks with 750 µg/ml G418 for transformant selection. For large-scale purification, 293F cells were cultured in 293F SFM (Life Technologies) supplemented with Glutamax (Life Technolgies) in a bioreactor.

Nuclear extraction and purification of Nurr1 interactants. Nuclear extracts (28) from 293F stable transformants expressing tagged Nurr1 and from SK-N-MC cells were loaded onto an anti-FLAG M2 affinity resin column and washed extensively (20 mM Tris-HCl [pH 8.0], 300 mM KCl, 0.2 mM EDTA, 0.05% NP-40, 10% glycerol, 0.5 mM phenylmethylsulfonyl fluoride, and 1 mM dithiothreitol). Bound proteins were eluted from the column by incubation with 133 g/ml FLAG peptide in washing buffer for 30 min at room temperature. The eluted solution was similarly applied to an HA resin column and washed, and Nurr1 complexes were eluted with the HA peptide.

Immunoprecipitation and GST pull-down assay. Cells were transfected with 5 µg of each expression vector and immunoprecipitated with anti-FLAG antibody (Sigma) for Western blotting with specific antibodies (28). For the GST pull-down assay, full-length human Nurr1, LEF-1, and ß-catenin were translated in vitro and incubated with GST-fused mutants of Nurr1, LEF-1, and ß-catenin immobilized on glutathione-Sepharose beads prior to analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Chromatin immunoprecipitation (ChIP). Soluble chromatin from 293F or SK-N-MC cells was prepared with an acetyl-histone H4 immunoprecipitation assay kit (Upstate Biotechnology) and immunoprecipitated with antibodies against the indicated proteins in the presence and absence of LiCl (28). LiCl stimulation was started 45 min before fixing the cells with formaldehyde (49). Specific primer pairs were designed to amplify the promoter region of cyclin D1 (5'-GGCTCCAGGACTTTGCAACTTC-3' and 5'-GGCGCCTCAGGGATGG-3'), TH (5'-GCTGTCTCAGCCCCCC-3' and 5'-CTGGGTCCCCCACCTTCC-3'), the KCNIP4 TCF/LEF site (5'-GTATTCTGCACCTCGGCCCT-3' and 5'-TACTGCTGCACAAAGTTAGGCTGAG-3'), and the KCNIP4 Nurr-responsive element (NBRE) (5'-CAGCCATAGGGAAGGCAAATAG-3' and 5'-AGAAGTCAAAATTAAAATGCAGATTTCTGTGTCC-3') from human genomic DNA. PCR conditions were optimized to allow semiquantitative measurement, and PCR products were visualized on 2% agarose-Tris-acetate-EDTA gels.

Microarray and quantitative RT-PCR analysis. Microarray and quantitative reverse transcription-PCR (RT-PCR) were performed as previously described (37, 42). Gene-specific primers and probes for human KCNIP4 and TH were purchased from Applied Biosystems. For cyclin D1, the following primers and probe were used: probe, 5'-AAGGAGACCATCCCCCTGACGGC-3'; forward primer, 5'-GCATGTTCGTGGCCTCTAAGA-3'; and reverse primer, 5'-CGGTGTAGATGCACAGCTTCTC-3'.

Immunofluorescence. SK-N-MC cells were seeded (40,000/cm2) on glass coverslips in six-well plates. Twenty-four hours after LiCl treatment, cells were washed with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde in PBS for 45 min at 4°C. Primary antibodies were used at a dilution of 1:100 to 1:250 with blocking reagent (Roche) and were incubated for 1 h at room temperature. After four PBS washes (5 min each), secondary antibodies coupled to fluorescein isothiocyanate, tetramethyl rhodamine isocyanate (both from Sigma), or Alexa Fluor488 (Molecular Probes) at a 1:100 dilution were incubated with the cells for 30 min. Cell nuclei were stained with Hoechst 33342 (Molecular Probes), and coverslips were mounted using Slow-fade reagent (Molecular Probes). Immunofluorescence signals were evaluated with a Nikon T300 microscope.

Transfection and luciferase assay. Human 293F cells and SK-N-MC cells were maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and, at 40 to 50% confluence, were transfected with plasmids, using Lipofectamine-Plus reagents (Invitrogen) in 12-well dishes. Total amounts of DNA were adjusted by supplementation with up to 1.0 µg of empty vector. Luciferase activities were determined using a luciferase assay system (Promega). As a reference plasmid to normalize transfection efficiency, 2 ng pRL-CMV plasmid (Promega) was cotransfected in all experiments (28, 58). All values are means ± standard deviations for at least three independent experiments. For RNA interference (RNAi), two-step transfection was performed with Trans IT-TKO and Trans IT-NTI transfection reagents (Mirus) following the manufacturer's recommendations. All siRNAs were evaluated for efficacy by immunoblotting (data not shown).


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RESULTS
 
Transcriptional cross talk between Nurr1 and Wnt signal transduction. We first determined if Wnt signaling regulates the function of Nurr1. Either Wnt-1 or LiCl was used to activate ß-catenin-mediated canonical Wnt signaling, and effects on Nurr1 transcriptional activity were measured. 293F cells stably overexpressing Nurr1 or parental control cells, which do not express detectable Nurr1, were transiently transfected with a luciferase reporter plasmid containing a consensus DNA binding site for Nurr1 (NBRE) (17). Overexpression of Nurr1 increased the activity of this reporter, as expected (Fig. 1A). Nurr1 transactivation function was enhanced when Wnt signaling was activated by either Wnt-1, LiCl, or a constitutively active mutant (S33A) of ß-catenin (41) (Fig. 1A). Similar results were observed in other clones from the same transfection (data not shown). We then tested for the opposite modulation by using a reporter construct harboring a consensus DNA binding site for TCF/LEF (TOPFLASH), which is a target of canonical Wnt signaling. Nurr1 overexpression produced a modest but reproducible inhibition of TOPFLASH activity in either the absence or presence of Wnt-1, LiCl, or S33A ß-catenin (Fig. 1B). These data suggested that Wnt signaling, specifically through ß-catenin, modulates Nurr1 activity and, conversely, that Nurr1 influences ß-catenin activity.


Figure 1
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  		FIG. 1. Convergence of Nurr1 and Wnt signaling. (A) Coactivation of Nurr1 transactivation by Wnt signaling. Luciferase assays were performed with 293F cells transfected with a consensus 2x-NBRE-containing luciferase reporter plasmid (2x NBRE-tk-luc) (400 ng), with or without 200 ng of the indicated expression vector (Nurr1, a constitutively active form of ß-catenin [ß-catenin S33A], or Wnt-1), in the presence or absence of LiCl (10 mM) or Wnt-1. (B) Nurr1 repression of TCF/LEF transactivation. Luciferase assays were performed with 293F cells transfected with a TOPFLASH luciferase reporter plasmid (containing a consensus TCF/LEF binding site) (400 ng), with or without the indicated expression vectors, in the presence or absence of LiCl (10 mM) or Wnt-1. (C) Signaling-dependent interaction between FLAG-Nurr1, LEF-1, and ß-catenin. Exogenous proteins were expressed in 293F cells, which were treated with vehicle, LiCl, or Wnt-1 for 24 h. Transfected cell extracts were subjected to IP with mouse anti-FLAG antibody and then immunoblotted. (D) Competitive binding between ß-catenin and Nurr1 for LEF-1. GST-tagged LEF-1 (top) or Nurr1 (bottom) was mixed with in vitro-translated Nurr1 (top) or LEF-1 (bottom) in the presence of increasing levels of unlabeled, in vitro-translated ß-catenin. Following GST pull-down, in vitro-translated Nurr1 and LEF-1 were visualized by immunoblotting. (E) Physical interaction and mapping of interaction domains of Nurr1, ß-catenin, and LEF-1. Associations of GST-fused, in vitro-translated Nurr1, LEF-1, and ß-catenin proteins with the indicated deletions were tested in a GST pull-down assay.

Direct physical interaction of Nurr1 with ß-catenin and Lef-1. To determine the molecular basis of this mutual coregulation, the physical interaction of Nurr1 with ß-catenin and LEF-1 was tested by coimmunoprecipitation of 293F cells overexpressing all three proteins (Fig. 1C). Lef-1, but not ß-catenin, was coimmunoprecipitated with FLAG epitope-tagged Nurr1 in nuclear extracts from unstimulated 293F cells. Following stimulation with Wnt-1 or LiCl, ß-catenin was recovered with anti-FLAG antibodies, while the amount of coprecipitating Lef-1 was reduced, suggesting that ß-catenin competes with Nurr1 for Lef-1 binding (Fig. 1C). GST pull-down experiments showed that this competition is direct. GST-Lef-1 and in vitro-translated ß-catenin were aliquoted into four assay tubes with increasing levels of recombinant ß-catenin protein. In the absence of recombinant ß-catenin, GST-Lef-1 pull-down recovered in vitro-translated Nurr1; the addition of ß-catenin protein to the reaction mix dose-dependently reduced the amount of Nurr1 recovered. Identical results were seen when GST-Nurr1 was used as the bait for Lef-1 (Fig. 1C).

To determine which of the protein-protein interaction sites in ß-catenin possess an affinity for Nurr1, the interacting domains were mapped by GST pull-down assay (Fig. 1E). A series of ß-catenin deletion mutants fused to GST were produced, diluted to equal concentrations, and incubated with either in vitro-translated Nurr1 or Lef-1. Constructs containing armadillo repeats 3 to 10 retained the binding affinity for both Nurr1 and Lef-1, suggesting that both proteins interact with ß-catenin in this region. Deletion of the Lef-1 N-terminal 70 amino acids caused a loss of binding of Lef-1 to both Nurr1 and ß-catenin. Finally, only amino acids 363 to 598 of Nurr1, encompassing the closed ligand-binding domain and AF2, interacted with Lef-1 or ß-catenin. These data suggest that the C terminus of Nurr1 binds to the N terminus of Lef-1 and that, in the presence of ß-catenin, these interaction sites compete for binding to the armadillo domains of ß-catenin.

Characterization of the Nurr1/ß-catenin complex. To further characterize the molecular properties of the Nurr1/ß-catenin complex, Nurr1 was purified from nuclear extracts of 293F cells stably expressing FLAG/HA-tagged Nurr1 in the presence of LiCl, with untransfected wild-type cells serving as a control (Fig. 2A). Endogenous proteins interacting with Nurr1 were purified through anti-FLAG and then anti-HA affinity columns, visualized by silver staining (Fig. 2B), and characterized by Western blotting for transcriptional cofactors (Fig. 2C). Silver staining showed that immunocapture of FLAG/HA-Nurr1 resulted in the recovery of Nurr1, at approximately 70 kDa (validated by mass spectrometry [data not shown]), as well as the copurification of many proteins, most of which were equally recovered in the presence or absence of LiCl. The protein mixture was then analyzed by Western blotting for cofactors selected from the literature. In the absence of LiCl, Lef-1 as well as the corepressors CtBP, TLE-1, HDAC-1, HDAC-3, and PIASy (18) were purified with Nurr1 but were not present in controls. LiCl treatment reduced the amounts of these proteins associated with Nurr1 and instead led to the recovery of endogenous ß-catenin and CBP, a key component of histone acetyltransferase complexes (9) (Fig. 2C). Thus, ß-catenin interaction with Nurr1 coincides with a switch from Nurr1 association with Lef-1 and corepressors to complex formation with CBP.


Figure 2
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  		FIG. 2. Functional interplay between Nurr1, ß-catenin, and LEF-1. (A) Schematic diagram of the biochemical purification procedure of FLAG/HA-Nurr1 interactants from a 293F cell stable transformant treated with and without LiCl. (B) Fractions eluted from anti-HA resin were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by silver staining. The arrow on the right indicates the Nurr1 protein. (C) Nurr1 and potential interacting proteins were detected by immunoblotting in eluates from parental and Nurr1-expressing cells treated with or without LiCl. (D) ChIP assays examining the association of the genomic regions containing the TCF/LEF site in cyclin D1 (left) and the NBRE site in TH (right) with Nurr1 and interacting proteins (indicated in the middle) in SK-N-MC cells. siRNAs (100 nM) were transfected prior to a 24-h treatment with LiCl or vehicle. Following cross-linking, sonication, and immunoprecipitation, genomic regions recovered with the indicated antibodies were detected by semiquantitative RT-PCR. (E) Effects of siRNAs on transcriptional regulation of the cyclin D1 promoter in Nurr1-expressing 293F cells. Each siRNA (100 nM) was transfected with a luciferase reporter containing the cyclin D1 promoter (cyclin D1-Luc) in the presence and absence of LiCl. (F) Effects of siRNAs on transcriptional regulation of the TH promoter in Nurr1-expressing 293F cells. siRNAs were transfected as described above with a luciferase reporter containing the TH promoter linked to luciferase (TH promoter-Luc) in the presence and absence of LiCl. (G) Schematic representation of the relationships among Nurr1, LEF-1, and ß-catenin under the control of activated canonical Wnt signaling at the TCF/LEF site (cyclin D1) and the NBRE (TH).

Identification and function of endogenous Nurr1/ß-catenin complexes. The proposed assembly of these factors on endogenous target promoters for TCF/LEF-1 (cyclin D1) and Nurr1 (TH) was then tested by ChIP with SK-N-MC cells, which express endogenous Nurr1 (26) (Fig. 2D). Cross-linked chromatin fragments averaging 250 bp in length were generated from SK-N-MC cells treated with nonsilencing control siRNA or Nurr1 siRNA and were immunoprecipitated with antibodies to Nurr1, Lef-1, and the cofactors identified above as being associated with Nurr1 in 293F cells. Semiquantitative PCR was used to detect the presence of the genomic region containing the TCF/LEF element in the cyclin D1 promoter. As expected, LEF-1 and its corepressors were associated with the cyclin D1 promoter in the absence of LiCl. Nurr1 was also associated with the TCF/LEF region in the cyclin D1 promoter in unstimulated cells. LiCl treatment reduced the amount of Nurr1 associated with the TCF/LEF region as well as the amounts of corepressor proteins, while ß-catenin and CBP association increased. These data suggest that the LiCl-mediated cofactor switch observed during purification of overexpressed Nurr1 in 293F cells also occurs on native promoters with endogenous proteins. Since Nurr1 overexpression modestly inhibits TCF/LEF responsiveness to Wnt-1 and LiCl (Fig. 1A), the same analysis was performed with cells transfected with siRNA targeting Nurr1. Nurr1 siRNA treatment caused a loss of Nurr1 association with the TCF/LEF element but did not alter the association of Lef-1 with the cyclin D1 promoter, indicating that Nurr1 is not essential for Lef-1 binding to TCF/LEF elements, as expected. Nurr1 siRNA slightly reduced the amounts of HDAC-1, HDAC-3, and PIASy associated with the TCF/LEF region under unstimulated conditions, but did not abolish them, consistent with Nurr1 modulating TCF/LEF repression. Following LiCl treatment, ß-catenin and CBP again were associated with the TCF/LEF element, with more ß-catenin association detected than that with control siRNA-treated cells. Like the case under basal conditions, less association between the TCF/LEF region and the corepressors was observed in the presence of Nurr1 siRNA. Thus, Nurr1 siRNA appears to enhance the association of ß-catenin with the TCF/LEF region of the cyclin D1 promoter and to reduce the amounts of associated corepressors.

Similar experiments were then performed to analyze endogenous Nurr1 association with the genomic region containing the NBRE in the TH promoter. In the absence of LiCl, Nurr1, Lef-1, and the corepressors were associated with the NBRE-containing region. LiCl promoted the association of ß-catenin and CBP and the loss of corepressors without altering the amount of associated Nurr1. Consistent with the requirement for Nurr1 for binding the NBRE, siRNAs targeting Nurr1 abolished Nurr1, corepressor, CBP, and ß-catenin association with the NBRE in the TH promoter. Thus, ß-catenin interaction with Nurr1 at the NBRE in the TH promoter is associated with a loss of corepressors and recruitment of CBP.

To determine the functional relevance of this ß-catenin-associated change in cofactor recruitment, promoter-reporter assays were performed to assess the effects of siRNAs targeting Nurr1, ß-catenin, and the cofactors on the response of the cyclin D1 and TH promoters in 293F Nurr1 stable transformants (Fig. 2E and F). siRNAs targeting Nurr1 and the corepressors caused a modest but reproducible increase in cyclin D1 promoter responsiveness to LiCl, consistent with the data obtained using the TOPFLASH reporter (Fig. 1B). Knockdown of ß-catenin or CBP nearly completely abolished LiCl responsiveness, confirming the known role for these factors in Wnt-stimulated cyclin D1 expression. As shown in Fig. 2F, TH promoter activity was stimulated by LiCl, similar to the effect observed using the 2x-NBRE reporter construct (Fig. 1A). siRNAs targeting Nurr1 inhibited promoter activity approximately fivefold and abolished LiCl responsiveness, while siRNAs targeting the corepressors tended to cause a small increase in activity of the TH promoter. In contrast, siRNAs targeting ß-catenin and CBP abrogated the LiCl responsiveness of the TH promoter. Together, these data confirm the observations that Nurr1 modestly inhibits the cyclin D1 promoter and that the TH promoter is stimulated by LiCl in a process requiring Nurr1, ß-catenin, and CBP. These data are summarized graphically in Fig. 2G.

Identification of KCNIP4 gene as a target gene coregulated by Nurr1 and ß-catenin. In order to determine if genes other than those for cyclin D1 and TH are mutually regulated by Nurr1/ß-catenin, we used microarrays to screen for genes that respond to both Nurr1 overexpression and Wnt-1 stimulation, particularly when both are present. Two clones of 293F cells stably expressing Nurr1 or wild-type parental cells were treated with LiCl or Wnt-1, and resulting changes in RNA levels were scrutinized for genes that responded to Nurr1 and LiCl treatment conditions more robustly than to either condition alone; in parallel, the same experiment was performed following LiCl or Wnt-1 stimulation of SK-N-MC cells transfected with Nurr1 or control vector (data not shown). One candidate gene, that encoding KCNIP4, was selected for further analysis based on these criteria and because we hypothesized it could be regulating Wnt signaling (see below). When analyzed by quantitative real-time RT-PCR, KCNIP4 RNA was approximately twofold lower in Nurr1+ 293F stable transformants than in parental cells, suggesting Nurr1 repression of KCNIP4 expression (Fig. 3A). Following LiCl treatment, KNCIP4 RNA was induced 2.5-fold more in the Nurr1 stable transformants than in controls by 24 h. In SK-N-MC cells, LiCl induced the expression of cyclin D1 RNA, as expected, but also significantly induced TH (2-fold) and KCNIP4 (5.5-fold) RNAs. Similar results were obtained using Wnt-1 (data not shown). Thus, KNCIP4 gene expression is regulated by Wnt-1/LiCl, and its expression is influenced by Nurr1.


Figure 3
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  		FIG. 3. Identification of KCNIP4 as a direct target of both Nurr1 and Wnt signaling. (A) Induction of the KCNIP4 gene by LiCl in 293F cells and SK-N-MC cells. The gene expression levels were measured in triplicate by quantitative RT-PCR and normalized to those of vehicle-treated parental cells. (B) Schematic presentation of putative binding sites for Nurr1 and TCF/LEF in the KCNIP4 promoter. Binding sites for Nurr1 (NBRE) and TCF/LEF/LY12 (TCF/LEF site) are shown. Two point mutations (NBRE mut and TCF/LEF mut) are displayed for reference. (C) The promoter region of the KCNIP4 gene is regulated by Nurr1 and TCF/LEF elements. Luciferase assays were performed with 293F cells transfected with reporters containing the KCNIP4 promoter or mutants (400 ng), with or without the indicated expression vectors (200 ng) (full-length Nurr1 or parent vector), in the presence and absence of LiCl (10 mM). Data were normalized to values for unstimulated parental cells expressing the wild-type KCNIP4-luciferase construct (first bar). (D) Recruitment of Nurr1 and TCF/LEF to the endogenous KCNIP4 promoter. ChIP analyses were performed with SK-N-MC cells, using specific antibodies for the indicated factors following 10 mM LiCl or vehicle treatment for 24 h. Genomic regions were tested for association with the indicated proteins by semiquantitative PCR.

To determine if the mechanism underlying these regulations involved direct transcriptional induction, the putative promoter region for KCNIP4 was searched, revealing potential binding sites for Nurr1 and ß-catenin/LEF-1 (Fig. 3B). In a promoter-reporter assay with 293F cells, the promoter region of the KCNIP4 gene (positions –1043 to –2) was responsive to both LiCl treatment and Nurr1 overexpression and, as with the native RNA, showed a greater response when both were present. Mutational analysis of the KCNIP4 promoter revealed that the putative TCF/LEF and NBRE are required for induction by LiCl and Nurr1, respectively (Fig. 3C). Mutation of the TCF/LEF-like sequence significantly inhibited the response to LiCl, whereas mutation of the potential NBRE inhibited LiCl responsiveness only in the Nurr1 stably transformed cells. Importantly, mutating both sites abolished the response to both LiCl and Nurr1 expression, suggesting that together these sites account for Nurr1 and LiCl sensitivity of the KCNIP4 promoter.

The TCF/LEF and NBRE regions of the KCNIP4 gene promoter were then analyzed by ChIP assays with both 293F and SK-N-MC cells (Fig. 3D). In both cell types, Nurr1 was associated with the genomic regions containing the TCF/LEF and NBRE sequences in the absence of LiCl. ß-Catenin was not associated with either sequence, and Lef-1 was associated only with the TCF/LEF-containing region, confirming that the assay distinguished between these elements, which are situated 344 bp apart. As observed with the cyclin D1 promoter, LiCl caused ß-catenin to associate with and Nurr1 to dissociate from the TCF/LEF region, while Lef-1 association remained constant. The response of the putative NBRE was similar to that observed with the TH promoter: LiCl caused a recruitment of ß-catenin with no change in Nurr1 association. These data, together with the promoter-reporter assay data, suggest that KCNIP4 gene expression is regulated by Nurr1 and ß-catenin interaction at or near sequences resembling TCF/LEF and NBRE sites.

PS1-mediated degradation of ß-catenin is regulated by KCNIP4. KCNIP4 is a protein that has been reported to interact with PS1 (34), a polytopic integral membrane protein that regulates ß-catenin degradation in the cytoplasm (24). To determine if KCNIP4 induction by Nurr1/ß-catenin influences PS1-mediated ß-catenin phosphorylation, we first examined the casein kinase I{alpha} (CKI{alpha}) and glycogen synthase kinase 3ß (GSK3ß) phosphorylation sites of ß-catenin (p45 and p33/37/41, respectively) in the presence and absence of Nurr1. CKI{alpha} phosphorylation precedes GSK3ß phosphorylation, which in turn triggers ß-catenin degradation; PS1 regulates the GSK3ß phosphorylation steps specifically (24). When cells were treated with the GSK3ß inhibitor LiCl in the absence of Nurr1, ß-catenin phosphorylated at the CKI{alpha} p45 site accumulated, whereas no accumulation of p33/37/41 was observed (Fig. 4A). However, in the presence of Nurr1 cDNA, p33/37/41 forms of ß-catenin accumulated and p45 forms did not. Thus, Nurr1 expression alters the GSK3ß-dependent phosphorylation status of ß-catenin.


Figure 4
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  		FIG. 4. Modulation of Wnt signaling by KCNIP4 depends on PS1. (A) Nurr1 regulation of the phosphorylation state of ß-catenin. Nurr1-expressing or parental 293F cells were treated with 10 mM LiCl and subjected to Western blotting with specific antibodies for the indicated proteins. (B) LiCl-induced assembly of KCNIP4 with PS1 and ß-catenin. Coimmunoprecipitation was performed with parental 293F cells or Nurr1 stable transformants treated with 10 mM LiCl. (C) Intracellular colocalization of PS1 with KCNIP4. Native coimmunoprecipitation was performed with SK-N-MC cells before and after LiCl stimulation (upper panels). Immunofluorescence was also performed with antibodies against KCNIP4 and PS1 (lower panels). (D) KCNIP4 and PS1 coregulate transcriptional activity of TCF/LEF in 293F cells. Luciferase assays were performed with 293F parental or Nurr1-expressing cells transfected with TOPFLASH (400 ng) and with the indicated siRNA (100 nM) for 24 h prior to treatment with 10 mM LiCl. (E) Effect of KCNIP4 on TCF/LEF-mediated transcription requires PS1. Luciferase assays were performed with wild-type or PS1–/– mouse embryonic fibroblasts (MEF) transfected with TOPFLASH (400 ng) and with the indicated expression vector (200 ng) in the presence or absence of LiCl (10 mM).

Next, coimmunoprecipitation experiments were used to examine the effects of Nurr1 expression and LiCl stimulation on the assembly of cytoplasmic ß-catenin complexes (Fig. 4B). 293F Nurr1 stable transformants or parental control cells were treated with LiCl, and the two major regulators of ß-catenin degradation, PS1 and axin, were collected by immunoprecipitation. In parental cells, LiCl caused the expected increase in ß-catenin coimmunoprecipitated with PS1 and axin. In contrast, the KCNIP4 protein displayed a time-dependent association specifically with PS1 but not with axin. Nurr1 expression increased the relative abundance of ß-catenin and KCNIP4 associated with PS1 but had no detectable effect on the axin/ß-catenin complex. Further evidence for LiCl-mediated KCNIP4 interaction with PS1 was obtained by examining the intracellular localization of KCNIP4 protein in SK-N-MC cells. LiCl treatment induced KCNIP4 protein expression, as seen by Western blotting and immunofluorescence (Fig. 4C). After 24 h, the KCNIP4 protein was coimmunoprecipitated with PS1 and appeared to be colocalized partially with PS1, as seen in the merged images of the KCNIP4 and PS1 fluorescent signals. Thus, Nurr1/ß-catenin induction of KCNIP4 is associated with remodeling of the PS1 signaling complex that regulates ß-catenin phosphorylation.

PS-dependent Nurr1 repression of ß-catenin signaling. The significance of PS1-KCNIP4 interaction was tested in 293F cells by RNAi and in mouse embryonic fibroblasts derived from PS1 knockout mice (PS1–/–) (3). As shown above, Nurr1 overexpression modestly reduced TOPFLASH reporter activity in 293F cells (Fig. 4D). siRNA targeting either PS1 or KCNIP4 restored TOPFLASH activity to that seen in parental cells not overexpressing Nurr1, suggesting that KCNIP4 and PS1 are required for Nurr1-mediated TOPFLASH repression. This hypothesis was then tested in PS1–/– fibroblasts transfected with KCNIP4 or PS1 cDNA (Fig. 4E). Transfection with either plasmid had no effect on TOPFLASH activity in unstimulated wild-type or PS1–/– cells. In wild-type fibroblasts, as in 293F cells, KCNIP4 overexpression repressed LiCl-induced TOPFLASH activity, confirming that KCNIP4 inhibits ß-catenin signaling. In PS1–/– embryonic fibroblasts, cells showed a slightly greater TOPFLASH response to LiCl than did wild-type cells, as reported previously (24), reflecting the relatively minor but biologically significant (57) contribution of PS1 to inhibiting ß-catenin activity. Interestingly, the inhibitory effect of KCNIP4 cDNA was absent in PS1–/– cells, suggesting that KCNIP4 repression of ß-catenin signaling requires PS1. Consistent with this hypothesis, PS1 cDNA cotransfection into PS1–/– cells inhibited TOPFLASH activity and restored the inhibitory effect of KCNIP4. These data together indicate that Nurr1/ß-catenin induction of KCNIP4 serves to inhibit ß-catenin signaling in a PS1-dependent feedback loop.

Nurr1 regulation of nuclear ß-catenin levels. A prediction of the above model is that Nurr1 regulates the accumulation of ß-catenin in the nucleus. To test this possibility, nuclei were prepared from cells treated with LiCl, with or without Nurr1 expression. In SK-N-MC cells (Fig. 5A, top two panels), LiCl caused a transient increase in nuclear ß-catenin; cotransfection of siRNA directed towards Nurr1 elevated the amount and duration of nuclear ß-catenin. Conversely, transfection of Nurr1 cDNA into 293F parental cells (Fig. 5A, bottom five panels) inhibited nuclear ß-catenin accumulation. Interestingly, functional human variants of Nurr1 that possess reduced transcriptional activity (31) also alter the amount of nuclear ß-catenin. We then tested the effect of these mutations on the Wnt signaling pathway by transiently transfecting Nurr1 cDNAs into 293F parental cells. As expected, the mutant forms of Nurr1 were less able to activate the NBRE-luciferase reporter than was wild-type Nurr1, but all were responsive to the additional stimulatory effects of LiCl (Fig. 5A). Nurr1 mutants had a generally reduced ability to inhibit LiCl-mediated stimulation of TOPFLASH activity, such that those most impaired in transactivation were also less able to suppress TOPFLASH activity. In contrast, the mutations did not affect KCNIP4 promoter activity in a consistent manner. Thus, human mutations in Nurr1 appear to affect not only transcriptional activation from an NBRE but also Nurr1 regulation of Wnt signaling pathways in a complex manner.


Figure 5
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  		FIG. 5. Nurr1 modulates the degradation of ß-catenin. (A) Duration of nuclear accumulation of ß-catenin in SK-N-MC cells transfected with control or Nurr1 siRNA and in 293F cells stably expressing wild-type or mutant Nurr1. Nuclear extracts were subjected to Western blotting at each time point after 10 mM LiCl stimulation. (B) Transcriptional properties of each Nurr1 mutant on NBRE, TOPFLASH, and KCNIP4 promoter. Luciferase assays were performed with 293F cells transfected with the indicated reporters (400 ng) and stably expressing wild-type or mutated Nurr1 in the presence or absence of LiCl (10 mM).


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DISCUSSION
 
Nurr1 is an orphan NR best known for its essential role in the development and maintenance of the midbrain dopaminergic neurons that regulate motor control and degenerate during Parkinson's disease. Here we tested the possibility that Wnt signaling regulates Nurr1, as Wnt is critical for the regional specification of the midbrain (5) and influences the proliferation and differentiation of Nurr1+ neuronal precursors in cell culture (6, 7). We found that ß-catenin acts as a transcriptional cofactor for Nurr1, most likely by direct physical association (although other possibilities, including interaction of ß-catenin with RXR or other Nurr1-associated factors, are not excluded by the current data). This interaction changes the transcriptional machinery associated with Nurr1 from a corepressor to a coactivator complex. Furthermore, we found that Nurr1 regulates Wnt signaling and propose that the major mechanism of inhibition is by inducing KCNIP4, a Nurr1/ß-catenin target that functions as a regulatory subunit of the PS1 complex promoting ß-catenin degradation. These findings are summarized in a model that incorporates these data (Fig. 6). It is important that the contribution of this proposed signaling system to dopaminergic cell differentiation and maintenance remains to be explored in vivo. It will be interesting to analyze mice that have a dopaminergic cell-specific deletion of ß-catenin, PS1, or KCNIP4.


Figure 6
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  		FIG. 6. Model for Nurr1 and Wnt cross-regulation. In the absence of Wnt signaling (top), Nurr1 is associated with corepressors in genes containing TCF/LEF binding sites, and Lef-1 is associated with Nurr1 on Nurr-responsive elements. Following Wnt-mediated ß-catenin accumulation in the nucleus (bottom), TCF/LEF genes become derepressed and ß-catenin switches Nurr1-associated proteins from corepressors to coactivators. Both ß-catenin and Nurr1 induce KCNIP4, which associates with a membrane-associated PS1 complex and promotes ß-catenin degradation in a negative feedback loop.

Several extracellular signals have been reported to modulate the transcriptional function of NRs through exchanging coregulators and cointegrators (14, 40). In this study, we show that Nurr1 can exist in corepressor complexes that are remodeled to CBP-containing coactivator complexes by ß-catenin. This finding extends the role that ß-catenin plays in NR signaling, as synergistic interactions with the androgen receptor and several other NRs have been described (35).

In vitro ß-catenin interacts predominantly with the C-terminal domain of Nurr1 (Fig. 1), which is significant considering that Nurr1, as well as the other members of the NR4A group, rely heavily on the N-terminal AF1 domain for transcriptional regulation, unlike many NRs (33, 38, 56). The Nurr1 C-terminal domain possesses a cell type-specific transactivation function that does not normally interact with common NR coactivators, such as SRC-1 (8), as it lacks the common NR coactivator binding site (12, 55). This C-terminal activity was proposed to be regulated instead by tissue-specific ligands or cofactors (8). Our data suggest that ß-catenin is one of these factors and that it acts somewhat similarly to an endogenous ligand in that it activates by interacting with the C-terminal domain. Given the unique function and structure of the Nurr1 C-terminal domain, it would be interesting to determine the structural changes in Nurr1 brought about by ß-catenin binding. The precise binding site for ß-catenin within the Nurr1 C-terminal domain has not been determined, but the recent discovery of a large, functionally important hydrophobic pocket opposite the place where the classic coactivator binding site would normally be (53) raises this location as a possibility.

Using microarrays to identify an endogenous gene controlled by this convergent signaling pathway, we found the KCNIP4 gene. The KCNIP4 promoter contains apparent Lef-1 and Nurr1 response elements, and interestingly, the KCNIP4 protein interacts with the cytoplasmic domain of PS1, which acts as a scaffold for ß-catenin signaling and degradation (24, 25, 39, 51, 57). We found that KCNIP4 promotes PS-mediated degradation of ß-catenin and thus appears to be a regulatory subunit for the PS complex. Transcriptional induction of KCNIP4 appears to be a primary mechanism by which Nurr1 inhibits ß-catenin activity, since the absence of KCNIP4 or PS1 abrogated the ability of Nurr1 to inhibit TOPFLASH. Although, as shown in Fig. 2, Nurr1 expression increases and Nurr1 siRNA decreases the levels of corepressors associated with the TCF/LEF region of the cyclin D1 promoter, these effects could also be indirect through the modulation of ß-catenin cytoplasmic stability via KCNIP4. Further experiments will be required to determine if Nurr1 plays a significant role in directly inhibiting the transcription of Wnt target genes or, rather, acts largely through the induction of KCNIP4. The physiological relevance of the modest inhibitor effect of Nurr1 on Wnt-1 signaling remains to be studied, but for cell culture our preliminary microarray data revealed that Wnt-1-regulated genes were, on average, 22% less responsive in 293F cells overexpressing Nurr1 than in parental controls (mean of two replicates with different subclones [n = 135 RNAs]; P < 0.001) (unpublished observations). It will be interesting to determine if Nurr1+ neuronal precursors respond differentially to Wnt signaling compared to Nurr1– cells in vivo.

In addition to the well-studied role for Wnt signaling during CNS development of the VM dopaminergic system, aberrant Wnt signaling has been implicated in several psychiatric and neurological disorders, such as bipolar disorder (19) and schizophrenia (16). Given the importance of the dopaminergic system in these diseases, it is conceivable that Wnt signaling could, in part, affect the activity of Nurr1. Interestingly, both LiCl and valproic acid, drugs useful in a number of mental disorders, inhibit GSK3ß, and recent data suggest that certain antipsychotics modulate this system as well (1, 2, 30). We show here that naturally occurring Nurr1 mutants have a reduced ability to inhibit ß-catenin transcription from a TCF/LEF element and appear to differentially regulate ß-catenin accumulation and KCNIP4 promoter activation. Future studies that determine if the functional interplay between ß-catenin and Nurr1 varies with disease state and is modulated by genetic factors will be of interest.


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ACKNOWLEDGMENTS
 
We thank T. Iwatsubo for the kind gift of KCNIP4 antibodies, Ung-il Chung for technical support with handling retrovirus, and J. Yanagisawa for critical reading of the manuscript. We also thank H. Higuchi and Y. Nagasawa for manuscript preparation.

This work was partially supported by the 19th Research Fellowship from the Naito Memorial Foundation (2003) and by a research fellowship from the Uehara Memorial Foundation (2004).


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FOOTNOTES
 
* Corresponding author. Present address: Wyeth Research, 500 Arcola Road N2283, Collegeville, PA 19426. Phone: (484) 865-2773. Fax: (484) 865-9393. E-mail: freedlp{at}wyeth.com Back

{triangledown} Published ahead of print on 20 August 2007. Back

{dagger} H.K. and W.J.R. gave equal contributions to this study. Back

{ddagger} Present address: Department of Alzheimer's Research, Merck Research Laboratories, West Point, PA 19486. Back


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Molecular and Cellular Biology, November 2007, p. 7486-7496, Vol. 27, No. 21
0270-7306/07/$08.00+0     doi:10.1128/MCB.00409-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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