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Previous Article | Next Article 
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.
William J. Ray ,1,,
Helmut Glantschnig,1
Pascale V. Nantermet,1
Yuanjiang Yu,1,
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 |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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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 |
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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 |
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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.
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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.
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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
(CKI) 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 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 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.
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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.
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DISCUSSION |
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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 |
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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 |
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Published ahead of print on 20 August 2007.
H.K. and W.J.R. gave equal contributions to this study.
Present address: Department of Alzheimer's Research, Merck Research Laboratories, West Point, PA 19486.
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REFERENCES |
|---|
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2. Alimohamad, H., L. Sutton, J. Mouyal, N. Rajakumar, and W. J. Rushlow. 2005. The effects of antipsychotics on beta-catenin, glycogen synthase kinase-3 and dishevelled in the ventral midbrain of rats. J. Neurochem. 95: 513-525.[CrossRef][Medline]
3. Armogida, M., A. Petit, B. Vincent, S. Scarzello, C. A. da Costa, and F. Checler. 2001. Endogenous beta-amyloid production in presenilin-deficient embryonic mouse fibroblasts. Nat. Cell Biol 3: 1030-1033.[CrossRef][Medline]
4. Cadigan, K. M., and Y. I. Liu. 2006. Wnt signaling: complexity at the surface. J. Cell Sci. 119: 395-402.
5. Castelo-Branco, G., and E. Arenas. 2006. Function of Wnts in dopaminergic neuron development. Neurodegener. Dis. 3: 5-11.[CrossRef][Medline]
6. Castelo-Branco, G., K. M. Sousa, V. Bryja, L. Pinto, J. Wagner, and E. Arenas. 2006. Ventral midbrain glia express region-specific transcription factors and regulate dopaminergic neurogenesis through Wnt-5a secretion. Mol. Cell. Neurosci. 31: 251-262.[CrossRef][Medline]
7. Castelo-Branco, G., J. Wagner, F. J. Rodriguez, J. Kele, K. Sousa, N. Rawal, H. A. Pasolli, E. Fuchs, J. Kitajewski, and E. Arenas. 2003. Differential regulation of midbrain dopaminergic neuron development by Wnt-1, Wnt-3a, and Wnt-5a. Proc. Natl. Acad. Sci. USA 100: 12747-12752.
8. Castro, D. S., M. Arvidsson, M. Bondesson Bolin, and T. Perlmann. 1999. Activity of the Nurr1 carboxyl-terminal domain depends on cell type and integrity of the activation function 2. J. Biol. Chem. 274: 37483-37490.
9. Chan, H. M., and N. B. La Thangue. 2001. p300/CBP proteins: HATs for transcriptional bridges and scaffolds. J. Cell Sci. 114: 2363-2373.
10. Chu, Y., K. Kompoliti, E. J. Cochran, E. J. Mufson, and J. H. Kordower. 2002. Age-related decreases in Nurr1 immunoreactivity in the human substantia nigra. J. Comp. Neurol. 450: 203-214.[CrossRef][Medline]
11. Chu, Y., W. Le, K. Kompoliti, J. Jankovic, E. J. Mufson, and J. H. Kordower. 2006. Nurr1 in Parkinson's disease and related disorders. J. Comp. Neurol. 494: 495-514.[CrossRef][Medline]
12. Codina, A., G. Benoit, J. T. Gooch, D. Neuhaus, T. Perlmann, and J. W. Schwabe. 2004. Identification of a novel co-regulator interaction surface on the ligand binding domain of Nurr1 using NMR footprinting. J. Biol. Chem. 279: 53338-53345.
13. Danielian, P. S., and A. P. McMahon. 1996. Engrailed-1 as a target of the Wnt-1 signalling pathway in vertebrate midbrain development. Nature 383: 332-334.[CrossRef][Medline]
14. Daniels, D. L., and W. I. Weis. 2005. Beta-catenin directly displaces Groucho/TLE repressors from Tcf/Lef in Wnt-mediated transcription activation. Nat. Struct. Mol. Biol. 12: 364-371.[CrossRef][Medline]
15. Darragh, J., A. Soloaga, V. A. Beardmore, A. D. Wingate, G. R. Wiggin, M. Peggie, and J. S. Arthur. 2005. MSKs are required for the transcription of the nuclear orphan receptors Nur77, Nurr1 and Nor1 downstream of MAPK signalling. Biochem. J. 390: 749-759.[Medline]
16. Dean, B. 2002. Understanding the pathology of schizophrenia: recent advances from the study of the molecular architecture of postmortem CNS tissue. Postgrad. Med. J. 78: 142-148.
17. Forman, B. M., K. Umesono, J. Chen, and R. M. Evans. 1995. Unique response pathways are established by allosteric interactions among nuclear hormone receptors. Cell 81: 541-550.[CrossRef][Medline]
18. Galleguillos, D., A. Vecchiola, J. A. Fuentealba, V. Ojeda, K. Alvarez, A. Gomez, and M. E. Andres. 2004. PIASgamma represses the transcriptional activation induced by the nuclear receptor Nurr1. J. Biol. Chem. 279: 2005-2011.
19. Gould, T. D., and H. K. Manji. 2002. The Wnt signaling pathway in bipolar disorder. Neuroscientist 8: 497-511.[Abstract]
20. Hermanson, E., B. Joseph, D. Castro, E. Lindqvist, P. Aarnisalo, A. Wallen, G. Benoit, B. Hengerer, L. Olson, and T. Perlmann. 2003. Nurr1 regulates dopamine synthesis and storage in MN9D dopamine cells. Exp. Cell Res. 288: 324-334.[CrossRef][Medline]
21. Holla, V. R., J. R. Mann, Q. Shi, and R. N. DuBois. 2006. Prostaglandin E2 regulates the nuclear receptor NR4A2 in colorectal cancer. J. Biol. Chem. 281: 2676-2682.
22. Jiang, C., X. Wan, Y. He, T. Pan, J. Jankovic, and W. Le. 2005. Age-dependent dopaminergic dysfunction in Nurr1 knockout mice. Exp. Neurol. 191: 154-162.[CrossRef][Medline]
23. Joseph, B., A. Wallen-Mackenzie, G. Benoit, T. Murata, E. Joodmardi, S. Okret, and T. Perlmann. 2003. p57(Kip2) cooperates with Nurr1 in developing dopamine cells. Proc. Natl. Acad. Sci. USA 100: 15619-15624.
24. Kang, D. E., S. Soriano, X. Xia, C. G. Eberhart, B. De Strooper, H. Zheng, and E. H. Koo. 2002. Presenilin couples the paired phosphorylation of beta-catenin independent of axin: implications for beta-catenin activation in tumorigenesis. Cell 110: 751-762.[CrossRef][Medline]
25. Killick, R., C. C. Pollard, A. A. Asuni, A. K. Mudher, J. C. Richardson, H. T. Rupniak, P. W. Sheppard, I. M. Varndell, J. P. Brion, A. I. Levey, O. A. Levy, M. Vestling, R. Cowburn, S. Lovestone, and B. H. Anderton. 2001. Presenilin 1 independently regulates beta-catenin stability and transcriptional activity. J. Biol. Chem. 276: 48554-48561.
26. Kim, K. S., C. H. Kim, D. Y. Hwang, H. Seo, S. Chung, S. J. Hong, J. K. Lim, T. Anderson, and O. Isacson. 2003. Orphan nuclear receptor Nurr1 directly transactivates the promoter activity of the tyrosine hydroxylase gene in a cell-specific manner. J. Neurochem. 85: 622-634.[Medline]
27. Kim, S. Y., K. C. Choi, M. S. Chang, M. H. Kim, Y. S. Na, J. E. Lee, B. K. Jin, B. H. Lee, and J. H. Baik. 2006. The dopamine D2 receptor regulates the development of dopaminergic neurons via extracellular signal-regulated kinase and Nurr1 activation. J. Neurosci. 26: 4567-4576.
28. Kitagawa, H., R. Fujiki, K. Yoshimura, Y. Mezaki, Y. Uematsu, D. Matsui, S. Ogawa, K. Unno, M. Okubo, A. Tokita, T. Nakagawa, T. Ito, Y. Ishimi, H. Nagasawa, T. Matsumoto, J. Yanagisawa, and S. Kato. 2003. The chromatin-remodeling complex WINAC targets a nuclear receptor to promoters and is impaired in Williams syndrome. Cell 113: 905-917.[CrossRef][Medline]
29. Kouzmenko, A. P., K. Takeyama, S. Ito, T. Furutani, S. Sawatsubashi, A. Maki, E. Suzuki, Y. Kawasaki, T. Akiyama, T. Tabata, and S. Kato. 2004. Wnt/beta-catenin and estrogen signaling converge in vivo. J. Biol. Chem. 279: 40255-40258.
30. Kozlovsky, N., S. Amar, R. H. Belmaker, and G. Agam. 2006. Psychotropic drugs affect Ser9-phosphorylated GSK-3 beta protein levels in rodent frontal cortex. Int. J. Neuropsychopharmacol. 9: 337-342.[CrossRef][Medline]
31. Le, W. D., P. Xu, J. Jankovic, H. Jiang, S. H. Appel, R. G. Smith, and D. K. Vassilatis. 2003. Mutations in NR4A2 associated with familial Parkinson disease. Nat. Genet. 33: 85-89.[CrossRef][Medline]
32. Liu, J., H. Wang, Y. Zuo, and S. R. Farmer. 2006. Functional interaction between peroxisome proliferator-activated receptor gamma and beta-catenin. Mol. Cell. Biol. 26: 5827-5837.
33. Maira, M., C. Martens, E. Batsche, Y. Gauthier, and J. Drouin. 2003. Dimer-specific potentiation of NGFI-B (Nur77) transcriptional activity by the protein kinase A pathway and AF-1-dependent coactivator recruitment. Mol. Cell. Biol. 23: 763-776.
34. Morohashi, Y., N. Hatano, S. Ohya, R. Takikawa, T. Watabiki, N. Takasugi, Y. Imaizumi, T. Tomita, and T. Iwatsubo. 2002. Molecular cloning and characterization of CALP/KChIP4, a novel EF-hand protein interacting with presenilin 2 and voltage-gated potassium channel subunit Kv4. J. Biol. Chem. 277: 14965-14975.
35. Mulholland, D. J., S. Dedhar, G. A. Coetzee, and C. C. Nelson. 2005. Interaction of nuclear receptors with the Wnt/beta-catenin/Tcf signaling axis: Wnt you like to know? Endocr. Rev. 26: 898-915.
36. Murphy, E. P., and O. M. Conneely. 1997. Neuroendocrine regulation of the hypothalamic pituitary adrenal axis by the nurr1/nur77 subfamily of nuclear receptors. Mol. Endocrinol. 11: 39-47.
37. Nantermet, P. V., J. Xu, Y. Yu, P. Hodor, D. Holder, S. Adamski, M. A. Gentile, D. B. Kimmel, S. Harada, D. Gerhold, L. P. Freedman, and W. J. Ray. 2004. Identification of genetic pathways activated by the androgen receptor during the induction of proliferation in the ventral prostate gland. J. Biol. Chem. 279: 1310-1322.
38. Nordzell, M., P. Aarnisalo, G. Benoit, D. S. Castro, and T. Perlmann. 2004. Defining an N-terminal activation domain of the orphan nuclear receptor Nurr1. Biochem. Biophys. Res. Commun. 313: 205-211.[CrossRef][Medline]
39. Palacino, J. J., M. P. Murphy, O. Murayama, K. Iwasaki, M. Fujiwara, A. Takashima, T. E. Golde, and B. Wolozin. 2001. Presenilin 1 regulates beta-catenin-mediated transcription in a glycogen synthase kinase-3-independent fashion. J. Biol. Chem. 276: 38563-38569.
40. Perissi, V., and M. G. Rosenfeld. 2005. Controlling nuclear receptors: the circular logic of cofactor cycles. Nat. Rev. Mol. Cell. Biol. 6: 542-554.[CrossRef][Medline]
41. Porfiri, E., B. Rubinfeld, I. Albert, K. Hovanes, M. Waterman, and P. Polakis. 1997. Induction of a beta-catenin-LEF-1 complex by wnt-1 and transforming mutants of beta-catenin. Oncogene 15: 2833-2839.[CrossRef][Medline]
42. Powzaniuk, M., S. McElwee-Witmer, R. L. Vogel, T. Hayami, S. J. Rutledge, F. Chen, S. Harada, A. Schmidt, G. A. Rodan, L. P. Freedman, and C. Bai. 2004. The LATS2/KPM tumor suppressor is a negative regulator of the androgen receptor. Mol. Endocrinol. 18: 2011-2023.
43. Sacchetti, P., R. Carpentier, P. Segard, C. Olive-Cren, and P. Lefebvre. 2006. Multiple signaling pathways regulate the transcriptional activity of the orphan nuclear receptor NURR1. Nucleic Acids Res. 34: 5515-5527.
44. Sacchetti, P., T. R. Mitchell, J. G. Granneman, and M. J. Bannon. 2001. Nurr1 enhances transcription of the human dopamine transporter gene through a novel mechanism. J. Neurochem. 76: 1565-1572.[CrossRef][Medline]
45. Sakurada, K., M. Ohshima-Sakurada, T. D. Palmer, and F. H. Gage. 1999. Nurr1, an orphan nuclear receptor, is a transcriptional activator of endogenous tyrosine hydroxylase in neural progenitor cells derived from the adult brain. Development 126: 4017-4026.[Abstract]
46. Salthun-Lassalle, B., E. C. Hirsch, J. Wolfart, M. Ruberg, and P. P. Michel. 2004. Rescue of mesencephalic dopaminergic neurons in culture by low-level stimulation of voltage-gated sodium channels. J. Neurosci. 24: 5922-5930.
47. Satoh, J., and Y. Kuroda. 2002. The constitutive and inducible expression of Nurr1, a key regulator of dopaminergic neuronal differentiation, in human neural and non-neural cell lines. Neuropathology 22: 219-232.[CrossRef][Medline]
48. Saucedo-Cardenas, O., J. D. Quintana-Hau, W. D. Le, M. P. Smidt, J. J. Cox, F. De Mayo, J. P. Burbach, and O. M. Conneely. 1998. Nurr1 is essential for the induction of the dopaminergic phenotype and the survival of ventral mesencephalic late dopaminergic precursor neurons. Proc. Natl. Acad. Sci. USA 95: 4013-4018.
49. Sierra, J., T. Yoshida, C. A. Joazeiro, and K. A. Jones. 2006. The APC tumor suppressor counteracts beta-catenin activation and H3K4 methylation at Wnt target genes. Genes Dev. 20: 586-600.
50. Singh, R., J. N. Artaza, W. E. Taylor, M. Braga, X. Yuan, N. F. Gonzalez-Cadavid, and S. Bhasin. 2006. Testosterone inhibits adipogenic differentiation in 3T3-L1 cells: nuclear translocation of androgen receptor complex with beta-catenin and T-cell factor 4 may bypass canonical Wnt signaling to down-regulate adipogenic transcription factors. Endocrinology 147: 141-154.
51. Soriano, S., D. E. Kang, M. Fu, R. Pestell, N. Chevallier, H. Zheng, and E. H. Koo. 2001. Presenilin 1 negatively regulates beta-catenin/T cell factor/lymphoid enhancer factor-1 signaling independently of beta-amyloid precursor protein and notch processing. J. Cell Biol. 152: 785-794.
52. Takayama, S., I. Rogatsky, L. E. Schwarcz, and B. D. Darimont. 2006. The glucocorticoid receptor represses cyclin D1 by targeting the Tcf-beta-catenin complex. J. Biol. Chem. 281: 17856-17863.
53. Volakakis, N., M. Malewicz, B. Kadkhodai, T. Perlmann, and G. Benoit. 2006. Characterization of the Nurr1 ligand-binding domain co-activator interaction surface. J. Mol. Endocrinol. 37: 317-326.
54. Volpicelli, F., C. Perrone-Capano, P. Da Pozzo, L. Colucci-D'Amato, and U. di Porzio. 2004. Modulation of nurr1 gene expression in mesencephalic dopaminergic neurones. J. Neurochem. 88: 1283-1294.[CrossRef][Medline]
55. Wang, Z., G. Benoit, J. Liu, S. Prasad, P. Aarnisalo, X. Liu, H. Xu, N. P. Walker, and T. Perlmann. 2003. Structure and function of Nurr1 identifies a class of ligand-independent nuclear receptors. Nature 423: 555-560.[CrossRef][Medline]
56. Wansa, K. D., J. M. Harris, and G. E. Muscat. 2002. The activation function-1 domain of Nur77/NR4A1 mediates trans-activation, cell specificity, and coactivator recruitment. J. Biol. Chem. 277: 33001-33011.
57. Xia, X., S. Qian, S. Soriano, Y. Wu, A. M. Fletcher, X. J. Wang, E. H. Koo, X. Wu, and H. Zheng. 2001. Loss of presenilin 1 is associated with enhanced beta-catenin signaling and skin tumorigenesis. Proc. Natl. Acad. Sci. USA 98: 10863-10868.
58. Yanagisawa, J., H. Kitagawa, M. Yanagida, O. Wada, S. Ogawa, M. Nakagomi, H. Oishi, Y. Yamamoto, H. Nagasawa, S. B. McMahon, M. D. Cole, L. Tora, N. Takahashi, and S. Kato. 2002. Nuclear receptor function requires a TFTC-type histone acetyl transferase complex. Mol. Cell 9: 553-562.[CrossRef][Medline]
59. Zetterstrom, R. H., L. Solomin, L. Jansson, B. J. Hoffer, L. Olson, and T. Perlmann. 1997. Dopamine neuron agenesis in Nurr1-deficient mice. Science 276: 248-250.
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