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Endocrinology Vol. 142, No. 2 663-670
Copyright © 2001 by The Endocrine Society

ARTICLES

Parathyroid Hormone Induces Expression of the Nuclear Orphan Receptor Nurr1 in Bone Cells1

Sotirios Tetradis, Olga Bezouglaia and Anastasia Tsingotjidou

Division of Diagnostic and Surgical Sciences, UCLA School of Dentistry, Los Angeles, California 90095-1668

Address all correspondence and requests for reprints to: Sotirios Tetradis, Division of Diagnostic and Surgical Sciences, Room 53-068 CHS, UCLA, School of Dentistry, Los Angeles, California 90095-1668. E-mail: sotirist{at}dent.ucla.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Following PTH treatment, immediate changes in osteoblast gene expression involve induction of primary response genes. Primary gene products subsequently mediate the osteoblast response to PTH. Using representational difference analysis (RDA) to isolate primary genes induced by PTH in osteoblasts, we identified Nurr1, a member of the NGFI-B nuclear orphan receptor subfamily. Nurr1 binds DNA as a monomer but also heterodimerizes with the 9-cis retinoic acid receptor (RXR). Nurr1’s importance in retinoic acid, vitamin D, and thyroid hormone signaling has been hypothesized. Nurr1 messenger RNA (mRNA) levels were maximal at 1 h and at 10 nM of PTH in primary mouse osteoblasts (MOB). Activation of the PKA and PKC pathways by 10 µM forskolin and 1 µM PMA, respectively, induced Nurr1 mRNA levels. However, inhibition of the PKA but not the PKC pathway significantly inhibited the PTH induction of Nurr1. Moreover, PTH(3–34) at 1–100 nM did not induce Nurr1 mRNA levels. Thus, PTH induction of Nurr1 in primary mouse osteoblasts is mediated primarily through the cAMP/PKA pathway. PTH also stimulated Nurr1 protein in MOB cells and Nurr1 mRNA in calvarial organ cultures. Nurr1 induction represents a potential cross-talk mechanism between PTH and steroid hormone signaling at the transcription factor level.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PTH HAS significant effects on bone metabolism, being both anabolic and catabolic, depending on the pattern of exposure (1, 2, 3, 4). PTH signaling starts with PTH binding to the highly conserved PTH/PTH-related peptide (PTHrP) receptor at the cell surface of osteoblasts (5, 6). The PTH/PTHrP receptor belongs to the seven-transmembrane domain family of receptors that signal through activated G proteins (5). Upon PTH binding to its receptor and activation of G proteins, a signal transduction cascade is initiated that ultimately leads to changes in osteoblastic function. Three members of the G protein family are coupled to the PTH/PTHrP receptor and are of particular importance in PTH-induced gene expression. Gs and Gi activate and inhibit adenylate cyclase activity, respectively, and regulate the cAMP/protein kinase A (PKA) pathway (7, 8, 9, 10). Gq stimulates phospholipase C, resulting in diacylglycerol (DAG) and inositol triphosphate (IP3) production. DAG and IP3 then activate the protein kinase C (PKC) pathway and increase intracellular calcium (11, 12, 13).

Signaling through the PKA, PKC, and calcium pathways leads to phosphorylation and activation of various transcription factors, which in turn induce transcription of several osteoblastic genes (14). These genes are the first to be affected by PTH treatment and thus they are classified as primary response genes. Their induction is rapid, transient, and does not require new protein synthesis. Osteoblastic primary response genes induced by PTH include the transcription factors c-fos, c-jun and inducible cAMP early repressor (15, 16, 17, 18), the cytokine interleukin-6 (19, 20), the growth factor leukemia inhibitory factor (20), the signaling molecule regulator of G proteins signaling (RGS2, 21) and the enzymes prostaglandin G/H synthase-2 (22, 23) and tissue plasminogen activator (24). The products of primary response genes, in turn, regulate the expression of late PTH-target genes such as COL1A1 and collagenase (25, 26, 27), mediate the restoration of primary gene transcription to control levels (23), and have been implicated in the anabolic and catabolic effects of PTH on bone (28). To understand the mechanisms controlling PTH-mediated regulation of bone metabolism, it is imperative to identify PTH-induced primary response genes and characterize the molecular signals that mediate their induction.

To isolate PTH-induced primary response genes in osteoblasts, we performed representational difference analysis (RDA) using MC3T3-E1 preosteoblastic cells treated with PTH in the presence of the protein synthesis inhibitor cycloheximide (CHX). One of the clones that we identified was the nuclear orphan receptor Nurr1.

Nurr1 was originally isolated in neonatal mouse brain (29) while the rat and human homologs were induced during liver regeneration (30) and activation of T cells (31). Nurr1 can bind promoter elements as a monomer and activate transcription in the absence of ligand binding (30). Nurr1 can also form a transcriptionally active heterodimer with the 9-cis retinoic acid receptor (RXR, 32, 33). Interestingly, RXR heterodimerizes with the retinoic acid receptor (RAR), the thyroid hormone receptor (T3R) and with the vitamin D receptor to bind DNA (34). In fact, the importance of Nurr1 in retinoic acid, vitamin D, and thyroid hormone signaling has been hypothesized (33).

Since its initial discovery, Nurr1 has been described in various areas of the developing and adult brain (35, 36, 37, 38) and is induced in the brain by various stimuli, including focal brain injury (39), global ischemia (40), phencyclidine treatment (41), and permanent middle cerebral artery occlusion (42). Nurr1’s importance for brain function is supported by the observation that Nurr1 knock-out mice fail to generate midbrain dopaminergic neurons, are hypoactive and die soon after birth, possibly due to an inability to suckle (43). In addition to the brain, Nurr1 is induced in neuroblastoma cells by forskolin and phorbol esters (TPA, 44), in the adrenal gland by pentylene tetrazole (45), in leukemic cells after mechanical agitation (46), and in MCF-7 breast cancer cells by all-trans and 9-cis retinoic acids (47).

In the present study, we report that PTH rapidly induced Nurr1 mRNA levels in MC3T3-E1 cells, in osteoblastic cells from neonatal mouse calvariae, and in mouse calvariae organ cultures. PTH induction of Nurr1 gene expression was mediated primarily through activation of the cAMP-PKA signaling pathway. PTH also induced Nurr1 protein levels in osteoblastic cells. Thus, Nurr1 induction may represent a very interesting cross-talk mechanism between PTH and steroid hormone signal-transduction in regulating bone metabolism.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
All reagents were purchased from Sigma (St Louis, MO) unless otherwise specified. The bovine PTH(1–34) fragment was used for all the experiments except the PTH analog experiment (see Fig. 8Go) where the amide bovine PTH(1–34) and (3–34) fragments were used.



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  		Figure 8. Effects of PTH analogs on the induction of Nurr1 mRNA levels. MOB cells were treated for 1 h with 10 nM PTH(1–34) or 1 to 100 nM PTH(3–34). Northern blot analysis of Nurr1 and GAPDH mRNA levels and ethidium bromide staining of RNA are shown.

 
Cell and organ culture
MC3T3-E1 cells were plated at 5000 cells/cm2 and grown to confluence for 8 days in DMEM supplemented with 10% FBS, 100 U/ml penicillin, and 50 µg/ml streptomycin in a humidified atmosphere of 5% CO2 at 37 C. Media were changed every 3 days.

All animals used in our studies were killed according to protocol approved by UCLA Institutional Animal Care and Use Committee (ARC No. 98–175-02). For isolation of primary osteoblasts, calvariae from 7-day-old CD-1 neonatal mice (Charles River Laboratories, Inc., Boston, MA) were digested with collagenase and trypsin as previously described (48). Briefly, calvariae were digested in 0.5 mg/ml collagenase (Roche Molecular Biochemicals, Indianapolis, IN), 0.01% trypsin, and 0.1 mM EDTA for 10 min at a time, on a rocking platform at 37 C. Cells from five sequential passages were collected. Cells from the first passage were discarded while cells from passages 2–5 were pooled, plated at 15,000 cells/cm2 and grown in DMEM supplemented with 10% FBS, 100 U/ml penicillin and 50 µg/ml streptomycin in a humidified atmosphere of 5% CO2 at 37 C. Media was changed every 3 days. After 1 week, the cells were trypsinized, pooled, and stored in liquid nitrogen. For experiments, cells were removed from liquid nitrogen, plated at 10,000 cells/cm2, grown to confluence (7–10 days) in DMEM supplemented with 10% FBS, and treated appropriately.

Calvariae from 7-day-old CD-1 neonatal mice (Charles River Laboratories, Inc., Boston, MA) were excised and cut along the saggittal suture to give two hemicalvariae per animal. Calvariae were cultured in BGJb medium containing 1 mg/ml BSA, 100 µg/ml ascorbic acid, 100 U/ml penicillin, and 50 µg/ml streptomycin.

Representational difference analysis
Representational difference analysis (RDA) was performed as previously described (49). Briefly, poly A mRNA was extracted from Control (CHX-treated) and Tester (CHX and PTH-treated) cells using poly A RNA extraction columns (5'-3'), converted to double-stranded DNA (dsDNA) using a complementary DNA (cDNA) synthesis kit (Life Technologies, Inc., Grand Island, NY), and was digested with DpnII (New England Biolabs, Inc., Beverly, MA). Three sets of linkers were designed (49). The first set was ligated to the DpnII digests. Because the linkers were not phosphorylated, only the 3' end of the linker was ligated to the 5' strand of the dsDNAs. The unligated linker was melted away and the 3' strand of the dsDNAs was filled in by Taq polymerase at 72 C for 3 min. The DpnII digested, linker-modified dsDNA fragments were amplified by PCR using the ligated strand of the first set of linkers as a primer.

The linkers of the two representations (Control and Tester) were removed by digestion with DpnII, and a second set of linkers was ligated only to the Tester representation. Then the two representations were mixed at a Tester:Control ratio of 1:100. The mixture of dsDNAs was denatured at 95 C for 5 min and allowed to renature at 67 C for 16 h. The 3' strand of the second set of linkers was melted away and the 3' strand of the dsDNAs was filled in by Taq polymerase at 72 C for 3 min. The renatured/denatured mixture of dsDNAs was amplified by PCR using the 5' strand of the second set of linkers as a primer. After 10 PCR cycles the PCR products were digested with Mung Bean Nuclease (New England Biolabs, Inc., Beverly, MA). The complexes were PCR amplified for 18 additional cycles to form the first difference product, DP1. The DP1 dsDNAs were digested with DpnII to remove the second set of linkers and a third set of linkers was ligated as described above. The denaturation/renaturation step described above was repeated but with the DP1:Control ratio set at 1:10,000. The hybridization products were amplified as described but using the ligated strand of the third set of linkers as a PCR primer. The final PCR product constituted the second difference product, DP2 (Fig. 1BGo). The hybridization and selective amplification was repeated once again using a fourth set of linkers and a DP2:Control ratio of 1:1,000,000 to generate the final third difference product, DP3. DP3 PCR products were directly subcloned to the TA vector (Invitrogen, Carlsbad, CA) and sequenced with the dideoxy sequencing method using the Sequenase version 2.0 kit (Amersham Pharmacia Biotech, Arlington Heights, IL).



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  		Figure 1. Representational difference analysis of mRNA from control (Cont, cycloheximide treated) and tester (PTH, cycloheximide + PTH-treated) cells. A, Initial representations were separated by 1% agarose gel electrophoresis. The representations appeared as a uniform streak of DNA molecules that ranged in size from 0.2–1.1 kb. Lane 1, M, Molecular weight marker. B, The first, second, and third Difference Products (DP1, DP2 and DP3, respectively) were separated by 5% PAGE. The representations ranged in size from 0.2–1.1 kb and contained several bands that became progressively more prominent. Lane 1, M, Molecular weight marker.

 
Northern blot analysis
Following the manufacturer’s protocol, total RNA was extracted using the TRIZOL reagent (Life Technologies, Inc., Grand Island, NY) and fractionated by electrophoresis on a 1% agarose gel containing 3.7% formaldehyde, then transferred to Gene Screen Plus hybridization membrane (NEN Life Science Products, Boston, MA). Filters were prehybridized for 20 min and hybridized for 1 h at 68 C using the QuikHyb hybridization solution (Stratagene, La Jolla, CA) in the presence of 2 x 107 counts of a [32]P labeled probe. Filters were washed and used to expose radiographic film (Fisher Scientific, Pittsburgh, PA). Nurr1 mRNA intensity was measured and corrected for GAPDH expression using phosphorimager screen (Molecular Dynamics, Inc., Sunnyvale, CA).

Extraction of total and nuclear proteins and Western blot analysis
Confluent MOB cells were collected in 10 ml ice-cold PBS and centrifuged at 2,000 x g for 5 min. For total protein extracts, cells were resuspended for 30 min in 1 ml triple detergent lysis buffer containing 50 mM Tris-Cl, 150 mM NaCl, 0.02% sodium azide, 0.1% SDS, 100 µg/ml PMSF 1 µg/ml aprotinin, 1% NP40 0.5% deoxycholate and 1x protease inhibitors. Lysates were spun at 15K for 15 min at 4 C, and supernatants were used for Western blot assays.

Nuclear protein extracts were prepared as previously described (50). Cells were resuspended in 400 µl buffer containing 10 mM HEPES buffer (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF, and 1x protease inhibitors (Roche Molecular Biochemicals, Indianapolis, IN) for 15 min on ice. Twenty-five microliters of 10% NP-40 were added, and cells were vortexed vigorously for 10 sec. Cells were spun at maximum speed in microfuge for 30 sec at 4 C and pellets were resuspended in ice-cold buffer containing 20 mM HEPES buffer (pH 7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.5 mM PMSF, and 1x protease inhibitors.

Fifty micrograms of total or 20 µg of nuclear proteins per sample were boiled for 5 min in 30 mM Tris-HCl (pH 6.8), 2.5% glycerol, 1% SDS, 350 mM 2-mercaptoethanol, and 0.0625% bromophenol blue (wt/vol). Samples were electrophoretically separated by a 10% polyacrylamide SDS-PAGE. Proteins were electrophoretically transferred to nitrocellulose membranes at 20 V for 16 h at 4 C. Membranes were stained with 0.1% Ponceau S (wt/vol) in 5% acetic acid (vol/vol) to determine equal loading. Immunoreactive species were determined using commercial rabbit polyclonal antibodies produced against the N- or C- terminus of rat Nurr1 proteins (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and antirabbit Ig, horseradish peroxidase linked whole antibody (Amersham Pharmacia Biotech) using the ECL western blotting detection reagent kit (Amersham Pharmacia Biotech).

Statistics
Data among groups were analyzed using one-way ANOVA and the Student-Newman-Keul’s post hoc test. Data between groups were analyzed using the Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PTH induction of primary response genes
We performed the RDA technique to identify PTH-inducible primary response genes in osteoblastic cells. MC3T3-E1 cells were pretreated with the protein synthesis inhibitor cycloheximide (CHX, 10 µg/ml) for 30 min and then treated with vehicle (Driver) or PTH (10 nM) for 90 min (Tester). Cycloheximide treatment of osteoblasts at 3 µg/ml inhibits more than 93% of total protein synthesis (48). Poly A mRNA from each group was extracted and converted to double stranded DNA (dsDNA), which was then digested with DpnII and PCR amplified. The mixture of molecules generated ranged in size from 0.2–1.1 kb and contained the representation population for each group. Both representations of control and PTH-treated groups contained a uniform population of DNA molecules that looked similar when subjected to gel electrophoresis (Fig. 1AGo).

For RDA, representations of the two populations were mixed at a Tester:Driver ratio of 1:100 and PCR amplified to form the first difference product, DP1 (Fig. 1BGo), which was enriched for sequences preferentially found in the Tester. DP1 was mixed with Driver at 1:10,000 to generate the second difference product, DP2 (Fig. 1BGo). Finally, DP2 was mixed with Driver at 1:1,000,000 to generate the third difference product, DP3 (Fig. 1BGo). Gel electrophoresis of DP1, DP2, and DP3 revealed a mixture of molecules that ranged in size from 0.2–1.1 kb, comparable to the initial representations (Fig. 1AGo). However, several distinct bands of various sizes could be identified in the DP3 group in contrast to the uniform smear seen in the Control and Tester representations (compare Fig. 1Go, panel A with panel B, lane 4). These bands became progressively more prominent in DP2 and DP3 groups whereas most of the smear appearance of the DNA molecules was greatly reduced. Ninety-six DP3 products were subcloned and sequenced. Several inserts had the same sequence and represented the same gene segment. Unique sequences were compared with those in the GenBank database using the BLAST program to determine whether they matched known genes. Eight clones, representing 21% of the sequenced fragments, matched segments of three genes in the data bank: the enzyme prostagandin G/H synthase 2 (PGS2, cyclooxygenase 2), the transcription factor fos B, and the nuclear orphan receptor Nurr1.

To confirm that the DNA fragments identified by RDA were among genes induced by PTH treatment of osteoblasts in the absence of protein synthesis, we treated confluent MC3T3-E1 cells with CHX (10 µg/µl) for 30 min followed by PTH (10 nM) for 90 min. Total RNA was extracted and subjected to Northern blot analysis using the RDA-isolated gene fragments as cDNA probes. Control cells showed very low levels of PGS2, fos B and Nurr1 mRNA, whereas PTH strongly induced their expression (Fig. 2Go).



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  		Figure 2. PTH induces prostaglandin G/H synthase-2 (cyclooxygenase-2, PGS-2), fos B and Nurr1 mRNA levels in the presence of cycloheximide. MC3T3-E1 cells were pretreated for 1 h with 10 µg/ml cycloheximide, then with vehicle (C) or 10 nM PTH for 90 min. Total RNA was extracted and mRNA levels of various genes were determined by Northern blot analysis. The last inset shows ethidium bromide (eth br) staining of 28S and 18S rRNA.

 
PTH induces Nurr1 in primary osteoblasts derived from neonatal mouse calvariae
MC3T3-E1 cells are spontaneously immortalized preosteoblastic cells. To ensure that PTH induction of Nurr1 is not unique to MC3T3-E1 cells and is, in fact, a normal osteoblastic response, we assayed cells derived from neonatal (7–8 days old) mouse calvariae (MOB cells) for Nurr1 expression following PTH treatment. Each experiment was performed at least three times with similar results.

PTH rapidly and transiently induced Nurr1 mRNA levels that peaked at 1–2 h (30-fold greater than untreated control), returned to almost control levels within 4 h of treatment (Fig. 3Go, A and B), and remained at basal levels up to 24 h after PTH treatment. Nurr1 transcript was approximately 3.5 kb in agreement with previously published data (29). PTH induction was detectable at 10 pM, and maximal at 10 and 1 nM PTH (Fig. 4Go, A and B). Half-maximal dose was approximately 0.1 nM PTH.



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  		Figure 3. PTH rapidly and transiently induces Nurr1 mRNA levels in primary osteoblasts. A, Time course of the effect of PTH. MOB cells were grown to confluence and treated for various times with 10 nM PTH(1–34). Nurr1 and GAPDH mRNA levels were determined by Northern blot analysis. Ethidium bromide staining of RNA is shown. B, Densitometric values for Nurr1 mRNA levels were expressed as percent maximal induction for time course experiments. Each value is the mean ± SEM of six different experiments. **, P < 0.001, *, P < 0.05.

 


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  		Figure 4. Dose response effect of PTH. A, MOB cells were grown to confluence and treated for 1 h with various concentrations of PTH. Nurr1 and GAPDH mRNA levels were determined by Northern blot analysis. Ethidium bromide staining of RNA is shown. B, Densitometric values for Nurr1 mRNA levels were expressed as percent maximal induction for dose-response experiments. Each value is the mean ± SEM of three different experiments.

 
PTH induces Nurr1 mRNA levels mainly through the cAMP-PKA pathway
PTH binds to the plasma membrane PTHR1 receptor and activates the cAMP-PKA, PKC, and calcium pathways (12). To examine which of these pathways can induces Nurr1 mRNA levels, MOB cells were treated with: forskolin (FSK, 10 µM), which activates adenylate cyclase; phorbol ester (PMA, 1 µM), which activates protein kinase C; or ionomycin (iono, 1 µM), a calcium ionophore that increases calcium influx and thereby raises the intracellular calcium concentration. Both FSK and PMA, but not ionomycin, increased Nurr1 mRNA levels above baseline at 1 h (Fig. 5Go). However, FSK induction was higher than PMA and similar to PTH induction of Nurr1 mRNA levels.



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  		Figure 5. Effect of agents that activate PKA and PKC signaling and increase intracellular calcium on Nurr1 mRNA levels. MOB cells were treated with 10 nM PTH(1–34), 10 µM FSK, 1 µM PMA or 1 µM ionomycin (iono) for 1 h. Northern blot analysis of Nurr1 and GAPDH mRNA levels and ethidium bromide staining of RNA are shown. C, Control cells.

 
To determine the role of the PKA signaling pathway, cells were pretreated for 1 h with the PKA inhibitor H89 at 10 or 30 µM and then treated with PTH (10 nM) or FSK (10 µM) for 1 h. H89 at 30 µM significantly inhibited PTH and FSK induction of Nurr1 mRNA levels by 75% and 83%, respectively. H89 at 10 nM showed a smaller inhibition of the PTH and FSK effect (Fig. 6Go, A and B).



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  		Figure 6. Effect of PKA inhibition on PTH induction of Nurr1 mRNA levels. A, MOB cells were pretreated with vehicle, 10 µM or 30 µM H89 for 1 h and then treated with 10 nM PTH, or 10 µM FSK for 1 h. Nurr1 and GAPDH mRNA levels were determined by Northern blot analysis. Ethidium bromide staining of RNA is shown. B, Densitometric values for Nurr1 mRNA levels were expressed as percent PTH-maximal induction. Each value is the mean ± SEM of three different experiments. *, Significantly different from the 0 µM H89-treated group, P < 0.01.

 
To assess the involvement of the PKC pathway, MOB cells were pretreated with PMA (1 µM) for 16 h, and then treated with PTH or PMA for 1 h. Prolonged treatment with phorbol esters down-regulates PKC activity (51, 52). Pretreatment with PMA blocked the ability of freshly added PMA to induce Nurr1 mRNA levels but did not affect the PTH induction (Fig. 7Go, A and B).



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  		Figure 7. Effect of PKC inhibition on PTH induction of Nurr1 mRNA levels. A, MOB cells were pretreated with vehicle or 1 µM PMA for 16 h and then treated with 10 nM PTH, or 1 µM PMA for 1 h. Nurr1 and GAPDH mRNA levels were determined by Northern blot analysis. Ethidium bromide staining of RNA is shown. B, Densitometric values for Nurr1 mRNA levels were expressed as percent maximal induction. Each value is the mean ± SEM of three different experiments. *, Significantly different from the vehicle (-PMA) treated group, P < 0.001.

 
To further examine the role of the cAMP-PKA pathway in the PTH induction of Nurr1 gene expression, cells were treated with the PTH analog PTH (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34). Deletion of the two N-terminal amino acids abolishes the ability of PTH to activate the cAMP-PKA pathway (53). As much as 100 nM PTH (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) did not induce Nurr1 mRNA levels, in contrast to 10 nM PTH (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) (Fig. 8Go).

PTH induces Nurr1 mRNA levels in mouse calvariae cultures
To determine whether PTH induced Nurr1 mRNA expression in bone tissue, neonatal mouse calvariae were treated with PTH (10 nM) for 0–8 h in serum-free media. Total RNA was extracted and was subjected to Northern blot analysis. Nurr1 mRNA levels were almost undetectable in the control group. PTH treatment resulted in rapid induction of Nurr1 mRNA levels that peaked at 1 h and declined thereafter (Fig. 9Go).



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  		Figure 9. PTH induces Nurr1 mRNA levels in mouse calvariae organ cultures. Neonatal mouse calvariae were dissected and cultured in serum-free BGJb medium. Calvariae were treated for various times with 10 nM PTH. Nurr1 and GAPDH mRNA levels were determined by Northern blot analysis. Ethidium bromide staining of RNA is shown.

 
PTH induces Nurr1 protein in MOB cells
To establish that PTH induction of Nurr1 mRNA results in induction of Nurr1 protein, MOB cells were treated with PTH for 0–6 h and nuclear proteins were extracted. Proteins were separated by 10% SDS-PAGE and subjected to Western blot analysis using a commercial Nurr1 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Nuclear and total protein extracts from cells treated with PTH for 2 h contained a Nurr1-immunoreactive doublet band around 66 kDa, which was absent in control cells and at low levels 4 h after treatment. Figure 10Go shows a representative experiment using nuclear protein extracts. The predicted and previously published size for Nurr1 protein is 66 kDa (29, 31).



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  		Figure 10. PTH induces Nurr1 protein expression in MOB cells. MOB cells were treated for 0–6 h with PTH (10 nM). Nuclear proteins were extracted and the level of Nurr1 immunoreactive proteins was measured by Western blot analysis. The migration of molecular weight markers (M) is indicated on the left.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
PTH is a major regulator of osteoblast function and has important effects on bone metabolism. Intermittent low-dose PTH stimulates bone formation, whereas continuous PTH decreases osteoblastic activity and increases bone resorption (54). It appears that initial events after PTH treatment are sufficient to induce an anabolic response, whereas late events lead to catabolic activity. Therefore, understanding early changes in osteoblast gene expression is important in deciphering the molecular signals that mediate the PTH effects on bone metabolism.

Identification of primary response genes induced by PTH in osteoblastic cells has been the subject of previous studies. A fragment of a novel PTH-responsive gene was isolated after treatment of SaoS-2/B10 osteoblastic cells with PTH for 1 h (55), and RGS-2 (the regulator of G protein signaling-2) was isolated from femurs of rats treated in vivo with PTH for 1 h (21). Both techniques used the differential display screening method (56). In our study, we pretreated MC3T3-E1 cells with cycloheximide to ensure that protein synthesis was not required for the induction of the identified genes. Also, we used RDA because of its high sensitivity, the kinetic enrichment achieved by the subtractive process, and low false positive ratio (49). Using this approach, we discovered that the nuclear orphan receptor Nurr1 is strongly induced by PTH in the absence of new protein synthesis. To our knowledge, PTH regulation of Nurr1 gene expression has not been reported previously.

PTH induction of Nurr1 gene expression was maximal at 1 h and at 10 nM of hormone. Although activation of both PKA and PKC pathways induced Nurr1 mRNA levels, the PTH effect appears to be mediated mainly through cAMP-PKA signaling as suggested by three lines of evidence. First, inhibition of PKA attenuated the ability of PTH and FSK to induce Nurr1 mRNA levels to a similar extent. Second, inhibition of PKC signaling, which blocked the PMA effect, did not affect PTH induction of Nurr1 gene expression. Finally, the PTH analog PTH (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) that does not activate the cAMP-PKA pathway (53), did not induce Nurr1 mRNA levels.

cAMP-PKA regulation of transcription is mediated by activation and binding of CREB/ATF transcription factors at cAMP response element (CRE) on the promoters of various genes (57). Isolation and sequencing of the 900 bp of the Nurr1 promoter revealed a consensus CRE, as well as glucocorticoid response element (GRE), SP1, and AP-1 binding sites (58, 59). Because our data indicate the cAMP-PKA pathway plays a major role in the PTH regulation of Nurr1 gene expression the CRE becomes a major candidate cis element to mediate potential regulation of the Nurr1 promoter by PTH. On the other hand, the AP-1 binding site has been implicated in the PTH induction of collagenase promoter activity (60, 61) suggesting a possible role for this site in PTH regulation of Nurr1 gene expression.

Nurr1 cDNA encodes a protein of 598 amino acids with a calculated molecular weight of 66 kDa (29). A Nurr1-immunoreactive doublet was observed in peripheral blood T cells treated with the combination of the calcium ionophore A23187 and PMA. The appearance of a doublet was attributed to the utilization of alternative translation initiation site (31). In our studies, PTH also induced a Nurr1 immunoreactive doublet with a molecular weight of approximately 66 kDa in MOB cells.

Nurr1 belongs to the family of nuclear receptors and activates transcription in the absence of ligand binding. Nurr1 binds DNA as a monomer and enhances 40-fold the activity of a reporter construct containing a NUR consensus binding-site upstream of the minimal globin promoter (30). Nurr1 binds to a GAAGGTCA cis-acting sequence and activates the promoter of the POMC gene. Nurr1 binding is specific and mutation of this element abolishes Nurr1’s ability to induce POMC promoter activity. Furthermore, this element confers Nurr1 regulation when placed upstream of a heterologous promoter (62).

We identified Nurr1 protein both in total and nuclear protein extracts of MOB cells. This suggests that newly synthesized Nurr1 is targeted in the nucleus, presumably where it will bind to promoter elements and regulate the transcription of late response osteoblastic genes. We performed computer analysis to identify potential Nurr1 binding sites in promoter and enhancer sequences of genes important for osteoblastic function (data not shown). Several sites that contained seven of the eight nucleotides of the consensus Nurr1 binding site were revealed. Genes with Nurr1 binding sites in their promoter/enhancer area included alkaline phosphatase, bone sialoprotein, osteocalcin, type I (a)1 and type I (a)2 collagen, insulin growth factor I, and collagenase.

Nurr1 is also involved in 9-cis retinoic acid receptor (RXR) signaling. RXR heterodimerization with the retinoic acid receptor (RAR) or the thyroid hormone receptor (T3R) abolishes RXR’s ability to bind ligand and activate transcription. However, RXR is able to heterodimerize with Nurr1 and activate transcription (32, 33). RXR-Nurr1 heterodimers bind specifically to direct DNA repeats spaced by five nucleotides. Dimerization with RXR appears to involve the carboxy-terminal domain of Nurr1 (33, 63). RXR can also heterodimerize with the vitamin D receptor to bind DNA (34). Regulation of RXR availability and activity by Nurr1 can modify vitamin D, retinoic acid, and thyroid hormone signaling (33). PTH and vitamin D, the two major calciotropic hormones, have synergistic effects on bone resorption (64) and induction of osteoblastic genes, such as vitamin D receptor, 24 hydroxylase and osteocalcin (65, 66). Therefore, induction of Nurr1 gene expression in bone suggests a potential cross-talk mechanism between PTH and vitamin D, or other steroid hormone signaling in regulating bone metabolism.


   Acknowledgments
 
We would like to thank Drs. Barbara Kream, Jeanne Nervina, and Sharon Hunt Gerardo for helpful and insightful suggestions during the preparation of the manuscript.


   Footnotes
 
1 This work was supported by NIH Grant R-01-DE-13316. Back

Received May 23, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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