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B Ligand, Osteoprotegerin, and Receptor Activator of NF-B in Mouse Calvariae1
* Department of Oral Cell Biology, Umea University, Umea, Sweden;
Center for Musculoskeletal Research, National Institute for Working Life, Umea, Sweden;
Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, AR 72205
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B ligand (RANKL), RANK, and
osteoprotegerin (OPG) were studied in neonatal mouse calvaria. When
tested separately, neither human (h) IL-6 nor the human soluble IL-6R
(shIL-6R) stimulated bone resorption, but when hIL-6 and the shIL-6R
were combined, significant stimulation of both mineral and matrix
release from bone explants was noted. Semiquantitative RT-PCR showed
that hIL-6 plus shIL-6R enhanced the expression of RANKL and OPG in
calvarial bones, but decreased RANK expression. Human LIF, hOSM, and
mouse OSM (mOSM) also stimulated 45Ca release and enhanced
the mRNA expression of RANKL and OPG in mouse calvaria, but had no
effect on the expression of RANK. In agreement with the RT-PCR
analyses, ELISA measurements showed that both hIL-6 plus shIL-6R and
mOSM increased RANKL and OPG proteins. 1,25-Dihydroxyvitamin
D3 (D3) also increased the RANKL protein level,
but decreased the protein level of OPG. OPG inhibited 45Ca
release stimulated by RANKL, hIL-6 plus shIL-6R, hLIF, hOSM, mOSM, and
D3. An Ab neutralizing mouse gp130 inhibited
45Ca release induced by hIL-6 plus shIL-6R. These
experiments demonstrated stimulation of calvarial bone resorption and
regulation of mRNA and protein expression of RANKL and OPG by
D3 and IL-6 family cytokines as well as regulation of RANK
expression in preosteoclasts/osteoclasts of mouse calvaria by
D3 and hIL-6 plus shIL-6R. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B ligand
(RANKL),5 and
osteoprotegerin (OPG) are key factors regulating osteoclastogenesis
(1, 2, 3, 4). M-CSF is a secreted product of stromal
cells/osteoblasts that enhances macrophage and osteoclast survival.
RANKL is a transmembrane protein produced in stromal cells/osteoblasts.
RANKL plays an essential role in osteoclast differentiation and
function by binding to receptor activator of NF-B (RANK) on
osteoclast progenitor cells. The interaction between RANKL and RANK can
be blocked by OPG, a soluble protein released from stromal
cells/osteoblasts that acts as a decoy receptor for RANKL. Mice that do
not express RANKL or RANK, or are deficient in M-CSF, do not have
functional osteoclasts and develop osteopetrosis (5, 6, 7, 8).
In contrast, mice with a targeted deletion of opg develop
multiple fractures and have decreased trabecular bone volume and
numerous osteoclasts (9). IL-6-type cytokines are a family of cytokines composed of IL-6, IL-11, leukemia inhibitory factor (LIF), oncostatin M (OSM), ciliary neurotrophic factor, cardiotrophin-1, and novel neutrophin-1/B cell stimulatory factor-3 (10, 11). These agents use the same receptor subunit, gp130, for signaling (12) and often have similar and overlapping functions. Signaling by the IL-6-type cytokines involves binding to specific receptors and activation of Janus kinases and transcription factors of the STAT family (10). The IL-6R, gp80, can exist either as a membrane-bound or a soluble protein (sIL-6R) (13). Binding of IL-6 to either the membrane-bound or soluble form of IL-6R promotes homodimerization of gp130 molecules, whereas the membrane receptors for LIF and OSM heterodimerize with gp130. Recent studies with dominant negative STAT3 and gp130 cells have shown that IL-6-type cytokines can stimulate RANKL expression in stromal cells/osteoblasts by mechanisms involving gp130 and increased STAT transcriptional activity (14).
Although few data currently exist defining the roles of LIF and OSM in skeletal turnover in the human, IL-6 has been suggested to be an important mediator of pathological bone loss (15, 16, 17, 18, 19, 20, 21, 22). Estrogen decreases the expression of IL-6 in stromal cells/osteoblasts (23), and the loss of bone caused by ovariectomy in mice can be prevented by infusion of Abs neutralizing IL-6 (24) or by IL-6 gene disruption (25). These observations have strongly implicated IL-6 in the decrease in bone mass that occurs in postmenopausal osteoporosis (15). In addition, enhanced IL-6 levels have been noted in a number of other conditions characterized by excessive loss of bone, such as periodontal disease (17, 18), Paget’s disease (19), multiple myeloma (20), rheumatoid arthritis (21), and hyperparathyroidism (22). However, IL-6 has not been noted to be an effective stimulator of osteoclast formation in mouse bone marrow cultures unless combined with sIL-6R (26). Furthermore, when sIL-6R and IL-6 are not combined in commonly employed in vitro bone culture assays, such as neonatal mouse calvarial bones, bone resorption does not occur (27, 28).
Studies in rodents have indicated that LIF and OSM are involved in the regulation of bone remodeling and bone cell function (reviewed in Refs. 29 and 30). Like IL-6, LIF is produced by osteoblasts in response to parathyroid hormone (PTH) stimulation (31, 32). Both in vivo and in vitro studies have shown that LIF stimulates osteoclastogenesis and bone resorption in mouse calvaria (33, 34). Increased osteoclastogenesis has also been observed in mouse bone marrow cultures following treatment with LIF and OSM (35, 36). Consistent with these observations, transgenic mice overexpressing LIF have been noted to exhibit increased osteoclastic bone resorption (37). However, when mice with targeted deletions of the LIF receptor gene have been studied, they have been found to have increased numbers of osteoclasts and decreased bone volume (38), findings more in line with reports that have indicated small, but significant, inhibition of basal bone resorption in fetal mouse and rat long bones treated with LIF and OSM (39, 40).
While it is known that cytokines in the IL-6 family can stimulate bone resorption, few data defining the effects of IL-6-type cytokines on the expression of RANKL, RANK, and OPG exist. The aims of the present study were to: 1) determine whether IL-6 together with sIL-6R could stimulate bone resorption in neonatal mouse calvaria, 2) determine whether OSM had a resorptive action similar to that of LIF in calvarial bones, and 3) assess the roles of RANKL, RANK, and OPG in the calvarial effects of IL-6, LIF, and OSM.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recombinant human IL-6 (hIL-6), recombinant mouse IL-6 (mIL-6),
recombinant human sIL-6R (shIL-6R), recombinant human LIF (hLIF),
recombinant human OSM (hOSM), recombinant mouse OSM (mOSM), recombinant
human IL-1
, recombinant mRANKL and mOPG fusion proteins,
neutralizing anti-mouse gp130 Ab, and mOPG and mRANKL immunoassay
kits were purchased from R&D Systems (Abingdon, U.K.); FBS was obtained
from ICN Pharmaceuticals (Costa Mesa, CA); acetazolamide, hydroxyurea,
and essentially acid free fetal BSA were purchased from Sigma-Aldrich
(St. Louis, MO); 
MEM, TRIzol LS reagent, and oligonucleotide primers
were obtained from Life Technologies (Paisley, U.K.); First Strand cDNA
Synthesis Kit and PCR Core Kit were purchased from Roche (Mannheim,
Germany); [45Ca]CaCl2,
RIA kit for PGE2; and
L-[3H]proline were obtained from
Amersham (Little Chalfont, U.K.); synthetic bovine PTH (PTH 1–34) was
obtained from Bachem (Bubendorf, Switzerland); Triton X-100 was
purchased from Merck Eurolabs (Darmstadt, Germany); culture dishes and
multiwell plates were obtained from Costar (Cambridge, MA).
Indomethacin was supplied by Merck, Sharp & Dohme (Haarlem, The
Netherlands), flurbiprofen by Boots (Nottingham, U.K.), and
3-amino-1-hydroxy-propylidene-1,1-bisphosphonate (AHPrBP) by Henkel
(Dusseldorf, Germany). Calcitonin was obtained from Sandoz (Basel,
Switzerland), and 1,25-dihydroxyvitamin D3
(D3) was supplied by Hoffmann-LaRoche (Basel,
Switzerland).
Bone organ culture
Mouse calvarial bone cultures were used as a bone resorption
assay and for PGE2 quantifications, PLR analysis
and ELISA measurements. Parietal bones from 6- to 7-day-old CsA mice
were dissected and cut into four pieces. The bones were preincubated
for 18–24 h in MEM containing 0.1% albumin and 1 µmol/L
indomethacin. Following preincubation, the bones were extensively
washed and subsequently cultured for up to 96 h in multiwell
culture dishes containing 1.0 ml indomethacin-free medium with or
without test substances (41, 42). The bones were incubated
in the presence of 5% CO2 in humidified air at
37°C.
Measurements of mineral release
Mineral mobilization was assessed by analyzing the release of 45Ca from bones prelabeled in vivo. In most experiments 2- to 3-day-old mice were injected with 1.5 µCi 45Ca, and the amount of radioactivity in bone and culture medium was analyzed by liquid scintillation at the end of the culture period. For the time-course experiments, the mice were injected with 12.5 µCi 45Ca, and radioactivity was analyzed at different time points by withdrawal of small amounts of culture medium. Isotope release was expressed as the percent release of the initial amount of isotope (calculated as the sum of radioactivity in medium and bone after culture) (40). In some experiments the data were recalculated, and the results were expressed as a percentage of the control, which was set at 100%. This allowed for accumulation of data from several experiments.
Measurements of matrix degradation
Bone matrix degradation was assessed by analyzing the release of
3H from bones dissected from mice prelabeled with
10 µCi [3H]proline 4 days before dissection.
The bones were dissected, preincubated, and cultured in MEM with or
without test substances as described above. The amount of
3H in the medium at the end of the experiments
was analyzed by liquid scintillation. At the end of the culture period,
the bones were hydrolyzed, and the radioactivity in the hydrolysates
and media was analyzed. The release of 3H
([3H]proline plus
[3H]hydroxyproline) parallels the release of
[3H]hydroxyproline and thus is a reliable
indicator of collagen breakdown.
Measurements of prostaglandin biosynthesis
PG formation in the calvarial bones was assessed by analyzing the concentration of PGE2 in the culture medium at the end of the cultures. PGE2 was determined using a commercially available RIA by following the instructions of the manufacturer.
Isolation of mouse calvarial osteoblasts
Bone cells were isolated from calvaria from 2- to 3-day-old mice
using the modified time sequential enzyme digestion technique described
by Boonekamp et al. (43). Cells from populations 6–10,
showing an osteoblastic phenotype as assessed by their cAMP
responsiveness to PTH, expression of alkaline phosphatase, osteocalcin
and bone sialoprotein expression, as well as the capacity to form
mineralized bone nodules (data not shown), were used. The cells were
incubated in MEM/10% FBS at a density of 10 x
103 cells/cm2 in culture
dishes and cultured for 48 h before RNA isolation.
RNA isolation and first-strand cDNA synthesis
Calvarial osteoblasts were incubated for 48 h in culture
dishes (60 cm2) containing MEM/10% FBS
without test substances, and then RNA was isolated. Calvarial bones, a
total of five calvarial halves per group, were incubated in 24-well
plates in the absence or the presence of hIL-6 plus shIL-6R,
D3, hLIF, hOSM, and mOSM for 24 h before RNA
isolation. The five bones belonging to the same group were pooled
before total RNA was extracted with TRIzol LS reagent following the
manufacturer’s protocol. RNA was quantified spectrophotometrically,
and the integrity of the RNA preparations was examined by agarose gel
electrophoresis. Only RNA preparations showing intact species were used
for subsequent analysis. One microgram of total RNA was reverse
transcribed into single-stranded cDNA with a First Strand cDNA
Synthesis Kit using oligo(dT)15 primers. After
incubation at 25°C for 10 min and at 42°C for 60 min, avian
myeloblastosis virus reverse transcriptase was denatured at 99°C for
5 min. cDNA was kept at -20°C until used for PCR.
Polymerase chain reactions
The synthesized cDNA was amplified by PCR using a PCR Core Kit and PC-960G Gradient Thermal Cycler (Corbett Research, Sydney, Australia). The PCR analyses for TRAP, GAPDH, RANKL, OPG, RANK, and OSM receptor (OSMR) were performed using PCR Core Kit standard protocol. In the PCR reactions for IL-6R the final MgCl2 concentration was changed from 1.5 to 1 mM, and to 2 mM for gp130 and LIF receptor (LIFR). The conditions for PCR of TRAP, GAPDH, gp130, IL-6R, LIFR, and OSMR were: denaturing at 94°C for 2 min, annealing for 40 s at 58°C for TRAP, at 57°C for GAPDH, gp130, IL-6R, and OSMR, and at 55°C for LIFR, followed by elongation at 72°C for 60 s; in subsequent cycles denaturing was performed at 94°C for 40 s. Reaction conditions for RANKL, OPG, and RANK were as follows: denaturation at 94°C for 35 s, annealing at 65°C for 35 s, and elongation at 72°C for 60 s for 10 cycles. In subsequent cycles the primer annealing temperature was decreased stepwise by 5°C every five cycles from 65 to 45°C. The sequences of primers used were: TRAP sense, 5'-AAATCACTCTTCAAGACCAG-3'; TRAP antisense, 5'-TTATTGAACAGCAGTGACAG-3'; GAPDH sense, 5'-ACTTTGTCAAGCTCATTTCC-3'; GAPDH antisense, 5'-TGCAGCGAACTTTATTGATG-3'; RANKL sense, 5'-GGTCGGGCAATTCTGAATT-3'; RANKL antisense, 5'-GGGAATTACAAAGTGCACCAG-3'; OPG sense, 5'-TGGAGATCGAATTCTGCTTG-3'; OPG antisense, 5'-TCAAGTGCTTGAGGGCATAC-3'; RANK sense, 5'-CACAGACAAATGCAAACCTTG-3'; RANK antisense, 5'-GTGTTCTGGAACCATCTTCCTCC-3'; gp130 sense, 5'-TCATCAACAGAACCACGTCC-3'; gp130 antisense, 5'-CCATACATGAAGTGCCATGC-3'; IL-6R sense, 5'-AAGAGTGACTTCCAGGTGCC-3'; IL-6R antisense, 5'-GGTATCGAAGCTGGAACTGC-3'; LIFR sense, 5'-GCTGTCATTGTTGGCGTGG; LIFR antisense, 5'-TTCATTTCCAATGTCTTAAGAGC; OSMR sense, 5'-GATGTACCCACTAAGCCGCC; and OSMR antisense, 5'-GAGGACCGTTGAGGTCAAGC. The GenBank accession numbers and the positions for the 5' and 3' ends of the nucleotides for the predicted PCR products are as follows: TRAP: NM 007388, 1072–1384; GAPDH: M32599, 957-1223; RANKL: AF013170, 929-1739; OPG: NM 008764, 428-1147; RANK: AF019046, 522–921; gp130: X62646, 330–632; IL-6R: X53802, 586–886; LIFR: NM 013584, 2595–2758; and OSMR: NM 011019, 2571–2990. The expression of these factors was compared at the logarithmic phase of the PCR reaction. The PCR products were electrophoretically size fractionated in 1.5% agarose gel and visualized using ethidium bromide. The estimated sizes of the PCR products were: TRAP, 313 bp; GAPDH, 267 bp; RANKL, 810 bp; OPG, 720 bp; RANK, 400 bp; gp130, 303 bp; IL-6R, 301 bp; LIFR, 164 bp; and OSMR, 420 bp. The identity of the PCR products was confirmed using a DYEnamic ET terminator cycle sequencing kit (Amersham Biosciences, Amersham, U.K.) with sequences analyzed on an ABI 377 XL DNA sequencer (PE Applied Biosystems, Foster City, CA).
Protein analysis
Measurements of OPG and RANKL protein synthesis were assessed by analyzing the levels of OPG and RANKL in mouse calvarial bones using commercially available ELISAs. Following preincubation, a total of eight calvarial halves per group were individually incubated in 24-well plates in the absence or the presence of hIL-6 plus shIL-6R (both 100 ng/ml), D3 (10 nmol/L), and mOSM (10 ng/ml) for 48 h. The bones were then treated with 0.2% Triton X-100 for 24 h at room temperature, and the samples were analyzed by ELISA according to the manufacturer’s protocol. The sensitivities for the immunoassay kits are 5 pg/ml. According to the manufacturer, the ELISA for RANKL might be affected by the presence of OPG, and similarly the ELISA for OPG might be affected by RANKL. The interference can be seen at concentrations of RANKL and OPG in the nanogram per milliliter range, which is not likely to be present in our samples (according to the ELISA measurements). D3 was used as a positive control and, as expected, resulted in an increased protein level of RANKL and a decreased protein level of OPG in the calvarial bones. These observations together with the results showing that our protein measurements after treatment with hIL-6 plus shIL-6R and mOSM were in agreement with the RT-PCR analyses suggest that the assessments of protein levels using the ELISAs reflect true changes in RANKL and OPG protein.
Statistics
Statistical analysis was performed using the nonparametric Kruskal-Wallis/Mann-Whitney U test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Treatment for 96 h with hIL-6, mIL-6 (both 2–200 ng/ml), or
shIL-6R (50–500 ng/ml) did not affect either
45Ca or 3H release from
neonatal mouse calvarial bones (Fig. 1
).
However, when the bones were exposed to the combination of hIL-6 (200
ng/ml) and shIL-6R (500 ng/ml), both 45Ca and
3H release were significantly stimulated. The
responses were less than those seen with either PTH or
D3 at maximally effective concentrations (10
nmol/L), but similar to release caused by a maximum concentration of
IL-1 (300 pg/ml).
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A). Similarly, when the level
of shIL-6R was kept constant (150 ng/ml), the release of
45Ca was related to the concentration of hIL-6
(5–50 ng/ml; Fig. 2B). In time-course experiments,
significant stimulation of 45Ca release caused by
hIL-6 plus shIL-6R (both 100 ng/ml) was observed at 24 h (the
first time point studied; Fig. 2C). This release was similar
to release caused by PTH (10 nmol/L) and IL-1 (300 pg/ml), although
clearly less pronounced than that produced by PTH.
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A).
Semiquantitative RT-PCR analysis showed that the mRNA expression of
TRAP, an osteoclast marker, was increased by hIL-6 plus shIL-6R (both
100 ng/ml) and D3 (10 nmol/L; Fig. 3B).
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and PTH could
stimulate IL-6 release (data not shown). This suggested that it was
possible that IL-6 might act in concert with these agents to increase
bone resorption in the presence of adequate amounts of IL-6R. However,
the release of IL-6 stimulated by IL-1 and PTH does not appear to be
a potentially important mechanism in calvaria, since
concentration-dependent stimulatory effects of IL-1 (10–100 pg/ml)
and PTH (10–100 pmol/L) were not potentiated by addition of shIL-6R
(150 ng/ml; Fig. 4).
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When calvarial bones exposed to hIL-6 plus shIL-6R (both 100
ng/ml) were treated with either indomethacin (1 µmol/L) or
flurbiprofen (1 µmol/L), two structurally different inhibitors of
cyclooxygenase activity, a significant (p < 0.01),
40% inhibition of cytokine-stimulated 45Ca
release was observed (data not shown). Treatment of mouse calvaria with
hIL-6 and shIL-6R (both 100 ng/ml) resulted in a significant
(p < 0.05), 4-fold stimulation of
PGE2 formation, while treatment with IL-1 (300
pg/ml) and PTH (10 nmol/L) increased PGE2 release
2000- and 40-fold, respectively (data not shown).
Effects of hIL-6 and shIL-6R on the expressions of RANKL, RANK, and OPG in neonatal mouse calvaria
Semiquantitative RT-PCR analysis of mRNA expressions of RANKL,
OPG, and RANK in bones treated with hIL-6 plus shIL-6R (both 100 ng/ml)
for 24 h revealed that the expression of RANKL was increased, but
the expression of RANK was decreased (Fig. 5A). The mRNA expression of
OPG in bone treated with hIL-6 plus sIL-6R was also increased, but less
than that of RANKL. Human IL-6, in the absence of shIL-6R, did not
affect the expression of RANKL, RANK, or OPG (data not shown).
Treatment of the bones with D3 (10 nmol/L)
resulted in enhanced mRNA expression of both RANKL and RANK, but
decreased expression of OPG (Fig. 5A). The semiquantitative
RT-PCR analyses were normalized with GAPDH, and identical results were
obtained in two independent experiments in which RT-PCR analyses were
repeated three times. No bands were seen in samples in which the RT
reactions were omitted, indicating no amplification of genomic DNA
(data not shown).
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B). The synthesis of OPG protein was also increased
by treatment with hIL-6 plus shIL-6R (both 100 ng/ml). In contrast,
D3 (10 nmol/L) decreased the synthesis of OPG
protein in calvarial bones (Fig. 5C). Similar data were
obtained in three independent experiments. Effects of OPG on bone resorption stimulated by RANKL, D3, and hIL-6 plus shIL-6R
RANKL (1–300 ng/ml) stimulated 45Ca release
from mouse calvarial bones in a concentration-dependent manner.
Half-maximal stimulation was found at 30 ng/ml, while maximum
stimulation (>2-fold) was noted at 200 ng/ml (Fig. 6A). OPG (300 ng/ml) abolished
45Ca release induced by RANKL (100 ng/ml; Fig. 6B). Similarly, OPG (3–300 ng/ml) significantly inhibited
45Ca release stimulated by both
D3 (10 nmol/L) and hIL-6 plus shIL-6R (both 100
ng/ml; Fig. 6, C and D). OPG did not affect
unstimulated release of 45Ca in control bones
(Fig. 6, B and C).
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Human LIF, hOSM, and mOSM (0.1–100 ng/ml) caused
concentration-dependent stimulation of 45Ca
release from mouse calvaria (data not shown). Stimulation was seen at
and above 0.3 ng/ml, with half-maximal effects at 
0.5–2 ng/ml.
Maximum stimulation was observed at 10 ng/ml; 1.5-fold increases were
noted with hOSM and hLIF, while a 1.9-fold increase was seen with
mOSM.
Treatment of calvarial bones with OPG (300 ng/ml) substantially
decreased 45Ca release stimulated with hLIF,
hOSM, and mOSM (all at 10 ng/ml; Fig. 7A). In these experiments OPG
also caused a small inhibition of 45Ca release in
the control group.
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Treatment of mouse calvaria with hLIF, hOSM, or mOSM (10 ng/ml,
respectively) for 24 h resulted in enhanced mRNA expressions of
RANKL and OPG, but neither agent changed the expression of RANK (Fig. 7, B–D). The analyses were normalized with GAPDH and
repeated three times using RNA from two experiments. No bands were seen
in samples in which the RT reactions were omitted, indicating that
genomic DNA was not amplified (data not shown).
ELISA measurements revealed that treatment of calvarial bones with mOSM
(10 ng/ml) for 48 h resulted in increased RANKL (Fig. 7E) and OPG (Fig. 7F) protein synthesis. In this
experiment D3 also caused increased RANKL and
decreased OPG protein levels. Similar data were obtained in three
independent experiments.
Expression of gp130, IL-6R, LIFR, and OSMR in neonatal mouse calvaria and calvarial osteoblasts
Calvarial bone as well as osteoblasts isolated from these bones
expressed mRNA for gp130, IL-6R, LIFR, and OSMR (Fig. 8). Comparison of mRNA expression levels
using a semiquantitative RT-PCR method revealed a lower expression
level of IL-6R compared with the expression of LIFR and OSMR in both
calvaria and isolated osteoblasts. No bands were seen in samples in
which the RT reactions were omitted, indicating no amplification of
genomic DNA (data not shown).
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Addition of anti-mouse gp130 (2 µg/ml) to calvarial bones
inhibited 45Ca release stimulated by hIL-6 plus
shIL-6R (50 ng/ml and 100 ng/ml, respectively). However, addition of
anti-mouse gp130 (2 µg/ml) did not inhibit either the stimulatory
effects of the IL-6-type cytokines, hLIF, hOSM, and mOSM (all at 10
ng/ml; Fig. 9A), or resorption
stimulated by PTH and D3 (both at 0.1 nmol/L;
Fig. 9B). In separate experiments the concentration of
anti-gp130 was increased to 4 µg/ml, but still no inhibition
could be seen in bones stimulated to resorb by mOSM (data not
shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Osteoclast formation stimulated by hIL-6 plus shIL-6R in mouse bone marrow cultures can be abolished by exposure to the cyclooxygenase inhibitor indomethacin (46). In neonatal mouse calvaria treated with hIL-6 plus shIL-6R, inhibition of resorption by indomethacin was 40%, and stimulation with hIL-6 plus shIL-6R caused only a small increase in PGE2, suggesting that the major portion of the resorptive effect of hIL-6 plus shIL-6R in calvarial explants was PG independent.
Members of the IL-6 family of cytokines, most notably IL-11 and IL-6,
have been suggested to play important roles as mediators of other
calciotrophic agents (47, 48, 49). In primary
hyperparathyroidism patients have been shown to have increased serum
levels of IL-6 that correlate with biochemical markers of bone
resorption (22). IL-6 release from cultured calvarial
bones was stimulated by PTH and IL-1 in the present study. However,
resorption stimulated by these agents was not increased by addition of
sIL-6R, suggesting that IL-6 is not a mediator of either PTH or IL-1
in neonatal mouse calvaria.
Similar to other IL-6-type cytokines, such as IL-11 and IL-6 (plus sIL-6R), both LIF and OSM were found to be stimulators of 45Ca release in neonatal mouse calvarial bones. Human LIF, hOSM, and mOSM were found to be equipotent, with half-maximal stimulation occurring at 0.5–2 ng/ml. This is the first report of OSM stimulating bone resorption in mouse calvaria. Our results showing that IL-6-type cytokines are potent resorptive agents in calvaria are in agreement with previous observations that have shown stimulation of osteoclast formation by IL-6 plus sIL-6R, IL-11, LIF, and OSM (14, 26, 35, 47). In the case of LIF the present study agrees with an investigation by Reid et al. (34) in which both human and mouse LIF were found to be in vitro stimulators of mouse calvarial bone resorption. In contrast and for unknown reasons, both LIF and OSM have also been found to exert small inhibitory effects on unstimulated (control) bone resorption in mouse and rat long bones without affecting PTH-stimulated resorption (39, 40).
Stimulation of biological activity by the IL-6 family of cytokines is dependent on the expression of biologically active cytokine binding proteins and functional gp130 protein. RT-PCR analysis demonstrated the expression of gp130 mRNA in both intact calvaria and calvarial osteoblasts. Since LIF and OSM as well as IL-11 stimulate bone resorption in mouse calvaria, a deficiency in functional gp130 cannot be the reason why IL-6 alone does not stimulate resorption. Other explanations for the depressed sensitivity to IL-6 include such possibilities as low expression of IL-6R or expression of a nonfunctional IL-6R (50). In the present study the former possibility seems to be the more plausible explanation, for RT-PCR analysis revealed only weak expression of IL-6R in whole calvaria and calvarial osteoblasts compared with mRNA expression of the receptors for LIF and OSM. This marginal expression of IL-6R, together with the inability of IL-6 to stimulate resorption without added IL-6R, or to alter mRNA expression of RANKL, RANK, or OPG in calvarial explants suggest that a sufficient quantity of IL-6R is not present in mouse calvaria. Similar results have been noted in human osteoblasts (51, 52) and gingival fibroblasts (53), where low levels of mRNA expression for IL-6R have been associated with the inability of the fibroblasts to bind IL-6 and of the osteoblasts to respond to IL-6.
Recent in vitro studies have indicated that hOSM does not activate the mOSMR, but probably functions by binding to the mLIFR. Thus, mLIFR recognizes not only mLIF, but hLIF and probably hOSM as well (54, 55, 56). This multifunctional ability of mLIFR may provide an explanation for the similarities in the actions of hOSM and hLIF on 45Ca release and the mRNA expression of RANKL and OPG in the present study.
Unlike hOSM, mOSM does not bind to mLIFR. In mouse cells, mOSM is thought to first form a low affinity complex with gp130, which then binds to the OSMR type II to form a high affinity signaling complex (56, 57). Because of the different receptors activated by human and mouse OSM, we studied the responses of both human and mouse OSM in the calvarial system. The actions of mOSM were indistinguishable from those of hOSM and hLIF, suggesting that activation of mouse OSM and LIF receptors results in similar effects on resorption.
An Ab neutralizing gp130 significantly inhibited
45Ca release stimulated by hIL-6 plus shIL-6R,
but failed to block resorption caused by hLIF, hOSM, and mOSM. The
possibility that this failure to suppress hLIF, hOSM, and mOSM was the
result of too low a concentration of Ab is not likely, for inhibition
did not occur when the concentration of Ab was increased. However, it
is possible that the activity of the anti-gp130 Ab is altered
depending on whether gp130 homodimerization or heterodimerization
occurs. In a recent study by Ahlén et al. (58) the
bone-resorptive effect of IL-11 was significantly decreased by addition
of the anti-gp130 Ab. Perhaps the ability of the anti-gp130 Ab
to interfere with both IL-6 and IL-11 signaling, but not with LIF or
OSM signaling, might be due to IL-6 and IL-11 binding to their
respective receptors before forming a signaling complex with a gp130
homodimer, whereas LIF and OSM bind to heterodimers consisting of their
respective receptor and gp130 (see Fig. 10). Still another possibility may be
related to the recent observations by O’Brien et al.
(36). These investigators showed that increasing the
expression of gp130 in a stromal/osteoblastic cell line resulted in
increased STAT3-dependent promotor activity and enhanced osteoclast
formation in response to IL-6 plus sIL-6R, but decreased STAT3 activity
and osteoclast formation in response to OSM.
|
Recent studies have shown that IL-11 stimulates RANKL and OPG mRNA
expression in osteoblasts and mouse calvaria (58, 59)
without affecting the mRNA expression of RANK in the calvarial bones
(58). Exogenous addition of OPG will also inhibit the
calvarial bone-resorptive response of IL-11 (58). In the
present study, three additional members of the IL-6 family of
cytokines, IL-6 (plus sIL-6R), LIF, and OSM, were found to be good
stimulators of resorption in neonatal mouse calvarial bones. These
cytokines as well as D3 also enhanced the mRNA
expression of RANKL. Moreover, IL-6 plus sIL-6R, OSM, and
D3 were all shown to stimulate RANKL protein
synthesis (see Fig. 10). The importance of increased RANKL expression
in the resorptive responses of D3 and the IL-6
family was demonstrated by showing that calvarial bone resorption
stimulated by D3, hIL-6 plus shIL-6R, hLIF, hOSM,
and mOSM as well as that stimulated by exogenous RANKL could be blocked
by OPG.
In addition to increasing the mRNA expression of RANKL, D3 decreased the expression of OPG and increased the expression of RANK. This alteration in the profile of RANKL, OPG, and RANK offers an explanation for why D3 caused greater stimulation of neonatal mouse calvarial bone resorption than the IL-6-type cytokines. In the case of LIF and OSM, these agents increased mRNA expression of the decoy receptor, OPG and had no effect on RANK. Enhanced stimulation of mRNA expression for OPG was additionally noted with hIL-6 plus shIL-6R, but hIL-6 plus shIL-6R decreased the expression of mRNA for RANK. In agreement with the RT-PCR analysis, hIL-6 plus shIL-6R and mOSM were found to increase OPG protein levels, whereas D3 caused a decrease. Although there has been a great deal of study of the regulation of RANKL and OPG in stromal cells/osteoblasts, there are very few data currently available on the regulation of RANK in preosteoclasts and osteoclasts. In preliminary studies we have observed that the mouse preosteoclastic cell line, Raw 264.7, expresses mRNA for both D3 receptors and gp130 (E. Persson and U. H. Lerner, unpublished observations). This suggests that the regulation of RANK by D3 and hIL-6 plus shIL-6R in neonatal mouse calvarial bones may be due to a direct effect of these agents on osteoclasts.
In summary, IL-6 was found to be a good stimulator of osteoclastic bone
resorption when combined in calvarial bones with sIL-6R. LIF and OSM
were also observed to be IL-6-type cytokines capable of stimulating
bone resorption in calvaria. In addition, neonatal mouse calvarial
bones proved to be a good model for comparing the resorptive effects of
D3 and the IL-6 family of cytokines with the mRNA
expression of RANK and mRNA expression and protein synthesis of RANKL
and OPG. The roles of RANKL, RANK, and OPG in osteoclast formation and
bone resorption stimulated by major calcium regulatory hormones and the
IL-6-type cytokines and their receptors are summarized in Fig. 10.
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Acknowledgments |
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Footnotes |
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2 P.P. and E.P. contributed equally to this study.
3 Address correspondence and reprint requests to Prof. Ulf H. Lerner, Department of Oral Cell Biology, Umea University, Umea SE-901 87, Sweden. E-mail address: ulf.lerner{at}odont.umu.se
4 Address correspondence and reprint requests to Prof. Ulf H. Lerner, Department of Oral Cell Biology, Umea University, Umea SE-90187, Sweden. E-mail address: ulf.lerner{at}odont.umu.se
5 Abbreviations used in this paper: RANKL, receptor activator of NF-B ligand; AHPrBP, 3-amino-1-hydroxy-propylidene-1,1-bisphosphonate; D3, 1,25-dihydroxyvitamin D3; h, human; LIF, leukemia inhibitory factor; LIFR, LIF receptor; m, mouse; OPG, osteoprotegerin; OSM, oncostatin M; OSMR, OSM receptor; PTH, parathyroid hormone; RANK, receptor activator of NF-B; shIL-6R, soluble human IL-6R.
Received for publication January 2, 2002. Accepted for publication July 18, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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