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Inhibitory effect of CGRP on osteoclast formation by mouse bone marrow cells treated with isoproterenol
Neuroscience Letters 379 (2005) 47–51
Inhibitory effect of CGRP on osteoclast formation by mouse bone marrow cells treated with isoproterenol Kyoko Ishizukaa,b , Koji Hirukawaa , Hiroshi Nakamurab , Akifumi Togaria,∗ a
Department of Pharamacology, School of Dentistry, Aichi-Gakuin University, Nagoya 464-8650, Japan Department of Endodontics, School of Dentistry, Aichi-Gakuin University, Nagoya 464-8651, Japan
b
Received 4 November 2004; received in revised form 20 December 2004; accepted 20 December 2004
Abstract The present study was designed to elucidate the mode of action of isoproterenol (Isp; adrenergic -agonist) and to characterize the effect of the calcitonin gene-related peptide (CGRP; sensory neuropeptide) on osteoclast formation induced by Isp in a mouse bone marrow culture system. Treatment of mouse bone marrow cells with Isp generated tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells (MNCs) capable of excavating resorptive pits on dentine slices, and caused an increase in receptor activator of NF-B ligand (RANKL) and a decrease in osteoprotegerin (OPG) production by the marrow cells. The osteoclast formation was significantly inhibited by OPG, suggesting the involvement of the RANKL–RANK system. CGRP inhibited the osteoclast formation caused by Isp or soluble RANKL (s-RANKL) but had no influence on RANKL or OPG production by the bone marrow cells treated with Isp, suggesting that CGRP inhibited the osteoclast formation by interfering with the action of RANKL produced by the Isp-treated bone marrow cells without affecting RANKL or OPG production. This in vitro data suggest the physiological interaction of sympathetic and sensory nerves in osteoclastogenesis in vivo. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Osteoclastogenesis; Isoproterenol (Isp); Calcitonin gene-related peptide (CGRP); Receptor activator of NF-B ligand (RANKL); RANK; Osteoprotegerin (OPG)
Bone envelopes have been demonstrated to be innervated by both myelinated and unmyelinated fibers belonging to the sensory and sympathetic nervous systems. These sympathetic and sensory innervations are required for regulating bone metabolism under physiological conditions [2,5,7,9,24]. Although bone-resorbing activity has been reported to be modulated by sensory neuropeptides and norepinephrine, little attention has been given to the physiological interaction of sensory neuropeptides with norepinephrine with respect to osteoclastic activity and osteoclast formation. The recent discovery of RANKL–RANK interaction confirms the wellknown hypothesis that osteoblasts play an essential role in osteoclast differentiation [10]. Osteoblasts/stromal cells express RANKL as a membrane-associated factor and OPG as a decoy receptor for RANKL. Osteoclast precursors that express RANK, a receptor for RANKL, recognize RANKL ∗
Corresponding author. Tel.: +81 52 751 2561; fax: +81 52 752 5988. E-mail address: [email protected] (A. Togari).
0304-3940/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2004.12.046
through cell–cell interaction and differentiate into osteoclasts. This RANKL–RANK system has been reported to underlie the effect of osteotropic factors on osteoclast differentiation. Epinephrine has been reported to increase the expression of osteotrophic factors such as interleukin (IL)6, IL-11, prostaglandin (PG)-E2 , and RANKL in osteoblastic cells [11,22], as well as the formation of osteoclast-like cells from mouse bone marrow cells by activating adrenergic -receptors [22]. These findings suggest that the adrenergic stimulation induces osteoclastogenesis via the RANKL–RANK system in culture systems. On the other hand, CGRP has been demonstrated to inhibit bone-resorbing activity of isolated osteoclasts [1,13], calcium release in bone tissue culture [18,23], and osteoclast formation from bone marrow cells treated with 1,25(OH)2 D3 [4,17]. In this study, we elucidated the involvement of RANKL and OPG in the osteoclast formation from mouse bone marrow cells treated with Isp, an adrenergic -agonist, for 7 days, and characterized the effect of the sensory neuropeptide CGRP on Isp-
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induced osteoclastogenesis to gain a better understanding of the physiological interaction between the sympathetic and sensory nerves in bone resorption. Mouse bone marrow cells were obtained from tibiae of 6to 8-week-old ddY male mice (Japan SLC Inc., Hamamatsu, Japan) as previously described [20] in accordance with the Guidelines for Animal Experiments at the School of Dentistry, Aichi-Gakuin University. Marrow cells were cultured in alpha-modified minimum essential medium (␣-MEM; Invitrogen Co., Carlsbad, CA, USA) containing 10% fetal calf serum (Sigma Co., St. Louis, MO, USA) at 1.5 × 106 cells in 24-well plates in a humidified air with 5% CO2 at 37 ◦ C. One-half of the culture medium was exchanged for fresh medium every two days, and Isp (Sigma Co.), recombinant mouse RANKL (s-RANKL), which is extracellular domain of RANKL and has an ability to induce osteoclast differentiation, OPG (R&D Systems, Minneapolis, MN, USA) or CGRP (Peptide institute Inc., Osaka, Japan) was added at the beginning of cultures and at each change of medium. To estimate the osteoclast formation induced by Isp or s-RANKL, we counted the number of TRAP-positive MNCs by the following method: adherent cells were fixed in a mixture of 10% formalin in phosphate-buffered saline and ethanol–acetone (50:50, v/v), and stained for TRAP by incubating the cells in 0.1 M sodium acetate buffer (pH 5) containing naphthol AS-MX phosphate, and red violet LB salt in the presence of 10 mM sodium tartrate, as described previously [15]. Cells containing three or more nuclei were designated as MNCs. Moreover, to confirm the bone-resorbing capability of the TRAP-positive MNCs, we used six or eight cultures grown on dentine slices in each group. To estimate RANKL protein expression, we lysed bone marrow cells in 6-cm tissue culture dishes with lysis buffer containing 50 mM phosphate buffer, 0.1% Triton X-100, phenylmethylsulfonylfloride, and protease inhibitor cocktail (Sigma Co.). The RANKL proteins in the lysate were measured by using an ELISA kit (R&D Systems); and for quantitative analysis, RANKL was normalized to the total protein content, as determined by the Bradford method (BioRad Laboratories, Hercules, CA, USA). The amount of OPG in the conditioned medium was quantified by performing an ELISA using commercially available antibodies and recombinant mouse OPG (R&D Systems). Data are presented as means ± S.D. The statistical significance of differences between control and experimental group was determined using Student’s t-test. The effect of OPG on osteoclast formation was evaluated by examining the formation of TRAP-positive MNCs generated by the treatment of bone marrow cells with Isp in the presence or absence of OPG. As shown in Fig. 1A, treatment with Isp (1 M) for 7 days significantly increased the formation of TRAP-positive MNCs. OPG (10 ng/ml) inhibited this osteoclast formation generated by Isp (1 M). The boneresorbing activity of osteoclasts was also evaluated based on the formation of pits on dentine slices. Osteoclasts newly generated by Isp (1 M) were capable of excavating resorptive
Fig. 1. Effect of isoproterenol on osteoclastogenesis and on the production of RANKL and OPG in mouse bone marrow cells. Mouse bone marrow cells were treated with vehicle (a), Isp (1 M; b), OPG (10 ng/ml; c) or Isp and OPG (d) for 7 days. (A) TRAP-positive MNC formation from mouse bone marrow cells. Bar = 2.0 mm. (B) Bone-resorbing activity of TRAPpositive MNCs. Bar = 1.0 mm. (C) Time-course of the effect of Isp (1 M) on RANKL production. (D) Time-course of the effect of Isp (1 M) on OPG production. The accumulation of RANKL and of OPG was determined by ELISA assays; and for quantitative analysis, RANKL was normalized to the total protein content. Values are the mean ± S.D. (n = 3). () Control, () 1 M Isp. Significantly different from the control values at * P < 0.01, and ** P < 0.001, respectively.
pits on dentine slices, but no excavated pits were observed when OPG (10 ng/ml) was also present (Fig. 1B). The timecourse of the effect of Isp (1 M) on osteoclast formation showed a sharp increase between days 4 and 7 in the cultures of mouse bone marrow cells (data not shown). Fig. 1C and D shows the time-course of alteration of both RANKL and OPG production in mouse bone marrow cells treated with Isp. Isp (1 M) caused a significant increase in RANKL production at days 2, 4 and 7, with a 192% increase at day 4. It also caused a significant decrease in OPG production, with a 33% inhibition at day 4.
K. Ishizuka et al. / Neuroscience Letters 379 (2005) 47–51
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Fig. 3. Effect of CGRP on the s-RANKL-induced TRAP-positive MNC formation. Mouse bone marrow cells were treated with vehicle (a), s-RANKL (1 ng/ml; b), CGRP (0.1 M; c) or s-RANKL and CGRP (d) for 7 days. Mouse bone marrow cells cultured for 7 days were fixed and stained for TRAP-positive MNCs (A), and the TRAP-positive MNCs were counted (B). Bar = 2.0 mm. Values are the mean ± S.D. (n = 8). Significantly different from the value for the cultures without s-RANKL at ** P < 0.001.
Fig. 2. Effect of CGRP on the Isp-induced increase in TRAP-positive MNC formation (A) and RANKL production (B) and on the decrease in OPG production (C). Mouse bone marrow cells were treated with vehicle or Isp (1 M) in the presence or absence of CGRP (0.1 M). (A) Mouse bone marrow cells cultured for 7 days were fixed and stained for TRAP-positive MNCs; and the TRAP-positive MNCs were then counted. Values are the mean ± S.D. (n = 8). The accumulation of RANKL (B) and of OPG (C) at 4 days was determined by ELISA assays; and for quantitative analysis, RANKL was normalized to the total protein content. Values are the mean ± S.D. (n = 6). Significantly different from the values of cultures without Isp at ** P < 0.001.
Next, the effect of the sensory neuropeptide CGRP on the osteoclast formation induced by Isp was examined in the mouse bone marrow culture system. As shown in Fig. 2A, in bone marrow cells treated with CGRP (0.1 M), Isp (1 M) could not induce the osteoclast formation. To elucidate the mode of action of CGRP, we examined the effect of CGRP on RANKL and OPG production in mouse bone marrow cells treated with Isp. As shown in Fig. 2B and C, in spite of the presence of CGRP, treatment with Isp (1 M) for 4 days significantly increased RANKL production and decreased OPG production in the bone marrow cells. Thus, although CGRP
significantly inhibited osteoclast formation, it did not alter RANKL or OPG production. To evaluate whether or not the action of CGRP on the osteoclast formation induced by Isp occurred by the direct action of the peptide on osteoclast precursor cells, we examined the effect of CGRP on osteoclast formation induced by s-RANKL. As shown in Fig. 3, the osteoclast formation induced by s-RANKL (1 ng/ml) was not observed in cells treated with CGRP (0.1 M) for 7 days. The present study demonstrated that treatment of mouse bone marrow cells with Isp generated osteoclast-like cells with bone-resorbing activity and that the osteoclast formation was not observed in the presence of OPG, a decoy receptor for RANKL. The osteoclast formation was also associated with an increase in RANKL production and a decrease in that of OPG. Osteoclast precursors are known to express RANK, a receptor that recognizes RANKL through cell–cell interaction and whose engagement causes the cells to differentiate into osteoclasts [10]. The evidence suggests that Isp generated osteoclasts from mouse bone marrow cells via RANKL–RANK interaction. Similarly, 1,25(OH)2 D3 has been shown to upregulate the mRNA expression of RANKL and to downregulate that of OPG in mouse bone marrow cultures for 7 days [16]. From these results, it may be concluded
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that the RANKL–RANK system is involved in osteoclastogenesis generated by Isp as well as by 1,25(OH)2 D3 in the bone marrow culture system. In the present study, the osteoclast formation induced by Isp was inhibited by CGRP, a sensory neuropeptide acting on osteoblastic and osteoclastic cells. CGRP has been demonstrated to inhibit bone resorption by isolated osteoclasts [1,13], and to inhibit osteoclastic bone resorption in bone tissue culture [18,23]. In mouse bone marrow cultures stimulated to generate osteoclasts by using 1,25(OH)2 D3 , Cornish et al. [4] demonstrated that CGRP acts at multiple stages in the osteoclast lineage, inhibiting the development of precursors as well as decreasing the fusion of committed precursors to form multinucleate cells. RANKL, expressed by osteoblasts/stromal cells, is known to stimulate the proliferation, differentiation, and fusion of osteoclast precursor cells [10]. On the other hand, CGRP has been reported to stimulate the proliferation of osteoblastic cells and the production of insulin-like growth factor in subconfluent primary fetal rat osteoblasts [25] while suppressing cytokine production [14]. These findings prompted us to investigate whether or not osteoblastic/stromal cells were involved in the effect of CGRP on the osteoclast formation induced by Isp. Although Isp increased the production of RANKL and decreased that of OPG in mouse bone marrow cells, CGRP had no influence on RANKL or OPG production. We also observed a significant inhibition of osteoclast formation by s-RANKL. These results suggest that CGRP directly inhibited the action of RANKL, expressed on mouse bone marrow cells by Isp, on osteoclast formation, without affecting RANKL and OPG production by osteoblasts/stromal cells. Since the mRNA encoding the calcitonin receptor-like receptor component of the CGRP1 has been reported to express in mature osteoclasts [24], it may be thought that CGRP is working through on the osteoclast precursors, pre-osteoclasts, and mature osteoclast. Recent studies in vivo indicated that sympathetic stimulation caused bone loss [21], and increased the synthesis in murine bone of IL-6 and PGE2 [8,12], well-known modulators of bone metabolism that act by regulating the development and function of osteoclasts and osteoblasts. Using guanethidine, which specifically destroys sympathetic adrenergic fibers, Cherruau et al. [3] showed that inactivation of the sympathetic system in adult rats significantly decreased both osteoclast number and activity in a synchronized model of bone resorption. They also reported inhibition of bone resorption with a concomitant increase in the number of CGRPimmunoreactive nerve fibers after sympathectomy. These in vivo findings suggest that bone resorption is increased by the activation of sympathetic nerves and decreased by the activation of sensory nerves. Our in vitro findings suggest that adrenergic stimulation of osteoclastogenesis by bone marrow cells is regulated negatively by the sensory neuropeptide CGRP, at least in part. However, conflicting results have been obtained for bone resorption after inactivation of the sympathetic system [6,19]. Further work is needed to determine the
physiological interaction between sympathetic and sensory nerves in bone resorption. In conclusion, Isp, a -adrenergic agonist, generated osteoclasts from mouse bone marrow cells via the RANKL–RANK system. CGRP, a sensory neuropeptide, significantly inhibited this RANKL-mediated osteoclastogenesis by interfering with the action of RANKL in osteoclast lineage cells.
Acknowledgments This study was partly supported by a grant-in-aid under the AGU High-Tech Research Center Project and by a grant-inaid from the Ministry of Education, Culture, Sports, Science and Technology of Japan (No. 14571782 to A.T.).
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Inhibitory effect of CGRP on osteoclast formation by mouse bone marrow cells treated with isoproterenol Kyoko Ishizukaa,b , Koji Hirukawaa , Hiroshi Nakamurab , Akifumi Togaria,∗ a
Department of Pharamacology, School of Dentistry, Aichi-Gakuin University, Nagoya 464-8650, Japan Department of Endodontics, School of Dentistry, Aichi-Gakuin University, Nagoya 464-8651, Japan
b
Received 4 November 2004; received in revised form 20 December 2004; accepted 20 December 2004
Abstract The present study was designed to elucidate the mode of action of isoproterenol (Isp; adrenergic -agonist) and to characterize the effect of the calcitonin gene-related peptide (CGRP; sensory neuropeptide) on osteoclast formation induced by Isp in a mouse bone marrow culture system. Treatment of mouse bone marrow cells with Isp generated tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells (MNCs) capable of excavating resorptive pits on dentine slices, and caused an increase in receptor activator of NF-B ligand (RANKL) and a decrease in osteoprotegerin (OPG) production by the marrow cells. The osteoclast formation was significantly inhibited by OPG, suggesting the involvement of the RANKL–RANK system. CGRP inhibited the osteoclast formation caused by Isp or soluble RANKL (s-RANKL) but had no influence on RANKL or OPG production by the bone marrow cells treated with Isp, suggesting that CGRP inhibited the osteoclast formation by interfering with the action of RANKL produced by the Isp-treated bone marrow cells without affecting RANKL or OPG production. This in vitro data suggest the physiological interaction of sympathetic and sensory nerves in osteoclastogenesis in vivo. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Osteoclastogenesis; Isoproterenol (Isp); Calcitonin gene-related peptide (CGRP); Receptor activator of NF-B ligand (RANKL); RANK; Osteoprotegerin (OPG)
Bone envelopes have been demonstrated to be innervated by both myelinated and unmyelinated fibers belonging to the sensory and sympathetic nervous systems. These sympathetic and sensory innervations are required for regulating bone metabolism under physiological conditions [2,5,7,9,24]. Although bone-resorbing activity has been reported to be modulated by sensory neuropeptides and norepinephrine, little attention has been given to the physiological interaction of sensory neuropeptides with norepinephrine with respect to osteoclastic activity and osteoclast formation. The recent discovery of RANKL–RANK interaction confirms the wellknown hypothesis that osteoblasts play an essential role in osteoclast differentiation [10]. Osteoblasts/stromal cells express RANKL as a membrane-associated factor and OPG as a decoy receptor for RANKL. Osteoclast precursors that express RANK, a receptor for RANKL, recognize RANKL ∗
Corresponding author. Tel.: +81 52 751 2561; fax: +81 52 752 5988. E-mail address: [email protected] (A. Togari).
0304-3940/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2004.12.046
through cell–cell interaction and differentiate into osteoclasts. This RANKL–RANK system has been reported to underlie the effect of osteotropic factors on osteoclast differentiation. Epinephrine has been reported to increase the expression of osteotrophic factors such as interleukin (IL)6, IL-11, prostaglandin (PG)-E2 , and RANKL in osteoblastic cells [11,22], as well as the formation of osteoclast-like cells from mouse bone marrow cells by activating adrenergic -receptors [22]. These findings suggest that the adrenergic stimulation induces osteoclastogenesis via the RANKL–RANK system in culture systems. On the other hand, CGRP has been demonstrated to inhibit bone-resorbing activity of isolated osteoclasts [1,13], calcium release in bone tissue culture [18,23], and osteoclast formation from bone marrow cells treated with 1,25(OH)2 D3 [4,17]. In this study, we elucidated the involvement of RANKL and OPG in the osteoclast formation from mouse bone marrow cells treated with Isp, an adrenergic -agonist, for 7 days, and characterized the effect of the sensory neuropeptide CGRP on Isp-
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induced osteoclastogenesis to gain a better understanding of the physiological interaction between the sympathetic and sensory nerves in bone resorption. Mouse bone marrow cells were obtained from tibiae of 6to 8-week-old ddY male mice (Japan SLC Inc., Hamamatsu, Japan) as previously described [20] in accordance with the Guidelines for Animal Experiments at the School of Dentistry, Aichi-Gakuin University. Marrow cells were cultured in alpha-modified minimum essential medium (␣-MEM; Invitrogen Co., Carlsbad, CA, USA) containing 10% fetal calf serum (Sigma Co., St. Louis, MO, USA) at 1.5 × 106 cells in 24-well plates in a humidified air with 5% CO2 at 37 ◦ C. One-half of the culture medium was exchanged for fresh medium every two days, and Isp (Sigma Co.), recombinant mouse RANKL (s-RANKL), which is extracellular domain of RANKL and has an ability to induce osteoclast differentiation, OPG (R&D Systems, Minneapolis, MN, USA) or CGRP (Peptide institute Inc., Osaka, Japan) was added at the beginning of cultures and at each change of medium. To estimate the osteoclast formation induced by Isp or s-RANKL, we counted the number of TRAP-positive MNCs by the following method: adherent cells were fixed in a mixture of 10% formalin in phosphate-buffered saline and ethanol–acetone (50:50, v/v), and stained for TRAP by incubating the cells in 0.1 M sodium acetate buffer (pH 5) containing naphthol AS-MX phosphate, and red violet LB salt in the presence of 10 mM sodium tartrate, as described previously [15]. Cells containing three or more nuclei were designated as MNCs. Moreover, to confirm the bone-resorbing capability of the TRAP-positive MNCs, we used six or eight cultures grown on dentine slices in each group. To estimate RANKL protein expression, we lysed bone marrow cells in 6-cm tissue culture dishes with lysis buffer containing 50 mM phosphate buffer, 0.1% Triton X-100, phenylmethylsulfonylfloride, and protease inhibitor cocktail (Sigma Co.). The RANKL proteins in the lysate were measured by using an ELISA kit (R&D Systems); and for quantitative analysis, RANKL was normalized to the total protein content, as determined by the Bradford method (BioRad Laboratories, Hercules, CA, USA). The amount of OPG in the conditioned medium was quantified by performing an ELISA using commercially available antibodies and recombinant mouse OPG (R&D Systems). Data are presented as means ± S.D. The statistical significance of differences between control and experimental group was determined using Student’s t-test. The effect of OPG on osteoclast formation was evaluated by examining the formation of TRAP-positive MNCs generated by the treatment of bone marrow cells with Isp in the presence or absence of OPG. As shown in Fig. 1A, treatment with Isp (1 M) for 7 days significantly increased the formation of TRAP-positive MNCs. OPG (10 ng/ml) inhibited this osteoclast formation generated by Isp (1 M). The boneresorbing activity of osteoclasts was also evaluated based on the formation of pits on dentine slices. Osteoclasts newly generated by Isp (1 M) were capable of excavating resorptive
Fig. 1. Effect of isoproterenol on osteoclastogenesis and on the production of RANKL and OPG in mouse bone marrow cells. Mouse bone marrow cells were treated with vehicle (a), Isp (1 M; b), OPG (10 ng/ml; c) or Isp and OPG (d) for 7 days. (A) TRAP-positive MNC formation from mouse bone marrow cells. Bar = 2.0 mm. (B) Bone-resorbing activity of TRAPpositive MNCs. Bar = 1.0 mm. (C) Time-course of the effect of Isp (1 M) on RANKL production. (D) Time-course of the effect of Isp (1 M) on OPG production. The accumulation of RANKL and of OPG was determined by ELISA assays; and for quantitative analysis, RANKL was normalized to the total protein content. Values are the mean ± S.D. (n = 3). () Control, () 1 M Isp. Significantly different from the control values at * P < 0.01, and ** P < 0.001, respectively.
pits on dentine slices, but no excavated pits were observed when OPG (10 ng/ml) was also present (Fig. 1B). The timecourse of the effect of Isp (1 M) on osteoclast formation showed a sharp increase between days 4 and 7 in the cultures of mouse bone marrow cells (data not shown). Fig. 1C and D shows the time-course of alteration of both RANKL and OPG production in mouse bone marrow cells treated with Isp. Isp (1 M) caused a significant increase in RANKL production at days 2, 4 and 7, with a 192% increase at day 4. It also caused a significant decrease in OPG production, with a 33% inhibition at day 4.
K. Ishizuka et al. / Neuroscience Letters 379 (2005) 47–51
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Fig. 3. Effect of CGRP on the s-RANKL-induced TRAP-positive MNC formation. Mouse bone marrow cells were treated with vehicle (a), s-RANKL (1 ng/ml; b), CGRP (0.1 M; c) or s-RANKL and CGRP (d) for 7 days. Mouse bone marrow cells cultured for 7 days were fixed and stained for TRAP-positive MNCs (A), and the TRAP-positive MNCs were counted (B). Bar = 2.0 mm. Values are the mean ± S.D. (n = 8). Significantly different from the value for the cultures without s-RANKL at ** P < 0.001.
Fig. 2. Effect of CGRP on the Isp-induced increase in TRAP-positive MNC formation (A) and RANKL production (B) and on the decrease in OPG production (C). Mouse bone marrow cells were treated with vehicle or Isp (1 M) in the presence or absence of CGRP (0.1 M). (A) Mouse bone marrow cells cultured for 7 days were fixed and stained for TRAP-positive MNCs; and the TRAP-positive MNCs were then counted. Values are the mean ± S.D. (n = 8). The accumulation of RANKL (B) and of OPG (C) at 4 days was determined by ELISA assays; and for quantitative analysis, RANKL was normalized to the total protein content. Values are the mean ± S.D. (n = 6). Significantly different from the values of cultures without Isp at ** P < 0.001.
Next, the effect of the sensory neuropeptide CGRP on the osteoclast formation induced by Isp was examined in the mouse bone marrow culture system. As shown in Fig. 2A, in bone marrow cells treated with CGRP (0.1 M), Isp (1 M) could not induce the osteoclast formation. To elucidate the mode of action of CGRP, we examined the effect of CGRP on RANKL and OPG production in mouse bone marrow cells treated with Isp. As shown in Fig. 2B and C, in spite of the presence of CGRP, treatment with Isp (1 M) for 4 days significantly increased RANKL production and decreased OPG production in the bone marrow cells. Thus, although CGRP
significantly inhibited osteoclast formation, it did not alter RANKL or OPG production. To evaluate whether or not the action of CGRP on the osteoclast formation induced by Isp occurred by the direct action of the peptide on osteoclast precursor cells, we examined the effect of CGRP on osteoclast formation induced by s-RANKL. As shown in Fig. 3, the osteoclast formation induced by s-RANKL (1 ng/ml) was not observed in cells treated with CGRP (0.1 M) for 7 days. The present study demonstrated that treatment of mouse bone marrow cells with Isp generated osteoclast-like cells with bone-resorbing activity and that the osteoclast formation was not observed in the presence of OPG, a decoy receptor for RANKL. The osteoclast formation was also associated with an increase in RANKL production and a decrease in that of OPG. Osteoclast precursors are known to express RANK, a receptor that recognizes RANKL through cell–cell interaction and whose engagement causes the cells to differentiate into osteoclasts [10]. The evidence suggests that Isp generated osteoclasts from mouse bone marrow cells via RANKL–RANK interaction. Similarly, 1,25(OH)2 D3 has been shown to upregulate the mRNA expression of RANKL and to downregulate that of OPG in mouse bone marrow cultures for 7 days [16]. From these results, it may be concluded
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that the RANKL–RANK system is involved in osteoclastogenesis generated by Isp as well as by 1,25(OH)2 D3 in the bone marrow culture system. In the present study, the osteoclast formation induced by Isp was inhibited by CGRP, a sensory neuropeptide acting on osteoblastic and osteoclastic cells. CGRP has been demonstrated to inhibit bone resorption by isolated osteoclasts [1,13], and to inhibit osteoclastic bone resorption in bone tissue culture [18,23]. In mouse bone marrow cultures stimulated to generate osteoclasts by using 1,25(OH)2 D3 , Cornish et al. [4] demonstrated that CGRP acts at multiple stages in the osteoclast lineage, inhibiting the development of precursors as well as decreasing the fusion of committed precursors to form multinucleate cells. RANKL, expressed by osteoblasts/stromal cells, is known to stimulate the proliferation, differentiation, and fusion of osteoclast precursor cells [10]. On the other hand, CGRP has been reported to stimulate the proliferation of osteoblastic cells and the production of insulin-like growth factor in subconfluent primary fetal rat osteoblasts [25] while suppressing cytokine production [14]. These findings prompted us to investigate whether or not osteoblastic/stromal cells were involved in the effect of CGRP on the osteoclast formation induced by Isp. Although Isp increased the production of RANKL and decreased that of OPG in mouse bone marrow cells, CGRP had no influence on RANKL or OPG production. We also observed a significant inhibition of osteoclast formation by s-RANKL. These results suggest that CGRP directly inhibited the action of RANKL, expressed on mouse bone marrow cells by Isp, on osteoclast formation, without affecting RANKL and OPG production by osteoblasts/stromal cells. Since the mRNA encoding the calcitonin receptor-like receptor component of the CGRP1 has been reported to express in mature osteoclasts [24], it may be thought that CGRP is working through on the osteoclast precursors, pre-osteoclasts, and mature osteoclast. Recent studies in vivo indicated that sympathetic stimulation caused bone loss [21], and increased the synthesis in murine bone of IL-6 and PGE2 [8,12], well-known modulators of bone metabolism that act by regulating the development and function of osteoclasts and osteoblasts. Using guanethidine, which specifically destroys sympathetic adrenergic fibers, Cherruau et al. [3] showed that inactivation of the sympathetic system in adult rats significantly decreased both osteoclast number and activity in a synchronized model of bone resorption. They also reported inhibition of bone resorption with a concomitant increase in the number of CGRPimmunoreactive nerve fibers after sympathectomy. These in vivo findings suggest that bone resorption is increased by the activation of sympathetic nerves and decreased by the activation of sensory nerves. Our in vitro findings suggest that adrenergic stimulation of osteoclastogenesis by bone marrow cells is regulated negatively by the sensory neuropeptide CGRP, at least in part. However, conflicting results have been obtained for bone resorption after inactivation of the sympathetic system [6,19]. Further work is needed to determine the
physiological interaction between sympathetic and sensory nerves in bone resorption. In conclusion, Isp, a -adrenergic agonist, generated osteoclasts from mouse bone marrow cells via the RANKL–RANK system. CGRP, a sensory neuropeptide, significantly inhibited this RANKL-mediated osteoclastogenesis by interfering with the action of RANKL in osteoclast lineage cells.
Acknowledgments This study was partly supported by a grant-in-aid under the AGU High-Tech Research Center Project and by a grant-inaid from the Ministry of Education, Culture, Sports, Science and Technology of Japan (No. 14571782 to A.T.).
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