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Department of Medicine and Clinical Science (T.M., Y.O., H.C., A.Y., N.T., Y.K., K.N.), Kyoto University Graduate School of Medicine, Sakyo-ku, Kyoto 606-8507, Japan; and Institut für Pharmakologie und Toxikologie der Technische Universität München (A.P., F.H.), D-80802 München, Germany
Address all correspondence and requests for reprints to: Yoshihiro Ogawa, Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan. E-mail: ogawa{at}kuhp.kyoto-u.ac.jp.
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Abstract |
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Introduction |
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The natriuretic peptide family consists of three structurally related peptides: atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) (6). These peptides can influence a variety of homeostatic processes by the intracellular accumulation of cyclic GMP (cGMP) through two different membrane-bound guanylyl cyclase (GC)-coupled receptors (GC-A and GC-B) (7, 8). ANP and BNP are cardiac hormones that are produced predominantly by the atrium and ventricle, respectively (9, 10, 11), and are thought to play important roles in the regulation of cardiovascular homeostasis, primarily through GC-A (12, 13). On the other hand, CNP occurs in a wide variety of tissues (7, 14, 15, 16), where it may act locally as an autocrine/paracrine regulator through GC-B (12, 13). Recently, we have generated CNP-deficient mice (Nppc-/- mice) and reported that they exhibit dwarfism as a result of impaired endochondral ossification (17). Targeted expression of CNP in the growth plate chondrocytes has rescued the skeletal defect of Nppc-/- mice (17). These observations indicate that CNP is a local positive regulator of endochondral ossification.
The increase in intracellular cGMP concentrations leads to the activation of several downstream mediators such as cyclic nucleotide phosphodiesterases (PDEs), cGMP-regulated ion channels, and cGMP-dependent protein kinases (cGKs) (18, 19, 20). For instance, at least two PDE isoforms (PDE1 and PDE5), which hydrolyze cGMP, are expressed during chondrogenesis in a mouse embryonal carcinoma-derived ATDC5 cell line (21). Two known cGK isoforms (cGKI and cGKII) are also present, but they are distributed differently in the mouse growth plate chondrocytes in vivo (22). We previously reported that cGKII-deficient mice (Prkg2-/- mice) show dwarfism because of impaired endochondral ossification (22). However, Nppc-/- mice differ remarkably from Prkg2-/- mice in the histology of the growth plate (17, 22). The Nppc-/- mice show a reduction of the growth plate in height with its chondrocytes arranged in regularly columnar array, whereas the growth plate of Prkg2-/- mice is characterized by increased height of the growth plate with proliferative chondrocytes intermingled in the hypertrophic zone. Which downstream mediator(s) of cGMP play key roles in CNP-mediated endochondral ossification is still an enigma.
We, therefore, postulate that the CNP/cGMP/cGKII pathway is important for endochondral ossification. In this study, we use both in vivo and in vitro genetic strategies to establish that cGKII plays a critical role in CNP-mediated longitudinal bone growth, chondrocytic proliferation and hypertrophy, and cartilage matrix synthesis during endochondral ossification.
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Materials and Methods |
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1 (II) collagen (Col2a1) promoter was reported previously (17). The CNP-Tg mice were crossed with heterozygous cGKII-deficient mice (Prkg2+/- mice) (22) to produce CNP-Tg mice with the disrupted cGKII allele (CNP-Tg/Prkg2+/- mice). Among the F1 generation, Prkg2+/- mice were intercrossed with CNP-Tg/Prkg2+/- mice to obtain nontransgenic Prkg2+/+ mice (wild-type mice), CNP-Tg Prkg2+/+ mice (CNP-Tg mice), nontransgenic Prkg2-/- mice (Prkg2-/- mice), and CNP-Tg Prkg2-/- mice (CNP-Tg/Prkg2-/- mice). In this study, we analyzed the skeletal phenotypes of wild-type, CNP-Tg, Prkg2-/-, and CNP-Tg/Prkg2-/- mice. Genotypes for the CNP transgene and disrupted cGKII allele were determined by Southern blot analysis using mouse tail DNAs. All the experimental procedures were approved by the Kyoto University Graduate School of Medicine Committee on Animal Research.
Skeletal preparation and histology
Skeletal preparation and histological analysis were performed as described (23). Briefly, mice were killed, skinned, eviscerated, and subjected to soft x-ray analysis. Tibias of 4-wk-old mice were fixed in 70% ethanol, decalcified in 5% formic acid and 5% formalin for 7 d, and embedded in paraffin. Five-micrometer-thick sections were cut and stained with alcian blue (pH 2.5) and hematoxylin-eosin (23). Immunohistochemical staining for Indian hedgehog (Ihh) and type X collagen was performed using a polyclonal goat anti-Ihh(C) antibody (1:10; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and a polyclonal rabbit anti-type X collagen antibody (1:400; LSL, Tokyo, Japan) as a primary antibody (24). Immunoreactions were visualized by a biotinylated antipolyvalent antibody, a streptavidin-biotin-horseradish peroxidase complex, and diaminobenzidine (Vector Laboratories, Inc., Burlingame, CA). The specificity of immunoreactions was controlled by omitting the primary antibody.
cGMP concentration measurements
The tail bone cGMP concentrations in 7-d-old neonates were determined by a RIA for cGMP as described (25).
Organ culture study
Tibial explants from wild-type and Prkg2-/- fetuses at 16.5 d post coitus were cultured for 4 d with vehicle or 10-7 M CNP as described (25). Before and after the culture, total longitudinal length of explants was measured by a light microscope with a linear ocular scale.
For histological analysis, cultured tibias were fixed in 10% formalin neutral buffer solution for 24 h, embedded in paraffin, and stained with alcian blue and hematoxylin-eosin. Immunohistochemical staining of type X collagen were performed as described above. The size of hypertrophic cells was measured on 5-µm-thick sections of cultured tibias for 4 d with or without 10-7 M CNP by computerized measurement system (KS400 Imaging System; Carl Zeiss, Eching, Germany).
Cell proliferation was assessed in the cultured tibias by measuring the incorporation of bromodeoxyuridine (BrdU) as previously described (25). After 3 d of culture with or without 10-6 M CNP, BrdU was added to the culture medium at a concentration of 10-5 M, and the tibias were incubated for an additional 3 h. Immunohistochemical staining of incorporated BrdU was performed using the 5-bromo-2'-deoxy-uridine labeling and detection kit II (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s protocol. The labeling index was calculated as the ratio of BrdU-labeled cell number to total cell number at the proliferative zones of growth plates.
Glycosaminoglycan synthesis was assessed in cultured tibias by measuring the incorporation of 35SO4 as previously described (26). After 3 d of culture with or without 10-6 M CNP, Na235SO4 (Amersham Pharmacia Biotech, Buckinghamshire, UK) was added to the culture medium at a concentration of 5 µCi/ml for an additional 6 h. The tibias were then rinsed three times for 10 min in Pucks saline solution (Sigma, St. Louis, MO) and digested in 0.8 ml of 0.3% papain (Sigma) at 60 C for 24 h. To this digest, 0.8 ml of 10% cetylpyridinium chloride (Sigma) in 0.2 M NaCl was added. After incubation at 22 C for 18 h, the precipitate was washed three times with 0.1% CPC in 0.2 M NaCl, and dissolved in 0.8 ml of 23 N formic acid. Total 35SO4 incorporation was measured by liquid scintillation counter.
Northern blot analysis
Total RNA was extracted from 1-wk-old mouse tibial epiphysis by the acid guanidinium phenol chloroform method (13). Northern blot analysis was performed using the mouse cetylpyridinium chloride (27), rat GC-B (13), mouse PDE1A, and mouse PDE5A (28) cDNA fragments.
Statistical analysis
Data were expressed as the mean ± SE. The statistical significance of differences in mean value was assessed by two-way ANOVA or Fisher’s test. Differences among mean values were considered significant as values of P < 0.05 and P < 0.01.
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Results |
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shows the gross appearance of 25-wk-old female wild-type, CNP-Tg, Prkg2-/-, and CNP-Tg/Prkg2-/- mice. The CNP-Tg mice exhibit kyphosis and elongated limbs, paws, and tails, which are similar to that found in BNP-Tg mice (23, 29). The Prkg2-/- mice develop dwarfism and short limbs, paws, and tails as reported (22). The CNP-Tg/Prkg2-/- mice are phenotypically indistinguishable from Prkg2-/- mice; they both show severe dwarfism characterized by short tails and extremities. At birth, there are no significant differences in the naso-anal length among genotypes (Fig. 1B). At 5 wk of age, CNP-Tg mice grow larger, whereas Prkg2-/- mice smaller, than wild-type mice. The naso-anal length differences among these animals increase progressively until 10 wk of age, which are unchanged thereafter. However, no significant difference in the naso-anal length is noted between Prkg2-/- and CNP-Tg/Prkg2-/- mice at any time points examined.
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). Both Prkg2-/- and CNP-Tg/Prkg2-/- mice show a reduction in the length of vertebral bodies, tails, and extremities relative to wild-type mice. The longitudinal lengths of femurs, tibias, vertebrae, and calvarium formed through endochondral ossification are 10–25% longer in CNP-Tg mice, whereas approximately 20% shorter in Prkg2-/- and CNP-Tg/Prkg2-/- mice, than wild-type mice (Fig. 1D). No significant difference in the longitudinal bone length is noted between Prkg2-/- and CNP-Tg/Prkg2-/- mice. There are no appreciable changes in the width of calvarium, which is determined by membranous ossification among genotypes.
Histological analysis
Histological examination of the tibial growth plate revealed no significant differences among genotypes at perinatal stage (data not shown). At 4 wk of age, the height of the proliferative and hypertrophic chondrocyte zones in tibias from CNP-Tg mice increases prominently (Fig. 2A). The tibial growth plate from 4-wk-old Prkg2-/- mice is characterized by irregular and broadened hypertrophic zones with nonhypertrophic cells intermingled with hypertrophic chondrocytes as reported (22). The growth plate histology of CNP-Tg/Prkg2-/- mice is similar to that of Prkg2-/- mice. The disorganized growth plate is not rescued by targeted expression of CNP in the growth plate chondrocytes in CNP-Tg/Prkg2-/- mice.
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). By contrast, type X collagen-positive extracellular space is not detected in either Prkg2-/- or CNP-Tg/Prkg2-/- mice. Furthermore, we examined the expression of Ihh. In the growth plate of wild-type and CNP-Tg mice, the expression of Ihh was limited to the prehypertrophic chondrocytes (Fig. 2C). On the other hand, in the growth plate of Prkg2-/- or CNP-Tg/Prkg2-/- mice, the expression of Ihh was detected in the prehypertrophic chondrocytes and disorganized hypertrophic zones.
Bone cGMP concentrations
To examine the cGMP production in the bone, we measured tail bone cGMP concentrations in 7-d-old neonates (Fig. 1E). Bone cGMP concentrations in CNP-Tg mice (32.1 ± 16.2 pmol/g tissue) are roughly equivalent to those in wild-type mice (24.4 ± 4.1 pmol/g tissue) (n = 3–7). Bone cGMP concentrations are elevated significantly in Prkg2-/- and CNP-Tg/Prkg2-/- mice (101.0 ± 19.9 and 251.1 ± 211.6 pmol/g tissue, respectively) relative to wild-type and CNP-Tg mice (n = 3–7; P < 0.01).
Organ culture
The organ culture of fetal mouse tibias provides an in vitro unique experimental model system with which to assess the process of endochondral ossification (25). In this study, using cultured tibias prepared from wild-type and Prkg2-/- mice, we examined the effect of CNP on the longitudinal bone growth in the absence of cGKII (Fig. 3A). Before the culture, there is no significant difference in the length of fetal tibias between wild-type and Prkg2-/- mice explants. Treatment with 10-7 M CNP for 4 d produces an approximately 40% increase in the total length of wild-type mice explants (25). On the other hand, Prkg2-/- mice tibias treated with 10-7 M CNP show only a 20% increase in the total length, which is roughly comparable to that found in vehicle-treated groups. Histological examination revealed a significant increase in the height of the proliferative and hypertrophic zones in the growth plate of wild-type mice tibias treated with 10-7 M CNP (Fig. 3B). No significant difference in the height of the growth plate of Prkg2-/- mice is noted between vehicle- and CNP-treated groups.
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Cell proliferation was assessed by BrdU incorporation into cultured tibias from wild-type and Prkg2-/- fetuses with or without 10-6 M CNP for 3 d (Fig. 4A). In wild-type fetal tibias, 10-6 M CNP increases significantly the labeling index (n = 5; P < 0.01), which is consistent with previous reports (25, 26). However, CNP is unable to increase the labeling index in Prkg2-/- fetal tibias (Fig. 4B). Next, we measured the size of hypertrophic chondrocytes in cultured tibias treated with or without 10-7 M CNP (Fig. 4C). In wild-type fetal tibias, CNP causes a significant increase in the size of hypertrophic chondrocytes (n = 10; P < 0.01). By contrast, in Prkg2-/- fetal tibias, CNP does not increase the size of hypertrophic chondrocytes (Fig. 4D). Third, we assessed cartilage matrix synthesis by immunohistochemical staining of type X collagen and measuring newly-synthesized glycosaminoglycans (Fig. 4, E and F). There is an appreciable increase in the extracellular space positive for type X collagen in wild-type fetal tibias treated with 10-7 M CNP, whereas no significant increase is noted in CNP-treated Prkg2-/- fetal tibias (Fig. 4E). Furthermore, wild-type fetal tibias treated with 10-6 M CNP exhibit an approximately 30% increase in 35SO4 incorporation relative to vehicle-treated groups (n = 4–5; P < 0.05). By contrast, in Prkg2-/- fetal tibias, CNP does not increase 35SO4 incorporation (Fig. 4F).
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Discussion |
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This study demonstrated that targeted expression of CNP in the growth plate chondrocytes does not rescue the skeletal defect of Prkg2-/- mice (CNP-Tg/Prkg2-/- mice). Using an in vitro organ culture of mouse tibias, we also found that CNP does not increase longitudinal bone growth in the absence of cGKII. Recently, we have reported both in vivo and in vitro that natriuretic peptides can affect endochondral ossification through a GC-coupled receptor other than GC-A (25, 31). It is likely that CNP acts as a positive regulator of endochondral ossification via cGMP mechanism; CNP may lead to the intracellular accumulation of cGMP via its cognate receptor (probably GC-B), which, in turn, activates cGKII in the growth plate chondrocytes. This notion is supported by the previous in situ hybridization analysis that Nppc, GC-B (Npr2), and Prkg2 mRNAs are all expressed in the proliferative and prehypertrophic zones of the growth plate (17, 22).
From 1 wk of age, Prkg2-/- and CNP-Tg/Prkg2-/- mice start to show histological changes in the growth plate. This histological change may affect cGMP generation, and thereby affect the process of endochondral ossification. In this study, we confirmed no significant difference in Nppc and Npr2 mRNA expression in the growth plate from 1-wk-old Prkg2-/- and CNP-Tg/Prkg2-/- mice relative to wild-type mice (data not shown). Furthermore, there was no impairment of bone cGMP generation in Prkg2-/- and CNP-Tg/Prkg2-/- mice (Fig. 1E). These findings suggest that the defect in endochondral ossification in Prkg2-/- and CNP-Tg/Prkg2-/- mice lies downstream of cGMP; it should be due to the disruption of cGKII per se.
It is generally understood that longitudinal bone growth is determined by a function of chondrocytic proliferation and hypertrophy, and cartilage matrix synthesis during the process of endochondral ossification (30). Because of the marked change in the growth plate histology in Prkg2-/- and CNP-Tg/Prkg2-/- mice, it is difficult to investigate in vivo the cellular mechanisms underlying the CNP-mediated endochondral ossification. We, therefore, examined the role of cGKII in CNP-mediated chondrocytic proliferation and hypertrophy, and cartilage matrix synthesis using an organ culture of fetal mouse tibias. The data of this study demonstrate that CNP can stimulate chondrocytic proliferation and hypertrophy, and cartilage matrix synthesis, which is consistent with previous reports (17, 25, 26). By contrast, in the absence of cGKII (or in Prkg2-/- tibias), CNP does not stimulate the above cellular functions compared with vehicle-treated groups. These observations indicate that cGKII is important for CNP-mediated chondrocytic proliferation and hypertrophy, and cartilage matrix synthesis, thus suggesting the notion that CNP signals through cGKII during the process of endochondral ossification.
The gross skeletal phenotype of Nppc-/- mice is similar to that of Prkg2-/- mice; they both develop severe dwarfism as a result of endochondral ossification. However, they differ remarkably in the growth plate histology. The Nppc-/- mice show a reduction of the growth plate in height with its chondrocytes arranged in regularly columnar array (17), whereas the growth plate of Prkg2-/- mice is characterized by increased height of the growth plate with BrdU-positive proliferative chondrocytes intermingled in the hypertrophic zone. These observations suggest the involvement of other mediator(s) in the CNP-mediated endochondral ossification. In this regard, there are no skeletal abnormalities reported in mice deficient in cGKI (32). Furthermore, we observed no significant difference in PDE1 (Pde1) and PDE5 (Pde5) mRNA expression among wild-type, CNP-Tg, Prkg2-/-, CNP-Tg/Prkg2-/- mice (data not shown). On the other hand, we have demonstrated that cGMP is produced, although reduced (approximately 40% of the wild-type littermates), in Nppc-/- mice tibias (17). We speculate that non-CNP-derived cGMP is capable of activating cGKII, thereby producing regularly columnar array of hypertrophic chondrocytes in Nppc-/- mice. However, in Prkg2-/- mice, cGMP, although overproduced in the bone, is unable to signal through cGKII, thus leading to the marked histological abnormality of the growth plate. The above discussion supports the notion that cGKII is a major mediator of the CNP-mediated endochondral ossification, but dose not rule out the possibility of other mediator(s), such as cGKI. To address this issue, it would be helpful to examine closely the growth plate of mice deficient in cGKI and to investigate whether double homozygous mutant mice for cGKI and cGKII would mimic the phenotype of Nppc-/- mice.
To obtain further insight into how the CNP/cGMP/cGKII pathway is involved in endochondral ossification, we examined the expression of Ihh and type X collagen immunohistochemically in the growth plate chondrocytes from 4-wk-old Prkg2-/- and CNP-Tg/Prkg2-/- mice. In this study, Ihh, a marker of prehypertrophic chondrocytes (33, 34, 35, 36), is expressed in the disorganized hypertrophic zone of the growth plate from Prkg2-/- and CNP-Tg/Prkg2-/- mice, where type X collagen, a marker of hypertrophic chondrocytes (37), is not detected. We have also found by Northern blot analysis and in situ hybridization analysis that type X collagen expression is markedly reduced in the growth plate from Prkg2-/- and CNP-Tg/Prkg2-/- mice relative to wild-type mice at 1 wk of age (Miyazawa, T., et al., unpublished data). These observations suggest the delay of chondrocytic differentiation (i.e. conversion from prehypertrophic to hypertrophic chondrocytes) in Prkg2-/- and CNP-Tg/Prkg2-/- mice. In this context, we have observed that the band width of Ihh- and type X collagen-expressing cells is narrowed in the growth plate from Nppc-/- mice relative to wild-type mice (17). Collectively, we postulate that CNP controls the rate of chondrocytic differentiation through cGKII during the process of endochondral ossification.
Targeted expression of a constitutively active receptor for PTH and PTHrP (PTH/PTHrP receptor) delays endochondral ossification through ligand-independent constitutive cAMP accumulation (38). Increased accumulation of cAMP may lead to the activation of cAMP-dependent protein kinase A (cAK). The growth plate histology of Tg mice expressing a constitutively active PTH/PTHrP receptor in the growth plate is characterized by the absence of Col10a1 mRNA expression and irregular and broadened hypertrophic zone, which is similar to that of Prkg2-/- mice. Furthermore, unlike cGMP, cAMP suppresses terminal differentiation of chondrocytes and cartilage matrix calcification in vitro (39). These findings suggest that the CNP/cGMP/cGKII and PTHrP/cAMP/cAK signaling pathways coordinate to control the rate of chondrocytic differentiation; it is accelerated by CNP via cGKII and decelerated by PTHrP via cAK. Further studies are needed to elucidate the mechanism of a delay in chondrocytic differentiation by the ablation of cGKII.
In conclusion, we have demonstrated that cGKII plays a critical role in longitudinal bone growth, chondrocytic proliferation and hypertrophy, and cartilage matrix synthesis during the CNP-mediated endochondral ossification. This study will provide further insight into the molecular mechanism of the CNP-mediated endochondral ossification.
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Acknowledgments |
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Footnotes |
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Abbreviations: ANP, Atrial natriuretic peptide; BNP, brain natriuretic peptide; BrdU, bromodeoxyuridine; cAK, cAMP-dependent protein kinase A; cGMP, cyclic GMP; CNP, C-type natriuretic peptide; cGKs, cGMP-dependent protein kinases; GC, guanylyl cyclase; Ihh, Indian hedgehog; Nppc, CNP gene; Nppc-/- mice, CNP-deficient mice; Npr2, GC-B gene; PDE, cyclic nucleotide phosphodiesterases; Prkg, cGK gene; Prkg2-/- mice, cGKII-deficient mice; Tg, transgenic.
Received March 18, 2002.
Accepted for publication May 24, 2002.
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