(Received for publication, March 9, 1995; and in revised form, March 31, 1995)
From the
Most agents that regulate osteoclast bone resorption exert their
effects indirectly, through the osteoblast. Nitric oxide, which
stimulates soluble guanylyl cyclase, has been reported to inhibit
osteoclast bone resorption directly, by a cGMP-independent
mechanism(1) . In this report, we demonstrate that C-type
natriuretic peptide (CNP), an activator of membrane-bound guanylyl
cyclase, stimulates bone resorption by osteoclast-containing
1,25-dihydroxyvitamin D (1,25-(OH)
D
)-stimulated mouse bone marrow
cultures. Quantitative reverse transcription polymerase chain reaction
assays and anti-CNP immunocytochemistry were used to demonstrate that
CNP is expressed in mouse marrow cells cultured in the presence, but
not the absence, of 1,25-(OH)
D
. mRNA for
guanylyl cyclase type B, the receptor for CNP, was expressed in
cultures independent of 1,25-(OH)
D
. CNP (1 and
10 µM) elevated cGMP production in marrow cultures to 350
and 870%, respectively, of control values. 10 µM CNP
increased osteoclast bone resorptive activity, measured by the
resorption area on whale dentine wafers, or by the
NH
Cl-inhibitable release of
[
H]proline from radiolabeled bone chips, to 214
and 557% of control, respectively, without affecting osteoclast
formation. Bone resorption by the marrow cultures was inhibited by
7F9.1, a monoclonal antibody raised against CNP, but not by control
antibodies. These results indicate that CNP is a potent activator of
osteoclast activity and may be a novel local regulator of bone
remodeling.
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) .
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.
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.
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
10
M 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.
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 NH
Cl to inhibit acidification-dependent
resorption(35) . Both the total
[
H]proline counts released and the component of
H release inhibited by treatment with NH
Cl 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 NH
Cl to prevent acidification at the osteoclast
attachment site. After 5 days,
H cpm in culture media was
determined. NH
Cl-sensitive activity is shown in the inset. Errorbars, S.E. from 3-12
wells. *, p < 05 versus -NH
Cl
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 10
M 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.
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.