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Am J Physiol Cell Physiol 291: C529-C537, 2006. First published April 12, 2006; doi:10.1152/ajpcell.00354.2005
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GROWTH, DIFFERENTIATION, AND APOPTOSIS

CGRP inhibits osteoprotegerin production in human osteoblast-like cells via cAMP/PKA-dependent pathway

I. Villa,1 E. Mrak,1,2 A. Rubinacci,1 F. Ravasi,1 and F. Guidobono2

1Bone Metabolic Unit, Scientific Institute H San Raffaele, Milan; and 2Department of Pharmacology, Chemotherapy, and Medical Toxicology, University of Milan, Milan, Italy

Submitted 14 July 2005 ; accepted in final form 4 April 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The osteoprotegerin (OPG)/receptor activator of nuclear factor-{kappa}B ligand (RANKL)/receptor activator of nuclear factor-{kappa}B (RANK) system was evaluated as a potential target of CGRP anabolic activity on bone. Primary cultures of human osteoblast-like cells (hOB) express calcitonin receptor-like receptor (CLR) and receptor activity modifying protein 1, and, because CGRP stimulates cAMP (one of the modulators of OPG production in osteoblasts), it was investigated whether it affects OPG secretion and expression in hOB. CGRP treatment of hOB (10–11 M–10–7 M) dose-dependently inhibited OPG secretion with an EC50 of 1.08 x 10–10 M, and also decreased its expression. This action was blocked by the antagonist CGRP8–37. Forskolin, a stimulator of cAMP production, and dibutyryl cAMP also reduced the production of OPG. CGRP (10–8 M) enhanced protein kinase A (PKA) activity in hOB, and hOB exposure to the PKA inhibitor, H89 (2 x 10–6 M), abolished the inhibitory effect of CGRP on OPG secretion. Conditioned media from CGRP-treated hOB increased the number of multinucleated tartrate-resistant acid phosphatase-positive cells and the secretion of cathepsin K in human peripheral blood mononuclear cells compared with the conditioned media of untreated hOB. These results show that the cAMP/PKA pathway is involved in the CGRP inhibition of OPG mRNA and protein secretion in hOB and that this effect favors osteoclastogenesis. CGRP could thus modulate the balance between osteoblast and osteoclast activity, participating in the fine tuning of all of the bone remodeling phases necessary for the subsequent anabolic effect.

receptor-activity-modifying proteins; protein kinase A; osteoclast; cathepsin K

BONE TISSUE CONTAINS CGRP-immunoreactive nerve fibers whose increased concentrations during bone development and regeneration suggest they are directly involved in the local regulation of bone remodeling (18–20). Further evidence shows that CGRP, which is derived from alternative splicing of calcitonin (CT) gene mRNA (2), plays a role in bone metabolism. It inhibits osteoclastic bone resorption by directly blocking osteoclast activation (1, 21, 49) or indirectly regulating the osteoblast release of cytokines, such as interleukin-1 (28) and TNF-{alpha} (31), that can affect osteoclast function. When administered to ovariectomized rats, CGRP inhibits bone resorption but not bone formation (44). Moreover, transgenic mice overexpressing CGRP in osteoblasts have increased bone density (3). Various human osteoblast cell lines and normal human osteoblast-like cells express mRNAs for CT and CGRP (10) and their receptors (46). On these cells, CGRP increases both cell proliferation (47) and the production of insulin growth factor 1 (45). Activated CGRP receptors stimulate both adenylyl cyclase activity (30) and phospholipase C (11), which respectively stimulates cAMP production and activation of protein kinase A (PKA), and increase intracellular free calcium levels (6) with subsequent protein kinase C activation (11). It has been shown that the activation of protein kinase C is involved in CGRP-induced osteoblast proliferation (46). CGRP also acts on murine osteoblast ATP-sensitive K+ channels to attenuate Ca2+ influx across the plasma membrane (22). The activation of these pathways leads to changes in osteoblast gene expression by means of transcriptional and/or posttranscriptional mechanisms.

In normal bone remodeling, osteoblastic bone formation follows osteoclastic bone resorption along a hierarchical sequence of events. Osteoblast function is intimately tied to osteoclast activity as a result of the osteoblast production of essential cytokines. The osteoprotegerin/receptor activator of nuclear factor-{kappa}B ligand/receptor activator of nuclear factor {kappa}B (OPG/RANKL/RANK) system plays a key role in the cross-talk between osteoblasts and osteoclasts (48). RANKL and OPG are members of a ligand-receptor system that directly regulates osteoclast differentiation and bone resorption. OPG is a member of the tumor necrosis factor receptor superfamily that is produced by osteoblastic lineage cells (16, 39) in the form of a 55-kDa monomer or 110-kDa dimer, and, because it lacks transmembrane and cytoplasmic domains, it is secreted by cells of osteoblastic lineage as a soluble protein (39, 48). The soluble and membranous forms of RANKL are preferentially produced by osteoblasts, whereas the specific receptor, RANK, is expressed on osteoclast progenitors. RANKL binds to RANK, which leads to osteoclast activation (25), or to OPG, thereby preventing osteoclast activation. RANKL stimulates osteoclast formation, fusion, and activation, and is essential for osteoclast survival (16). Both osteoblasts and osteoclasts individually contribute to bone remodeling, but the type and extent of the remodeling depends on their cell-to-cell interactions. Increased RANKL/RANK signaling leads to high bone turnover with stimulated osteoblastic bone formation (34). Hormones, growth factors, cytokines, and prostaglandins regulate these processes through their direct effects on bone cells and the OPG/RANKL/RANK system (7, 40). Several osteotropic hormones, cytokines, and drugs affect this system by activating the cAMP pathway (5, 12, 24). Because CGRP stimulates cAMP production in human osteoblasts (47), it was investigated whether the effect of CGRP on bone is, at least in part, mediated by the OPG/RANKL/RANK system. For this purpose, we studied the effects of CGRP on OPG and RANKL expression and production in cultured human osteoblast-like cells (hOB). We analyzed the presence of calcitonin receptor-like receptor (CLR) and receptor-activity-modifying proteins (RAMPs), which are important for the ligand specificity of CGRP receptors (29). Furthermore, we evaluated whether or not CGRP, by modulating the OPG/RANKL/RANK system in hOB, could influence osteoclastogenesis in cultured human peripheral blood mononuclear cells (PBMCs).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Drugs. Human {alpha}CGRP was from Phoenix Pharmaceuticals, Belmont, CA; CGRP8–37 from Peninsula, St. Helen, UK; human recombinant macrophage colony-stimulating factor (hM-CSF) and human recombinant RANKL (Peprotech, Rocky Hill, NJ); 1,25(OH)2D3 from Hoffmann-LaRoche, Basel, Switzerland; and dibutyryl cAMP (dBcAMP), forskolin (FSK), and H89 from Sigma, Milan, Italy.

Osteoblast-like cell cultures. Human bone cells were established in culture by means of a modified version of the Gehron Robey and Termine procedure (14) using trabecular bone samples obtained from waste materials during orthopedic surgery for degenerative diseases or traumatic fractures of the femoral neck requiring osteotomy. None of the operated patients (aged 51–73 yr) had any metabolic or malignant bone diseases and all of them gave their written consent for the use of the waste material. No significant trend related to donor age was observed in any of the effects studied.

Briefly, the trabecular bone was cut into small pieces (2 x 2 x 2 mm) and thoroughly washed with commercial standardized Joklik's modified MEM (Sigma, Milan, Italy) serum-free medium, to remove nonadherent marrow cells. The pieces were incubated with rotation at 37°C for 30 min with the same medium containing 0.5 mg/ml type IV collagenase (Sigma), and collagenase digestion was stopped by the addition of Iscove's modified Dulbecco's medium (Eurobio, Les Ulis, France) containing 10% fetal bovine serum (FBS; HyClone, Logan, UT). Between eight and ten pieces for each patient were then placed in 25 cm2 flasks and cultured in Iscove's modified Dulbecco's medium containing 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, 50 U/ml mycostatin, and 0.25 µg/ml amphotericin B; the culture medium was changed every 2–3 days. The cells began to migrate within 1–2 wk and reached confluence after 1 mo. They were tested for alkaline phosphatase and osteocalcin production after 1,25(OH)2D3 10–8 M to ensure that they were endowed with osteoblast characteristics: alkaline phosphatase was determined in the cell layer solubilized with 0.5 ml 0.1% SDS by measuring the reduction in p-nitrophenol phosphate (Roche Diagnostics, Basel, Switzerland); osteocalcin was measured by chemiluminescence assay (Nichols Advantage, San Juan Capistrano, CA). All of the cells were used at first passage to reduce the possibility of phenotype changes. Conditioned media were collected after 48 h of CGRP treatment of hOB cultures, filtered, and stored at –80°C until use.

Isolation of cells from human peripheral blood. Human PBMCs were isolated by Ficoll-Paque sedimentation and adherence from the peripheral blood of different healthy donors (13). Approximately 20 ml of blood were diluted 1:1 with {alpha}MEM (Sigma, Milan, Italy), layered over 20 ml of lymphocyte separation medium (Cambrex Bio Science, Walkersville, MD) and centrifuged at 2,300 rpm for 20 min. The mononuclear cell-rich layer at the interface was removed and washed twice in {alpha}MEM, and the pellet was then resuspended in {alpha}MEM with 10% FBS. The number of cells in the suspension was finally counted in a Burker chamber after red blood cell lysis using a 5% (vol/vol) acetic acid solution. The PBMCs (3 x 105 cells/well) were seeded on glass coverslips (13 mm diameter) in 24-well plates. After incubation at 37°C for 2 h, they were removed from the wells, washed vigorously in {alpha}MEM to remove nonadherent cells, and placed in new wells containing 1 ml of conditioned media with 10% FBS. These cultures were incubated for 21 days in the presence of 25 ng/ml hM-CSF and 0.1 ng/ml or 30 ng/ml hRANKL in conditioned media obtained from hOB treated or not with 10–8 M {alpha}CGRP for 48 h. The culture medium was changed every 3–4 days.

Characterization of generated osteoclasts. After 21 days, the coverslips were removed and stained histochemically for the expression of tartrate-resistant acid phosphatase (TRAP), an osteoclast-associated marker (32). The cells were fixed in citrate/acetone solution and stained for acid phosphatase using a commercially available kit (Sigma, St. Louis, MO). Multinucleated TRAP-positive (TRAP+) cells were then counted.

RT-PCR. Total RNA was extracted from osteoblast-like cells from eight different donors using TRIzol according to the manufacturer's instructions (Invitrogen Life Technology, Paisley, UK). The RNA pellets were dissolved in sterile distilled water and their concentrations were assessed by spectrophotometric analysis (OD260/280). One microgram of total RNA was retrotranscribed in a total volume of 25 µl using an oligo(dT) primer (0.5 µM), 200 units of Moloney murine leukemia virus reverse transcriptase, deoxynucleotides (0.5 mM), Moloney murine leukemia virus reaction buffer (1x), and rRNasin ribonuclease inhibitor (1 U/µl) (Promega, Madison, WI). Specific primers were constructed for the human CRLR, RAMP1, RAMP2, RAMP3, OPG, and RANKL on the basis of the sequences published in GenBank (Table 1). PCR was performed in a final volume of 20 µl containing cDNA (4 µl of the RT-PCR solution), 1 mM primers, 10 mM of each deoxynucleotide triphosphate, Taq polymerase (0.5 units), and PCR buffer (1x) supplied with MgCl2 (2 mM) (Promega), using a thermal cycler (Tpersonal; Whatman Biometra, Goettingen, Germany) and the conditions are listed in Table 2. The PCR products were finally analyzed on 2% Tris-acetate-EDTA agarose gel. The DNA ladder was the PCR Marker 50–2000 bp and the PCR Low Ladder Set (100-bp ladder) (Sigma, Milan).


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  		Table 1. Primer sequences for CRL, OPG, and receptor activity modifying proteins

 

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  		Table 2. PCR conditions for amplification of CLR, RAMP1–3, OPG, and RANKL

 
Real-time PCR. Relative OPG mRNA expression was evaluated by means of Real-time PCR in hOB obtained from different donors. After 48 h of culture in a serum-free medium, confluent hOB were treated with 10–8 M CGRP for 3, 6, and 24 h in the presence or absence of the CGRP receptor antagonist, CGRP8–37 (10–8 M), or with 5 x 10–5 M dBcAMP. Total RNA was isolated and reverse transcribed as previously described. OPG gene expression was relatively quantified on an ABI PRISM 7700 sequence detector (Applied Biosystems, Foster City, CA) using 10 ng cDNA for the RT-PCR solution in a final volume of 25 µl. The primer-probe sets were purchased from Applied Biosystems. Real-time PCR was performed using FAM-labeled specific probes for OPG detection and VICTM-labeled probes for detection of the GAPDH housekeeping gene used as an internal control. All of the primers were chosen to span exon junctions or to lie in different exons to prevent the amplification of genomic DNA. The real-time PCR protocol included an initial step of 2 min at 50°C and 10 min at 95°C, followed by 40 cycles of 15 s at 95°C and 1 min at 60°C. The data were collected during each extension phase of the PCR reaction, and analyzed using the SDS software package (Applied Biosystems). Threshold cycles were determined for each gene, with the relative expression of each target gene of interest being normalized against GAPDH.1 Three replicates were performed for each experimental point, and the experiments were repeated four times using cells from different donors.

PKA assay. CGRP 10–8 M was used for different incubation times (2, 15, 30, 60, and 90 min), and the preincubations with H89 (2 x 10–6 M), a specific PKA inhibitor, took place 30 min before each CGRP (10–8 M) treatment. The cells were then rinsed twice in cold PBS and solubilized in a lysis buffer containing 50 mM HEPES, 250 mM NaCl, 5 mM MgCl2, 1% Triton, 10% glycerol, and 10 µg/ml aprotinin, leupeptin, and phenylmethylsulfonyl fluoride, pH 7.4. PKA activity was evaluated using the PepTag assay (Promega): briefly, this assay uses a fluorescent peptide substrate highly specific for PKA whose phosphorylation alters its net charge from positive to negative, thus allowing its phosphorylated and nonphosphorylated forms to be rapidly separated on agarose gel. When the electrophoresis was completed, the bands were excised, solubilized, and evaluated by spectrofluorometry (excitation 568 nm; emission 592 nm). The results were expressed as ratios of the emitted fluorescence intensity of the treated and untreated cells.

OPG assay. Primary hOB cultures were seeded into 6-well multiwell plates and allowed to grow to confluence. After 48 h of culture in serum-free medium, they were treated for 48 h with increasing concentrations of CGRP (10–11 M–10–8 M) in the presence or absence of the CGRP antagonist, CGRP8–37 (10–8 M), or the PKA inhibitor H89 (2 x 10–6 M). The same procedure was used for dBcAMP (5 x 10–5 M) and FSK (5 x 10–6 M). OPG concentrations were measured in the conditioned media using a commercial kit (OPG, Immundiagnostik, Bensheim, Germany), and the results (fmol/105 cells) expressed as the ratio of OPG secretion from treated and untreated cells.

RANKL assay. Soluble RANKL was measured using a commercial kit (Biomedica, Vienna, Austria) in the conditioned media of hOB cultured for 48 h in the presence or absence of CGRP (10–8 M), or 1,25(OH)2D3 (10–8 M).

Cathepsin K assay. Cathepsin K was measured in the supernatants of cultured human PBMCs after 21 days of treatment with the conditioned media of hOB cultured for 48 h with or without CGRP (10–8 M) using a commercial kit (Biomedica).

Statistical analysis. The data were statistically analyzed with Prism version 4.00 for Windows (GraphPad Software, San Diego, CA; www.graphpad.com). The significance of the within group differences was assessed by means of one-way ANOVA for nonparametric values (Kruskal-Wallis test) and a multiple-comparison test (Dunn's test), and that of the between-group differences by means of the two-tailed Student's t-test. The CGRP dose-response curve of OPG production was analyzed using nonlinear regression analysis (sigmoidal dose-response curve). The significance of the effect of H89 on CGRP-induced PKA activity was evaluated by means of two-way ANOVA and Bonferroni's multiple-comparison test.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
The hOB expressed the components of a functioning CGRP receptor (CLR and RAMP1); RAMP2 was also present, but no RAMP3 expression was detected (Fig. 1). Forty-eight hours of treatment with different concentrations of CGRP (10–11 M–10–8 M) significantly inhibited OPG secretion in a dose-dependent manner, with an EC50 of 1.08 x 10–10 M (Fig. 2). The inhibitory effect was greatest (~45%) at the concentration of 10–9 M, and reached a plateau at 10–8 M; a higher concentration of 10–7 M was less effective (data not shown). Decreased OPG secretion was accompanied by an inhibitory effect on OPG expression in hOB. Figure 3A shows that OPG mRNA was constitutively expressed in primary hOB; Real-time PCR revealed that CGRP significantly inhibited OPG mRNA transcription after 6 h, and this effect was still present after 24 h (Fig. 3B) but had disappeared after 48 h, when OPG mRNA expression returned to baseline levels (data not shown). The inhibitory action of CGRP (10–8 M) on OPG secretion (Fig. 4A) and expression (Fig. 4B) in hOB was prevented by the peptide antagonist CGRP8–37 (10–8 M), which per se had no effect. The treatment of hOB with dBcAMP (5 x 10–5 M), a nonhydrolyzable analog of cAMP, or FSK (5 x 10–6 M), an activator of adenylyl cyclase, significantly inhibited OPG secretion (Fig. 5A), and dBcAMP also led to a decrease in OPG expression (Fig. 5B). As a consequence of its well-known stimulation of cAMP production (28, 42), CGRP significantly increased PKA activity in hOB, starting after 2 min and lasting up to 90 min, with maximum activation after 15 min (Fig. 6A); preincubation with the PKA inihibitor H89 (2 x 10–6 M) 30 min before the addition of 10–8 M CGRP at each time point (2, 15, 30, 60, and 90 min) prevented CGRP-induced PKA activation (Fig. 6B). Preincubation of hOB with H89 (30 min before CGRP) inhibited the effect of 10–8 M CGRP on OPG secretion after 48 h of treatment (Fig. 7).


Figure 1
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  		Fig. 1. A: expression of the gene for calcitonin receptor-like receptor (CRL) in human osteoblast-like cells (hOB). Lanes 1, 3, and 5: PCR products obtained from hOB from three different donors; lanes 2, 4, and 6: negative PCR control samples with no RNA to test possible contamination of reagents; lane M: DNA ladder. B and C: expression of the genes for receptor activity modifying protein (RAMP)1 and RAMP2 in hOB. Lanes 1, 3, and 5: PCR products obtained from cells from three donors; lanes 2, 4, and 6: negative PCR controls, lane M: DNA ladder. D: expression of the gene for RAMP3 in hOB and human kidney tissue. Lanes 1, 3, and 5: PCR products obtained from hOB from three donors; lane 7: PCR product from human kidney (positive control); lanes 2, 4, 6, and 8: negative PCR controls; lane M: DNA ladder. These data were confirmed in hOB from eight different donors.

 

Figure 2
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  		Fig. 2. Dose-dependent decrease in osteoprotegerin (OPG) secretion in hOB-like cells after 48 h of treatment with CGRP concentrations ranging from 10–11 M to 10–8 M. Data are means ± SE of 6 experiments using cells from six different donors. The results are expressed as the ratio of the OPG (fmol/105 cells) produced by treated and untreated cells. The EC50, calculated using the nonlinear regression analysis sigmoidal dose-response curve, was 1.08 x 10–10 M. **P < 0.001, *P < 0.01 vs. untreated cells (Kruskal-Wallis test for nonparametric data, and Dunn's multiple-comparison test).

 

Figure 3
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  		Fig. 3. A: OPG mRNA expression in untreated hOB-like cells. Lane 1, DNA ladder (100 bp); lanes 2–4: PCR products from cells from three different donors. B: OPG mRNA expression measured by real-time PCR 3, 6, and 24 h after treatment with CGRP 10–8 M. The results were normalized to GAPDH, and the mRNA in the samples from treated cells is expressed as a ratio of the amount measured in samples from untreated cells (see MATERIALS AND METHODS for details). The dotted line represents OPG mRNA expression in untreated cells. Results are the mean ± SE of 4 experiments performed in triplicate using cells from four different donors. *P < 0.01 vs. untreated cells (Kruskal-Wallis test for nonparametric data, and Dunn's multiple-comparison test).

 

Figure 4
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  		Fig. 4. A: effect of pretreatment with the CGRP antagonist CGRP8–37 (10–8 M, 15 min before CGRP) on CGRP (10–8 M) inhibition of OPG secretion measured 48 h after CGRP administration in hOB. The results are expressed as the ratio of the OPG (fmol/105cells) produced by treated and untreated cells; the dotted line represents the OPG production (26.83 ± 4.52 fmol/105 cells) of untreated cells. Data are means ± SE of four experiments using cells from different donors. B: real-time PCR showing the effect of pretreatment with the CGRP antagonist CGRP8–37 (10–8M, 15 min before CGRP) on CGRP (10–8 M) inhibition of OPG mRNA expression in hOB. OPG mRNA was determined 24 h after CGRP treatment; the results were normalized to GAPDH, and the mRNA in the samples from treated cells was expressed as a ratio of the amount measured in untreated samples (see MATERIALS AND METHODS for details). The dotted line represents OPG mRNA expression in untreated cells. Results are the means ± SE of 4 experiments performed in triplicate using cells derived from 4 different donors. **P < 0.01 vs. untreated cells; bulletP < 0.05 vs. CGRP + CGRP8–37 (Kruskal-Wallis test for nonparametric values, and Dunn's test multiple-comparison test).

 

Figure 5
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  		Fig. 5. A: effect of 48 h treatment with dibutyryl cAMP (dBcAMP, 5 x 10–5 M) or forskolin (FSK; 5 x 10–6 M) on OPG secretion in human osteoblast-like cells (hOB). The results are expressed as the ratio of the OPG (fmol/105 cells) produced by treated and untreated cells; the dotted line represents OPG production (20.68 ± 0.40 fmol/105 cells) by untreated cells. Data are the means ± SE of 4 experiments using cells from four different donors. B: real-time PCR showing the effect of dibutyryl cAMP (dBcAMP, 5 x 10–5 M) on OPG mRNA expression in hOB after 6- and 24-h incubation. The results were normalized to GAPDH, and the mRNA in the samples from treated cells was expressed as a ratio of the amount measured in the samples from untreated cells (see MATERIALS AND METHODS for details). The dotted line represents OPG mRNA expression in untreated cells. The results are the mean ± SE of 4 experiments performed in triplicate using cells from 4 different donors. *P < 0.05 vs. untreated cells (Kruskal-Wallis test for nonparametric data, and Dunn's multiple-comparison test).

 

Figure 6
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  		Fig. 6. A: PKA activity induced by 10–8 M CGRP (n = 8) in hOB cells 2, 15, 30, 60, and 90 min after treatment; n = number of experiments performed using cells from 8 different donors. B: effect of the pretreatment with the PKA inhibitor H89 (2 x 10–6 M, n = 6) on PKA activity induced by 10–8 M CGRP in hOB 2, 15, 30, 60, and 90 min after CGRP treatment. The results are the means ± SE of the ratios of the emitted fluorescence intensity of the treated (Ftreated) and untreated cells (Funtreated). Insets, typical gels obtained from hOB cell lysates at various times after treatment with CGRP (A), and the combination of CGRP and H89 (B). The fluorescent bands represent the amounts of the phosphorylated PKA substrate that migrates to the cathode (+) and the unphosphorylated substrate that migrates to the anode (–). a and b: positive and negative controls of the reaction.

 

Figure 7
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  		Fig. 7. Effect of pretreatment with H89 (2 x 10–6 M, 30 min before CGRP) on the inhibition of OPG secretion by CGRP (10–8 M, 48 h treatment). The results are expressed as the ratio of the OPG (fmol/105 cells) produced by the treated and untreated cells. The dotted line represents OPG production (10.91 ± 0.39 fmol/105 cells) by untreated cells. Data are the means ± SE of 4 experiments using cells from four different donors. **P < 0.01 vs. untreated cells; bulletP < 0.05 vs. H89 + CGRP (Kruskal-Wallis test for nonparametric data, and Dunn's multiple-comparison test).

 
In hOB, no detectable levels of RANKL mRNA or secreted protein were observed in the conditioned media under baseline conditions or after 10–8 M 1,25(OH)2D3 treatment. Cytofluorimetry showed that the percentage of RANKL-positive cells in the hOB culture was very low and not significant (data not shown).

To examine the possible effect of the reduced OPG secretion induced by CGRP in hOB on osteoclastogenesis, PBMCs were cultured for 21 days in conditioned media obtained from hOB treated with or without CGRP 10–8 M for 48 h, and in the presence of M-CSF (25 ng/ml) and RANKL (0.1 or 30 ng/ml). The presence of osteoclast-like cells was evaluated by counting the number of multinucleated TRAP+ cells per well.

In the presence of the subeffective dose of RANKL (0.1 ng/ml), the conditioned media of untreated hOB did not induce the formation of TRAP+ multinucleated cells. However, the conditioned media of CGRP-treated hOB induced the formation of few TRAP+ multinucleated cells (Fig. 8). In the presence of the effective dose of RANKL (30 ng/ml), the conditioned media of CGRP-treated hOB induced a significant increase (P < 0.01) in the number of TRAP+ multinucleated cells compared with the number of TRAP+ multinucleated cells formed in the presence of the conditioned media of the untreated hOB (Fig. 9C). Similarly, a significant increase (P < 0.05) in cathepsin K secretion was observed in the supernatants of PBMC exposed to the conditioned media of CGRP-treated hOB in presence of the effective dose of RANKL (Fig. 9D).


Figure 8
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  		Fig. 8. Effect of conditioned media obtained from hOB treated without (A) or with (B) CGRP (10–8M for 48 h) on osteoclast formation in presence of 0.1 ng/ml RANKL. A: no multinucleated tartrate-resistant acid phosphatase positive cells (TRAP+) were observed in the peripheral blood monocyte cells (PBMCs) cultured with conditioned media of untreated hOB. B: few multinucleated TRAP+ cells were present in the peripheral blood monocyte cells (PBMCs) cultured with conditioned media obtained from CGRP-treated hOB. Red arrow: multinucleated TRAP+ cells.

 

Figure 9
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  		Fig. 9. Top: effect of conditioned media obtained from hOB treated without (A) and with (B) CGRP (10–8M for 48 h) on osteoclast formation in presence of 30 ng/ml RANKL. Red arrows: multinucleated tartrate resistant acid phosphatase positive cells (TRAP+). Bottom, C: number of multinucleated TRAP+ cells formed after 21 days culture with conditioned media obtained from treated (CGRP; 10–8M) and untreated hOB. D: cathepsin K values measured in the supernatants of PBMC after 21 days culture with conditioned media obtained from treated (CGRP; 10–8M) and untreated hOB. Data are the means ± SE of 3 experiments performed with cells from three different donors; **P < 0.01, *P < 0.05 vs. conditioned media of untreated cells, Student's t-test.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The results of this study show that hOB express CLR, RAMP1 and RAMP2, but not RAMP3. The expression of CLR and RAMP1 indicates that hOB have functional receptors for CGRP insofar as CLR and RAMP1 seem to behave as a unitary complex acting as a CGRP receptor. In fact, the coexpression of the two proteins is required to transfer the receptor to the cell surface and for the endocytosis of the ligand-receptor complex after CGRP binding and receptor activation (15). The coexpression of CLR and RAMP2 gives rise to a receptor with greater affinity for adrenomedullin (23, 29), a member of the calcitonin peptide family, which, like CGRP, has mitogenic effects on osteoblasts (9). At variance with published findings of the expression of all three RAMPs in primary cultures of fetal rat osteoblasts (35), the lack of RAMP3 expression in hOB indicates that the expression of this family of proteins is species specific.

Our results also show that CGRP inhibits OPG production in hOB in a dose-dependent manner, and that this occurs at both mRNA and protein levels. Pretreatment with the CGRP receptor antagonist CGRP8–37 abolished this inhibition of OPG expression and production, thus indicating that the effects are specific and receptor mediated. The inhibitory action of CGRP requires the activation of the cAMP/PKA pathway, as suggested by the finding that impaired PKA activity prevented the action of CGRP, and the fact that the action of CGRP is mimicked by dBcAMP and forskolin further indicates that the ability of CGRP to inhibit OPG expression and production depends on the induction of cAMP and subsequent downstream events mediated by PKA.

Analysis of the human OPG promoter has revealed putative consensus cAMP response element-binding sites at positions –289 and –104 (4). Because cAMP response element-binding activity is regulated by protein kinases (such as PKA, calmodulin-dependent protein kinase, and MAPK), it is possible that, by activating the cAMP/PKA cascade, CGRP inhibits OPG gene expression by means of a direct genomic mechanism.

The suppression of OPG secretion was ~45%, and so it is possible that other intracellular pathways in addition to cAMP may be involved. Bergh et al. (4) have described a model in which the calcium signals generated by the regulatory activity of L-type voltage-sensitive channels can regulate OPG expression and secretion by means of calmodulin-sensitive protein kinase signaling; blocking the activity of these channels reduces the level of OPG expression and secretion in primary calvarial organ (4).

This study shows that the reduction of OPG secretion and expression induced by CGRP favors the development of multinucleated TRAP+ cells from PBMC, as expected from the actual understanding of the molecular triad OPG/RANK/RANKL (42). It follows that CGRP, by modulating at least one element of the triad, enhances the differentiation of committed precursors into mature cells to induce bone resorption. This pro-osteoclastogenic effect of CGRP may be the prerequisite to exert the generally acknowledged bone anabolic action of the peptide. It is indeed well established that the hierarchical nature of bone remodeling to increase bone mass implies osteoclast activation and formation as first mandatory events (33). CGRP appears therefore to be a member of the family of anabolic factors that displays a complex biphasic activation of bone remodeling. Other hormones that increase cAMP accumulation, such as PTH and the prostaglandins, reduce OPG expression in osteoblasts (5, 12, 40), and are simultaneously potent stimulators of bone formation (37). In fact, PTH inhibits OPG expression and stimulates RANKL production through PKA activation in murine bone marrow cells (26) thus leading to enhanced osteoclastogenesis (17). Similarly, prostaglandins activate bone resorption by stimulating RANKL expression (27), an effect mediated by the specific prostaglandin EP1 receptor (43).

Measurements of RANKL in hOB after exposure to CGRP may have given a better insight into the peptide's effect on the cross-talk between bone forming and bone resorbing cells but, as recently reported by others (41), neither the expression nor the secretion of RANKL reached detectable levels thus hampering the possibility of evaluating the cross-talk signal in coculture of human osteoblasts and osteoclasts.

The finding that CGRP favors osteoclast formation via its pro-osteoclastogenic effect on the OPG/RANK/RANKL triad apparently conflicts with its direct inhibitory action on bone resorption (1, 8, 21, 49). However, the cellular events (formation, activation, and survival of osteoclasts) involved in the bone resorption phases, are subjected to a complex of other factors, besides OPG/RANK/RANKL, that may have an impact on bone mass outcome at each remodeling cycle (42). Therefore, it can be suggested that CGRP by activating bone remodeling, contributes to the fine adjustment of this process, favoring the gain or loss of bone mass depending on the local demand. In particular, this modulatory function may be important in bone fractures, where an increase of CGRP-containing nerve fibers (18) and circulating CGRP levels have been shown (36). When bone formation must offset bone resorption, as in the case of bone repair with highly activated bone remodeling, CGRP activity may help fulfil the requirements of regeneration processes. The anabolic role of CGRP in bone is further supported by the fact that mice with the selective deletion of {alpha}-CGRP have osteopenia caused by a reduced rate of bone formation (38).

In conclusion, this study suggests that CGRP acts as a modulator of the OPG/RANKL/RANK system by reducing OPG release and expression by hOB, thus favoring osteoclast formation with subsequent activation of the bone remodeling events. CGRP may play a role in modulating the fine balance between bone resorption and bone formation, thus leading to the generally acknowledged anabolic outcome.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by Italian Ministry of Education, University and Research Grants FIRB 2001 and COFIN 2004.


   ACKNOWLEDGMENTS
 
We are grateful to Hoffmann-LaRoche (Basel, Switzerland) for the kind gift of 1,25(OH)2D3.


   FOOTNOTES
 

Address for reprint requests and other correspondence: F. Guidobono, Dept. of Pharmacology, Chemotherapy and Medical Toxicology, Univ. of Milan, Via Vanvitelli 32, 20129 Milan, Italy (e-mail: francesca.guidobono{at}unimi.it)

1 mRNA levels were quantified using the comparative threshold cycle (CT) method in which the reactions are characterized by the point in time during cycling when the amplification of a PCR product is first detected. The amount of target mRNA in each sample directly correlates with the CT. For each sample, the amount of the target mRNA, CT(target gene), was normalized to the amount of housekeeper mRNA, CT(GAPDH), designated as a calibrator, to give {Delta}CT (CTtarget gene – CTGAPDH). The amounts of target mRNA in the samples were expressed using the formula, 2–{Delta}{Delta}CT, where {Delta}{Delta}CT = {Delta}CT(treated sample) – {Delta}CT(untreated sample). Back

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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