 |
INTRODUCTION |
Osteoclastic bone resorption is regulated by the differentiation,
function, and survival of osteoclasts. The tumor necrosis factor family
molecule receptor activator of nuclear factor
(NF)1-
B ligand/osteoclast
differentiation factor (RANKL/ODF) was identified as the
membrane-associated molecule regulating osteoclast differentiation (1-3); however, the signaling pathways in regulating mature osteoclast function and survival are still controversial. Recent study of random
sequence analysis of PCR-amplified cDNA clones identified 14 distinct kinase-related genes in purified rabbit mature osteoclasts, and 8 of them were identified as receptor tyrosine kinases (RTKs) (4).
RTKs expressed on mature osteoclasts include fibroblast growth factor
receptor type 1 (FGFR1), c-Fms, and Tyro 3, whose ligands: FGF-2,
macrophage-colony stimulating factor (M-CSF), and the growth
arrest-specific gene 6 (Gas6), respectively, are known to regulate
osteoclast differentiation, function, or survival. We recently reported
that FGF-2 stimulates mature osteoclast function directly through the
activation of FGFR1 on mature osteoclasts (5, 6). M-CSF is also
reported to stimulate the survival and chemotactic behavior of
osteoclasts through the activation of its receptor, c-Fms, on
osteoclasts (7-10). Another RTK, Tyro 3, is the RTK most frequently
cloned in isolated rabbit osteoclasts in the random sequence study
above (4). Tyro 3, also known as Sky, Rse, Brt, and Tif, is a member of
the Axl, Ufo, and Sky family RTKs (11-14) containing characteristic
extracellular ligand binding domain composed of two immunoglobulin-like
domains and two fibronectin type III repeats (15, 16). The ligand for Tyro 3 is known to be Gas6, which is a vitamin K-dependent
protein acting as a growth-potentiating factor for thrombin-induced
cell proliferation (17-19).
We now propose the possibility that signaling through RTKs on
osteoclasts contributes not only to osteoclastic function but also to
the pathophysiology of osteopenic disorders. In this regard, we
recently reported that endogenous FGF-2 in the synovial fluid contributes to joint destruction in rheumatoid arthritis (RA) patients
through a direct action on mature osteoclasts (5, 6, 20). M-CSF has
been implicated in the pathophysiology of the bone loss of ovariectomy
(21-24). Hence, in this study we examined the mechanism whereby
Gas6/Tyro 3 signaling stimulates osteoclastic bone resorption using
several mouse culture systems, and investigated the possible
physiological relevance of this signaling to the bone loss of estrogen deficiency.
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EXPERIMENTAL PROCEDURES |
Materials--
Neonatal, 5-week-old, and 8-week-old ddY mice
were purchased from Shizuoka Laboratories Animal Center (Shizuoka,
Japan). Rat recombinant Gas6 and human recombinant FGF-2 were
generously provided by Shionogi Research Laboratory (Osaka, Japan) and
Kaken Pharmaceutical Co., Ltd. (Kyoto, Japan), respectively.
-Modified minimum essential medium (
MEM) was purchased from Life
Technologies, Inc., and fetal bovine serum (FBS) was from the Cell
Culture Laboratory (Cleveland, OH). Recombinant human M-CSF was
purchased from R&D Systems Inc. (Minneapolis, MN). Recombinant human
soluble RANKL/ODF was purchased from PeproTech, Inc. (London, United
Kingdom (UK)). Bacterial collagenase, 1,25(OH)2 vitamin
D3, and ISOGEN were purchased from Wako Pure Chemicals Co.
(Osaka, Japan), and dispase from Nitta Gelatin Co. (Osaka). Monoclonal
mouse antibody against phosphotyrosine was obtained from Upstate
Biotechnology Inc. (Lake Placid, NY), and monoclonal mouse antibody
against p60v-src (mAb 327) from Oncogene
Research Products (Cambridge, MA). This antibody recognizes
specifically both p60v-src and
p60c-src, and has been used to determine the
expression of p60c-src in various primary cells
and clonal cell lines. Polyclonal goat antibody against mouse Tyro 3 and nonimmune IgG as well as blocking peptides for respective
antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa
Cruz, CA). [32P]dCTP and [
-32P]ATP were
obtained from Amersham Pharmacia Biotech (Buckinghamshire, UK).
Herbimycin A and
4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1) were from Alexis Biochemicals (San Diego, CA).
2'-Amino-3'-methoxyflanone (PD98059), monoclonal mouse antibody against
phospho-p44/42 MAPK, polyclonal rabbit antibody against phospho-p38
MAPK, and mouse monoclonal against phospho-JNK MAPK, were obtained from
New England Biolabs, Inc. (Beverly, MA).
4-(4-Fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole (SB203580) was purchased from Calbiochem-Novobiochem Co. (La Jolla, CA). Other chemicals were obtained from Sigma.
Mouse Primary Osteoblast Culture--
All animal experiments
were performed according to the guidelines of the International
Association for the Study of Pain (25). In addition, the experimental
work was reviewed by the committee of Tokyo University charged with
confirming ethics. Calvariae dissected from 1-4-day-old mice were
washed in phosphate-buffered saline (PBS) and digested with 1 ml of
trypsin/EDTA (Life Technologies, Inc.) containing 10 mg of collagenase
(Sigma, type 7) for 10 min five times, and cells from fractions 3-5
were pooled. Cells were plated in six-multiwell dishes at a density of
5,000 cells/cm2 and grown to confluence in
MEM
containing 10% FBS.
Resorbed Pit Formation Assay in the Coculture of Mouse Bone
Marrow Cells and Osteoblasts--
Mouse osteoblasts (1 × 106 cells/dish) prepared as described above and bone marrow
cells (2 × 107 cells/dish) from tibiae of 8-week-old
ddY mice were cocultured on 10-cm culture dishes coated with 0.24%
collagen gel matrix (Nitta Gelatin, Tokyo, Japan) containing
MEM
with 10% FBS, 1,25(OH)2 vitamin D3
(10
8 M), and PGE2
(10
6 M) for 6 days with a medium
change every 3 days, and for 1 additional day in
MEM with 10% FBS.
After culture for 7 days, non adherent cells were washed with PBS and
adherent cells were stripped by 0.2% bacterial collagenase. An aliquot
of crude osteoclast preparation (0.1 ml) was further cultured on a
dentine slice placed in each well of 96-well dishes containing
MEM
and 10% FBS in the presence or absence of Gas6
(10
14 to 10
8
M) and/or PD98059 (1-30 µM) and SB203580 (30 µM). After 48 h of culture, cells were removed with
1 N NH4OH solution, and stained with 0.5%
toluidine blue. Total area was estimated under a light microscope with
a micrometer to assess osteoclastic bone resorption using an image
analyzer (System Supply Co., Nagano, Japan). At the same time, cells on
a dentine slice in the independent culture were fixed with 3.7% (v/v)
formaldehyde in PBS and ethanol-acetone (50:50, v/v), and stained at pH
5.0 in the presence of L(+)-tartaric acid using naphthol
AS-MX phosphate (Sigma) in N,N-dimethyl formamide as the substrate. Tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells containing more than three nuclei were counted as osteoclasts.
TRAP-positive Multinucleated Cell Formation Assay in the Mouse
Coculture System--
Mouse osteoblasts (3 × 104
cells/well) and bone marrow cells (1 × 106
cells/well) were cocultured in 24-multiwell dishes containing
MEM
and 10% FBS in the presence and/or absence of Gas6
(10
14 to 10
8
M) for 6 days with a medium change at 3 days. After 6 days
of culture, the cells were fixed and stained for TRAP as described above. TRAP-positive multinucleated cells containing more than three
nuclei were counted as osteoclasts.
Analysis of Osteoclast Survival--
Coculture of mouse
osteoblasts and marrow cells was performed on 10-cm culture dishes
coated with 0.24% collagen gel matrix to obtain osteoclasts as
described above. After 7 days of culture, dishes were treated with 4 ml
of 0.2% bacterial collagenase in
MEM for 20 min at 37 °C, and
collected cells were suspended in 170 ml of
MEM and 10% FBS. An
aliquot of crude osteoclast preparation (2 ml) was replaced in 12-well
dishes, and further cultured. After incubation for 2 h, the plates
were treated with PBS containing 0.001% Pronase E and 0.02% EDTA to
remove stromal cells. After purification, osteoclasts were cultured in
the presence or absence of Gas6 (10
8
M) or M-CSF (100 ng/ml) for various periods up to 72 h, stained with trypan blue and TRAP. Trypan blue-negative and
TRAP-positive cells were counted as living osteoclasts.
Reverse Transcriptase-PCR (RT-PCR) for Gas6 and Tyro
3--
Mouse osteoblasts and marrow cells were cocultured in 10-cm
dishes as described above. After 6 days of culture, osteoclasts were
formed and isolated by 0.001% Pronase E and 0.02% EDTA in PBS. In
addition, to obtain osteoclastic cells in various differentiation stages, spleen cells (2 × 108 cells/dish) from
8-week-old mice were cultured on 10-cm culture dishes containing
MEM
and 10% FBS with soluble RANKL/ODF (100 ng/ml) and M-CSF (10 ng/ml)
for 6 days. Total RNA was extracted from mouse brain, bone marrow
cells, osteoblasts, osteoclasts, and cultured spleen cells harvested
every day using ISOGEN following the manufacturer's instructions, and
2 µg of RNA was reverse-transcribed and amplified by PCR using
Ampli-Taq Gold (PerkinElmer Life Sciences). The primers for Gas6 were
sense (5'-CCATCAACCACGGCATGTGG-3') and antisense
(5'-TCGCACACCTTGATTTCCAT-3') and for Tyro 3 were sense (5'-GGAAGAGACGCAAGGAGAC-3') and antisense (5'-ATGGGAATGGGGAGACGAC-3'). The cycling parameters were 1 min at 96 °C, 30 s at 58 °C, 1 min 30 s at 72 °C for 25 cycles. The PCR products for Gas6 and
Tyro 3 were 589 and 445 base pairs, respectively.
Western Blot Analysis for Tyro 3--
Mouse osteoclasts and
osteoblasts were lysed with TNE buffer (10 mM Tris-HCl, 150 mM NaCl, 1% Nonidet P-40, 1 mM EDTA, 10 mM NaF, 2 mM Na3VO4, 1 mM aminoethylbenzenesulfonyl fluoride, and 10 µg/ml
aprotinin). The protein concentration in the cell lysate was measured
using a Protein Assay Kit II (Bio-Rad). Equivalent amounts (60 µg) of
cell lysates were electrophoresed by 8% SDS-PAGE and transferred to
nitrocellulose membrane. After blocking nonspecific binding with 5%
skim milk, Tyro 3-containing proteins were stained using the ECL
chemiluminescence reaction (Amersham Pharmacia Biotech) following the
manufacture's instructions. After this visualization, the antibodies
on the membrane were stripped in a buffer consisting of 62.5 mM Tris-HCl (pH 6.7), 2% SDS, and 100 mM
2-mercaptoethanol at 50 °C for 40 min. To ascertain the specificity
of these blottings, the stripping membrane was further immunoreacted
with polyclonal anti-Tyro 3 and respective blocking peptide, and the
immunoreactive bands were again visualized under the above conditions.
The immunoreactivity to anti-Tyro 3 was not lost by this stripping procedure.
Assay for Tyrosine Phosphorylation of Cellular
Proteins--
Isolated mouse osteoclasts were incubated in
MEM,
0.1% FBS for 2 h and treated with Gas6 (5 × 10
9 M) for various periods (0-10
min). Cell lysates containing equal amounts of protein (20 µg) were
analyzed by Western blot as described above. After blocking with 5%
bovine serum albumin, the membrane was incubated with monoclonal mouse
antibody against phosphotyrosine and with peroxidase-conjugated
anti-mouse IgG antibody. Phosphotyrosine-containing proteins were
visualized using the ECL chemiluminescence reaction following the
manufacturer's instructions. After the antibody was stripped from the
membrane, membranes were incubated with 5% skim milk to block
nonspecific binding, and then with monoclonal mouse antibody against
p60v-src, monoclonal mouse antibodies against
phospho-p44/42 MAPK, -JNK MAPK, polyclonal rabbit antibodies against
phospho-p38 MAPK, and the immunoreactive bands were visualized as
described above.
In Vitro Kinase Assay--
Equal amounts of protein (100 µg)
from osteoclasts stimulated with Gas6 for various periods were
immunoprecipitated with 1 µg of polyclonal goat antibody against
mouse Tyro 3 for 4 h at 4 °C, and the immune complexes were
recovered with protein G-Sepharose (Life Technologies, Inc.). The
immune complex was washed three times with TNE buffer and three times
with kinase buffer (20 mM HEPES-NaOH (pH 7.4), and 10 mM MgCl2); the samples were then resuspended in
60 µl of kinase buffer with 1 µCi (37 kBq) of
[
-32P]ATP, and incubated for 15 min at 30 °C. The
reaction was stopped by adding 20 µl of 4× sample buffer (250 mM Tris-HCl (pH 6.8), 8 mM EDTA, 12% SDS, 500 mM 2-mercaptoethanol, 15% glycerol, and 0.01% bromphenol
blue) and subjected to 10% SDS-PAGE under reducing conditions followed
by autoradiography.
Northern Blotting for Bone and Bone Marrow Cells of
Sham-operated, Ovariectomized (OVX), and Estrogen-replaced OVX
Mice--
Eight-week-old female ddY mice were subjected to either
dorsal ovariectomy or sham operation under general anesthesia. The OVX
mice were implanted with either a placebo pellet (n = 6) or a slow release (21 days) pellet containing 10 µg of
17
-estradiol (Innovative Research of America, Toledo, OH)
(n = 6). The sham-operated mice were also implanted
with a placebo pellet (n = 6). Mice were sacrificed 3 weeks after surgery, and the whole tibiae and femora were excised. To
extract RNA from cells of bone marrow cells and residual bone from
which bone marrow was removed, epiphyses at both ends were cut off,
bone marrow was flushed with PBS, collected cells were suspended in
ISOGEN extraction buffer, and total RNA was extracted. The residual
bones were washed with PBS and immediately put into ISOGEN
extraction buffer. Twenty µg and 5 µg of total RNA from bone marrow
cells and residual bones, respectively, were run on a 1.2% agarose,
2.2 M formaldehyde gel, transferred to a nitrocellulose
membrane by positive pressure, and fixed to the membrane by ultraviolet
irradiation. After 1 h of prehybridization in GMC buffer (0.5 M Na2HPO4, 1% bovine serum
albumin, 1 mM EDTA, and 7% SDS, pH 7.2) at 60 °C,
filters were hybridized overnight in GMC buffer at 65 °C with a
random primer [32P]dCTP-labeled cDNA probe for Gas6,
Tyro 3, FGF-2, FGFR1, M-CSF, and c-Fms. cDNA probes were generated
by RT-PCR using the template of total RNA from mouse brain. The primers
for probes were as follows: Gas6, sense (5'-CCATCAACCACGGCATGTGG-3')
and antisense (5'-TCGCACACCTTGATTTCCAT-3'); Tyro 3, sense
(5'-GGAAGAGACGCAAGGAGAC-3') and antisense (5'-ATGGGAATGGGGAGACGAC-3');
FGF-2, sense (5'-CAAGCAGAAGAGAGAGGAGTTGTGTC-3') and antisense
(5'-CAGTTCGTTTCAGTGCCACATACC-3'); FGFR1, sense
(5'-TGGAGTTCATGTGCAAGGTG-3') and antisense
(5'-ATAGAGAGGACCATCCTGTG-3'); M-CSF, sense
(5'-ACAACACCCCCAATGCTAAC-3') and antisense
(5'-ACTGCCTGCGTCCTCTATGC-3'); c-Fms, sense (5'-TGCTAAAGTCCACGGCTCAT-3') and antisense (5'-CAGTCCAAAGTCCCCAATCT-3').
Filters were washed in 1× SSC (0.15 M NaCl, 15 mM Na3 citrate, pH 7.0) plus 0.1% SDS twice
for 15 min at 65 °C, then once for 15 min in 0.1× SSC plus 0.1%
SDS at 65 °C. Signals were quantitated by densitometry (Bio-Rad).
Filters were stripped by boiling in 0.1% SDS + 0.1× SSC between hybridizations.
Statistical Analysis--
Means of groups were compared by
analysis of variance and significance of differences was determined by
post hoc testing using Bonferroni's method.
 |
RESULTS |
Effects of Gas6 on the Function, Differentiation, and Survival of
Osteoclasts--
To examine the effect of Gas6 on the function of
mouse osteoclasts, the pit area on a dentine slice resorbed by crude
osteoclastic cells formed in the coculture of mouse osteoblasts and
bone marrow cells in the presence of 1,25(OH)2 vitamin
D3 and PGE2 was measured. Gas6
(
10
11 M)
dose-dependently stimulated the resorbed pit area up to
2.1-fold of the control culture (Fig. 1,
top panel). This stimulation was not due to the
increase in the number of osteoclasts but due to the activation of each
osteoclast function because the number of TRAP-positive multinucleated
osteoclasts on a dentine slice was not affected by Gas6 (Fig. 1,
middle panel). In fact, the effect of Gas6 on the
pit area per osteoclast (resorbed pit area/osteoclast number in a
dentine slice) showed a similar pattern to that on the resorbed pit
area (Fig. 1, bottom panel).

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Fig. 1.
Dose response of effects of Gas6 on resorbed
pit area (top), osteoclast number
(middle), and the pit area per osteoclast
(bottom) on a dentine slice. Osteoblasts from
neonatal mouse calvariae and bone marrow cells from 8-week-old mice
were cocultured on a collagen gel to form osteoclasts. A crude fraction
of cells including osteoclasts was then released from the gel and
further cultured on a dentine slice with or without Gas6
(10 14-10 8
M). After 48 h of culture, the total pit area and the
osteoclast number were measured by toluidine blue and TRAP staining,
respectively. Data are expressed as means (bars) ± S.E. (error bars) for 8 cultures/group. *,
p < 0.01, significantly different versus
control.
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To confirm the action of Gas6 on osteoclast differentiation, dose
response of effects of Gas6 (10
14 to
10
8 M) on TRAP-positive
multinucleated osteoclast formation in the coculture for 6 days on a
plastic dish was examined. Gas6 did not increase osteoclast formation
at any concentration, whereas FGF-2 and 1,25(OH)2 vitamin
D3, positive controls, stimulated it potently (Fig.
2A). Gas6 also did not affect
TRAP-positive multinucleated osteoclast formation in the culture of
mouse bone marrow cells alone, although FGF-2 and 1,25(OH)2
vitamin D3 did (data not shown).

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Fig. 2.
Effects of Gas6 on the differentiation
(A) and the survival (B) of
osteoclasts. A, dose response of effects of Gas6,
FGF-2, and 1,25(OH)2 D3
(Vit.D3) on TRAP-positive multinucleated cell
formation in the coculture system. Mouse bone marrow cells and
osteoblasts were cocultured with or without Gas6
(10 14 to 10 8
M), FGF-2 (10 8 M),
and 1,25(OH)2 D3 (10 8
M) on plastic dishes for 6 days. TRAP-positive
multinucleated cells containing more than three nuclei were counted as
osteoclasts. Data are expressed as means (bars) ± S.E.
(error bars) for 8 cultures/group. B,
effect of Gas6 and M-CSF on the survival of isolated osteoclasts. Mouse
osteoclasts isolated from the coculture on a plastic dish were further
cultured with or without Gas6 (10 8
M) or M-CSF (100 ng/ml) for various periods up to 72 h. Trypan blue-negative and TRAP-positive osteoclasts were counted as
living osteoclasts. Data are expressed as means
(symbols) ± S.E. (error bars)
for 6 cultures/group. *, p < 0.01, significant
difference from control culture at each time point.
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We further investigated the effect of Gas6
(10
8 M) on the survival of
osteoclasts. Mouse osteoclasts isolated from the coculture were
cultured on a plastic dish for up to 72 h with or without Gas6 and
M-CSF (100 ng/ml) as a positive control (Fig. 2B). The survival rates decreased similarly with time in the control and Gas6-treated cultures. At 24 h only 32.5% and 22.5% of initially surviving cells still adhered to the dish in control and Gas6-treated cultures, respectively, and by 72 h all cells had died in both cultures. On the contrary, M-CSF increased the survival rate of osteoclasts as reported previously (6-10); the survival rates were
83.3% at 24 h and 17.8% of cells were still alive after 72 h. Hence, these studies using mouse culture systems revealed that Gas6
did not affect the differentiation or survival of osteoclasts, but did
stimulate their bone resorptive function.
Expression Pattern of Gas6 and Tyro 3 in Bone Cells--
To study
the expression of Gas6 and Tyro 3 in cells of osteoblastic and
osteoclastic lineages, mRNA levels in osteoblasts, osteoclasts,
spleen cells, bone marrow cells, and brain were examined by RT-PCR.
Gas6 was expressed in all cells examined; however, the expression of
Tyro 3 was detected only in osteoclasts and brain (Fig.
3A). Western blot analysis
confirmed that the protein level of Tyro 3 was detected in osteoclasts,
but not in osteoblasts (Fig. 3B). To investigate the
expression of Tyro 3 during the differentiation of osteoclastic cells,
Tyro 3 mRNA levels were examined in spleen cells cultured in the
presence of soluble RANKL/ODF and M-CSF without support of
osteoblastic/stromal cells (Fig. 3C). Because we extracted
mRNA from cultured spleen cells every day, various differentiation
stages of osteoclastic cells were assumed to be included. TRAP-positive
multinucleated osteoclasts became detectable at 5 days of culture and
increased at 6 days, and Tyro 3 expression was detected slightly at 5 days and abundantly at 6 days of culture. These findings confirm that
Tyro 3 is expressed predominantly in mature osteoclasts, but not in
osteoclast precursors. We therefore propose that this localization of
Tyro 3 may explain the selective action of Gas6 on the function of
mature osteoclasts.

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Fig. 3.
Expression patterns of Gas6 and Tyro 3 in
bone cells. A, messenger RNA levels of Gas6 and Tyro 3 in mouse osteoblasts (OB), osteoclasts (OCL),
spleen cells, bone marrow, and brain (RT-PCR). B,
protein levels of Tyro 3 in mouse osteoclasts (OCL) and
osteoblasts (OB) (Western blotting). C, Tyro 3 mRNA level and TRAP-positive osteoclast formation during
differentiation of osteoclastic cells in the mouse spleen cell culture
(RT-PCR). Total RNA was extracted from osteoblasts, osteoclasts, spleen
cells, bone marrow, and brain as described under "Experimental
Procedures." The PCR products for Gas6 and Tyro 3 were 589 and 445 base pairs, respectively. For Western blotting, cellular proteins
extracted with TNE buffer were subjected to SDS-PAGE, and immunoblotted
with polyclonal anti-goat Tyro 3 antibody or nonimmune IgG. To confirm
the specificity of these blottings, stripped membranes were
immunoreacted with each polyclonal anti-Tyro 3 and respective blocking
peptide. For spleen cell cultures, spleen cells (2 × 108 cells/dish) were cultured in the presence of soluble
RANKL/ODF and M-CSF for 6 days, and mRNA was extracted every day.
TRAP-positive multinucleated cells containing more than three nuclei
were counted as osteoclasts (# of
OCL). G3PDH, glyceraldehyde-3-phosphate
dehydrogenase.
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Intracellular Signaling through Gas6/Tyro 3 in Isolated
Osteoclasts--
To learn the mechanism of Gas6/Tyro 3 signaling in
mature osteoclasts, we examined the time course of effects of Gas6
(5 × 10
9 M) on tyrosine
phosphorylation of cellular proteins in isolated mouse osteoclasts
(Fig. 4A). Several proteins
were phosphorylated by Gas6 as early as 1 min, and this activation was
maintained for 10 min. The c-Src signal was used as an internal
control. Western blot analyses using antibodies against specific
proteins related to MAPKs revealed that phosphorylation of p42/p44 MAPK was seen at 1 min and maintained for 10 min, while neither p38 nor JNK
MAPK phosphorylation was seen (Fig. 4A). To investigate the
autophosphorylation of Tyro 3 by Gas6, the kinase activity of
immunoprecipitated Tyro 3 was examined by in vitro kinase
assay (Fig. 4B). Gas6 induced the kinase activity of Tyro 3 at 1 min, reached maximum at 2 min, and decreased considerably after
5 min.

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Fig. 4.
Intracellular signaling through Gas6/Tyro 3 in isolated osteoclasts. A, effects of Gas6 on
phosphorylation of cellular proteins and MAPKs in isolated osteoclasts.
Mouse osteoclasts isolated from the coculture were cultured with or
without Gas6 (5 × 10 9 M)
for the indicated period (0-10 min) and lysed with TNE buffer. Twenty
µg of cell lysates was subjected to 7.5% SDS-PAGE, and immunoblotted
with antibodies against phosphotyrosine, Src, phospho-p44/42 MAPK,
phospho-p38 MAPK, and phospho-JNK MAPK. Arrowhead indicates
170-kDa protein (the same size as Tyro 3). B, tyrosine
kinase activity of immunoprecipitated Tyro 3 in isolated osteoclasts.
Isolated osteoclasts were cultured with and without Gas6 (5 × 10 9 M) for various periods (1-10
min), lysed with TNE buffer, and 100 µg of cell lysates was
immunoprecipitated with polyclonal anti-Tyro 3 antibody. The samples
were incubated in kinase buffer with [ -32P]ATP and
subjected to SDS-PAGE. C, effects of PD98059 (PD)
and SB203580 (SB) on resorbed pit formation stimulated by
Gas6 on a dentine slice. Osteoblasts from neonatal mouse calvariae and
bone marrow cells from 8-week-old mice were cocultured on a collagen
gel to form osteoclasts. Crude osteoclastic cells released from the
coculture on a collagen gel were further cultured on a dentine slice
with or without Gas6. Gas6 (10 8
M), PD98059 (1, 3, 10, and 30 µM), and
SB203580 (30 µM) were added to the culture at 1 h
after the seeding. After 48 h of culture, total pit area in a
dentine slice was measured. Data are expressed as means
(bars) ± S.E. (error bars) for 8 cultures/group. *, p < 0.01, significant stimulation
by Gas6; #, p < 0.01, significant inhibition by
PD98059.
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To examine the functional relevance of the activation of p42/p44 MAPK
by Gas6 in osteoclasts, PD98059, a specific inhibitor of upstream
kinase of p42/p44 MAPK (26, 27), was added to the pit formation assay
system (Fig. 4C). PD98059 (1-30 µM)
dose-dependently inhibited the stimulation of Gas6 on pit
formation resorbed by mouse osteoclasts to the levels of the control
culture, while SB203580 (30 µM), a specific inhibitor of
p38 MAPK (28, 29), did not affect the Gas6 stimulation. Although
PD98059 at the highest concentration (30 µM) did not
decrease the resorbed pit formation in the control culture, inhibitors
of Src kinase, herbimycin (1 µM) and PP1 (10 µM), abrogated pit formation not only in the
Gas6-stimulated culture but also in the control culture (data not
shown), suggesting the essential role of Src kinase signaling in the
basal function of osteoclasts.
Messenger RNA Levels of RTKs and Their Ligands in Bone and Bone
Marrow of Sham-operated, OVX, and Estrogen-replaced OVX Mice--
The
physiological relevance of this Gas6/Tyro 3 signaling to the
pathophysiology of bone loss by estrogen deficiency was investigated by
examination of mRNA levels of Gas6 and Tyro 3 in cells of bone marrow and residual long bones of sham-operated, OVX, and
estrogen-replaced OVX mice (Fig. 5).
Since we assume it possible that signaling through RTKs on osteoclasts
may contribute to the pathophysiology of osteopenic disorders, we also
examined mRNA levels of other RTKs and their ligands: FGFR1 and
FGF-2, and c-Fms & M-CSF, which are known to regulate osteoclast
function or survival (5-10). Among them, Gas6 mRNA level in bone
marrow cells was up-regulated by OVX and was reduced by estrogen
replacement. No Tyro 3 expression could be detected by Northern
blotting either in bone or bone marrow of any mice, although it was
detected as a single band when mRNA from mouse brain was used (data
not shown). Among other RTKs and ligands, M-CSF was up-regulated by OVX
as reported previously (21-24), although it was little reduced by
estrogen replacement. Messenger RNA levels of other molecules were not
regulated by OVX or estrogen replacement. It is therefore proposed that
the up-regulation of Gas6 expression in bone marrow may possibly
contribute to the bone resorptive status by estrogen deficiency.

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Fig. 5.
Messenger RNA levels of RTKs and their
ligands in bone and bone marrow of sham-operated
(Sham), OVX, and estrogen-replaced OVX
(OVX+E) mice. Eight-week-old mice were subjected
to either ovariectomy (n = 12) or sham operation
(n = 6). The OVX mice were implanted with either
placebo pellets (n = 6) or slow release pellets of
17 -estradiol (n = 6). Mice were sacrificed 3 weeks
after surgery, the whole tibiae and femora were excised, and total RNA
was extracted from bone marrow and the residual bone as described under
"Experimental Procedures." Twenty µg and 5 µg of total RNA from
bone marrow and residual bone, respectively, were run on a gel, and
mRNA levels of Gas6, Tyro 3, FGF-2, FGFR1, M-CSF, and c-Fms were
analyzed by Northern blotting using cDNA probes produced by PCR.
The number under each band is the treated/control ratio of
the intensity of each band normalized to that of
glyceraldehyde-3-phosphate dehydrogenase (G3PDH) measured by
densitometry.
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 |
DISCUSSION |
This study demonstrated that Gas6, although ubiquitously expressed
in bone cells, did not affect the differentiation or survival of
osteoclasts, but stimulated osteoclast bone resorptive function using
several mouse culture systems. This may be due to the restricted localization of its receptor Tyro 3 on mature osteoclasts. Gas6 was
further shown to activate osteoclasts through the phosphorylation of
Tyro 3 and p42/p44 MAPK in mature osteoclasts. This Gas6/Tyro 3 signaling in osteoclasts was suggested to be involved in bone loss by
estrogen deficiency because Gas6 mRNA level was up-regulated by OVX
and reduced by estrogen replacement.
Gas6 is reported to be expressed ubiquitously in heart, lung, stomach,
kidney, muscle, brain, spleen, liver, ovary, and testis (30), although
little is known about its function in these organs. It is reported to
increase the proliferation and the survival of fibroblasts and vascular
smooth muscle cells (31, 32). The expression pattern of Tyro 3, on the
other hand, is limited to the central nervous system, kidney, ovary,
and testis (11-16). The downstream pathway of Gas6/Tyro 3 signaling
has been little investigated. The possible mediation by p42/p44 MAPK
shown here in osteoclasts has also been reported in a previous study
using human embryonic kidney 293 cells (33), while that of
phosphatidylinositol 3-kinase pathway is indicated in NIH3T3 cells
(34). However, functional or physiological relevance of the signaling
through Gas6/Tyro 3 in these cells remains elusive.
FGF-2, M-CSF, and Gas6 are potent regulators of osteoclastic bone
resorption whose receptors are RTKs expressed on mature osteoclasts. We
recently reported that FGF-2 acts directly on mature osteoclasts
through the signaling pathway similar to that of Gas6: activation of
its receptor FGFR1 and p42/p44 MAPK (6). M-CSF is reported to activate
its receptor, c-Fms, followed by the activation of not only p42/p44
MAPK pathway (10) but also c-Src-dependent pathway (8).
c-Src, a ubiquitous cellular tyrosine kinase, which is highly expressed
in osteoclasts, is essential for osteoclasts to form a ruffled border
and to resorb bone (35), and the contribution of c-Src kinase to
Gas6/Tyro 3 signaling has been suggested (36). In this study,
inhibitors of the Src family kinases, herbimycin and PP1, abrogated the
osteoclast function in control cultures as well as in Gas6-stimulated
cultures. Hence, we assume that the Src kinase signal may be essential
for the basal osteoclast function, while p42/p44 MAPK is the major
pathway for the Gas6 action.
Although the essential component of signaling regulating osteoclast
differentiation and function is controversial, the roles of NF-
B and
JNK have been extensively investigated (37). The stimulation by
RANKL/ODF has been demonstrated to be mediated by the activation of
NF-
B, which is dependent on the interaction with tumor necrosis
factor receptor-associated factor 6 (TRAF6) or TRAF2 (38). In fact,
knockout mice of both NF-
B1 and NF-
B2, and of TRAF6 exhibited
severe osteopetrosis due to the impaired osteoclast differentiation and
function (39, 40). Although RANKL/ODF also activates JNK in osteoclasts
(37), the role of osteoclast function is still controversial. In this
study, JNK does not seem to mediate the Gas6 effect judging from the
lack of JNK phosphorylation by Gas6. The essential signal pathway for the survival of osteoclasts is also controversial. NF-
B pathway (9)
and p42/p44 MAPK pathway (10) have been reported to be important in
sustaining the survival of osteoclasts. In the present study, although
Gas6 induced activation of p42/p44 MAPK, it did not promote the
survival of osteoclasts. This result, however, cannot rule out
possibility of involvement of p42/p44 MAPK in osteoclast survival
because the p42/p44 MAPK activation by Gas6 might be insufficient to
sustain the survival while sufficient to activate osteoclast function.
In regard to the possible physiological relevance, this study
demonstrated that OVX increased and estrogen replacement decreased the
Gas6 mRNA expression in bone marrow. OVX animal is a model for the
postmenopausal osteoporosis caused by estrogen withdrawal in humans.
Joint destruction in RA patients is also accompanied by the
acceleration of osteoclastic bone resorption. We recently reported that
increased FGF-2 in the synovial fluid contributed to joint destruction
through a direct action on mature osteoclasts (20). Expressions of Gas6
and Tyro 3 have been recently demonstrated in the synovium of RA
patients (41). These observations suggest that Gas6 as well as FGF-2
play some roles in the pathological bone disorders in these diseases.
In addition, Gas6 and FGF-2 have a common action in induction of
angiogenesis. Regarding other angiogenic growth factors besides Gas6
and FGF-2, vascular endothelial growth factor has been reported to
enhance the osteoclastic bone resorption directly and indirectly in
cultures (42-44). In our recent study to identify RTKs expressed on
rabbit mature osteoclasts, we cloned an RTK, TIE, whose ligands are
angiopoietins, other potent angiogenic growth factors (4). These
findings indicate that several angiogenic factors have a common action
to stimulate osteoclastic function as well as angiogenesis, suggesting
a positive interrelationship between the bone resorption and the
invasion of blood vessels during bone modeling and remodeling.
The results in this study demonstrate that Gas6/Tyro 3, an RTK
signaling, may play an important role in the mature osteoclast function. This direct action of Gas6 on osteoclasts might possibly have
a key function in bone loss by estrogen deficiency. Further studies
will reveal the contribution of RTK signalings like Gas6/Tyro 3 to the
pathophysiology of osteopenic disorders.