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B Ligand and Osteoprotegerin in Osteoblasts
Department of Biochemistry, School of Dentistry, Showa University (M.T., N.T., N.U., T.S.), Tokyo 142-8555, Japan; Department of Bioengineering, Tokyo Institute of Technology (K.S., J.-T. W., K.N.), Yokohama 226-8501, Japan; Department of Biochemistry, School of Pharmacy, Tokyo University of Pharmacy and Life Science (C.M.), Tokyo 192-0392, Japan; and St. Vincent’s Institute of Medical Research (T.J.M.), Fitzroy, Victoria 3065, Australia
Address all correspondence and requests for reprints to: Dr. Tatsuo Suda, Department of Biochemistry, School of Dentistry, Showa University, 1–5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan. E-mail: suda{at}dent.showa-u.ac.jp
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B ligand (RANKL) and
osteoprotegerin (OPG) produced by osteoblasts/stromal cells are
involved as positive and negative regulators in osteoclast formation.
Three independent signals have been proposed to induce RANKL expression
in osteoblasts/stromal cells: vitamin D receptor-, cAMP-, and
gp130-mediated signals. We previously reported that intracellular
calcium-elevating compounds such as ionomycin, cyclopiazonic acid, and
thapsigargin induced osteoclast formation in cocultures of mouse bone
marrow cells and primary osteoblasts. Increases in calcium
concentration in culture medium also induced osteoclast formation in
cocultures. Treatment of primary osteoblasts with these compounds or
with high calcium medium stimulated the expression of both RANKL and
OPG messenger RNAs (mRNAs).
1,2-Bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid)-tetra(acetoxymethyl)ester, an intracellular calcium
chelator, suppressed both ionomycin-induced osteoclast formation in
cocultures and expression of RANKL and OPG mRNAs in primary
osteoblasts. Phorbol 12-myristate 13-acetate (PMA), an activator of
protein kinase C, also stimulated osteoclast formation in these
cocultures and the expression of RANKL and OPG mRNAs in primary
osteoblasts. Protein kinase C inhibitors such as calphostin and
staurosporin suppressed ionomycin- and PMA-induced osteoclast formation
in cocultures and expression of RANKL and OPG mRNAs in primary
osteoblasts. Ionomycin stimulated RANKL mRNA expression in ST2 and
MC3T3-G2/PA6 cells, but not in MC3T3-E1 or NIH-3T3 cells. These effects
were closely correlated with osteoclast formation in response to
ionomycin in cocultures with these stromal cell lines. OPG strongly
inhibited osteoclast formation induced by calcium-elevating compounds
and PMA in cocultures, suggesting that RANKL expression in osteoblasts
is a rate-limiting step for osteoclast induction. Forskolin, an
activator of cAMP signals, also stimulated osteoclast formation in
cocultures. Forskolin enhanced RANKL mRNA expression but suppressed OPG
mRNA expression in primary osteoblasts. These results suggest that the
calcium/protein kinase C signal in osteoblasts/stromal cells is the
fourth signal for inducing RANKL mRNA expression, which, in turn,
stimulates osteoclast formation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,25-dihydroxyvitamin D3
[1,25-(OH)2D3], PTH,
PGE2, and interleukin-6 (IL-6) plus soluble IL-6
receptors (4). Cell to cell contact between
osteoblast/stromal cells and osteoclast progenitors is required for the
induction of osteoclast formation (4). Subsequent
experiments established that the target cells of bone-resorbing factors
for inducing osteoclast formation in such cocultures are
osteoblasts/stromal cells, but not osteoclast precursors
(5, 6, 7, 8).
The discovery of receptor activator of nuclear factor-B ligand
(RANKL, also known as osteoclast differentiation
factor/osteoprotegerin ligand/tumor necrosis factor-related
activation-induced cytokine) allowed elucidation of the precise
mechanism by which osteoblasts/stromal cells regulate osteoclast
differentiation (9, 10, 11, 12). Osteoblasts/stromal cells express
RANKL as a membrane-associated factor in response to several
bone-resorbing factors. Osteoclast precursors that possess RANK, a
receptor for RANKL, recognize RANKL through cell to cell interaction
with osteoblasts/stromal cells and differentiate into osteoclasts
(13, 14). Disruption of the gene encoding RANKL or RANK in
mice resulted in severe osteopetrosis due to the absence of osteoclasts
(15, 16, 17). These findings indicate that RANK-mediated
signals are essential for osteoclast formation.
Osteoprotegerin (OPG, also known as osteoclastogenesis inhibitory factor), produced by many types of cells including osteoblasts, is a negative regulator of osteoclast formation (18, 19). This factor functions as a decoy receptor for RANKL and inhibits osteoclast formation by interrupting the RANKL-RANK interaction. Analyses of transgenic mice overexpressing OPG and of animals injected with OPG have demonstrated that OPG suppresses bone resorption and increases bone mass (18). OPG knockout mice exhibited severe osteoporosis due to the stimulation of osteoclastic bone resorption (20, 21). These results suggest that OPG is an important physiological regulator of osteoclast formation.
Three independent intracellular signals in osteoblasts/stromal cells
have been proposed to regulate RANKL expression: vitamin D receptor
(VDR)-, cAMP-, and gp130-mediated signals (4). The
expression of RANKL messenger RNA (mRNA) is stimulated by
1,25-dihydroxyvitamin D3
[1,25-(OH)2D3] in
osteoblasts/stromal cells through VDR. In fact, no osteoclasts were
formed in cocultures of normal spleen cells and VDR-deficient
osteoblasts in response to
1,25-(OH)2D3, but many
osteoclasts were formed in cocultures in response to PTH
(8). PTH and PGE2 stimulate cAMP
production and RANKL mRNA expression in osteoblasts/stromal cells
(22, 23). Subtypes of PGE2
receptors, EP4 and EP2, are responsible for
PGE2-induced cAMP production in the target cells,
and signals mediated by EP4 and EP2 in osteoblasts are crucial for
osteoclast formation induced by PGE2 in
cocultures (23). Using transgenic mice expressing the
human IL-6 receptor, we have shown that cytokines, which use gp130 as a
common signal transducer, act directly on osteoblasts to induce
osteoclast formation (5). O’Brien et al.
recently reported that the expression of dominant negative STAT3
(signal transducer and activator of transcription-3) and gp130 in
stromal cells specifically abolished the ability to support osteoclast
formation in response to IL-6 plus soluble IL-6 receptors in cocultures
(24). These results suggest that VDR-, cAMP-, and
gp130-mediated signals independently stimulate RANKL expression in
osteoblasts/stromal cells. It is interesting that both VDR knockout
mice and gp130 knockout mice have osteoclasts in bone. This implies
that there is a redundancy in osteoclast formation in response to these
three signaling pathways, there are other important signaling pathways,
or both.
Cyclopiazonic acid [CPA; calcium-adenosine triphosphatase (calcium-ATPase) inhibitor] and ionomycin (calcium ionophore) have been shown to elevate intracellular calcium concentrations in various types of cells. We previously reported that CPA and ionomycin induced osteoclast formation in cocultures of bone marrow cells and primary osteoblasts (25). In support of this, Lorenzo and Raisz reported that calcium ionophores such as A23187 and ionomycin stimulated 45Ca release from fetal rat long bones prelabeled with 45Ca, an effect accompanied by an increase in the number of osteoclasts in ionomycin-treated long bones (26). These findings indicate that the increase in the intracellular calcium concentration acts as a signal for inducing osteoclast formation.
In this study we examined the mechanism of calcium signals in osteoclast formation in such cocultures. Osteoclast formation induced by CPA and ionomycin in the cocultures was completely inhibited by adding OPG. CPA and ionomycin increased the expression of RANKL and OPG mRNAs in primary osteoblasts. We also found that phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC), stimulated osteoclast formation in cocultures though up-regulation of RANKL expression in primary osteoblasts. Both intracellular calcium chelators and PKC inhibitors suppressed ionomycin-induced osteoclast formation in cocultures and expression of RANKL and OPG mRNAs in osteoblasts. Calcium/PKC-mediated signals appeared to be different from cAMP-mediated signals in inducing osteoclast formation, as the former stimulated expression of OPG mRNA but the latter inhibited it. We report here that the calcium/PKC-mediated signal in osteoblasts/stromal cells is the fourth signal for inducing RANKL, which induces osteoclast formation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cells and coculture systems
The established stromal cell lines such as ST2 (bone
marrow-derived preadipocyte cell line), MC3T3-G2/PA6 (preadipocyte cell
line), MC3T3-E1 (osteoblastic cell line), and NIH-3T3 (fibroblastic
cell line) were obtained from RIKEN Cell Bank (Tsukuba, Japan). Primary
osteoblasts were obtained from calvariae of newborn ddY mice by a
conventional method using collagenase (27). Bone marrow
cells were collected from the femora and tibiae of 5- to 8-week-old
male mice. Primary osteoblasts (2 x 104
cells) and bone marrow cells (2 x 105
cells) were cocultured for 6 days in MEM and 10% FCS (CSL Lt.,
Victoria, Australia) in 48-well culture plates (Corning, Inc., Corning, NY; 0.4 ml/well). Cocultures were treated with
various concentrations of CPA, ionomycin, thapsigargin, PMA, or
forskolin for the last 3 days, but not for the entire 6 days of the
coculture period because of the toxicity of these compounds. Some of
the cocultures were pretreated with BAPTA-AM (50 µM) for
30 min before adding these intracellular calcium-elevating compounds.
Some of the cocultures were also treated with increasing concentrations
of extracellular calcium (CaCl2) for the last 3
days. The final concentrations of calcium in the culture medium were
adjusted to 1 mM (no addition of
CaCl2), 3 mM, and 10 mM
by adding CaCl2. Bone marrow cells (2 x
105 cells) were also cocultured with 2 x
104 cells of ST2, MC3T3-G2/PA6, MC3T3-E1, or
NIH-3T3 for 6 days, and the cocultures were treated with ionomycin for
the last 3 days of coculture. After culture for 6 days, cells were
fixed and stained for tartrate-resistant acid phosphatase (TRAP; a
marker of osteoclasts) (27). The numbers of TRAP-positive
cells containing more than 3 nuclei were counted as osteoclast-like
multinucleated cells (MNCs). The results obtained from a typical
experiment are expressed as the mean ± SD of three
cultures. Significance of the differences was determined using
Student’s t test (P < 0.05).
Northern blot analysis
Primary osteoblasts (1 x 106 cells)
were plated in cell culture dishes (60 mm in diameter; Corning, Inc.) and cultured for 3 days. Cells were treated with various
concentrations of ionomycin, PMA, forskolin, or vehicle alone (ethanol)
for the indicated periods (usually 3 h). Some of the cultures were
pretreated with BAPTA-AM (50 µM), calphostin (100
nM), or staurosporin (2 nM) for 30 min before
adding intracellular calcium-elevating compounds or PMA. ST2
cells, MC3T3-G2/PA6 cells, MC3T3-E1 cells, or NIH-3T3 cells (1 x
106 cells) were also plated in dishes (60 mm in
diameter), cultured for 3 days, and treated with
10-6 M
ionomycin for 3 h. Total RNA was then isolated from cultures using
TRIzol (Life Technologies, Inc., Grand Island, NY).
Northern blotting analysis was performed using denaturing
formaldehyde/agarose gels as described. Double stranded complementary
DNA (cDNA) fragments encoding mouse RANKL and OPG were provided by Dr.
Yasuda (Snow Brand Milk Products). cDNA probes (RANKL, OPG, and
ß-tubulin) were labeled with 32P using a cDNA
labeling kit (Takara Shuzo, Kyoto, Japan). The RANKL, OPG, and
ß-tubulin probes were hybridized with membranes to which total RNA
isolated from osteoblasts/stromal cells was transferred.
Signals of RANKL, OPG, and ß-tubulin mRNAs were quantitated using a radioactive image analyzer (BAS2000, Fuji Photo Film Co., Ltd., Tokyo, Japan). Signals of RANKL and OPG mRNAs were normalized with the respective ß-tubulin mRNA expression levels to calculate the relative intensity.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, A and B). Ionomycin
dose dependently stimulated osteoclast formation in the cocultures,
which was strongly inhibited by simultaneous addition of 100 ng/ml OPG.
Northern blot analysis showed that treatment of primary osteoblasts
with ionomycin increased the expression of RANKL mRNA in a
dose-dependent manner (Fig. 1C). The dose-response effect of ionomycin
on RANKL mRNA expression in primary osteoblasts was similar to that on
osteoclast formation in the cocultures (Fig. 1, A and C). An increase
in RANKL mRNA expression occurred as early as 1 h after the
addition of ionomycin and reached a plateau at 3 h (Fig. 1D). The
expression level was still higher than the basal level even 24 h
after treatment (Fig. 1D). The expression of OPG mRNA in primary
osteoblasts was also enhanced by ionomycin in a dose-dependent manner
(Fig. 1C). The expression of OPG mRNA in primary osteoblasts reached a
plateau at 1 h after treatment and decreased thereafter (Fig. 1D).
Expression of OPG mRNA was also observed at 24 h (Fig. 1D).
Expression levels of RANKL and OPG mRNAs in bone marrow cells were
lower than those in primary osteoblasts and were unchanged by treatment
with 1 µM ionomycin for 3 h (data not shown).
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). Consistent with the stimulatory
effect of ionomycin on osteoclast formation, A23187 (calcium
ionophore), CPA (calcium-ATPase inhibitor), and thapsigargin
(calcium-ATPase inhibitor) all stimulated osteoclast formation in
cocultures. Addition of 100 ng/ml OPG to the cocultures completely
inhibited osteoclast formation induced by these compounds (Fig. 2A).
When primary osteoblasts were treated for 3 h with A23187, CPA, or
thapsigargin at concentrations necessary for induction of osteoclast
formation in cocultures, RANKL mRNA expression was increased in primary
osteoblasts (Fig. 2B). These compounds also enhanced the expression of
OPG mRNA in primary osteoblasts (Fig. 2B). Dose-dependent effects of
these intracellular calcium-elevating compounds were observed in the
osteoclast formation in cocultures and the expression of RANKL and OPG
mRNAs in primary osteoblasts (data not shown). When the cocultures were
treated for the last 3 days of culture with high calcium medium,
osteoclasts were formed in the culture medium containing calcium
concentrations of 3 mM and higher (Fig. 2C). OPG again
inhibited osteoclast formation induced by high calcium medium (Fig. 2C). Both RANKL and OPG mRNA expression in primary osteoblasts were
stimulated by high calcium medium (Fig. 2D). When cocultures were
pretreated with BAPTA-AM, an intracellular calcium chelator, for 30 min
just before adding ionomycin or CaCl2 (10
mM of the final concentration), the number of TRAP-positive
MNCs was decreased significantly (Fig. 2E). Pretreatment of osteoblasts
with BAPTA-AM also suppressed ionomycin- or high calcium medium-induced
up-regulation of RANKL and OPG mRNA expression in osteoblasts (Fig. 2F).
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,25-(OH)2D3, whereas
other stromal cell lines, such as MC3T3-E1 and NIH-3T3, did not. Bone
marrow cells were cocultured with ST2, MC3T3-G2/PA6, MC3T3-E1, or
NIH-3T3 cells. When cocultures were treated with ionomycin for the last
3 days, osteoclasts were formed in cocultures with ST2 or MC3T3-G2/PA6
cells (Fig. 3A). Neither MC3T3-E1 nor
NIH-3T3 cells supported osteoclast formation in the coculture even in
the presence of ionomycin (Fig. 3A). Addition of OPG to the cocultures
with ST2 or MC3T3-G2/PA6 cells inhibited ionomycin-induced osteoclast
formation (Fig. 3A). The expression of RANKL mRNA was stimulated by
ionomycin in ST2 and MC3T3-G2/PA6 cells, but not in MC3T3-E1 or NIH-3T3
cells (Fig. 3B). Expression of OPG mRNA, however, was up-regulated by
ionomycin in all four cell lines examined (Fig. 3B).
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). Addition of OPG to the coculture
strongly inhibited osteoclast formation induced by PMA (Fig. 4A).
Treatment of primary osteoblasts with PMA at concentrations necessary
for induction of osteoclast formation in cocultures stimulated the
expression of both RANKL and OPG mRNAs in primary osteoblasts (Fig. 4B). Thus, both calcium-mediated signals and PKC-mediated signals
stimulated not only osteoclast formation in coculture, but also RANKL
and OPG mRNA expression in osteoblasts. To determine whether PKC is
involved in ionomycin-induced osteoclast formation, cocultures
were pretreated with PKC inhibitors such as calphostin and staurosporin
before ionomycin treatment. Both calphostin and staurosporin
significantly reduced the number of osteoclasts formed in the coculture
treated with ionomycin (Fig. 4C). The PMA-induced osteoclast formation
was also suppressed by pretreatment with PKC inhibitors (data not
shown). Pretreatment of osteoblasts with these PKC inhibitors similarly
suppressed the ionomycin- and PMA-induced expression of RANKL and OPG
mRNAs in osteoblasts (Fig. 4D). 4-Phorbol, an inactive phorbol
ester, stimulated neither osteoclast formation in the coculture nor
expression of RANKL and OPG mRNAs in osteoblasts (data not shown).
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). Whereas
expression of RANKL mRNA in primary osteoblasts was increased by
treatment with forskolin (Fig. 5B), OPG mRNA expression in these cells
was suppressed (Fig. 5B). Similarly, PTH enhanced RANKL mRNA expression
and inhibited OPG mRNA expression in primary osteoblasts (Fig. 5B).
Thus, the regulation of RANKL and OPG mRNA expression by PTH in
osteoblasts was similar to that by forskolin, but not that by PMA.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Other intracellular calcium-elevating compounds, such as A23187, CPA, and thapsigargin, also stimulated osteoclast formation in cocultures and the expression of RANKL mRNA in primary osteoblasts. In addition, increases in calcium concentrations in the culture medium induced both osteoclast formation in cocultures and RANKL mRNA expression in primary osteoblasts. Addition of OPG strongly inhibited osteoclast formation induced by not only calcium-elevating compounds but also high calcium. Furthermore, pretreatment of osteoblasts with BAPTA-AM reduced both the number of osteoclasts formed in cocultures and the expression of RANKL mRNA in osteoblasts induced by ionomycin and a high concentration of calcium in the medium. These observations suggested that calcium signals in osteoblasts stimulate the expression of RANKL mRNA, which, in turn, induces osteoclast formation in the coculture. These results also suggest that the increase in cytosolic calcium is involved in RANKL expression.
Ionomycin-induced expression of RANKL mRNA was transient. The RANKL mRNA expression induced by ionomycin returned to a basal level after 48 h. However, treatment of cocultures with ionomycin for 24 h on day 4 was not enough to induce maximal osteoclast formation (data not shown). These results suggest that the continuous expression of RANKL even at a low level in osteoblasts is important for inducing osteoclast differentiation.
Many bone-resorbing hormones and cytokines have been reported to inhibit OPG mRNA expression in osteoblasts (11, 23, 28). Unexpectedly, calcium signals stimulated the expression of OPG mRNA in primary osteoblasts; nevertheless, calcium signals induced osteoclast formation in the cocultures. It is apparent from earlier work that the difference between RANKL and OPG production is likely to determine whether osteoclast formation occurs (29). The present results suggest that the stimulatory effect of RANKL on calcium signal-induced osteoclast formation is predominant over the inhibitory effect of OPG in coculture. As OPG is a secreted protein, OPG produced by osteoblasts might be diluted in the culture medium. In contrast, RANKL is expressed in osteoblasts as a cell surface protein because it contains a transmembrane domain (9, 10, 12). This structural difference between RANKL and OPG proteins together with the relative amounts produced may explain the predominant effect of RANKL over OPG in osteoclast formation in cocultures. Further studies are necessary to measure protein levels of RANKL and OPG produced by osteoblasts.
Similar to calcium-elevating compounds, PMA (an activator of PKC) stimulated osteoclast formation in the cocultures and the expression of RANKL and OPG mRNAs in primary osteoblasts. The PKC family has been classified into three groups: conventional PKCs (cPKCs), novel PKCs (nPKCs), and atypical PKCs (aPKCs) (30, 31). The sensitivities of these PKCs to PMA and intracellular calcium are different due to the presence of binding sites for PMA and calcium in the PKC proteins. Intracellular calcium signals activate only cPKCs, whereas PMA activates both cPKCs and nPKCs. Neither calcium nor PMA affects the activity of aPKCs. These findings suggest that osteoclast formation induced by PMA is mediated by cPKCs and/or nPKCs, whereas that by calcium-elevating compounds is mediated by cPKCs. The effects of PMA and calcium-elevating compounds on RANKL and OPG mRNA expression in osteoblasts/stromal cells were similar to each other. Furthermore, PKC inhibitors also suppressed both ionomycin-induced osteoclast formation in cocultures and expression of RANKL and OPG mRNAs in osteoblasts. These results suggest that cPKC-mediated signals play a central role in osteoclast formation induced by PMA and calcium-elevating compounds.
Three independent signals have been proposed to induce RANKL expression in osteoblasts/stromal cells: VDR-, cAMP-, and gp130-mediated signals. Forskolin, an activator of cAMP-mediated protein kinase A (PKA) signals, stimulated both RANKL mRNA expression in primary osteoblasts and osteoclast formation in cocultures. However, in contrast to calcium/PKC signals, forskolin inhibited OPG mRNA expression in primary osteoblasts. PTH/PTH-related protein (PTHrP) receptors have been shown to be coupled to both PKA- and PKC-mediated signals (32). Regulation of RANKL and OPG mRNA expression by PTH was similar to that by forskolin-induced signals in primary osteoblasts. Previous studies showed that cAMP production by mouse bone marrow cultures treated with several N-terminal fragments of PTHrP was well correlated with the potency of PTHrP fragments in inducing osteoclast formation in the marrow cultures (33). This suggests that the calcium/PKC signals are different from the cAMP/PKA signals in inducing RANKL expression in osteoblasts/stromal cells, and PTH-induced osteoclast formation is mainly mediated by cAMP/PKA signals. We propose that the calcium/PKC-mediated signal is the fourth signal for inducing RANKL expression in osteoblasts/stromal cells.
It is well known that there is heterogeneity in osteoblasts/stromal cells in terms of supporting activity of osteoclast formation in coculture (34). Some stromal cell lines, such as ST2 and MC3T3-G2/PA6, support osteoclast formation in response to osteotropic factors in coculture, whereas other stromal cell lines, such as NIH-3T3 or MC3T3-E1, do not (35). Calcium signals stimulated RANKL mRNA expression in ST2 and MC3T3-G2/PA6 cells, but not in NIH-3T3 and MC3T3-E1 cells. In contrast, OPG mRNA expression was stimulated by calcium signals in all of the cell lines examined. This indicates that calcium signals are similarly active in up-regulating OPG production in these stromal cell lines. It may be that RANKL expression is more tightly regulated by a cell-specific factor(s), which may be attributed to the intrinsic nature of stromal cells. Further studies will elucidate the role of such a cell-specific factor(s) in RANKL mRNA expression in osteoblasts/stromal cells.
The characteristics of bone resorption induced by osteotropic hormones and inflammatory cytokines appear to be different from those induced by calcium/PKC signals. Bone resorption induced by osteotropic hormones and cytokines is accelerated by decreasing the amount of OPG, because they often inhibit OPG production in osteoblasts (11, 29). In contrast, calcium/PKC signals stimulated OPG mRNA expression in osteoblasts, suggesting that bone resorption induced by calcium/PKC signals is regulated in a manner different from that by osteotropic factors. During embryonic bone development, osteoclasts appear just after the onset of mineralization in bone (36). Implantation of bone morphogenetic proteins into muscle or sc tissues induces ectopic bone formation at the site of implantation (37). In bone morphogenetic protein implantation, osteoclasts are also formed after the beginning of mineralization in bone tissues (38). These results suggest that physiological expression of RANKL in osteoblasts is mainly regulated by factors present in mineralized tissues. Calcium is one of the candidates that may induce physiological osteoclast formation in calcified bone. Further studies are necessary to elucidate the involvement of calcium/PKC signals in physiological regulation of osteoclast formation.
In conclusion, calcium/PKC signals stimulate the expression of both RANKL and OPG mRNAs in osteoblasts/stromal cells, and osteoclast formation in cocultures. The calcium/PKC signal is now proposed as the fourth signal for inducing RANKL expression in osteoblasts/stromal cells, in addition to VDR-, cAMP-, and gp130-mediated signals. The physiological and pharmacological significance of calcium/PKC signals in osteoclast formation needs further investigation, particularly to determine whether this signaling pathway can be used to modulate osteoclast formation in vivo.
Received May 3, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B ligand and
stimulation of osteoclastogenesis by gp130-utilizing cytokines or
interleukin-1 but not 1,25-dihydroxyvitamin D3 or
parathyroid hormone. J Biol Chem 274:19301–19308,25-dihydroxyvitamin D3 in osteoclast
differentiation and function. Methods Enzymol 282:223–235[Medline]
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) or without (
) 100 ng/ml OPG
on day 3. Cells were fixed and stained for TRAP on day 6. The number of
TRAP-positive MNCs was counted as osteoclasts. B, TRAP-staining of
cocultures treated with 1 µM ionomycin with (lower
panel) or without (upper panel) 100 ng/ml OPG
for the last 3 days. TRAP-positive cells appeared as red cells. Note
that TRAP-positive cell formation induced by ionomycin in the
cocultures was inhibited by adding OPG. C, Effects of ionomycin on the
expression of RANKL and OPG mRNA in osteoblasts. Primary osteoblasts
were treated with increasing concentrations of ionomycin for 3 h.
Total RNA was isolated from osteoblasts, and the expression of RANKL
and OPG mRNA was analyzed by Northern blotting. D, Time courses of
changes in the expression of RANKL and OPG mRNAs in osteoblasts.
Primary osteoblasts were treated with 1 µM ionomycin for
the indicated periods. Total RNA was isolated from osteoblasts, and the
expression of RANKL and OPG mRNAs was analyzed by Northern blotting.
Figures above the signals represent the relative intensities of RANKL
and OPG mRNA expression normalized with ß-tubulin mRNA expression (C
and D). Similar results were obtained from two additional sets of
experiments (A–D).
