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Molecular Endocrinology, doi:10.1210/me.2003-0247
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Molecular Endocrinology 18 (6): 1546-1557
Copyright © 2004 by The Endocrine Society

Regulation of the Osteopontin Gene by the Orphan Nuclear Receptor NURR1 in Osteoblasts

Johanna Lammi, Johanna Huppunen and Piia Aarnisalo

Institute of Biomedicine (J.L., J.H., P.A.), Biomedicum Helsinki, University of Helsinki, 00014 Helsinki, Finland; Department of Clinical Chemistry (P.A.), University of Helsinki and Helsinki University Central Hospital, 00014 Helsinki, Finland

Address all correspondence and requests for reprints to: Piia Aarnisalo, Institute of Biomedicine, Biomedicum Helsinki, University of Helsinki, P.O. Box 63, 00014 Helsinki, Finland. E-mail: piia.aarnisalo{at}helsinki.fi.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The orphan nuclear receptor Nurr1 is mainly expressed in the central nervous system but is also detected in certain peripheral tissues such as bone. To elucidate the role of Nurr1 in bone, we examined the ability of Nurr1 to regulate osteopontin (OPN) expression in osteoblastic cell lines. Transfection of Nurr1 in osteoblastic cells increased OPN mRNA expression. A dominant negative Nurr1 variant abolished the ability of PTH to induce OPN expression, suggesting that Nurr1 is involved in mediating the regulation of OPN by PTH. Nurr1 efficiently transactivated a luciferase reporter construct driven by the –857/+191 fragment of the mouse OPN promoter. The activation of the OPN promoter was mediated by the monomeric form of Nurr1, required direct binding of Nurr1 to the OPN promoter, and was dependent on the amino-terminal transactivation function-1. The OPN promoter is also regulated by vitamin D receptor and estrogen-related receptors. We show that Nurr1 and vitamin D activate the OPN promoter in a synergistic fashion, whereas Nurr1-mediated transactivation of the OPN promoter is repressed by estrogen-related receptors. In conclusion, Nurr1 activates the OPN promoter directly in osteoblastic cells, suggesting a role for Nurr1 in the regulation of bone homeostasis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
NUCLEAR RECEPTORS ARE ligand-inducible transcription factors including the receptors for steroid hormones, retinoids, and vitamin D. In addition, a large number of evolutionally conserved proteins with unknown ligands resemble the ligand-regulated nuclear receptors and are referred to as orphan nuclear receptors (1, 2). Nurr1 (NR4A2) and the closely related NGFI-B (NR4A1) and Nor1 (NR4A3) belong to the category of orphan nuclear receptors (3, 4, 5). The recently published crystal structure of the Nurr1 ligand-binding domain demonstrated that Nurr1 is a ligand-independent nuclear receptor as there is no cavity for ligand binding (6). Thus, the mechanisms modulating Nurr1 activity are unknown. Nurr1 is predominantly expressed in the central nervous system (CNS) (1) where it plays a crucial role in the development of the midbrain dopaminergic neurons (7, 8). It has also been suggested that Nurr1 is involved in the pathogenesis of certain diseases involving dopamine transmission such as Parkinson’s disease, schizophrenia, and manic-depressive disorder (9, 10). In addition to the CNS, Nurr1 is expressed in certain peripheral tissues such as adrenals, liver, synovium, arterial wall, and bone (11, 12, 13, 14, 15, 16), yet the role of Nurr1 in these tissues remains elusive.

An interesting feature of Nurr1 is that it is encoded by an immediate early gene (4). Therefore, Nurr1 expression is rapidly induced in response to various stimuli in several tissues (11, 12, 13, 14, 15). In osteoblasts of bone, Nurr1 expression is increased as an immediate early gene in response to PTH (13).

Nurr1 binds DNA as a monomer and recognizes an extended nuclear receptor binding site referred to as NGFI-B response element [NBRE; 5'-AAAGGTCA-3' (17)]. As a monomer, Nurr1 promotes constitutive transcriptional activation that is dependent on two distinct activation functions (AF1 and AF2) localized in the amino- and carboxyl-terminal regions of Nurr1, respectively (18). Nurr1 also forms heterodimers with the retinoid X receptor (RXR). In contrast to most heterodimers formed by RXR and other nuclear receptors, RXR-Nurr1 dimers are efficiently activated by RXR ligands (19, 20), suggesting that Nurr1 may be important for signaling in response to RXR ligands. Importantly, the closely related receptor NGFI-B is equally efficient in promoting RXR activation as Nurr1, whereas Nor1 is unable to form heterodimers with RXR (21).

The aim of the current study was to identify genes regulated by Nurr1 in osteoblasts. We have analyzed promoter regions of several osteoblastic genes for potential Nurr1 response elements, NBREs. We identified a functional NBRE in the mouse osteopontin (OPN) gene that mediates activation of the OPN promoter by Nurr1, NGFI-B, and Nor1 in osteoblastic cells. OPN is a major noncollagenous bone matrix protein produced by osteoblasts, osteoclasts, and hypertrophic chondrocytes. OPN functions both as a cell attachment protein and as a cytokine acting on cells via a number of receptors including certain integrins and CD44 (reviewed in Ref.22). Analyses of mice with targeted disruption of the OPN gene have shown that OPN is not essential for normal skeletal development (23). However, OPN is required for postmenopausal and PTH-induced bone resorption and has been implicated in the callus formation process during fracture healing (24, 25, 26). The OPN promoter is regulated by various transcription factors including nuclear receptors (27, 28, 29, 30, 31, 32, 33, 34, 35). In addition to Nurr1 reported here, previous studies have demonstrated OPN promoter regulation by estrogen receptor-{alpha}, estrogen-related receptor-{alpha} (ERR{alpha}), vitamin D receptor (VDR), and peroxisome proliferator-activated receptor-{gamma} (27, 28, 30, 31, 35). We show here that Nurr1 binds directly to the OPN promoter and activates it as a monomer. Nurr1 increases the expression of OPN mRNA in osteoblasts. Dominant negative Nurr1 abolishes PTH-induced OPN expression, suggesting that Nurr1 has a role in mediating the regulation of OPN by PTH. Furthermore, Nurr1 and VDR transactivate the OPN promoter in a synergistic fashion. In contrast, ERR{alpha} and ERR{gamma} inhibit the activating effect of Nurr1. In summary, we have identified OPN as the first Nurr1 target gene in osteoblasts.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Nurr1 Regulates OPN Expression in Osteoblasts
Nurr1 is expressed in osteoblasts and osteoblastic cell lines (Fig. 1Go; Refs.13 and 16). The osteoblastic cell lines and primary osteoblasts used in this study also express the related orphan receptors NGFI-B and Nor1. The expression of Nurr1 in both primary osteoblasts and in the osteoblastic U2-OS, SaOs-2, and MC3T3-E1 cell lines was dramatically induced in response to PTH (data not shown and Ref.13). To identify genes regulated by Nurr1 in osteoblasts, we analyzed the promoter sequences of several osteoblastic genes for potential Nurr1 response elements, NBREs. The mouse osteopontin promoter contains at least two potential NBREs (30, 31). Therefore, we examined whether Nurr1 regulated OPN expression in osteoblastic cells. Because Nurr1 lacks a ligand that could be used to stimulate its activity in cells, we expressed Nurr1 ectopically by transient transfection in human U2-OS and mouse MC3T3-E1 osteoblastic cells and subsequently analyzed its effect on OPN mRNA expression using RT-PCR. As shown in Fig. 2Go, Nurr1 increased OPN expression in these cells.



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  		Fig. 1. Nurr1 Is Expressed in Primary Mouse Osteoblasts and in Osteoblastic Cell Lines

RNA extracted from human osteoblastic U2-OS, SaOs-2, and MG63 cells and from mouse primary osteoblasts (PMO) was analyzed for Nurr1 mRNA expression by RT-PCR. Primers specific for G3PDH were used as controls.

 


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  		Fig. 2. Nurr1 Induces the Expression of Endogenous OPN in Osteoblasts

A, Human U2-OS and mouse MC3T3-E1 osteoblastic cells were transfected with the expression vector for Nurr1 as indicated, and the expression level of OPN mRNA was subsequently analyzed by RT-PCR. G3PDH was used as a control. B and C, The PCR products were quantified using the Kodak Image station 440 CF. The results were normalized with respect to G3PDH. The graphs represent the fold increase in OPN (B) or Nurr1 (C) mRNA levels induced by transfected Nurr1 (control cells = 1). The experiment has been repeated twice with essentially identical results. The results of one representative experiment are shown.

 
The expression of Nurr1 mRNA in osteoblasts increases very rapidly (in 30 min) in response to PTH (13). PTH also induces OPN mRNA expression, but the induction occurs later, after about 6 h (36). This suggests that Nurr1 may act as a mediator of PTH-induced OPN expression. To study this possibility, we used a dominant negative Nurr1 variant (Nurr1DN) that encodes Nurr1 residues 94–356 including part of the amino-terminal domain and the entire DNA-binding domain (DBD) fused to the Drosophila repressor protein Engrailed (37). We transfected Nurr1DN in MC3T3-E1 cells and measured its effect on PTH-induced OPN mRNA expression by RT-PCR. Expression of Nurr1DN abolished the ability of PTH to induce OPN mRNA (Fig. 3Go). These data suggest that Nurr1 plays a role in mediating PTH-induced OPN expression.



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  		Fig. 3. PTH-Induced OPN mRNA Expression Is Abolished by Dominant Negative Nurr1

A, MC3T3-E1 cells were transfected with the expression vector for dominant negative Nurr1 (Nurr1 DN) and treated with 100 nM PTH for 8 h as indicated. The expression of OPN was subsequently analyzed by RT-PCR. The experiment has been repeated three times with similar results. The results of one representative experiment are shown. B, The PCR products were quantified using the Kodak Image station 440 CF. The results were normalized with respect to G3PDH. The graph represents the fold increase in OPN mRNA level (control cells = 1). Values are mean ± SD of two independent experiments.

 
Nurr1 Activates the OPN Promoter in Osteoblastic Cell Lines
The two potential NBREs in the mouse OPN promoter have previously been shown to mediate regulation of the promoter by estrogen-related receptor-{alpha} (ERR{alpha}), another orphan member of the nuclear receptor superfamily (30, 31). One of the potential response elements, referred to as S1 element, is a consensus NBRE, whereas the other, S2 element, is similar to the consensus sequence of the ERR{alpha} response element, ERRE (Fig. 6AGo). To examine regulation of the OPN promoter by Nurr1, we amplified the –857/+191 fragment of the mouse OPN promoter containing the S1 and S2 elements by PCR from mouse genomic DNA and cloned this fragment upstream of a luciferase reporter gene (OPN-LUC). Nurr1 transactivated OPN-LUC reporter when cotransfected in U2-OS (Fig. 4AGo) but not in SaOs-2 cells (Fig. 4BGo). Similarly, Nurr1 activated an artificial NBRE-driven reporter in U2-OS cells but not in SaOs-2 cells. The reason for this difference is elusive but could result from differential expression of Nurr1-specific coactivator proteins in these two osteoblastic cell lines. However, cotransfection of Nor1 and NGFI-B elicited activation of the OPN promoter in both U2-OS and SaOs-2 cells (Fig. 4Go, A and B). Nurr1, NGFI-B, and Nor1 transactivated the OPN promoter also in MC3T3-E1 and MG-63 osteoblastic cells (data not shown). In contrast, no activation was detected in kidney-derived 293 cells or human cervical carcinoma HeLa cells, although all three receptors were capable of transactivating an artificial NBRE-driven reporter in these cell lines (Fig. 4CGo and data not shown). These results suggest that the activation of the OPN promoter by Nurr1 and the related receptors requires the presence of additional factors specific for osteoblastic cells.



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  		Fig. 6. Nurr1 Regulates the OPN Promoter by Directly Binding to the S1 Element

A, Sequences of the oligonucleotides used in this study corresponding to the S1 and S2 elements of the mouse OPN promoter. The mutated nucleotides in OPN S1mut and OPN S2mut are underlined. B, The ability of in vitro produced Nurr1 protein to bind to OPN S1 and S2 elements was assessed by EMSA as described in the legend to Fig. 5Go. OPN S1 and OPN S2 oligonucleotides were used as probes. C, The ability of OPN S1 and OPN S2 elements to compete with NBRE for Nurr1 binding was examined using EMSA. In vitro produced Nurr1 protein was incubated with 32P-labeled NBRE probe in the absence or presence of 10- or 100-fold molar excess of unlabeled NBRE, OPN S1, OPN S1mut, OPN S2, or OPN S2mut oligonucleotides as depicted. D, U2-OS cells were transfected with the Nurr1 expression vector together with the OPN-LUC reporter or its variants harboring mutations in either the S1 element (OPN S1mut-LUC) or S2 element (OPN S2mut-LUC) as indicated.

 


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  		Fig. 4. Nurr1 Transactivates the Mouse OPN Promoter in Osteoblastic Cells Osteoblastic U2-OS (A) and SaOs-2 (B) cells and kidney-derived 293 cells (C) were cotransfected with 50 ng of the expression vectors for Nurr1, NGFI-B, or Nor1 along with 100 ng of pCMX-ßgal internal control and 300 ng of NBRE3tk-LUC or OPN-LUC reporters as indicated. The cells were harvested 42 h later and analyzed for luciferase and ß-galactosidase activities. Values are presented as fold induction after normalization to ß-galactosidase activities. Each experiment has been repeated in triplicate dishes at least three times with similar results. The mean ± SD of one representative experiment is shown.

 
Nurr1 Binds Directly to the OPN Promoter
Nurr1 binds DNA via the highly conserved DBD. DNA binding is mediated by two zinc-finger structures and the so-called A box region in the DBD (17, 38). To examine whether transactivation of the OPN promoter required an intact Nurr1 DBD, two point mutations were introduced. The first mutant was a substitution of glycine for the zinc-coordinating cysteine 283 in the first zinc finger (Nurr1C283G). This mutant completely lacked the ability to bind to the NBRE element in vitro and to activate an artificial NBRE-reporter gene in transfected cells (Fig. 5Go, A and B). The second DBD mutant was a substitution of alanine for arginine 334 in the A box, leaving the zinc-finger region intact (Nurr1R334A). This mutation has previously been shown to prevent NBRE binding and subsequently the ability to transactivate NBRE-driven reporters (Fig. 5BGo and Ref.37). The lack of transcriptional activation by Nurr1C283G and Nurr1R334A was not due to reduced expression as both mutants were expressed at levels comparable to the wild-type Nurr1 in U2-OS cells (data not shown). Next, the mutants were analyzed for their ability to activate OPN-LUC in U2-OS cells. Both mutants failed to transactivate OPN-LUC (Fig. 5CGo), suggesting that regulation of the OPN promoter requires an intact Nurr1 DBD.



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  		Fig. 5. Nurr1 DNA Binding Is Required for OPN Promoter Activation

A, Nurr1 and Nurr1C283G proteins were produced by coupled in vitro transcription and translation in reticulocyte lysates, and their ability to bind DNA was studied using EMSA. Translation reactions (5 µl) were incubated with 32P-labeled NBRE element, and the protein-DNA complexes were resolved by electrophoresis on 4% nondenaturing polyacrylamide gel in 0.25x Tris-borate, EDTA. The asterisk indicates the Nurr1-DNA complex. B, Nurr1, Nurr1C283G, and Nurr1R334A expression plasmids were transfected in U2-OS cells along with the NBRE3tk-LUC reporter as described in the legend to Fig. 4Go. C, Expression vectors encoding Nurr1, Nurr1C283G, and Nurr1R334A were cotransfected in U2-OS cells along with the OPN-LUC reporter.

 
We next investigated whether Nurr1 bound directly to the OPN promoter using EMSA. Radioactively labeled oligonucleotides corresponding to the OPN S1 and S2 elements were used as probes (Fig. 6AGo). When the probes were incubated with in vitro translated Nurr1 protein, strong Nurr1 binding to the S1 element was observed (Fig. 6BGo). In addition, the complex formed between Nurr1 and an artificial NBRE element could be displaced by addition of an unlabeled oligonucleotide corresponding to the OPN S1 element (Fig. 6CGo). In contrast, no binding to the mutated S1 element or to the S2 element was detected. Thus, Nurr1 is able to bind directly to the S1 element of the OPN promoter.

The role of the OPN S1 and S2 elements in mediating OPN promoter regulation by Nurr1 was then investigated. We mutated the S1 and S2 elements (OPN S1mut-LUC and OPN S2mut-LUC, respectively) and examined the effects of the mutations on Nurr1-induced OPN-LUC transactivation in U2-OS cells. When the S1 element was mutated, the ability of Nurr1 to transactivate the OPN promoter was almost completely abolished (Fig. 6DGo). Mutating the S2 element, in contrast, had no effect. This suggests that activation of the OPN promoter by Nurr1 is mediated mainly by the S1 element.

Functional Requirements of Nurr1 for OPN Regulation
Nurr1 regulates transcription both as a monomer and as a heterodimer with RXR (19, 20). Therefore we next examined whether Nurr1 transactivated the OPN promoter as a monomer or as a heterodimer using a Nurr1 mutant [Nurr1DIM; Nurr1 KLL(554–556)AAA] that is fully active as a monomer but is unable to heterodimerize with RXR (39). In transfected U2-OS cells, Nurr1DIM activated OPN-LUC as efficiently as the wild-type Nurr1, suggesting that Nurr1 regulates the OPN promoter as a monomer (Fig. 7AGo). This is in agreement with our finding that the related receptor Nor1 that is unable to interact with RXR is able to activate OPN-LUC (Fig. 4Go, A and B). RXR ligands, however, have been shown to enhance transactivation induced by Nurr1 via the monomeric response element NBRE (20, 39). To examine whether RXR ligands could enhance Nurr1-mediated OPN promoter activation, U2-OS cells were cultured in the presence of the synthetic RXR agonist SR11237. Treatment of the cells with SR11237 enhanced Nurr1-mediated OPN-LUC activation by 2.5-fold (Fig. 7AGo). When the Nurr1-responsive S1 element was mutated, the ability of Nurr1 to induce OPN-LUC was almost completely abolished, and the stimulatory effect of SR11237 was also significantly reduced (Fig. 7BGo). When U2-OS cells were transfected with Nurr1DIM, the ability of SR11237 to stimulate Nurr1-induced OPN-LUC activity was lost. These results indicate that the RXR ligand is activating the OPN promoter via RXR-Nurr1 heterodimers (Fig. 7AGo).



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  		Fig. 7. Nurr1 Transactivates the OPN Promoter as a Monomer in an AF1-Dependent Manner

U2-OS cells were transfected with Nurr1 and Nurr1 KLL(554–556)AAA (Nurr1DIM) expression vectors together with OPN-LUC (A) and OPN S1mut-LUC (B) reporters. Subsequently, the cells were treated with vehicle or 1 µM SR11237 for 24 h as indicated. The contributions of Nurr1 AFs in OPN promoter regulation were addressed by cotransfecting U2-OS cells with the expression plasmids for the wild-type Nurr1 (Nurr1), Nurr1 {Delta}1–84/D589A (Nurr1AF1/AF2mut), Nurr1 {Delta}1–84 (Nurr1AF1mut), Nurr1 D589A (Nurr1AF2mut), and dominant negative Nurr1 (Nurr1DN) along with OPN-LUC (C) or NBRE3tk-LUC (D) reporters.

 
Nurr1, as do most nuclear receptors, harbors two transactivation functions: the amino-terminal AF1 and the carboxyl-terminal AF2 (18). To examine the role of Nurr1 AFs in OPN promoter regulation, a Nurr1 mutant lacking both AF1 and AF2 activity (Nurr1AF1/AF2) (18) was tested in U2-OS cells. Nurr1AF1/AF2 was unable to activate OPN-LUC, indicating that the Nurr1 AFs were required (Fig. 7CGo). To examine which of the two AFs was more important for OPN regulation, the AF1 and AF2 were mutated individually. Deletion of amino acids 1–84 (Nurr1{Delta}AF1) (18) severely reduced the ability to activate the OPN promoter, whereas substitution of alanine for the conserved aspartic acid 589 in AF2 (Nurr1AF2) (18) had no effect. Interestingly, efficient activation of the reporter driven by three NBREs required both AF1 and AF2 in U2-OS cells, suggesting that the contributions of the AFs may vary in a promoter-specific manner within the same cell type (Fig. 7DGo). Finally, the dominant negative Nurr1 variant (Nurr1DN) was tested. Nurr1DN reduced basal OPN promoter activity in U2-OS cells. It also abolished the ability of the cotransfected wild-type Nurr1 to activate OPN-LUC (Fig. 7CGo and data not shown).

Regulation of the OPN Promoter by Nurr1, Vitamin D, and Estrogen-Related Receptors
OPN expression is stimulated by vitamin D in osteoblasts. This stimulation has been shown to be mediated by VDR binding directly to its response element in the OPN promoter (27). We therefore investigated whether Nurr1 and VDR cooperate in OPN promoter regulation. U2-OS cells were transfected with the OPN-LUC reporter along with either the Nurr1 expression plasmid or empty vector, and the cells were subsequently treated with vitamin D. Vitamin D alone induced OPN-LUC as reported earlier (Fig. 8AGo and Ref.27), and the combined effect of vitamin D and Nurr1 was synergistic (Fig. 8AGo). Mutation of the S1 element that mediates most of the activating effects of Nurr1 had no influence on the ability of vitamin D to activate the OPN promoter. Nurr1 alone accomplished low transactivation of OPN S1mut-LUC (Figs. 6DGo and 8BGo). Interestingly, Nurr1 and vitamin D together were able to efficiently stimulate OPN S1mut-LUC activity (Fig. 8BGo). Vitamin D, however, had no effect on Nurr1-induced transactivation of the artificial reporter NBRE3tk-LUC (data not shown). Thus, Nurr1 and vitamin D regulate the OPN promoter in a synergistic fashion.



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  		Fig. 8. Regulation of the OPN Promoter by Nurr1 and Vitamin D

U2-OS cells were transfected with pCMX-Nurr1 and OPN-LUC (A) or OPN S1mut-LUC (B) reporters as described in the legend to Fig. 4Go. The cells were subsequently treated with 100 nM vitamin D as indicated.

 
ERR{alpha}, another orphan nuclear receptor expressed in osteoblasts, has previously been reported to activate the mouse OPN promoter via the S1 and S2 elements (28, 30, 31). Because our results demonstrated OPN promoter regulation by Nurr1 via the S1 element, we next examined whether there was cross-talk between these two orphan nuclear receptors. We first examined activation of OPN-LUC by ERR{alpha} and the related orphan receptor ERR{gamma}, which is also expressed in osteoblasts (Huppunen, J., and P. Aarnisalo, unpublished observations). Surprisingly, neither ERR{alpha} nor ERR{gamma} activated OPN-LUC when transfected in U2-OS or SaOs-2 cells. However, ERR{gamma} was able to efficiently transactivate reporters driven by either estrogen response elements (EREs) or ERREs in these cell lines. ERR{alpha}, in contrast, failed to activate any of these reporters under our experimental conditions (Fig. 9AGo and data not shown). Next, the effect of ERRs on Nurr1-mediated transactivation of OPN-LUC was examined. Interestingly, coexpression of ERR{gamma} repressed Nurr1-mediated OPN promoter activation in a dose-dependent manner (Fig. 9BGo). A similar repression was also observed with ERR{alpha} (Fig. 9CGo) and on the NBRE3tk-LUC reporter (data not shown).



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  		Fig. 9. Cross-Talk between Nurr1 and ERRs on the OPN Promoter

A, SaOs-2 cells were cotransfected with 50 ng of the expression vectors for ERR{alpha} or ERR{gamma} along with 300 ng OPN-LUC or ERE2tk-LUC reporters as indicated. B, pCMX-Nurr1 (25 ng) was cotransfected with increasing amount of pCMX-ERR{gamma} (5 ng, 10 ng, 15 ng, 20 ng, and 25 ng) and 300 ng OPN-LUC in U2-OS cells. C, 25 ng pCMX-Nurr1 were transfected in U2-OS cells along with 25 ng pCMX-ERR{alpha} and 300 ng OPN-LUC. D, The ability of ERR{alpha}, ERR{gamma}, and Nurr1 to bind to the OPN S1 element was examined using EMSA. Nurr1, ERR{alpha}, and ERR{gamma} proteins were produced by coupled in vitro transcription and translation in reticulocyte lysates. In the upper panel, 5 µl of the translation mixtures were incubated with 32P-labeled OPN S1 element, and the protein-DNA complexes were resolved by electrophoresis on 4% nondenaturing polyacrylamide gel in 0.25x Tris-borate, EDTA. The asterisk depicts unspecific binding. In the lower panel, 0.5 µl of Nurr1-programmed reticulocyte lysate was incubated with increasing amounts of ERR{alpha}- or ERR{gamma}-lysates (2 µl, 4 µl, and 6 µl) and subsequently analyzed for binding to 32P-labeled OPN S1 element. E, U2-OS cells were transfected with 25 ng ERR{gamma} and 25 ng Nurr1 expression plasmids along with 300 ng OPN-LUC. Subsequently, the cells were treated with 1 µM 4-hydroxytamoxifen (4OHT) for 16 h as indicated.

 
To study the mechanism of ERR-mediated Nurr1 repression, the influence of ERRs on Nurr1 DNA binding was assessed by EMSA using the OPN S1 element as the probe. The receptor proteins were expressed at comparable levels when produced in vitro in reticulocyte lysates (data not shown). In agreement with previous reports, ERR{alpha} and ERR{gamma} bound the S1 element. However, binding of Nurr1 to the S1 element was clearly more efficient (Fig. 9DGo). Coincubation of Nurr1 with increasing amounts of ERR{alpha} or ERR{gamma} proteins did not reduce the ability of Nurr1 to bind to the S1 element, suggesting that repression of Nurr1 by ERRs is not merely a result of competition for a common binding element. This is further supported by the observation that treatment of the cells with 4-hydroxytamoxifen, an inverse agonist of ERR{gamma} that does not impair ERR{gamma} DNA binding (40), completely abolished the repressive effect of ERR{gamma} on Nurr1 (Fig. 9EGo). Coincubation of Nurr1 with ERRs did not change the mobility of the Nurr1-DNA complex, indicating that ERRs are not forming transcriptionally inactive heterodimers with Nurr1 (Fig. 9DGo). Thus, the mechanism of ERR-mediated Nurr1 repression requires further examination.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Nurr1 is an orphan nuclear receptor, the functions of which have previously been investigated mainly in the CNS (7, 8, 37, 41). An increasing amount of evidence suggests, however, that Nurr1 as well as the related receptors NGFI-B and Nor1, also have important roles in several peripheral tissues (11, 12, 13, 14, 15, 42, 43). To elucidate the role of Nurr1 in osteoblasts, we have analyzed the promoter regions of several osteoblastic genes for potential Nurr1 response elements. In this study, we show that the mouse OPN promoter contains a functional Nurr1 response element. Nurr1 binds directly to this element and is subsequently able to transactivate the OPN promoter. Transactivation depends on Nurr1 AFs but does not require heterodimerization with RXR. A synthetic RXR ligand did, however, further enhance Nurr1-mediated OPN activation, implying that retinoids can modulate OPN expression via Nurr1.

PTH induces the expression of Nurr1 and OPN in osteoblasts (13, 36). We report here that expression of a dominant negative Nurr1 variant abolishes PTH-induced OPN expression. This observation suggests that Nurr1 is involved in mediating PTH-induced OPN expression. In addition to Nurr1, PTH also induces the expression of the related receptors NGFI-B and Nor1 in osteoblasts (42, 43). The dominant negative Nurr1 variant used in our study inhibits the transcriptional activities of Nurr1, NGFI-B, and Nor1 (Castro, D. S., personal communication). According to our cotransfection experiments, all three receptors are also able to transactivate OPN-LUC (Fig. 4Go). Further studies are thus required to distinguish the role of each of these receptors in OPN regulation.

OPN comprises about 2% of the noncollagenous protein in bone. It is produced by osteoblasts, osteoclasts, and hypertrophic chondrocytes. Studies on OPN gene-targeted mice have revealed that OPN is not required for bone formation during development (23). It is, however, an important mediator of bone remodeling in response to hormonal or physical stimuli, e.g. PTH-induced bone resorption does not occur in the absence of OPN (25). OPN-deficient mice are also resistant to ovariectomy-induced bone loss, suggesting that OPN has an essential role in postmenopausal osteoporosis (24). Furthermore, OPN has been suggested to have an important role in triggering bone remodeling in response to mechanical stress (44). Regulation of the OPN promoter activity by Nurr1 suggests a role for Nurr1 in bone remodeling.

OPN was originally cloned from bone but has later been shown to be expressed in several other tissues, including the immune system, vascular system, and kidney (reviewed in Ref.22). OPN has also been associated with several pathological conditions such as inflammation, pathological calcification, tumorigenesis, and metastases (22, 45, 46). Intriguingly, Nurr1 and the related orphan receptors NGFI-B and Nor1 have been suggested to have roles in inflammatory processes such as rheumatoid arthritis and glomerulonephritis, in atherosclerotic lesions, in development of extraskeletal myxoid chondrosarcoma, and in regulation of cell proliferation (14, 15, 37, 47, 48, 49). In this study, OPN promoter activation by Nurr1 was only detected in osteoblastic cells but not in kidney-derived 293 cells or in human cervical carcinoma HeLa cells. However, Nurr1 transactivates the NBRE-driven artificial reporter efficiently in both 293 and HeLa cells. The inability of Nurr1 to activate the OPN promoter in 293 and HeLa cells is most likely due to lack of some cell-specific factors present in osteoblasts that are required for OPN regulation. Whether Nurr1 regulates the OPN promoter in OPN-expressing cells other than osteoblasts remains to be determined.

The OPN promoter is subject to regulation by several transcription factors including Cbfa1, ETS1, TP53, Smads, Hoxa-9, and nuclear receptors (27, 29, 30, 32, 34). Regulation of OPN expression involves potentially complex interactions between various transcription factors. Hoxa-9 has been shown to repress OPN expression by directly binding to the OPN promoter. However, upon TGFß stimulation and subsequent Smad activation, Hoxa-9 is displaced from DNA due to the formation of Smad4/Hoxa-9 complexes (32). We show here that ERRs repress Nurr1’s ability to activate the OPN promoter. Further studies are required to reveal the detailed mechanism of ERR-mediated Nurr1 repression and to elucidate the significance of this cross-talk in other tissues in which these orphan receptors are coexpressed.

The function and biological role of Nurr1 have been intensively examined during the last 10 yr. Studies with Nurr1 gene-targeted mice (Nurr1 –/– mice) have revealed a crucial role for this orphan nuclear receptor in the dopaminergic neurons of developing midbrain (8, 41, 50, 51, 52). However, little is known of the mechanisms by which Nurr1 regulates the development of the dopamine cells. Unfortunately, Nurr1 –/– mice die during the first postnatal day. Therefore, the role of Nurr1 in the adult dopaminergic system has remained elusive. Nurr1 mutations have been identified in patients with Parkinson’s disease and manic-depressive disorder, indicating that Nurr1 plays an important role in maintenance of the normal dopamine cell functions in the adult (9, 10). Very little is known about the genes Nurr1 regulates to exert its biological functions. In dopaminergic cells, tyrosine hydroxylase and dopamine transporter have been reported to be regulated by Nurr1 (53, 54, 55). Recently, aromatic L-amino acid decarboxylase and vesicular monoamine transporter-2 were reported to be induced by Nurr1 in the MN9D dopaminergic cell line and to be deregulated in Nurr1 –/– mice (56). Whether aromatic L-amino acid decarboxylase and vesicular monoamine transporter-2 are direct Nurr1 target genes remains to be addressed. In the pituitary, Nurr1 and the related receptors have been shown to regulate the expression of the proopiomelanocortin gene by directly binding to the proopiomelanocortin promoter (57). The skeletal phenotype of Nurr1 –/– mice has not been examined, and the role of Nurr1 in bone during development has remained elusive. In this report, we describe OPN as the first Nurr1 target gene in bone. Identification of Nurr1 as a regulator of OPN expression demonstrates that Nurr1 is able to modulate gene expression in osteoblasts and suggests that via enhancing OPN expression Nurr1 regulates bone homeostasis. Nurr1, an orphan nuclear receptor originally isolated from the brain, also has important functions in tissues outside the CNS.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Plasmid Constructions
The expression vectors for full-length Nurr1 (pCMX-Nurr1), NGFI-B (pCMX-NGFI-B), and Nor1 (pCMX-Nor1) were kind gifts from Dr. Thomas Perlmann (Ludwig Institute for Cancer Research, Stockholm, Sweden). pCMX-ERR{alpha} and pCMX-ERR{gamma} encoding mouse ERR{alpha} and ERR{gamma}, respectively, were generously provided by Dr. Vincent Giguere (McGill University, Montreal, Quebec, Canada). NBRE3tk-LUC, ERRE3tk-LUC, and MH100tk-LUC reporters as well as the pCMX-ßgal control plasmid were received from Dr. Ronald Evans (Howard Hughes Medical Institute, The Salk Institute for Biological Studies, La Jolla, CA). pCMX-Nurr1 C283G variant was created using the QuikChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer’s instructions. Other Nurr1 mutants were provided by Dr. Thomas Perlmann and have been described elsewhere (18, 37, 39). To create the OPN luciferase reporter construct (OPN-LUC), the –857/+191 fragment of the mouse osteopontin (OPN) promoter was amplified by PCR from mouse genomic DNA and cloned in the pGL3-Basic plasmid (Promega Corp., Madison, WI). OPN S1mut-LUC and OPN S2mut-LUC reporters were created by mutating the S1 and S2 elements of the OPN promoter, respectively, using the QuikChange Site-Directed Mutagenesis kit (Stratagene). ERE2tk-LUC was a kind gift from Dr. Pekka Kallio (Orion Pharma, Turku, Finland).

Cell Culture and Transfections
Mouse osteoblast MC3T3-E1 cells, human osteosarcoma cell lines SaOs-2, U2-OS, and MG63, human embryonic kidney 293 cells, and human cervical cancer HeLa cells were obtained from American Type Culture Collection (Manassas, VA). MC3T3-E1 cells were maintained in {alpha}-MEM. Other cells were maintained in DMEM. The media were supplemented with 10% fetal bovine serum, L-glutamine, penicillin, and streptomycin. Primary osteoblasts were isolated from femurs and tibias of 10-wk-old NMRI mice as described previously (58). Primary osteoblasts were maintained in {alpha}-MEM supplemented with 15% fetal bovine serum, 50 µg/ml ascorbic acid (Sigma Chemical Co., St. Louis, MO), 10–8 M dexamethasone (Sigma), 100 U/ml of penicillin, 100 µg/ml of streptomycin, and 2.5 µg/ml of amphotericin B (Sigma).

Transfections for reporter assays were performed in 12-well plates using the FuGene transfection reagent (Roche Clinical Laboratories, Basel, Switzerland) according to the manufacturer’s instructions. Briefly, 35,000–50,000 cells per well were plated 24 h before the transfection. Each well was transfected with 50 ng of the expression vectors, 300 ng of the luciferase reporter plasmids, and 100 ng of pCMX-ßgal, which was used as an internal control for transfection efficiency. MC3T3-E1 cells were transfected on 12-well plates with 220 ng of expression vectors, 1300 ng of reporter plasmids, and 440 ng pCMX-ßgal using jetPEI transfection reagent (Polyplus-Transfection, Illkirch Cedex, France) according to the manufacturer’s instructions. Eighteen hours after transfection, the cells received fresh medium supplemented with the synthetic RXR ligand SR11237 (a kind gift from Dr. Thomas Perlmann) or vitamin D (Sigma) where indicated. The cells were harvested 24 h later and assayed for luciferase and ß-galactosidase activities. All transfection experiments were performed in triplicate dishes, and each experiment was repeated at least twice with essentially identical results. Results of one representative experiment are shown.

For RNA isolation, 150,000 cells were plated on a six-well plate 24 h before transfection. U2-OS cells were transfected with 1.5 µg pCMX-Nurr1 using the FuGene transfection reagent. MC3T3-E1 cells were transfected with 3 µg of the Nurr1 or Nurr1DN expression vectors using jetPEI transfection reagent. Total RNA was isolated 42 h later.

In Vitro DNA-Binding Assay
The DNA-binding experiments were carried out essentially as described (18, 39). Nurr1, ERR{alpha}, and ERR{gamma} proteins were produced by coupled in vitro transcription and translation in reticulocyte lysates according to the instructions provided by the manufacturer (TNT Quick Coupled Transcription/Translation System, Promega Corp.). The following response elements and their complements were end labeled with 32P using T4 polynucleotide kinase (Amersham Biosciences, Uppsala, Sweden) and used as probes: NBRE, agcttgagttttaAAAGGTCAtgctcaattt; OPN S1, gatcctctctAAAGGTCAgtggaa; OPN S1mut, gatcctctctAAATTTCAgtggaa; OPN S2, gatccggaatTCAGGGTCActgtga; and OPN S2mut, gatccggaatTCAGTTTCActgtga.

RNA Extraction and RT-PCR
Total RNA was extracted from cells grown on six-well plates using Trizol reagent (Invitrogen, San Diego, CA). After DNase I treatment (Invitrogen), 1 µg of RNA was used for cDNA synthesis with Superscript II (Invitrogen). RT-PCR was performed with Taq polymerase (Amersham Pharmacia Biotech). PCRs were denatured at 92 C for 2 min and then amplified at 92 C for 60 sec, 55 C for 60 sec, and 72 C for 60 sec. The PCRs were originally set up for 24 cycles. The reaction products were tested on agarose gel, and the number of cycles was subsequently increased until signal saturation. Only one of the steps from the linear range of amplification is displayed [28 cycles for OPN and glyceraldehyde 3-phosphate dehydrogenase (G3PDH); 40 cycles for Nurr1]. The PCR products were fractionated on 1.5% agarose gels, stained with ethidium bromide, and quantified using the Kodak Image station 440 CF system (Eastman Kodak, Rochester, NY). OPN and Nurr1 mRNA intensities were corrected for G3PDH expression. The following primers were used to amplify target cDNAs:

Human Nurr1 (GenBank NM006186)

sense 5'-CGA CAT TTC TGC CTT CTC C-3'

antisense 5'-GGT AAA GTG TCC AGG AAA AG-3'

Mouse Nurr1 (GenBank S53744)

sense 5'-CGA CAT TTC TGC CTT CTC C-3'

antisense 5'-AGG TAA GGT GTC CAG GAA AAG-3'

Dominant negative Nurr1

sense 5'-CTA CGG TGT TCG CAC TT-3'

antisense 5'-GTA AAC GAC CTC TCC GG-3'

Human NGFI-B (GenBank NM002135)

sense 5'-GAA AAA CGC CAA GTA CAT CTG-3'

antisense 5'-GGA CAC GCT GCC CTT CTG A-3'

Human Nor1 (GenBank NM006981)

sense 5'-TCT GCC TTC CAA ACC AAA G-3'

antisense 5'-GTC CTC AGA CTT TCC ATC A-3'

Human OPN (GenBank NM000582)

sense 5'-TGA GAG CAA TGA GCA TTC CGA TG-3'

antisense 5'-CAG GGA GTT TCC ATG AAG CCA C-3'

Mouse OPN (GenBank AF515708)

sense 5'-TCA CCA TTC GGA TGA GTC TG-3'

antisense 5'-ACT TGT GGC TCT GAT GTT CC-3'

G3PDH (CLONTECH Laboratories, Inc., Palo, Alto, CA)

sense 5'-ACC ACA GTC CAT GCC ATC AC-3'

antisense 5'-TCC ACC ACC CTG TTG CTG TA-3'


   ACKNOWLEDGMENTS
 
Professor Olli A. Jänne is acknowledged for support, comments on the manuscript, and for providing excellent research facilities. Professor Thomas Perlmann is warmly thanked for discussions and advice. We acknowledge James Thompson for reviewing the language of the manuscript and Dr. Marika Linja for her technical help. We also thank Drs. Ronald Evans, Vincent Giguere, Pekka Kallio, and Thomas Perlmann for providing plasmids.


   FOOTNOTES
 
This work was supported by grants from the Medical Research Council (Academy of Finland), the Sigrid Juselius Foundation, and Biocentrum Helsinki.

Abbreviations: AF, Activation function; CNS, central nervous system; DBD, DNA-binding domain; ERE, estrogen response element; ERRE, estrogen-related receptor response element; ERR, estrogen-related receptor; G3PDH, glyceraldehyde 3-phosphate dehydrogenase; NBRE, NGFI-B response element; OPN, osteopontin; RXR, retinoid X receptor; VDR, vitamin D receptor.

Received for publication June 24, 2003. Accepted for publication February 22, 2004.


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NURSA Molecule Pages Link:

Nuclear Receptors:   VDR  |  ERRα  |  ERRγ  |  NURR1
Ligands:   Calcitriol



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G. Benoit, A. Cooney, V. Giguere, H. Ingraham, M. Lazar, G. Muscat, T. Perlmann, J.-P. Renaud, J. Schwabe, F. Sladek, et al.
International Union of Pharmacology. LXVI. Orphan Nuclear Receptors
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M. Schroeter, P. Zickler, D. T. Denhardt, H.-P. Hartung, and S. Jander
Increased thalamic neurodegeneration following ischaemic cortical stroke in osteopontin-deficient mice
Brain, June 1, 2006; 129(6): 1426 - 1437.
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V. R. Holla, J. R. Mann, Q. Shi, and R. N. DuBois
Prostaglandin E2 Regulates the Nuclear Receptor NR4A2 in Colorectal Cancer
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F. Q. Pirih, A. Tang, I. C. Ozkurt, J. M. Nervina, and S. Tetradis
Nuclear Orphan Receptor Nurr1 Directly Transactivates the Osteocalcin Gene in Osteoblasts
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