Bone is renewed by the bone remodeling cycle, in which bone resorption by osteoclasts is tightly coupled to new bone formation by osteoblasts(2, 3) . Most factors that affect osteoclast bone resorption exert their effects indirectly, stimulating the osteoblast to produce intermediary messengers that act on the osteoclast(4) . Although numerous factors participate in bone remodeling(3, 5, 6) , the coupling of bone resorption to formation remains poorly understood.

Rodan et al.(7) suggested that intracellular cGMP is involved in the control of bone remodeling. Cellular cGMP is synthesized by two general classes of guanylyl cyclases: soluble cytoplasmic guanylyl cyclases and cyclases associated with the plasma membrane (for review, see (8) and (9) ). Soluble guanylyl cyclases are heterodimers whose activity is stimulated by nitric oxide (NO), a gaseous signaling molecule that is produced by constitutive and inducible NO synthases(10) .

Several lines of evidence support a role for NO in bone remodeling. Osteoclast bone resorptive activity is suppressed by agents such as nitroprusside that generate NO(1, 11, 12, 13) . Osteoclasts generate NO, and treatment of isolated chicken osteoclasts or intact rats with nitric oxide synthase inhibitors causes them to resorb more bone(12) . Osteoblasts possess NO synthase activity that is regulated by cytokines, which affect bone metabolism(13) . Although these data suggest that NO is an important inhibitor of osteoclast bone resorption, MacIntyre et al.(1) found evidence that its effects may not be mediated by cGMP.

The other principal cellular sources of cGMP are the three membrane-bound receptor guanylyl cyclases: guanylyl cyclase type A (GC-A), type B (GC-B), and type C (GC-C). Atrial natriuretic factor (ANP or atriopeptin) and C-type natriuretic peptide (CNP) are the specific ligands of GC-A and GC-B, respectively, and guanylin is the endogenous ligand for GC-C(8, 9, 14) .

The involvement of natriuretic peptides and receptor guanylyl cyclases in bone is only beginning to emerge. Guanylyl cyclase activity has been detected by histochemical methods in the plasma membrane of osteoblasts, but it has not been demonstrated in osteoclasts(15) . Osteoblasts respond to ANP by increasing cGMP production(16) , but ANP has only minimal effects on bone remodeling(17) .

CNP, first identified in 1990 in the central nervous system(18) , has been found in a growing list of tissues. The CNP receptor, GC-B, has been detected in bone marrow(19) . Although CNP has not been demonstrated in marrow(20) , CNP and GC-B have been found in cell types closely related to bone cells. Both CNP and GC-B are expressed in cultured chondrocytes(21) , which originate from the same stromal lineage as osteoblasts(22, 23) . Osteoclasts originate from cells of monocytic lineage, and CNP is produced by the monocytic cell line, THP-1(24) .

These observations suggested to us the possibility that CNP might function as a local regulator of bone resorptive activity through a cGMP-mediated pathway. In this paper, we demonstrate that CNP is a potent stimulator of osteoclast bone resorption in mouse bone marrow cultures, a well-characterized model system for studying osteoclasts (25) .

EXPERIMENTAL PROCEDURES

Materials

Mouse Bone Marrow Culture

Polymerase Chain Reaction (PCR) Assays

For preparation of the competitive plasmid standards for CNP, a 100-bp oligonucleotide was synthesized corresponding to bases 151-173 and 319-397, omitting a 146-bp internal segment of the cDNA sequence. Plasmid standards for GC-B and glyceraldehyde-3-phosphate dehydrogenase were constructed by selective restriction enzyme digestion of amplified target DNA to remove a fragment of DNA between the 5` and 3` primer sites. A fragment of GC-B spanning bases 1511-1865 was cleaved with TaqI and ligated to yield a 130-bp fragment from which bases 1587-1812 were excised. A fragment of glyceraldehyde-3-phosphate dehydrogenase (bases 40-1017) was cleaved with AccI and ligated to yield an 817-bp fragment from which bases 156-316 were excised. The resulting products were amplified by PCR, and ligated into the vector pCRII (Invitrogen, San Diego, CA). Amplification of these competitive plasmid standards resulted in products that were 100, 132, and 814 bp in size for CNP, GC-B, and glyceraldehyde-3-phosphate dehydrogenase, respectively.

PCR-amplified products for CNP, GC-B, and glyceraldehyde-3-phosphate dehydrogenase were verified by sequence analysis. The levels of CNP, GC-B, and glyceraldehyde-3-phosphate dehydrogenase mRNA were measured by competitive RT-PCR using the protocol of Siebert and Larrick(32) . Briefly, total RNA was isolated from cell cultures using RNAzol B (Cina/Biotecx, Friendswood, TX). Equal amounts of RNA were taken to prepare cDNA using Moloney murine leukemia virus reverse transcriptase as described previously(29) . Equal volumes of the resulting cDNAs were added to PCR tubes containing PCR buffer, 0.8 µM primers, and a single concentration of competitive plasmid standard (0.001, 0.001, and 0.01 amol for CNP, GC-B, and glyceraldehyde-3-phosphate dehydrogenase, respectively) and amplified using conditions as described previously(29) . After PCR amplification, samples were fractionated by gel electrophoresis (CNP and GC-B, 1.5% agarose; glyceraldehyde-3-phosphate dehydrogenase, 5% nondenaturing polyacrylamide), visualized by ethidium bromide staining, and photographed using Polaroid 55 positive/negative film (Cambridge, MA). Target and plasmid standard amplified products were easily distinguished by size. Negatives were densitometrically scanned (E-C Apparatus Corp.; St. Petersburg, FL), and band intensities were analyzed using Beckman System Gold software (Beckman Instrumentation Inc., Fullerton, CA). The densitometric ratios of target CNP and GC-B to their plasmid standard were measured and normalized to glyceraldehyde-3-phosphate dehydrogenase ratios.

Immunocytochemistry

cGMP Assays

cGMP was measured by ELISA using a kit from Cayman Chemicals (Ann Arbor, MI) according to the manufacturer's instructions. Levels were determined from a standard curve, and concentrations of cGMP were normalized for cell protein/well determined using a commercial kit (Bio-Rad) for the Bradford protein assay(33) .

For the assays in MDCT cells, 2 10 cells were plated in each well of a 24-well plate in Dulbecco's modified Eagle's medium/Ham's F-12 medium containing 10% fetal bovine serum. After approximately 24 h, when the cells were nearly confluent, the cells were washed with PBS and incubated for 10 min in 1 ml containing 0.5 ml of MEM D10, 0.5 ml of hybridoma supernatant (antibody concentration approximately 10 µg/ml) or control medium, 1 µM CNP or ANP, and 0.5 mM IBMX. The cells were then washed, and cGMP/mg of cell protein was measured as described above.

Bone Resorption Assays

Resorption pit-forming assays were performed essentially as described by Boyde et al.(36) . Sperm whale teeth were obtained from the United States Department of Fisheries, and 100-µm-thick sections with surface area of about 1 cm were cut using a low speed diamond saw (Buehler, Lake Bluff, IL). Slices were washed by agitation in 50 ml of sterile PBS and then stored in MEM D10. 2 days prior to assays, dentine slices were transferred to 24-well plates and incubated in MEM D10. Mouse bone marrow cells were cultured in tissue culture plates for 5 days and then scraped free using a disposable cell scraper (Costar, Cambridge, MA), washed with MEM D10 3 times, and plated on bone slices at a concentration of 1 10 total cells/well. Cells were maintained on the dentine wafers for 5 days; medium was replaced after 3 days. Dentine slices were then rinsed with 1% SDS to remove cells and debris, fixed with 2.5% glutaraldehyde, dehydrated through an ethanol series, air dried, sputter coated with gold, and examined using a Hitachi H-400 scanning electron microscope (Tokyo, Japan) operated at 15 kV. For quantitation, photos of slices were taken at 100 with no tilt angle. Overlays that divided micrographs into 42-µm grid spaces were placed over photos, and grid spaces with or without pits were counted to determine the surface area resorbed. For the experiments shown in Table2, resorption pit number and area was determined in three representative fields from each of six separate dentine slices. For enumeration, a single pit was counted as any contiguous area of bone resorption, even if it contained more than one scalloped area.

Where indicated in the text, 1 10M 1,25-(OH)D, 1 µM CNP, or 1 µM ANP, and inhibiting antibodies (20 µg/ml) were added on the first day of the resorption pit-forming assay and replenished after 3 days. 7F9.1 is a monoclonal antibody that was raised against CNP and binds both CNP and ANP (see ``Results''). MRW is a monoclonal antibody that is specific for ANP(37) . Monoclonal anti--galactosidase was purchased from Sigma.

Production and Characterization of Monoclonal Antibody 7F9.1

Statistics

RESULTS

To determine whether CNP and its receptor are expressed in mouse bone marrow cultures, we tested for the presence of CNP and GC-B mRNA by quantitative RT-PCR. mRNA for CNP was found in freshly isolated mouse bone marrow cells maintained in culture for 7 days in the presence of 1,25-(OH)D (Fig.1). CNP mRNA was not detected in marrow cells cultured in the absence of 1,25-(OH)D (Fig.1). In the same cells, mRNA for the CNP receptor GC-B was detectable, and approximately the same level of GC-B mRNA was found in cells incubated with 1,25-(OH)D.

Figure 1: A, CNP and GC-B expression in mouse marrow cultures by RT-PCR. RNA was isolated from day 1 (lanes1 and 2) and day 7 bone marrow cultures treated either with 10M 1, 25-(OH)D (lanes3 and 4) or vehicle (lanes5 and 6). cDNA was prepared and amplified by PCR as described ``Experimental Procedures.'' The appropriate competitive DNA standard was added to each reaction, and the amplification products from these are indicated as follows: CNPstd, 100 bp; GC-B std, 132 bp; G3DPHstd, 814 bp. Lane7, positive controls (standard only); lane8, negative controls (no template). B, quantitation of CNP and GC-B mRNA levels in mouse marrow cultures by RT-PCR. Amplification products from A were densitometrically scanned as described under ``Experimental Procedures,'' and quantified as the ratio of the CNP or GC-B fragment to its plasmid standard; in the rightpanels, ratios were normalized to glyceraldehyde-3-phosphate dehydrogenase ratios. Errorbars = S.E.

To confirm that the CNP protein was expressed in the marrow cell cultures and to determine which cell types express CNP, we performed fluorescent immunocytochemistry on 1,25-(OH)D-stimulated or unstimulated cultures, using an anti-CNP polyclonal antibody. In 1,25-(OH)D-stimulated cultures, all of the giant, multinucleated osteoclasts were intensely labeled in areas surrounding nuclei (Fig.2, A and B). Many mononuclear and stromal cells were also stained. Cultures not stimulated with 1,25-(OH)D did not label with anti-CNP antibody (Fig.2, C and D). Low levels of background staining were found in 1,25-(OH)D-stimulated cultures when the primary antibody was omitted (Fig.2, E and F).

Figure 2: Expression of CNP protein in 1,25-(OH)D-stimulated mouse bone marrow cultures. Mouse bone marrow cultures were grown for 7 days on coverslips, and stained with rabbit anti-CNP antiserum (A-D) or nonimmune rabbit antiserum (E and F) as described under ``Experimental Procedures.'' Cells were incubated with (A, B, E, and F) or without (C and D) 10 nM 1,25-(OH)D. A, C, and E, phase contrast; B, D, and F, corresponding fluorescent micrographs. Arrows indicate osteoclasts. Bar = 10 µm.

We assayed for the presence of functional GC-B by testing whether the cultures respond to CNP by producing cGMP. Mouse bone marrow was plated in six-well plates at 1 10 nucleated cells/well and incubated for 7 days in the presence of 1,25-(OH)D, as described under ``Experimental Procedures''; the cells were then washed and incubated in solutions containing IBMX and varying concentrations of CNP, and cGMP was measured by radioimmunoassay. The marrow cultures stimulated with CNP produced cGMP in a concentration-dependent manner (Fig.3). This result and the observation of GC-B mRNA in the cultures indicate that the cells likely express functional GC-B receptors that are responsible for cGMP production in response to CNP stimulation.

Figure 3: CNP increases cGMP production in mouse bone marrow cells. Marrow cultures were incubated for 7 days in the presence of 1,25-(OH)D, washed, and incubated with indicated concentrations of CNP and 0.5 mM IBMX, and cGMP was determined by ELISA as described under ``Experimental Procedures.'' Errorbars = S.E. *, p < 05 versus control by analysis of variance.

Since cGMP is suspected to be an important regulator of bone remodeling, we examined whether CNP alters osteoclast resorptive activity. Two different resorption assays were used. First, the ability of osteoclasts to release soluble [H]proline from [H]proline-radiolabeled bone chips was examined. 1,25-(OH)D-stimulated marrow cells were maintained in culture for 5 days to ensure development of mature osteoclasts, and labeled bone particles were added. The cells were incubated for an additional 5 days in the presence or absence of CNP, and with or without NHCl to inhibit acidification-dependent resorption(35) . Both the total [H]proline counts released and the component of H release inhibited by treatment with NHCl were determined.

Fig.4shows that 1 µM CNP added to cultures between day 6 and day 10 increased resorption by 34%, and 10 µM CNP increased resorption by 118%. CNP had little or no effect on the number of NHCl-insensitive H counts released. In marked contrast, 10 µM CNP increased the ammonium chloride-sensitive component of resorptive activity 457% (Fig.4, inset).

Figure 4: CNP stimulates bone resorption assayed by [H]proline release. Mouse bone marrow cells were incubated for 5 days in the presence of 1,25-(OH)D. [H]proline-labeled bone particles (1500 counts/well) were then added with the indicated concentrations of CNP. Sets of cultures were incubated either without (filledbars) or with (hatchedbars) 5 mM NHCl to prevent acidification at the osteoclast attachment site. After 5 days, H cpm in culture media was determined. NHCl-sensitive activity is shown in the inset. Errorbars, S.E. from 3-12 wells. *, p < 05 versus -NHCl control by analysis of variance.

To confirm that CNP stimulates osteoclast bone resorption, we used a second method for assaying resorptive activity. Mouse bone marrow cells maintained in culture for 5 days were scraped free and plated on sperm whale dentine slices; 1,25-(OH)D, with or without 10 µM CNP, was added on the first day, and after 5 days the surface area of the dentine slices that was resorbed was quantified by scanning electron microscopy. Incubation of the marrow cultures on the dentine slices produced multiple resorption lacunae characteristic of osteoclasts (Fig.5). In cultures treated with 1 µM CNP, the surface area of dentine resorbed and area per pit were 207% and 233% of control values, respectively, but the number of pits formed was unaffected (see Table2).

Figure 5: CNP stimulates bone resorption assayed by resorption pit formation. Mouse bone marrow cells were incubated on tissue culture plates in the presence of 1,25-(OH)D for 5 days to induce osteoclast formation, and the cells were scraped and plated onto dentine wafers. After 5 days, cells were removed with 1% SDS, and wafers were examined for resorption pit formation by scanning electron microscopy as detailed under ``Experimental Procedures''; representative fields are shown. A and B, cells treated with 1 µM CNP; C and D, controls. Bar = 50 µM.

Since the osteoclast cultures express the CNP message and contain CNP detectable in immunocytochemical assays, we examined whether the bone resorptive activity of the marrow cells was altered by CNP produced endogenously in the cultures. For these experiments, we generated a monoclonal antibody, 7F9.1, that binds both CNP and ANP (Fig.6). Antibody 7F9.1 inhibited the ability of CNP to stimulate cGMP generation in cultured MDCT renal epithelial cells (Table1).

Figure 6: Monoclonal antibody 7F9.1 binds both CNP and ANP by competitive ELISA. 96-well plates were coated with CNP peptide (2 µg/ml) overnight, and then incubated with 7F9.1 culture supernatants. Wells were assayed for antibody binding by ELISA in the presence of indicated concentrations of CNP and ANP, as described under ``Experimental Procedures.''

The effect of endogenously produced CNP on bone resorption by mouse marrow cultures was examined in the dentine wafer bone resorption assay by performing the 5-day incubations in the presence or absence of 7F9.1 and of CNP. When added to marrow cultures, antibody 7F9.1 inhibited 100% of the CNP-stimulated resorption activity and further reduced the bone resorption activity of the cultures to 44% of control levels (Fig.7). 7F9.1 also inhibited bone resorption in marrow cultures not stimulated with CNP to 30% of control (Fig.7). Since 7F9.1 binds both CNP and ANP, we performed control experiments using MRW, an antibody that binds only ANP, to exclude the possibility that 7F9.1 functions by binding endogenously produced ANP. Neither 1 µM ANP nor anti-ANP antibody MRW had any detectable effect on bone resorption (Fig.7). A second control monoclonal antibody, directed against -galactosidase, also caused no change in the amount of bone resorbed by cultures. These results indicate that endogenously produced CNP accounts for as much as 70% of bone resorption in the mouse marrow cultures.

Figure 7: Endogenously produced CNP stimulates bone resorption. Marrow cultures on dentine slices were incubated with vehicle (control), 1 µM CNP, 1 µM CNP + 4 µM antibody 7F9.1, 4 µM 7F9.1, 1 µM ANP, 4 µM antibody MRW, or 4 µM anti--galactosidase. Percent resorption was determined from 3 random 12.9-mm fields taken from three different dentine wafers for each condition used. Errorbars = S.E. *, p < 0.05 versus control by analysis of variance.

In principle, CNP could act by increasing the number of osteoclasts in the cultures or by activating osteoclasts already present. To distinguish between these possibilities, we examined the effect of CNP on osteoclast formation in the mouse marrow cultures. Cells were cultured for 7 days in the presence and absence of 10M 1,25-(OH)D and 10M CNP, and the number of cells with histochemical staining for tartrate-resistant acid phosphatase, a marker for osteoclasts, was quantified (Fig.8). CNP had no significant effect on the number of osteoclasts formed, indicating that it stimulates bone resorption by activating existing osteoclasts rather than by promoting their formation. This result is supported by the data in Table2, showing that CNP increases the area of bone resorbed without increasing pit number.

Figure 8: CNP does not affect osteoclast formation. Mouse bone marrow cultures were grown for 7 days in 24-well plates in the presence and absence of 10 1,25-(OH)D and 10 CNP (n = 4-6 wells for each condition). Cultures were fixed and stained for tartrate-resistant acid phosphatase activity, and the number of mononuclear, multinucleated (2-10 nuclei), and giant (>10 nuclei) tartrate-resistant acid phosphatase (TRAP+) cells/well was determined.

DISCUSSION

Our results indicate that CNP increases osteoclast bone resorption in 1,25-(OH)D-stimulated mouse bone marrow cultures. To our knowledge, this is the first demonstration that both CNP and its receptor GC-B are present in these cultures. The expression of CNP required 1,25-(OH)D, while GC-B was present in both 1,25-(OH)D-stimulated and unstimulated cultures. CNP added to cultures increased the amount of bone resorbed, as measured by two different types of assays, and resorption was inhibited by inclusion of an antibody that binds CNP. The increase in bone resorption was a result of activation of existing osteoclasts rather than increased formation of osteoclasts.

The 1,25-(OH)D-induced expression of CNP in the mouse bone marrow cells likely arises from the genomic actions of 1,25-(OH)D. In in vitro models of murine osteoclast development, 1,25-(OH)D is required for differentiation of osteoclast precursors to mononuclear cells expressing markers of mature osteoclasts(41) . Since receptors for 1,25-(OH)D are present in the osteoclast precursor(41) , it is possible that the 1,25-(OH)D-induced expression of CNP represents a direct effect. CNP expression in endothelial cells, however, is inducible by cytokines, including interleukin-1, interleukin-1, and tumor necrosis factor (42) . Since all of these factors are also known to affect osteoclast resorptive activity (5) , it is conceivable that the 1,25-(OH)D-induced increase in CNP expression is an indirect effect mediated by cytokines.

We demonstrated by immunocytochemistry that CNP protein is present in 1,25-(OH)D-stimulated bone marrow cultures. The osteoclasts were heavily labeled, and CNP was localized to regions around nuclei, suggesting that the osteoclasts synthesize CNP. A majority of mononuclear cells in the culture was also labeled. At the present time, it is not possible to determine which cell types were the principal source of secreted CNP in the cultures.

The receptor for CNP, GC-B, was demonstrated in these cultures by RT-PCR. Exogenously added CNP elicited an increase in cGMP production in the marrow cultures, indicating that functional GC-B is present in the bone marrow cultures. The increase in cGMP production was concentration-dependent, with a response curve similar to that observed in other systems(9, 14) .

Adding CNP to cultures increased bone resorption as measured by two separate assays. The increase in bone resorption in response to CNP was concentration-dependent and correlated well with CNP-stimulated increases in cGMP production. 1 µM CNP induced 2-3-fold increase in bone resorption by either the NHCl-sensitive H release from bone chips or the surface area resorbed of dentine slices. A monoclonal antibody raised against CNP, 7F9.1, decreased bone resorption in cultures stimulated by CNP as well as in cultures not stimulated by CNP. Thus CNP produced endogenously by the cultures seems to play a role in stimulating osteoclast activity. The inhibition of activity by 7F9.1 is most likely due to inhibition of CNP, as neither MRW, a monoclonal antibody specific for ANP, nor a monoclonal antibody against -galactosidase affected bone resorption. Exogenous ANP also failed to elicit a response in this system.

Our results demonstrate that CNP is produced by 1,25-(OH)D-treated mouse bone marrow cultures and that it increases osteoclast bone resorptive activity. These findings suggest the possibility that CNP may be one of the ``coupling factors'' that control the bone remodeling unit(43) . Although the immunocytochemical results indicate that osteoclasts may produce CNP, the cultures contain a mixed population of cells, and the principal source of secreted CNP remains unresolved. It is also unclear if CNP acts directly on osteoclasts or indirectly, such as by releasing coupling factors that activate the osteoclasts(44) , by preventing the release of inhibitory factors(44) , or by allowing osteoclasts increased access to the bone matrix. It will be important, in future studies, to determine if GC-B resides on osteoclasts.

Surprisingly, the effect of CNP on bone resorption was opposite to that reported for nitric oxide, an agent that increases cellular cGMP levels by stimulating soluble guanylyl cyclases (9) but inhibits bone resorption(1, 12, 13) . These ostensibly disparate results suggest either that nitric oxide acts by a cGMP-independent pathway, as initially proposed(1) , or that other factors modify the effects of cGMP levels on osteoclast activity. A previous study showed that ANP had a slight inhibitory effect on bone resorption under certain conditions(17) . We detected no significant effect on bone resorption by ANP. Since both GC-A and GC-B are thought to initiate signaling by elevating cytosolic cGMP, it is likely that GC-A, if present in these cultures, is located on different cells than GC-B. Further studies will be required to determine which cell types within this complex system produce and respond to the natriuretic peptides and nitric oxide and to determine the precise role of cGMP in the responses.

Finally, it may be useful to examine whether agents that inhibit CNP or its receptor, such as HS-142-1, an inhibitor of GC-B and GC-A (45, 46, 47) , are effective in the treatment of osteoporosis.

FOOTNOTES

*

This work was supported by National Institutes of Health Grants AR32087, DK38848, DK09976, and DK45181 and Training Grant DK07126. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

Contributed equally to this project.

¶

To whom correspondence should be addressed: Renal Division, Washington University School of Medicine, 660 S. Euclid Ave., Box 8126, St. Louis, MO 63108. Tel.: 314-362-8762; Fax: 314-362-8237; sgluck{at}imgate.wustl.edu.

The abbreviations used are: NO, nitric oxide; ANP, atrial natriuretic peptide (atriopeptin); CNP, C-type natriuretic peptide; GC-A, receptor guanylyl cyclase type A; MEM D10, -modified minimal essential medium; PCR, polymerase chain reaction; RT, reverse transcription; bp, base pair(s); 1,25-(OH)D, 1,25-dihydroxyvitamin D; BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; IBMX, isobutylmethylxanthine.

ACKNOWLEDGEMENTS

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