From the Department of Medicine and Clinical Science,
Kyoto University Graduate School of Medicine, 54 Shogoin
Kawahara-cho, Sakyo-ku, Kyoto 606, Japan and the ¶ Department of
Anatomy and Developmental Biology, Kyoto University Graduate School of
Medicine, Yoshida Konoe-cho, Sakyo-ku, Kyoto 606, Japan
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ABSTRACT |
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The natriuretic peptide family consists of three structurally related endogenous ligands: atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP). The biological actions of natriuretic peptides are thought to be mediated through the activation of two guanylyl cyclase (GC)-coupled receptor subtypes (GC-A and GC-B). In this study, we examined the effects of ANP and CNP, which are endogenous ligands for GC-A and GC-B, respectively, on bone growth using an organ culture of fetal mouse tibias, an in vitro model of endochondral ossification. CNP increased the cGMP production much more potently than ANP, thereby resulting in an increase in the total longitudinal bone length. Histological examination revealed an increase in the height of the proliferative and hypertrophic chondrocyte zones in fetal mouse tibias treated with CNP. The natriuretic peptide stimulation of bone growth, which was mimicked by 8-bromo-cGMP, was inhibited by HS-142-1, a non-peptide GC-coupled natriuretic peptide receptor antagonist. The spontaneous increase in the total longitudinal bone growth and cGMP production was also inhibited significantly by HS-142-1. CNP mRNA was expressed abundantly in fetal mouse tibias, where no significant amounts of ANP and BNP mRNAs were detected. A considerable amount of GC-B mRNA was present in fetal mouse tibias. This study suggests the physiologic significance of the CNP/GC-B pathway in the process of endochondral ossification.
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INTRODUCTION |
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Endochondral ossification requires the sequential formation and degradation of cartilaginous structures that serve as molds for the developing bones (1). Longitudinal bone growth is determined by the process of endochondral ossification in the cartilaginous growth plate, which is located at both ends of vertebrae and long bones (2). The control mechanisms that allow for coordinated bone growth throughout the body are poorly understood, but probably involve many systemic hormones and local regulators.
Natriuretic peptides organize a family of three structurally related
peptides: atrial natriuretic peptide
(ANP),1 brain natriuretic
peptide (BNP), and C-type natriuretic peptide (CNP) (3). ANP and BNP
act as cardiac hormones that are produced predominantly by the atrium
and ventricle, respectively (4-8). CNP occurs in a wide variety of
tissues (9-12), where it acts as a neuropeptide as well as a local
regulator. The biological actions of natriuretic peptides are thought
to be mediated by intracellular accumulation of cGMP through the
activation of particulate guanylyl cyclases (13). Two subtypes of
guanylyl cyclase (GC)-coupled natriuretic peptide receptors (GC-A and
GC-B) have been cloned so far (13). The rank order of ligand
selectivity for GC-A and GC-B is ANP BNP
CNP and CNP > ANP
BNP, respectively (14, 15). Thus, ANP and BNP are
thought to be endogenous ligands for GC-A, whereas CNP is selective for
GC-B. A third natriuretic peptide receptor has been implicated in the
metabolic clearance of ligands and thereafter named the clearance
receptor (C-receptor) (16). Recent studies using transgenic mice with
overexpression of ANP or BNP (17, 18) and mice with disruption of ANP
or GC-A (19, 20) showed that natriuretic peptides play critical roles
in the regulation of body fluid homeostasis and blood pressure control.
It is well recognized that natriuretic peptide receptors are expressed not only in the cardiovascular system, but in a variety of extracardiovascular tissues (13, 15), suggesting that natriuretic peptides play important roles outside the cardiovascular system as well. Evidence has accumulated indicating that natriuretic peptides modulate cellular functions in several cell lineages in vitro. Previous studies using primary cultures of osteoblast-like cells and chondrocytes and osteoclast-containing bone marrow cultures or cultured cell lines such as osteoblastic MC3T3-E1 cells revealed that natriuretic peptides can regulate the proliferation and differentiation of osteoblasts, chondrocytes, and osteoclasts (21-25). Recently, we observed the longitudinal growth of vertebrae and long bones from transgenic mice with overexpression of BNP in the liver and suggested that natriuretic peptides activate chondrogenesis in the process of endochondral ossification (26). However, the molecular mechanisms for natriuretic peptide regulation of bone formation in vivo still remain elusive. In this study, we examined the effects of natriuretic peptides on bone formation using an organ culture of fetal mouse tibias, an in vitro model system with which to assess the process of endochondral ossification (27). We also examined expression of mRNAs for natriuretic peptides and their receptors in fetal mouse tibias.
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EXPERIMENTAL PROCEDURES |
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Materials-- Mouse ANP and CNP were purchased from Peptide Institute, Inc. (Minoh, Japan). C-ANF-(4-23), a synthetic ring-deleted analog of ANP (16) that binds with a high affinity to the C-receptor, was donated by Dr. T. Maack (Cornell University Medical College, New York). HS-142-1, a non-peptide GC-coupled natriuretic peptide receptor antagonist (28), was a generous gift of Dr. S. Nakanishi (Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., Tokyo).
Organ Culture-- Pregnant female ICR mice were purchased from Shimizu Experimental Supplies (Kyoto, Japan). Between 10 and 12 h a.m. on day 16 of pregnancy, mice were killed by cervical dislocation. Bilateral tibias were dissected aseptically from mouse fetuses. Fetuses significantly smaller or bigger compared with the average size were discarded. Tibias were left intact, and care was taken not to damage the perichondrium. Organ culture of fetal mouse tibias was performed by the suspension culture technique in a chemically defined medium (Bigger's BJG medium, Life Technologies, Inc.) containing 50 units/ml penicillin and 50 µg/ml streptomycin (Life Technologies, Inc.) (29). No serum was added to the medium. Tibial explants were incubated in a 50-ml penicillin bottle with 10 ml of the culture medium. Four to eight explants were incubated in one bottle. The bottle was sealed airtight with a rubber stopper and a metal clamp. The bottle was flushed for ~2 min with a gas mixture of 95% atmosphere and 5% CO2 using a 23-gauge syringe needle. A second needle was used to balance the gas pressure in the bottle. Bottles were incubated at 37 °C on a roller device (Taiyo Scientific Industrial Co., Tokyo) at a speed of 20-25 rpm. The culture bottles were flushed every 24 h with the same gas mixture. Organ culture of fetal mouse tibias was continued for 6 days with ANP, CNP, 8-bromo-cGMP (8-Br-cGMP), HS-142-1, and C-ANF-(4-23) at the indicated doses.
Morphometric Analysis-- Before and after the 6-day culture, fetal mouse tibias were measured using a linear ocular scale mounted on a dissecting microscope at 10× magnification (see Fig. 1A). The maximal longitudinal length of the whole long bone was measured as the total length, and maximal longitudinal lengths of proximal and distal cartilaginous primordia (CP) and the osteogenic center (OC) were also determined by measuring the length of the developing light zone of chondrocytes as well as the dark zone of calcification.
Histology--
Fetal mouse tibias were fixed in 4%
paraformaldehyde in phosphate-buffered saline for 24 h and
embedded in paraffin. Five-µm-thick sections were cut from
paraffin-embedded specimens, stained with Alcian blue (pH 2.5), and
counterstained with hematoxylin/eosin. Immunohistochemistry for type X
collagen was performed using a polyclonal rabbit antiserum (500-fold
dilution; LSL, Tokyo) as a primary antibody as described (30).
Immunoreactions were visualized by a biotinylated anti-polyvalent
antibody, a streptavidin-biotin-horseradish peroxidase complex, and
diaminobenzidine (Sensi Tek HRP (anti-polyvalent) Ready-to-Use kit,
SCYTEK, Logan, UT). Immunohistochemical reactivity with nonimmune serum
was used as control. For bromodeoxyuridine (BrdUrd) staining, after the
3-day culture, fetal mouse tibias were incubated with 105
M BrdUrd (Sigma) for 6 h. Immunohistochemical staining
of incorporated BrdUrd was performed using the 5-bromo-2'-deoxyuridine
labeling and detection kit II (Boehringer Mannheim GmbH, Mannheim,
Germany) according to the manufacturer's protocol.
cGMP Measurements-- Fetal mouse tibias were preincubated for 30 min in Bigger's BJG medium (one tibia/500 µl/24-well plate) containing 0.5 mM isobutylmethylxanthine and 6 mg/ml bovine serum albumin, after which they were treated for 45 min with ANP and CNP at the indicated doses. After adding 500 µl of 12% trichloroacetic acid, tibias were homogenized with a Physcotron® homogenizer (NITI-ON Medical Supply, Chiba, Japan). The cGMP concentrations were determined using a radioimmunoassay as described (15).
RNA Extraction and Northern Blot Analysis-- Total RNA was extracted from fetal mouse tibias, adult mouse brains and lungs, cultured rat aortic smooth muscle cells, and rat pheochromocytoma PC12 cells by the acid guanidinium/phenol/chloroform method (8, 15). Bones were removed microscopically to avoid the contamination of surrounding tissues. The tibial epiphysis (or CP) and diaphysis (or OC) were also separated carefully. Poly(A)+ RNA was purified using OligotexTM dT30 <super> (Japan Synthetic Rubber, Tokyo). After reverse transcription of 10 µg of total RNA from mouse brain, the 383-base pair mouse GC-A and 634-base pair mouse GC-B cDNA fragments were obtained by polymerase chain reaction with primers (5'-AGCTAAAACTCTTGGCTGACAAGA-3' (sense) and 5'-ACATTTAGGGATGTCAGGAGGTGG-3' (antisense) for mouse GC-A and 5'-GGAGATGCTTCATGAGATCCTGCTTCA-3' (sense) and 5'-AAAGGCACAGGGGGGGTTGTCCAAAGG-3' (antisense) for rat GC-B) selected to amplify sequences corresponding to the extracellular regions of mouse GC-A and rat GC-B (31, 32). Northern blot analysis was performed as described (8) using the mouse ANP and CNP genomic fragments (33, 34), the mouse BNP cDNA fragment (18), the mouse GC-A and GC-B cDNA fragments, and the rat C-receptor cDNA fragment (15) as probes.
Statistical Analysis-- Data were expressed as the mean ± S.D. of the indicated number of individual cultures (n = 5-8). The statistical significance of differences in mean values was assessed by Student's t test. Differences among means were considered significant as values of p < 0.05 and p < 0.01.
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RESULTS |
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Organ Culture-- Using the organ culture of fetal mouse tibias, an in vitro model of endochondral ossification (Fig. 1A) (27), we examined the effects of natriuretic peptides on longitudinal bone growth. Without treatment of natriuretic peptides, the total longitudinal length of fetal mouse tibias was increased time-dependently during the course of the 6-day culture (45% relative to that before the culture) (p < 0.01) (Fig. 1B), as reported previously (27). After the 6-day culture, the lengths of CP and OC were also increased by 65 and 25%, respectively, relative to those before the culture (p < 0.01).
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Effects of ANP and CNP on Bone Growth and cGMP
Production--
Fig. 2A shows
a typical gross appearance of cultured fetal mouse tibias treated with
107 M ANP or CNP for 6 days. Both ANP and CNP
increased the total longitudinal length of mouse tibias significantly
and dose-dependently compared with vehicle-treated groups
(Fig. 2B). Treatment of mouse tibias with 10
7
M CNP for 6 days produced an ~25% increase in the total
longitudinal bone length compared with vehicle-treated groups
(p < 0.01). CNP was much more potent than ANP in
increasing the total longitudinal length of cultured fetal mouse
tibias. CNP at a dose of 10
7 M increased the
length of CP relative to vehicle-treated groups (~35% increase)
(p < 0.01), whereas ANP at the same dose produced only
a 15% increase in the length of CP (p < 0.05). No
significant increases in the length of OC were observed in cultured
fetal mouse tibias treated with ANP or CNP at the doses tested.
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Effects of cGMP on Bone Growth--
Treatment of mouse tibias with
107 to 10
4 M 8-Br-cGMP for 6 days increased the total bone growth significantly and
dose-dependently (p < 0.01) (Fig.
3). Treatment with 10
4
M 8-Br-cGMP showed an ~25% increase in the total
longitudinal bone length relative to vehicle-treated groups. The
effects of 8-Br-cGMP were found in CP, but not in OC. The length of CP
of mouse tibias treated with 10
4 M 8-Br-cGMP
was increased significantly (~50%) compared with that of
vehicle-treated tibias (p < 0.01). Histological
changes observed in fetal mouse tibias treated with 10
4
M 8-Br-cGMP were essentially the same as those observed
when treated with 10
7 M CNP (data not
shown).
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Effects of HS-142-1 on Bone Growth and cGMP Production-- HS-142-1 decreased dose-dependently a spontaneous increase in the total longitudinal bone growth (Fig. 4A). HS-142-1 at a dose of 50 mg/liter inhibited significantly the longitudinal bone growth (~40% of vehicle-treated groups) (p < 0.01). Only the length of CP was affected by treatment with HS-142-1. No significant effects were observed in the length of OC when treated with HS-142-1. HS-142-1 also decreased dose-dependently the CNP-induced increase in the total longitudinal bone growth (~35% of vehicle-treated groups when treated with 50 mg/liter HS-142-1) (Fig. 4A). HS-142-1 decreased significantly and dose-dependently the CNP-induced increase in the length of CP, but not that in the length of OC. HS-142-1 at a dose of 50 mg/liter inhibited significantly the length of CP (~35% of vehicle-treated groups) (p < 0.01).
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Effects of C-ANF-(4-23) on Bone Growth--
The effects of
C-ANF-(4-23), the C-receptor agonist (16), on bone growth were also
examined (Fig. 5). Treatment with
109 to 10
7 M C-ANF-(4-23) for
6 days did not affect the total longitudinal bone length or the lengths
of CP and OC.
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Expression of mRNAs for Natriuretic Peptides and Their Receptors-- Northern blot analysis revealed a single CNP mRNA species of ~1.2 kilobase in size found abundantly in fetal mouse tibias (Fig. 6, left). The amount of CNP mRNA in mouse tibias was larger than in mouse brain, where CNP is known to be expressed most abundantly (9, 10). By contrast, no appreciable amounts of ANP and BNP mRNAs were detected in fetal mouse tibias. We also examined natriuretic peptide receptor mRNA expression in fetal mouse tibias (Fig. 6, right). Northern blot analysis revealed that a considerable amount of GC-B mRNA is present in both CP and OC from fetal mouse tibias, which was roughly comparable to that found in rat smooth muscle cells and mouse lung. By contrast, GC-A mRNA was expressed only slightly in both CP and OC from fetal mouse tibias. No significant amount of C-receptor mRNA was detected in fetal mouse tibias.
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DISCUSSION |
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In this study, using an organ culture of fetal mouse tibias, we examined the effects of natriuretic peptides on bone growth. Several previous studies using cultured cells showed that natriuretic peptides can regulate the proliferation and differentiation of osteoblasts, chondrocytes, and osteoclasts in vitro (21-25). Recently, we observed marked skeletal overgrowth in transgenic mice with elevated plasma BNP concentrations and suggested that natriuretic peptides can affect the process of endochondral ossification in vivo (26). BNP transgenic mice exhibit overgrowth of the growth plate cartilage in long bones and vertebrae, which histologically resembles cultured fetal mouse tibias treated with natriuretic peptides. Therefore, the organ culture of fetal mouse tibias provides a useful in vitro model system with which to assess the functional significance of natriuretic peptides in the process of endochondral ossification.
This study demonstrates that natriuretic peptides increase the longitudinal length of cultured fetal mouse tibias. The natriuretic peptide-induced increase in the longitudinal bone length is observed in the length of CP. Histological examinations revealed that natriuretic peptides increase the height of the proliferative and hypertrophic chondrocyte zones in CP from cultured fetal mouse tibias. These results suggest that natriuretic peptides activate chondrogenesis in the process of endochondral ossification. In this study, in fetal mouse tibias treated with natriuretic peptides, hypertrophic chondrocytes were enlarged in size and surrounded by increased extracellular spaces stained positively with type X collagen. Furthermore, BrdUrd-positive proliferative chondrocytes tended to be increased in number in fetal mouse tibias treated with natriuretic peptides. Collectively, these findings suggest that natriuretic peptides activate the proliferation and differentiation of chondrocytes in the process of endochondral ossification.
The chondrogenic responses to natriuretic peptides are reproduced in fetal mouse tibias treated with 8-Br-cGMP. Furthermore, HS-142-1 inhibits the chondrogenic responses to natriuretic peptides. In this study, no significant changes in the length of cultured fetal mouse tibias were observed when treated with C-ANF-(4-23), suggesting that the C-receptor is not involved in the process. These observations, taken together, indicate that natriuretic peptides activate the process of endochondral ossification via GC-coupled natriuretic peptide receptors. Pfeifer et al. (36) recently reported that mice deficient in type II cGMP-dependent protein kinase develop impaired endochondral ossification and that type I and type II cGMP-dependent protein kinase mRNAs are expressed in the growth plate chondrocytes from mouse long bones. Therefore, the natriuretic peptide-induced cGMP accumulation might lead to the activation of cGMP-dependent protein kinases in the growth plate chondrocytes, thereby regulating the process of endochondral ossification.
In this study, CNP activated chondrogenesis much more potently than ANP. Considering that CNP is selective for GC-B, whereas ANP is an endogenous ligand for GC-A (14, 15), the data of this study strongly suggest that natriuretic peptide activation of chondrogenesis is mediated primarily by GC-B. This notion is consistent with our observation that GC-B is expressed abundantly in CP from long bones (Fig. 6, right). Furthermore, no skeletal abnormalities have been reported so far in GC-A-deficient mice (20). We therefore postulate that in BNP transgenic mice (26), BNP is secreted in large quantities from the liver into the circulation and cross-react with GC-B in long bones and vertebrae, thereby activating endochondral ossification.
In this study, HS-142-1 inhibited significantly a spontaneous increase in the longitudinal bone growth and cGMP production in cultured fetal mouse tibias. These results suggest the involvement of GC-coupled natriuretic peptide receptors in the process of endochondral ossification. In this study, among natriuretic peptides, CNP was expressed abundantly in fetal mouse tibias, where ANP and BNP were not expressed (Fig. 6, right). Taken together, the data of this study strongly suggest the physiologic significance of the CNP/GC-B pathway in the process of endochondral ossification; CNP is an endogenous activator of chondrogenesis via its cognate receptor, GC-B. Further studies using in situ hybridization and immunohistochemical analyses are ongoing in our laboratory to determine the precise site of production of CNP in long bones in vivo.
The molecular mechanisms by which the CNP/GC-B pathway is involved in the process of endochondral ossification are unclear at present. Recent studies using transgenic or knockout mice have implicated several molecules such as fibroblast growth factor receptor 3, parathyroid hormone-related peptide, and Indian hedgehog in the process of endochondral ossification (37-42). It is important to determine the signaling molecules that are located upstream and downstream of the CNP/GC-B pathway in the process of endochondral ossification.
In conclusion, this study provides the first in vitro evidence that natriuretic peptides are involved in the process of endochondral ossification. This study also suggests the physiologic significance of the CNP/GC-B pathway in the process of endochondral ossification.
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ACKNOWLEDGEMENTS |
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We thank Dr. N. Amizuka (Niigata University School of Dentistry) for discussions, Dr. T. Maack for C-ANF-(4-23), and Dr. S. Nakanishi for HS-142-1. We also acknowledge A. Yonemitsu for secretarial assistance.
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FOOTNOTES |
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* This work was supported in part by research grants from the Japanese Ministry of Education, Science, Sports, and Culture; the Japanese Ministry of Health and Welfare; the Yamanouchi Foundation for Research on Metabolic Disorders; the Smoking Research Foundation; the Salt Science Research Foundation; and the Research Society for Metabolic Bone Disorders.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.
§ To whom correspondence should be addressed. Tel.: 81-75-751-3173; Fax: 81-75-771-9452; E-mail: ogawa{at}kuhp.kyoto-u.ac.jp.
To whom correspondence should be addressed. Tel.:
81-75-753-7511; Fax: 81-75-753-7504.
1 The abbreviations used are: ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; CNP, C-type natriuretic peptide; C-ANF, des-[Gln18,Ser19,Gly20,Leu21,Gly22]ANP(4-23)-NH2; GC, guanylyl cyclase; C-receptor, clearance receptor; 8-Br-cGMP, 8-bromo-cGMP; CP, cartilaginous primordia/primordium; OC, osteogenic center; BrdUrd, bromodeoxyuridine.
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REFERENCES |
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