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Mitogenic Action of Calcium-Sensing Receptor on Rat Calvarial Osteoblasts

Division of Endocrinology, Diabetes and Hypertension (N.C., S.Y., J.T.-H., P.R., D.K., E.M.B.), Department of Medicine and Membrane Biology Program, Brigham and Women’s Hospital, Harvard Medical School, and Division of Experimental Medicine (X.R., E.T.), Beth Israel Deaconess Medical Center, Harvard Institutes of Medicine, Boston, Massachusetts 02115; and Genetics and Aging Unit (S.B.), Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts 02129

Address all correspondence and requests for reprints to: Naibedya Chattopadhyay, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, 221 Longwood Avenue, Boston, Massachusetts 02115. E-mail: Naibedya{at}rics.bwh.harvard.edu.

	Abstract

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The parathyroid calcium-sensing receptor (CaR) plays a nonredundant role in systemic calcium homeostasis. In bone, Ca²⁺_o, a major extracellular factor in the bone microenvironment during bone remodeling, could potentially serve as an extracellular first messenger, acting via the CaR, that stimulates the proliferation of preosteoblasts and their differentiation to osteoblasts (OBs). Primary digests of rat calvarial OBs express the CaR as assessed by RT-PCR, Northern, and Western blot analysis, and immunocolocalization of the CaR with the OB marker cbfa-1. Real-time PCR revealed a significant increase in CaR mRNA in 5- and 7-d cultures compared with 3-d cultures post harvesting. High Ca²⁺_o did not affect the expression of CaR mRNA during this time but up-regulated cyclin D (D1, D2, and D3) genes, which are involved in transition from the G1 to the S phase of the cell cycle, as well as the early oncogenes, c-fos and early growth response-1; high Ca²⁺_o did not, however, alter IGF-I expression, a mitogenic factor for OBs. The high Ca²⁺_o-dependent increase in the proliferation of OBs was attenuated after transduction with a dominant-negative CaR (R185Q), confirming that the effect of high Ca²⁺_o is CaR mediated. Stimulation of proliferation by the CaR involves the Jun-terminal kinase (JNK) pathway, as high Ca²⁺_o stimulated the phosphorylation of JNK in a CaR-mediated manner, and the JNK inhibitor SP600125 abolished CaR-induced proliferation. Our data, therefore, show that the parathyroid/kidney CaR expressed in rat calvarial OBs exerts a mitogenic effect that involves activation of the JNK pathway and up-regulation of several mitogenic genes.

	Introduction

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BONE IS INTIMATELY involved in systemic calcium homeostasis by virtue of its interplay with the parathyroid glands and kidneys. The extracellular ionic calcium concentration (Ca²⁺_o) under actively resorbing osteoclasts can reach levels as high as 8–40 mM (1). Indeed, certain malignancies that metastasize to bone promote uncontrolled release of skeletal Ca²⁺_o resulting in hypercalcemia. Therefore, during bone remodeling, it is likely that the bone-forming osteoblasts (OBs), via the calcium-sensing receptor (CaR) (2), sense high Ca²⁺_o generated within the immediate microenvironment of resorbing osteoclasts (OCs). In fact, high Ca²⁺_o induces chemotaxis of human peripheral blood monocytes (3) and induces both chemotaxis and proliferation of mouse stromal cells (ST2) (4) and osteoblastic MC3T3-E1 cells (5). These cell types, respectively, have the capacity to differentiate into mature OCs (6) and OBs (7) under appropriate culture conditions.

OBs are mesenchymal-derived cells. Other mesenchymal-derived cells known to express a functional CaR include fibroblasts (8) and chondrocytes (9). However, studies of a murine preosteoblast (MC3T3-E1) cell line (5, 10) and human OB-like (MG-63) cells (11, 12) have yielded conflicting results regarding the molecular nature of the CaR that they express. Such discrepancies are not unusual given the reported phenotypic switch in MC3T3-E1 cells that may occur during cell passage (13, 14) and the incomplete resemblance between MG-63 cells and actual OBs at the level of gene expression (15). The existence of species-specific variation in CaR expression has also been reported. For example, murine OBs do not appear to express the CaR cloned from parathyroid/kidney (16, 17) but rather use an EF hand-containing calcium-binding protein, calcyclin, for Ca²⁺_o-sensing (18); CaR-like immunoreactivity has been shown in OBs of bovine and rat bone sections (19). However, expression of calcyclin and /or other EF hand proteins do not necessarily preclude, expression of a CaR in OB cells, similar to that expressed in parathyroid/kidney. For example, calcium-binding proteins belonging to the superfamily of EF-hand proteins are abundantly expressed in neuron (for review, see Ref. 20) and yet, a CaR similar to that in parathyroid and kidney has been cloned from rat striatum and localized in nerve terminals (21). In addition, accumulating evidence suggest that Ca²⁺_o acts as a major extracellular factor generated during bone remodeling, inducing the action of OBs while inhibiting those of OCs (3). Therefore, determining the molecular target and mode of action of Ca²⁺_o deserves careful study, because it might hold the potential for developing an anabolic therapy for states of bone loss.

With this as background, we used rat calvarial cells to investigate the molecular nature and function of the CaR expressed in bona fide osteoblasts. We found that the CaR expressed in the parathyroid (2) and kidney (22) is also expressed in calvarial OBs where it induces proliferation via the activation of stress-activated protein kinase (SAPK)/Jun-terminal kinase (JNK) pathway and up-regulation of c-fos, egr-1 (early growth response-1), and cyclin D genes.

	Materials and Methods

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Materials
All routine culture media were obtained from Invitrogen Life Technologies (Grand Island, NY). Collagenase P and the bromodeoxyuridine (BrdU) proliferation kit were from Roche (Indianapolis, IN); the supersignal, enhanced chemiluminescence kit was from Pierce (Rockford, IL); protease inhibitors were from Roche; and other reagents were purchased from Sigma Chemical (St. Louis, MO).

Calvarial OB cultures
For each experiment, about 25–30 calvariae were harvested at room temperature from 21-d fetal rats (Sprague Dawley). Humane handling of rats was carried out according to the guidelines of the Center for Animal Resources & Comparative Medicine of Harvard Medical School. A previously described method of repeated digestion of the calvariae with 0.05% trypsin and 0.1% collagenase P (23) was used to release cells. After discarding the cells from the first two digestions, cells from next three digestions were pooled and cultured in DMEM containing 10% heat-inactivated FBS and 1% penicillin-streptomycin in 5% CO₂ at 37 C. For immunocytochemical determination of the CaR, cells were placed directly on coverslips. For the proliferation assay and Western blot analysis, cells were plated on 96- and six-well plates, respectively. For preparing RNA, cells were plated in 75-cm² flasks. After 3 d of culture, cells were serum starved in DMEM (0.5 mM Ca²⁺, 4 mM L-glutamine, 1% penicillin-streptomycin, and 0.2% BSA) for 4 h before any experiments. All experiments were done within 7 d after beginning the culture.

Gene delivery by recombinant adeno-associated virus (rAAV)
High-efficiency gene transfer into OB cells was accomplished using a rAAV-based method as described previously (24). Both the human CaR sequence with a naturally occurring, dominant-negative mutation (R185Q) (25, 26) and the same vector containing the cDNA for the ß-gal protein (referred to hereafter as BG) were under the control of a cytomegalovirus immediate-early (CMV-IE) promoter element and packaged as described previously. The BG served as the control for any nonspecific effects of rAAV infection. Cells were seeded (500 cells/well) in 96-well plates in 0.1 ml of growth medium and cultured overnight. About 1000-virus particles/cell (as optimized by pilot studies) were used to infect each well.

Immunoperoxidase and immunofluorescence
Cells cultured on coverslips for 3 d were fixed with 4% formaldehyde. After blocking endogenous peroxidase, cells were incubated in parallel with 5 µg/ml mouse monoclonal anti-CaR antibody LRG (raised to amino acids 374–391 in the human CaR) and affinity-purified anti-CaR antibody 4637 (raised to amino acids 344–358 in the human CaR) (27, 28, 29). Negative controls were carried out by incubating the cells with the respective antisera after they had been preabsorbed with 10 µg/ml of the specific peptide to which each had been raised. After washing of cells incubated with the primary antibodies, appropriate incubations with secondary antibodies were carried out and the color reaction was developed with the 3-amino-9-ethyl-carbazole substrate system (Dako, Carpinteria, CA).

Two-color immunofluorescence to detect CaR- and cbfa-1-positive cells (thus osteoblastic) was performed with monoclonal anti-CaR LRG antibody (peptide sequence used to raise this antibody was LRGHEESGDRFSNSSTAF) and polyclonal cbfa-1 antibody as previously described (27, 28). Alexa Fluor 488 conjugated to goat antimouse secondary antibody was used for the CaR and Alexa Fluor 546 conjugated to goat antirabbit secondary antibody was used for cbfa-1 (the Alexa antibodies were both from Molecular Probes, Eugene, OR). Fluorescence images were collected with a Zeiss LSM 510 Meta Confocal microscope (Jena, Germany) at Harvard Center for Neurodegeneration and Repair (see Acknowledgments). Alexa 546 was excited at 546 nm, which produces a red signal, whereas fluorescein and Alexa 488 was excited at 488 nm, which yields a green signal. The autofluorescence of the samples was minimal and was subtracted from the values obtained during measurements. Because peptide blocking of LRG antibody was performed for both immunoperoxidase staining and Western blotting to establish its specificity, this control was not repeated in the studies using immunofluorescence.

Western analysis
Dispersed bovine parathyroid cells or confluent monolayers of HEKCaR or calvarial OB cells that had been cultured in six-well plastic cluster plates were rinsed with ice-cold PBS and scraped on ice into lysis buffer that contained 10 mM Tris-HCl (pH 7.4), 1 mM EGTA, 1 mM EDTA, 0.25 M sucrose, 1% Triton X-100, 1 mM dithiothreitol, and a cocktail of protease inhibitors (10 µg/ml each of aprotinin, leupeptin, and calpain inhibitor as well as 100 µg/ml Pefabloc). The cell lysates were then sonicated for 30 sec. Nuclei and cell debris were removed by centrifugation at 10,000 x g for 10 min, and the resultant total cellular lysate in the supernatant was used either directly for SDS-PAGE or stored at –80 C.

Immunoblot analysis was performed essentially as described previously (12). Aliquots of supernatant fractions containing the total cellular lysate (20 µg of protein from HEKCaR and bovine parathyroid cells and 40 µg from calvarial OB cells) were mixed with an equal volume of 2x SDS-Laemmli gel loading buffer containing 100 mM dithiothreitol (DTT), incubated at 65 C for 30 min, and resolved electrophoretically on 6.5% acrylamide gels. The separated proteins were then transferred to nitrocellulose filters (Schleicher and Schuell, Keene, NH) and incubated with blocking solution (PBS with 0.25% Triton X-100 and 5% dry milk) for 1 h at room temperature. The blots were subsequently incubated overnight at 4 C with affinity-purified polyclonal antiserum 4637 at 1 µg/ml with or without preincubation with twice the concentration (e.g. 2 µg/ml) of the peptide to which the antiserum had been raised (as a control for nonspecific binding) in blocking solution with 1% dry milk. The blots were subsequently washed five times with PBS that contained 1% Triton X-100 and 0.15% dry milk (washing solution) at room temperature for 10 min each. The blots were further incubated with a 1:2000 dilution of horseradish peroxidase-coupled goat antirabbit IgG (Sigma) in PBS containing 1% Triton X-100 for 1 h at room temperature. The blots were then washed five times with the washing solution, and bands were visualized by chemiluminescence according to the manufacturer’s protocol (Supersignal, Pierce Chemical).

Immunoprecipitation
Cell lysates from HEKCaR and calvarial OBs prepared as described above were centrifuged at 10,000 x g for 10 min. Supernatant protein (500 µg total lysate) was incubated with monoclonal LRG anti-CaR antibody overnight at 4 C. Protein A/G-agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA) were then added for an additional 1 h at 4 C. Bound immunocomplexes were washed three times with immunoprecipitation buffer, and the pellet was eluted by boiling with 2x Laemmli sample buffer without DTT. After SDS-PAGE, Western analysis was performed, as described above, using affinity-purified polyclonal anti-CaR antibody 4637.

Kinase assay
For the determination of JNK phosphorylation, cells were harvested at 80–90% confluence. After overnight culture in growth medium, cells were transfected with either dominant-negative CaR (DNCaR) or BG as described before using rAAV technology. Cells were then cultured for another 48–72 h when protein expression of the transduced CaR was determined in pilot studies to be maximal. A previously described protocol for studying SEK-1 phosphorylation in H-500 Leydig tumor cells was modified and used to study JNK phosphorylation (24). Briefly, cells were incubated for 18 h in serum-free, Ca²⁺-free DMEM containing 4 mM L-glutamine, 0.2% BSA, and 0.5 mM CaCl₂. The medium was removed and replaced with the same medium supplemented with 3.5 mM CaCl₂. At the end of the incubation period, the medium was removed, the cells were washed twice with ice-cold PBS containing 1 mM sodium vanadate and 25 mM NaF, and 100 µl of ice-cold lysis buffer was then added [20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 25 mM NaF, 1% Triton X-100, 10% glycerol, 1 mM dithiothreitol, 1 mM sodium vanadate, 50 mM glycerolphosphate, and a cocktail of protease inhibitors which contain aprotinin, leupeptin, soybean trypsin inhibitor, pepstatin, and calpain inhibitor (10 µg/ml of each), as well as 100 µg/ml of Pefabloc; all were added from frozen stocks except for the Pefabloc]. The sodium vanadate, NaF, and Pefabloc were freshly prepared on the day of the experiment. Aliquots of 20 µg of protein were separated by SDS-PAGE and electrotransferred onto nitrocellulose membranes. Membranes were incubated with phosphorylated JNK antibodies (1:1000 dilution of the stock antibody from the supplier) overnight at 4 C in blocking buffer as described previously and above. The subsequent protocol for detection and development of western signals was similar to that described above. The same membrane was used after stripping (Restore Western Blot Stripping, Pierce) to measure total JNK. Protein concentrations were measured with the Micro BCA (bicinchoninic acid) protein kit (Pierce). National Institutes of Health Image 1.62 was used to quantify the intensity of each band.

RT-PCR
Total RNA was prepared from 3- to 5-d-old cultures as described previously (30, 31, 32). For RT-PCR analysis of the CaR mRNA, 2 µg of total RNA were subjected to a one-step protocol according to the manufacturer’s instructions (QIAGEN, Valencia, CA) using previously published primer pairs for the CaR that were derived from the RaKCaR sequence (22). Table 1 shows the list of primers used for this purpose. The optimal temperature cycling protocol was determined to be 94 C for 30 sec, 56 C for 30 sec, and 72 C for 30 sec for 40 cycles with a programmable thermal cycler (PCR system 9700, Applied Biosystems). PCR products obtained in this manner were subjected to direct, bidirectional sequencing, employing the same primer pairs used for amplification of the products after purification of the respective DNA fragments from the PCRs with the QiaQuick kit (QIAGEN).

Quantitative real-time PCR
SYBR green chemistry was used to perform quantitative determination of CaR, IGF-I, c-fos, egr-1, and the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA following an optimized protocol described before (33). The design of sense and antisense oligonucleotide primers was based on published cDNA sequences using the Primer Express version 2.0.0 (Applied Biosystems, Foster City, CA). Primer sequences are listed in Table 1. cDNA was synthesized with the Omniscript Reverse Transcription Kit (QIAGEN) using 2 µg of total RNA in a 20-µl reaction volume. For real-time PCR, the cDNA was amplified using an ABI PRISM 7000 Sequence Detection System (PE Applied Biosystems, Foster City, CA). The double-stranded DNA-specific dye SYBR Green I was incorporated into the PCR buffer QuantiTech SYBR PCR (QIAGEN) to allow for quantitative detection of the PCR product in a 25-µl reaction volume. The temperature profile of the reaction was 95 C for 15 min, 40 cycles of denaturation at 94 C for 15 sec, annealing, and extension at 60 C for 1 min. An internal housekeeping control gene, GAPDH, was used to normalize differences in RNA isolation, RNA degradation, and the efficiencies of the reverse transcription. The size of the PCR product was first verified on a 1.5% agarose gel, followed thereafter by melting-curve analysis.

Proliferation assay
Subconfluent calvarial OB cells on 96-well plates were serum starved for 4 h in Ca²⁺-free DMEM containing 4 mM L-glutamine, 0.2% BSA, and 0.5 mM CaCl₂. Cells were then stimulated in the same medium with various concentrations of Ca²⁺ (0.5–7.5 mM) for 18 h. Cell proliferation was measured using the BrdU ELISA from Roche according to the manufacturer’s instructions. For the last 3 h of the 18-h stimulation period, the cells were pulsed with BrdU. Absorbance at 450 nm was measured with a microplate reader (Model 550, Bio-Rad, Hercules, CA).

Statistics
The data are presented as the mean ± SE of the indicated number of experiments. Data were analyzed by one-way ANOVA followed by Dunnett’s multiple comparison test or Student’s t test when appropriate. P < 0.05 indicates a statistically significant difference.

	Results

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Immunoreactivity of CaR protein in calvarial OBs with CaR-specific antibodies
We first determined the specificity of the two antibodies used in this study: the monoclonal (LRG) and the affinity-purified polyclonal (4637) antibodies. These studies employed cells derived from calvaria that were greater than 90% alkaline phosphatase (ALP) positive. Figure 1A shows strong immunocytochemical staining of the CaR with monoclonal LRG and elimination of the staining after preincubation of the antibody with the peptide to which it was raised (Fig. 1B). The 4637 antibody also stained CaR protein but to a lesser extent (Fig. 1C). However, the staining with 4637 was specific as revealed by the abolition of staining upon preincubating the antibody with the peptide (FF7) against which it was raised (Fig. 1D).

View larger version (112K): [in this window] [in a new window]   FIG. 1. Determination of specificity of immunoreactivity of rat calvarial OBs with anti-CaR antibodies. A and C, Cells incubated with monoclonal LRG and affinity-purified 4637 antibodies against the CaR, respectively. B and D, Abolition of CaR staining after preincubation of the antibodies with corresponding peptides to which the antibodies were raised.

View larger version (54K): [in this window] [in a new window]   FIG. 2. Immunofluorescent localization of the CaR in cbfa-1-positive cells. A, CaR staining (green) with LRG (monoclonal). B, cbfa-1 Staining (red) with a polyclonal antibody. C, A composite image resulting from the superimposition of A and B reveals CaR immunoreactivity in cbfa-1-positive cells.

View larger version (64K): [in this window] [in a new window]   FIG. 3. Determination of CaR protein and the specificity of its immunoreactivity in rat calvarial OBs. A, Western blot with (lane 2) and without (lane 1) blocking peptide against affinity-purified anti-CaR polyclonal antibody 4637. Strong immunoreactive bands (indicated by *) were detected between 140 and 160 kDa and were abolished by preincubation with the immunogenic peptide in lane 2, indicating expression of the CaR protein. Protein samples were prepared under reducing condition by denaturing in the presence of DTT. B, Immunoprecipitation with LRG anti-CaR antibody followed by immunoblotting with 4637 antibody. CaR-transfected HEK cells (lane 1), and calvarial OB (lane 3). Lane 2, Simple Western blot of bovine parathyroid protein lysate with 4637. Arrows (140 kDa and 160 kDa) represent the comparative sizes among these samples. The uppermost band, marked with arrowhead, represents the putative CaR dimer.

View larger version (37K): [in this window] [in a new window]   FIG. 4. Determination of CaR mRNA in rat calvarial OBs. A, Amplification of CaR transcripts by RT-PCR. Lanes from left are 100-bp ladder followed by amplified PCR fragments obtained with four different primer pairs designed from RaKCaR as described in Materials and Methods. B, Northern analysis showing CaR mRNAs in kidney, total calvaria, and calvarial OBs. A 4.1-kb band was detected in calvaria and calvarial OB samples.

View larger version (24K): [in this window] [in a new window]   FIG. 5. A, Time course of CaR mRNA expression in calvarial OBs in culture. RNA samples from primary digests of calvarial OBs were prepared on d 3, 5, and 7 from the day of harvesting. Real-time PCR performed on these RNA samples with CaR primer pairs shows higher expression of CaR mRNA in cultures at d 5 and 7 than at d 3 (n = 3, P < 0.05). B, Calvarial OBs were treated with 5.0 mM Ca2+o on d 5 and 7 in culture and compared with those treated with 0.5 mM Ca2+o for CaR mRNA expression by real-time PCR. Data show no significant alteration in CaR mRNA due to high Ca2+o (n = 3). *, Significantly more than d 3.

View larger version (20K): [in this window] [in a new window]   FIG. 6. High Ca2+ rapidly induces c-fos and egr-1 mRNAs. 5.0 mM Ca2+o induced c-fos (A) and egr-1 mRNAs (B) in calvarial OBs, as assessed by real-time PCR and the time courses of induction are shown. Due to the very high level of induction of the c-fos mRNA by high Ca2+o at the 15- and 30-min time points, the data in panel A are presented using a log scale. For c-fos (A), a > c > b. For egr-1 (panel B) b > a. Data pooled from three independent experiments (P < 0.05).

View larger version (24K): [in this window] [in a new window]   FIG. 7. High Ca2+ induces cyclin D genes. A, Representative Northern blot showing up-regulation of cyclin D1 (4.2 kb), D2 (6.8 kb), and D3 (2.3 kb) in calvarial OBs by 5.0 mM Ca2+o compared with 0.5 mM Ca2+o. B, Densitometric analysis of cyclin D Northern blot data (pooled from three independent experiments; P < 0.05). *, Significantly more than 0.5 mM Ca2+.

View larger version (28K): [in this window] [in a new window]   FIG. 8. High Ca2+o stimulates proliferation of calvarial OBs. Cells were plated directly in 96-well plates after collagenase digestion at 3–4 x 103/well and cultured for 3–5 d, at which point they were approximately 70% confluent. Alternatively, confluent OBs were trypsinized from 10 cm2 plates and seeded in 96-well plates with similar density and were cultured similarly. The cells were starved for 2 h and then incubated for 16 h in serum-free medium containing increasing concentrations of Ca2+o starting from 0.5 mM. After 16 h of incubation, cells were pulsed with BrdU for 3 h and color was developed as described in Materials and Methods. A, Dose-dependent increase of proliferation of calvarial OBs by Ca2+o. B, Elevated Ca2+o had no effect on IGF-I mRNA in OB cells. Real-time RT-PCR showing no significant change in the expression of IGF-I gene in the presence of 5.0 mM Ca2+o compared with basal level in 0.5 mM Ca2+o. *, Significantly higher than 0.5 mM Ca2+; **, significantly higher than 2.5 mM Ca2+.

High Ca²⁺_o-induced proliferation of calvarial OBs is CaR mediated
Using calvarial digests without any passaging, we next tested whether the CaR mediated high Ca²⁺_o-induced proliferation. We introduced the human DNCaR-(R185Q) or BG into these cells using rAAV as the delivery system. Whereas the proliferative response induced by high Ca²⁺_o was retained in cells transduced with BG, it was significantly attenuated in cells with DNCaR (Fig. 9). Such attenuation is not a nonspecific event because DNCaR cells were still responsive to BMP-2-induced proliferation (Fig. 9). These data confirm that the CaR mediates the proliferation induced by high Ca²⁺_o.

View larger version (18K): [in this window] [in a new window]   FIG. 9. CaR mediates high Ca2+o-induced proliferation of calvarial OBs. Calvarial OBs were infected with rAAV expressing either DNCaR or BG for 48 h before stimulation as described in Materials and Methods. The cells were then starved for 2 h in serum-free medium followed by addition of 0.5, 2.5, and 5.0 mM Ca 2+ or 0.5 mM Ca2+o + 50 ng/ml BMP-2 in serum-free medium and incubation for 16 h. The remainder of the experiment was similar to that described in Fig. 7. *, Significantly greater than 0.5 mM Ca2+; **, significantly greater than 2.5 mM Ca2+. P < 0.05; n = 4.

View larger version (13K): [in this window] [in a new window]   FIG. 10. High Ca2+o-induced proliferation requires activation of the JNK pathway. Cells were incubated in either 0.5 or 5.0 mM Ca2+o containing the JNK inhibitor SP600125 (10 µM), and the BrdU incorporation assay was performed as described previously. SP600125 (10 µM) had no effect on the basal state of proliferation but reduced high Ca2+o-induced proliferation to the basal level (P < 0.05, n = 3). *, Significantly more than 0.5 mM Ca2+.

View larger version (74K): [in this window] [in a new window]   FIG. 11. CaR induces JNK phosphorylation. The upper panel shows the time course of high Ca2+o (5.0 mM)-induced phosphorylation (p-) of JNK in calvarial OBs at the indicated time points in empty vector (BG) and DNCaR (DN) infected cells. Total (t)-JNK in the lower panel shows equal loading of cell lysate. This is representative of three independent experiments with similar results. The bottom panel shows an approximately 3.0-fold increase in p-JNK in BG-infected cells due to high Ca2+o (5.0 mM) compared with the significantly attenuated increase observed in DN infected cells. *, Significantly lower than BG at 5 min; **, significantly lower than BG at 15 min (P < 0.05). Pooled from the data obtained from three independent experiments where p-JNK bands were determined by densitometry and normalized with t-JNK band intensity and expressed in arbitrary units (P < 0.05).

	Discussion

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To date, our laboratory and others have shown that an elevated level of Ca²⁺_o promotes OB proliferation and chemotaxis (5, 10). However, the molecular basis for these actions is contentious, given the reported inconsistency in documenting the expression of a CaR in OBs similar to that in parathyroid and kidney (10, 11). An important factor contributing to this inconsistency may be the osteoblastic cell lines that have been used for these studies. MC3T3-E1 cells (5, 10) are known to undergo a phenotypic switch over a number of passages (13, 14) and are not readily available (e.g. not obtainable through the ATCC). Furthermore, MG-63 cells (11, 12) don’t completely resemble their normal OB counterpart with regard to gene expression and other patterns of cytokine/growth factor responses (15). Furthermore, species variation in its expression could also contribute, as expression of the CaR was undetectable in mouse osteoblasts (16). Furthermore, osteoblasts from the rescued, CaR-deficient mouse, which lack of a bone phenotype, function normally in their response to Ca²⁺_o in vitro (42). However, expression of the CaR has been documented in osteoblasts of bovine tibia and rat femur by in situ hybridization (18). Moreover, using the type II CaR agonist in organ culture (fetal rat metatarsal bones) for bone growth, Wu et al. (43) showed that CaR regulates growth plate chondrogenesis and longitudinal bone growth. In addition, CaR knockout mouse bone showed rickets, not a phenotype expected with hyperparathyroidism (17). Therefore, we have pursued these observations in rat calvarial OBs and determined its molecular nature and showed that it regulates cellular proliferation of calvarial OBs, which appear to be mediated by the parathyroid CaR, i.e. through use of the DNCaR (R185Q) (25, 26).

We obtained an approximately 90% pure population of osteoblastic cells, as assessed by ALP staining (data not shown), from the primary digests of calvariae. Well-characterized antibodies raised to two different regions of the CaR protein showed specific immunoreactivity in these cells. In addition, immunoreactivity of the CaR in cbfa-1-positive cells was observed with an immunofluorescent detection method combined with confocal microscopy. Interestingly, functional expression of the CaR has been found in ameloblast cells and developing tooth organ (44, 45), which express cbfa-1 (46) and regulate the production, resorption, and degradation of enamel matrix as well as the transport of Ca²⁺ into the extracellular matrix.

Western analysis with affinity-purified antibody 4637 showed labeling of bands of sizes consistent with CaR monomers and dimers with varying degrees of glycosylation, based on their being similar or identical in size to the corresponding bands in extracts of bovine parathyroid glands labeled with the same antibody. The specificity of the antibody was demonstrated by the elimination of these bands after preincubation of the antibody with the peptide to which it was raised. To provide additional evidence for the presence of the CaR in calvarial OBs, we performed immunoprecipitation with anti-CaR antiserum LRG (monoclonal) and then subjected the immunoprecipitate to Western blotting using anti-CaR antiserum 4637. Because these two antibodies were raised to two different epitopes of the CaR protein, the presence of labeled bands obtained from calvarial OB that are comparable in size to those of the CaR in parathyroid cells provides additional evidence for its expression in OBs.

Use of nucleotide-based approach such as RT-PCR and Northern analysis revealed expression of a CaR gene that is similar to the one expressed in rat kidney. A combination of intron-spanning primers representing the extracellular domain of RaKCaR and primer pairs representing transmembrane and intracellular domains amplified cDNA products identical with the corresponding segments of RaKCaR. These data strongly suggest that calvarial OBs express a CaR gene similar to that previously cloned from rat kidney (22) and striatum (21). High-stringency Northern analysis with a cRNA probe for RaKCaR revealed a 4.1-kb band in both whole calvaria and calvarial OBs. A differential pattern of expression of CaR transcripts has been reported in various rat/mouse tissues and cell types (30, 31, 32, 47, 48). Expression of only a 4.1-kb band has been reported in stomach and microglia (30, 31), whereas in the duodenum, the 4.1-kb transcript is much more abundantly expressed than is the 7.5-kb transcript (30). Although the physiological significance of such cell type-specific expression of CaR transcript(s) is presently unknown, the 4.1-kb transcript that is expressed in OBs encodes the entire functional CaR protein (22).

These results provide irrefutable evidence that calvarial OBs express a CaR that is similar to the CaR previously cloned from rat kidney and brain. Furthermore, unlike parathyroid cells, which lose expression of the CaR in vitro (34), expression of the CaR is retained in cultures of calvarial OBs and is up-regulated as a function of time. Possible reasons for the increased CaR expression might include increased confluency and increased secretion of autocrine factor(s): however, ascertaining the reason(s) for this temporal increase is beyond the scope of this report. Our data on the temporal increase in CaR gene expression in culture allowed us to use a window of 5–7 d post harvesting to perform functional studies. In addition, CaR expression did not differ between low (0.5 mM) and high (5.0 mM) Ca²⁺_o, suggesting that CaR expression does not undergo ligand-induced alteration. These data are useful for optimizing conditions for carrying out studies on the functional impact of activation of the CaR in OBs.

Calvarial OBs derived from a fetal/neonatal source are predominantly highly proliferative preosteoblastic cells. Numerous lines of evidence suggest that c-fos may be involved in several aspects of OB function, such as proliferation, differentiation, and ultimately bone formation (49, 50, 51). The c-fos has been shown to be expressed in regions of fetal bone having the highest growth potential (35) and is regulated in vitro and in vivo by PTH (1–34) (52, 53). In addition, c-fos-deficient mice develop osteoporosis and lack osteoclasts (54, 55), which provide compelling evidence for its role in OB function. Egr-1, a nuclear Zn²⁺ finger protein and transcriptional regulator, was shown to be coregulated with c-fos in developing mouse organs and has an overlapping pattern of expression in mesenchymally derived cells at sites of ossification (36, 37). Therefore, we investigated whether high Ca²⁺_o, possibly acting via the CaR, regulates these proto-oncogenes. We observed a rapid induction in the expression of these genes by high Ca²⁺_o, consistent with previously published rapid activation in response to PTH (51) and PGI₂ (37). An interesting feature of the time course of high Ca²⁺_o-induced c-fos mRNA level is a late (18 h) surge in this gene, albeit to a much lesser extent than that observed at very early time points (15 and 30 min). It is possible that high Ca²⁺_o, acting via the CaR, could induce PTHrP secretion in OB cells as observed in the case of other cells (24), which could then act in an autocrine manner to induce c-fos expression (52). Thus, it is conceivable that the CaR might exert PTH-like effects in OB cells.

In addition, proliferation of mammalian cells is strictly regulated by extracellular signals, which exert their effects on cells primarily during the G1 phase of the cell cycle and regulate progression to the S-phase. D-type cyclins are some of the major mediators of the G1-S-phase progression, serving as targets of growth factors that integrate extracellular signals into the core cell-cycle regulators (38). Because high Ca²⁺_o down-regulates cyclin D genes in the parathyroid gland (39) and overexpression of the cyclin D1 gene has been associated with excessive parathyroid cell proliferation (56), we postulated a regulatory role for high Ca²⁺_o in the expression of this gene in calvarial OBs. We observed that high Ca²⁺_o induced cyclin D genes, with a much greater induction of D1 than D2 and D3. It is possible that induction of cyclin D1 could be physiologically more significant than the other two cyclin D genes owing to its much greater level of induction by high Ca²⁺_o. However, it is also possible that it is the cumulative effect of increased cyclin D expression by high Ca²⁺_o that stimulates the proliferation of OBs. Further studies are needed to ascertain the relative contributions of individual cyclin D genes in mediating high Ca²⁺_o-induced proliferation of calvarial OBs. Nevertheless, activation of cyclin D by high Ca²⁺_o represents a possible mechanism for the growth promoting action of Ca²⁺_o, probably acting via the CaR. This is physiologically important because it could result in amplification of the population of preosteoblasts in response to the high Ca²⁺_o signal generated by osteoclastic bone resorption.

Not surprisingly, we observed that increasing concentrations of Ca²⁺_o promoted the proliferation of OBs. That a CaR similar to that in parathyroid and kidney mediates this effect was shown by the attenuation of the high Ca²⁺_o-induced mitogenic response after transduction of a DNCaR (R185Q) via rAAV. A similar approach revealed CaR’s involvement in PTHrP secretion in other cells expressing this receptor (24, 57). Therefore, the molecular nature of the CaR displayed by calvarial osteoblasts in this study is functionally consistent with that of the cloned G protein-coupled CaR from rat kidney (22) that is homologous with parathyroid CaR (2).

Interesting in this regard is the failure of high Ca²⁺_o to stimulate IGF-I mRNA in these cells. IGF-I is a potent mitogen for OBs, and PTH stimulates its secretion (58). The report of abolition of high Ca²⁺_o-induced proliferation of MC3T3-E1 cells, a mouse osteoblastic cell line, by IGF-I-neutralizing antibody suggests a mediatory role of IGF-I in this process (40), whereas such a role for IGF-I was not found in the rat osteoblast like cell line, PyMS (59). However, the report using PyMS cells used concentrations of Ca²⁺ between 0.2 and 1.0 mM, well below those generally used to activate the CaR, and the effect of Ca²⁺ on DNA synthesis was blocked by nifedipine, a L-type channel blocker. In our study, we not only found no change in IGF-I mRNA level in response to high Ca²⁺ (5.0 mM) but also observed that IGF-I expression level is extremely low as assessed by real-time PCR.

IGF-I expression by osteoblastic cells is a controversial issue, with evidence both for (60, 61, 62) and against (63, 64, 65) its expression. Our method of assessing IGF-I expression by real-time RT-PCR indicates a low copy number in calvarial OBs because of the very late appearance of the PCR product, which was unchanged by high Ca²⁺_o. Therefore, our data suggest that CaR-induced proliferation of calvarial OB is independent of IGF-I.

Induction of proliferation by the CaR has been shown in variety of cell types including fibroblasts (8), ovarian surface epithelial cells (66), H-500 Leydig tumor cells (67), and CaR-transfected CCL3 hamster fibroblasts (which lack endogenous CaR expression) (68). CaR-induced proliferation involves a diverse array of signaling pathways (for review see Ref. 69). Whereas activation of mRNAs for c-fos, egr-1, and cyclin D by high Ca²⁺_o in our study, represent a more distal, post-receptor event; activation of the SEK1/JNK pathway is a more proximal event by which the CaR promotes proliferation of these cells. Although involvement of ERK1/2 has been implicated in high Ca²⁺-induced proliferation of fibroblast (8) and MG-63 human osteoblast-like cells (70), JNK activation has been implicated in PTHrP secretion from H-500 Leydig tumor cells (24). Our demonstration that the CaR mediates JNK activation and that interruption of JNK signaling inhibits high Ca²⁺_o-induced proliferation of OB cells shed new light on the growth-regulatory characteristics of these cells in general and the extracellular signal-induced proliferation (e.g. by the CaR) in particular. In addition, recent reports have shown that cyclin D1 (71) and D2 (72) are the downstream targets of JNK in regenerating liver cells and murine hybridoma 7TD1 cells, respectively. It is possible that similar regulation of cyclin D by JNK, resulting in CaR-induced proliferation, takes place in rat calvarial OBs; however, this awaits further study.

In conclusion, our study reveals expression of a kidney/parathyroid CaR in rat calvarial osteoblasts, which promotes mitogenesis in these cells.

	Acknowledgments

 
We gratefully acknowledge Dr. Lawrence G. Raisz (University of Connecticut Health Center, Farmington, CT) for his helpful discussion and kindly sharing expertise of his laboratory in preparing calvarial osteoblast culture. We are thankful to Dr. C. J. Sherr (St. Jude Children’s Research Hospital, Memphis, TN) for generously providing cyclin D1, D2, and D3 cDNA probes. We thank Mark Chafel and Michelle L. Ocana at Harvard Center for Neurodegeneration and Repair for helping us with confocal imaging.

	Footnotes

 
This work was supported by grants from the National Institutes of Health (AR02215), Pfizer/American Federation for Aging Research, and Research Career Enhancement Award from the American Physiological Society (to N.C.); National Institutes of Health (DK41415, DK48330, and DK52005), NPS Pharmaceuticals, and the St. Giles Foundation (to E.M.B.).

N.C. and S.Y. contributed equally to this work.

Abbreviations: ALP, Alkaline phosphatase; BG, ß-gal; BrdU, bromodeoxyuridine; CaR, calcium-sensing receptor; DNCaR, dominant-negative CaR; DTT, dithiothreitol; egr-1, early growth response-1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; JNK, Jun-terminal kinase; OBs, osteoblasts; OCs, osteoclasts; rAAV, recombinant adeno-associated virus; RaKCaR, rat kidney CaR; SEK, stress-activated protein kinase; ST2, mouse stromal cells.

Received August 27, 2003.

Accepted for publication March 30, 2004.

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