From the Department of Medicine, University of Melbourne, St. Vincent's Hospital, and St. Vincent's Institute of Medical Research, Fitzroy, Victoria 3065, Australia
Received for publication, December 21, 2000, and in revised form, February 6, 2001
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ABSTRACT |
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We have cloned and expressed murine
osteoclast inhibitory
lectin (mOCIL), a 207-amino acid type II transmembrane
C-type lectin. In osteoclast formation assays of primary murine
calvarial osteoblasts with bone marrow cells, antisense
oligonucleotides for mOCIL increased tartrate-resistant acid
phosphatase-positive mononucleate cell formation by 3-5-fold,
whereas control oligonucleotides had no effect. The extracellular
domain of mOCIL, expressed as a recombinant protein in
Escherichia coli, dose-dependently inhibited
multinucleate osteoclast formation in murine osteoblast and spleen cell
co-cultures as well as in spleen cell cultures treated with RANKL and
macrophage colony-stimulating factor. Furthermore, mOCIL acted directly
on macrophage/monocyte cells as evidenced by its inhibitory action on
adherent spleen cell cultures, which were depleted of stromal and
lymphocytic cells. mOCIL completely inhibited osteoclast formation during the proliferative phase of osteoclast formation and resulted in
70% inhibition during the differentiation phase. Osteoblast OCIL
mRNA expression was enhanced by parathyroid hormone, calcitriol, interleukin-1 The interactions of the recently discovered proteins RANKL
(Receptor Activator of
NF- OPG is a soluble member of the tumor necrosis factor receptor family
that is secreted by osteoblastic stromal cells. It acts as a decoy
receptor for RANKL, antagonizing its biological actions by preventing
it from binding to and activating its receptor, RANK (16).
Overexpression of OPG in transgenic mice results in severe
osteopetrosis, with impaired formation of marrow cavity and profound
depletion of osteoclasts. Furthermore, OPG blocks ovariectomy-associated bone loss in the rat. In addition to inhibition of osteoclast formation, OPG also inhibits resorption pit formation by
mature osteoclasts and antagonizes the induction of bone resorption by
1,25-dihydroxyvitamin D3, parathyroid hormone,
PGE2, and IL-1 In pursuit of a factor that regulates the formation of osteoclasts, we
have identified an osteoblast-derived membrane protein with structural
homology to the C-type lectin family that limits osteoclast formation.
The full-length cDNA of osteoclast
inhibitory lectin (OCIL) predicts OCIL to be a
207-amino acid type II membrane protein with a 143-amino acid
extracellular domain, a 21-amino acid transmembrane domain, and a
43-amino acid cytoplasmic domain.
Materials--
Rat UMR 201 cells, rat UMR 106 cells, and primary
mouse calvarial osteoblasts were routinely grown in cDNA Cloning from Rat and Mouse cDNA Libraries--
We
hypothesized that mature osteoblasts might express osteoclastogenic
inhibitors, based on the observation that mature osteoblasts have
limited potential to support osteoclast formation (11). The
preosteoblastic cell line UMR 201 was differentiated with 1 µM retinoic acid to enable the expression of a more
mature osteoblast phenotype (25, 26), and mRNA profiles were
compared between untreated and retinoic acid-treated cells by
differential display-polymerase chain reaction as previously described
(27).
Briefly, total RNA was isolated from untreated and retinoic
acid-treated preosteoblastic UMR 201 cells using guanidine thiocyanate (28). First strand cDNA was synthesized from 2 µg of total RNA by
incubation for 1 h at 42 °C with 15 units of avian
myeloblastosis virus reverse transcriptase (Promega, Madison, WI)
following T12VA, T12VC, or T12VG
priming. The primers used in the PCR were 5'-ATG CTG GGC ACG TAC ACA
CAA-3' and either T12VA, T12VC, or
T12VG (CLONTECH, CA). The PCR
conditions utilized a touchup PCR protocol with denaturation at
94 °C for 5 min and then five cycles of 94 °C for 1 min, 37 °C
for 1 min, and 72 °C for 1 min, followed by 35 cycles of 94 °C 1 min, 49 °C for 1 min, and 72 °C for 1 min. For these experiments, the Expand High Fidelity PCR system (Roche Molecular Biochemicals, Mannheim, Germany) was used in a PerkinElmer Life Sciences 480 thermal
cycler. A 321-bp PCR product was obtained. To extend its sequence,
anchored PCR was used to screen a rat ROS 17/2.8 cDNA library with
The full-length rat OCIL cDNA was obtained from total RNA isolated
from human parathyroid hormone-(1-34)-treated rat osteoblast-like UMR
106 cells. A 3'-rapid amplification of cDNA ends strategy was used
to obtain the 3'-end of rat OCIL using the 3'-adaptor primer 5'-GGC CAC
GCG TCG ACT AGT ACT TTT TTT TTT TTT TTT T-3' (CLONTECH) for the reverse transcription, followed
by amplification with universal adaptor primer 5'-GGC CAC GCG
TCG ACT AGT-3' and the sense primer 5'-GAA ACA TCC CCC TGG AGT ATC
C-3', which was complementary to sequences within rOCIL402. A
5'-rapid amplification of cDNA ends strategy was used to obtain the
5'-ends of the rat OCIL cDNA using the SMART RACE cDNA
amplification kit (CLONTECH). The antisense primer
5'-CTC AGT GTT GTC TGT CCA CTT CCA AGG G-3' was complementary to
sequences within rOCIL402. The full-length rat OCIL cDNA
sequence is 1628 bp.
Osteoclast Formation Assays--
Osteoclast formation was
assessed either by culturing mouse bone marrow cells with calvarial
osteoblasts or by culturing mouse spleen cells with soluble RANKL and
M-CSF.
Mouse bone marrow cell cultures were prepared as described previously
(29, 30). Bone marrow cells from long bones of 6-9-week-old male
C57BL/6J mice were co-cultured with calvarial osteoblasts obtained from
newborn mice in the absence or presence of 10 nM 1,25-dihydroxyvitamin D3 and 10 nM
PGE2. The expression of mOCIL was inhibited by concurrently
treating the co-cultures with antisense oligonucleotides added with
medium changes on days 0 and 3 of a 7-day co-culture. The following
phosphorothioate oligodeoxynucleotides for mOCIL were used at 5 µM with the co-cultures: 5'-GAG TGT TGT CTG TCC ACT
TCC-3', complementary to the extracellular domain, and 5'-GGT AGG GAA
GCC TTT GTG AC-3', complementary to the intracellular domain. Scrambled
phosphorothioate oligonucleotides with the same base composition as the
antisense oligonucleotides were used as controls for these experiments:
5'-CGC TCG CTG TAT GTC TGC TAT-3' for the extracellular domain and
5'-AGT CGT GCG TAG TGC GAG TA-3' for the intracellular domain. After 7 days, cells were fixed and stained for tartrate-resistant acid
phosphatase (TRAP) using an acid phosphatase leukocyte commercial
kit (Sigma). TRAP-positive multinucleate cells were further
characterized as osteoclasts by the presence of calcitonin receptors,
demonstrated by immunohistochemistry (30).
Mouse spleen cells were obtained from adult mice and cultured for 7 days in culture medium containing 10% fetal bovine serum, 25 ng/ml
M-CSF, and 50 ng/ml soluble RANKL in the absence or presence of
maltose-binding protein (MBP) vector control or recombinant MBP-mOCIL
fusion protein at various concentrations. To obtain cultures of
adherent spleen cells that were depleted of non-adherent erythrocytes
and lymphocytes, spleen cell suspensions were added to 10-mm diameter
culture wells (2 × 106 cells/well) containing 6-mm
diameter glass coverslips. The cells were allowed to settle and attach
for 40-60 min at 37 °C before the coverslips were removed and
rinsed vigorously in Expression of Recombinant mOCIL--
A DNA fragment encoding the
extracellular domain (residues 76-207) of mOCIL was obtained by PCR
and cloned into the EcoRI and HindIII sites of
pMAL-c2 (New England Biolabs Inc., Beverly, MA), creating a gene fusion
with the MBP-encoding malE gene. PCR was performed using
mOCIL cDNA as a template. The reaction used a sense primer
representing nucleotides 285-303 of mOCIL, encoding amino acids 76-81
(TYAACP), with an EcoRI site (5'-TCA GAA TTC ACC TAT GCT GCT
TGC CCG C-3') and an antisense primer representing nucleotides 711 to
690 of mOCIL after the termination codon with a HindIII site
(5'-GGT TAA GCT TCA GGC TAA AAA GCG TCT CTT GG-3'). The PCR product was
digested with EcoRI and HindIII and cloned into
pMAL-c2. E. coli BL21 cells were transformed with this
construct, and the fusion protein was induced with 0.3 mM
isopropyl-1-thio- Northern Blots--
UMR 106 osteoblast-like cells and primary
mouse calvarial osteoblasts were treated for 24 h with osteotropic
factors: retinoic acid, parathyroid hormone-(1-34), IL-1
A 713-bp mOCIL riboprobe was generated by reverse transcription PCR
using RNA derived from mouse calvarial osteoblasts with primers
mOCIL-17 (5'-TGG AAA CTC AGC TCC TCA GCT CTG-3', representing nucleotides 34-57) and mOCIL-12 (5'-GGG ACC ATA GGG GAA AGA GTA G-3',
representing nucleotides 725-746) and cloned into pGEM-T (Promega,
Madison, WI). The plasmid was linearized with ApaI or PstI and transcribed with T7 or SP6 RNA polymerase to
generate antisense and sense riboprobes, respectively. The riboprobes
were labeled with digoxigenin during RNA transcription using a RNA labeling kit (Roche Molecular Biochemicals) according to the
manufacturer's instructions.
Expression of OCIL mRNA during Osteoclast
Formation--
OCIL mRNA expression during osteoclast formation in
mouse bone marrow cultures was investigated by reverse
transcription-PCR. Mouse bone marrow cells were cultured for 10 days in the presence of 10 nM 1,25-dihydroxyvitamin
D3 as described (32). Total RNA was isolated at each 24-h
time point. Reverse transcriptase-PCR was carried out using mOCIL-17
and mOCIL-12 as the sense and antisense primers, respectively. The PCR
was run at 94 °C for 5 min and then for 30 cycles of 94 °C for
30 s, 60 °C for 30 s, and 72 °C for 1 min, followed by
a final extension step of 72 °C for 10 min. Southern blot analysis
was carried out as reported (33). 20 µl of each PCR mixture was run
on a 2% agarose gel and transferred to nylon membranes, and products
were authenticated by probing with an internal antisense strand
oligonucleotide complementary to mOCIL, labeled with digoxigenin-dUTP
using a 3'-tailing kit (Roche Molecular Biochemicals). Hybridization
was carried out with 2 pmol/ml labeled oligonucleotides in buffer
containing 5× SSC, 0.02% SDS, 0.1% sarcosine, and 100 ng/ml poly(A)
at 55 °C for 14 h. Detection was by chemiluminescence using CDP
(Roche Molecular Biochemicals) according to the manufacturer's instructions.
The time course of OCIL mRNA expression during osteoclast formation
was compared with mRNA expression of OPG. A set of sense and
antisense primers were used as reported (11) with nucleotide sequences
represented by OPG-7 (5'-TGA GTG TGA GGA AGG GCG TTA C-3', nucleotides
405-426) and OPG-3 (5'-TTT CTC GTT CTC TCA ATC TC-3', nucleotides
1021-1040), respectively. The PCR was run at 94 °C for 5 min and
then for 35 cycles of 94 °C for 30 s, 57 °C for 30 s,
and 72 °C for 1 min, followed by a final extension step of 72 °C
for 10 min. Hybridization was carried out using the digoxigenin-labeled internal sense strand oligonucleotide OPG-1 (5'-ACC AAA GTG AAT GCC
GAG-3') under the same conditions described above. To ensure equal
starting quantities of RNA in each sample, the reverse-transcribed material was also amplified using oligonucleotide primers specific for
rat GAPDH as described (33).
Tissue Distribution of OCIL Using Northern Blot
Analysis--
Total RNA was extracted from adult mouse tissues for the
detection of OCIL mRNA expression using Northern blot analysis as described above. Relative mRNA levels were normalized for loading variability by comparison with 18 S rRNA levels in the same filters.
In Situ Hybridization and Immunohistochemistry--
Skeletal and
extraskeletal tissues were obtained from C57BL/6J male mice. Long bones
from the femurs and tibiae of embryo (15 days of gestation), newborn
(days 1, 3, and 7), and adult (week 7) mice and calvarial bones from
newborn (day 1) and adult (week 5) mice were removed and dissected free
of tissue. For the study of extraskeletal tissue distribution, the
following tissues were obtained from embryonic (15 days of gestation),
newborn (day 1), and adult (week 8) mice: brain, kidney, lung, heart,
liver, small intestine, skin, skeletal muscle, and spleen (week 5 adult only).
Tissue specimens were fixed immediately by immersion in 4%
paraformaldehyde in diethyl ester pyrocarbonate/phosphate-buffered saline and maintained overnight at 4 °C (34). Long bones obtained from the 7-day-old and 7-week-old mice were decalcified with 15% EDTA
in 0.5% paraformaldehyde/phosphate-buffered saline solution (pH 8.0)
for 4 and 10 days, respectively, at 4 °C. The decalcifying solution
was changed daily. All tissue specimens were processed and embedded in
paraffin under sterile conditions. In situ
hybridization using the 713-bp mOCIL antisense and sense riboprobes was
carried out as described previously (34, 35).
For immunohistochemistry, the peptide
H-Cys-Met-Ala-Gln-Glu-Ala-Gln-Leu-Ala-Arg-Phe-Asp-Asn-Gln-Asp-Glu-Leu-Asn-Phe-OH,
located in the extracellular domain of OCIL, was synthesized and used to immunize rabbits following standard protocols (36). The standard peroxidase-labeled streptavidin/biotin detection method was used according to the manufacturer's instructions (Dako Corp., Carpinteria, CA) with minor modifications. The dilution of the antiserum used was
optimized in preliminary experiments. Incubation of tissue sections
with a 1:100 dilution of the primary antiserum was carried out
overnight at 4 °C in a humidified chamber. Peroxidase activity was
detected with 3',3'-diaminobenzidine tetrahydrochloride (Sigma) and
0.15% H2O2. Slides were counterstained with
hematoxylin, dehydrated, and mounted with a coverslip. To
confirm specificity of immunostaining, the primary antiserum was
substituted with preimmune rabbit serum at the same dilution.
Statistical Analysis--
Statistics were performed using
Student's t test and expressed as means ± S.E.
OCIL--
The full-length mOCIL cDNA was 1206 bp in length.
The open reading frame encodes a putative 207-amino acid peptide whose
structure has characteristics typical of a type II membrane protein,
with a predicted 143-amino acid extracellular domain, a 21-amino acid transmembrane domain, and a 43-amino acid cytoplasmic domain. The
extracellular domain has 5 cysteine residues and three predicted N-linked glycosylation sites at residues 74, 100, and 158. A
myristoylation motif is also predicted in the intracellular domain
(Fig. 1).
Comparison of the putative protein sequences derived from rat and mouse
OCIL cDNA sequences with the Swiss Protein Database indicated that
the mOCIL protein sequence included a 113-amino acid C-type lectin
motif from positions 80 to 192 in the mOCIL protein sequence (Fig. 1).
This C-type lectin motif is similar to that of CD69, a membrane-bound
lectin expressed by bone marrow hematopoietic cells and thought to be
involved in monocyte differentiation (37, 38). C-type lectin motifs are
also involved in cell-cell contact and lipid binding (39-41).
Homologies between the putative amino acid sequences of mouse and rat
OCIL and human and mouse CD69 were noted exclusively in the respective
extracellular domains of the different peptides. This is shown in Fig.
2.
Effect of mOCIL Antisense Oligonucleotides on Osteoclast
Formation--
Primary calvarial osteoblasts were co-cultured with
mouse bone marrow cells to generate osteoclasts. After 7 days, there
was a 3-5-fold increase in the number of mononucleate TRAP-positive cells in the co-cultures treated with antisense oligonucleotides for
the intracellular and extracellular domains of mOCIL compared with
co-cultures performed in the absence of oligonucleotides or in the
presence of OCIL scrambled oligonucleotides of the same base
composition as the antisense oligonucleotides (Fig.
3A). Under these experimental
conditions, multinucleate osteoclasts are generally not formed unless
stimulated by 1,25-dihydroxyvitamin D3 and
PGE2. A small number of multinucleate osteoclasts were observed in co-cultures treated with OCIL antisense oligonucleotides for 7 days (eight ± five/well), but none were observed in control co-cultures that were unstimulated or in those treated with OCIL sense
or scrambled oligonucleotides. These experiments were performed three
times, and representative results are shown. When the co-cultures were
stimulated with 10 nM 1,25-dihydroxyvitamin D3
and 10 nM PGE2, multinucleate TRAP-positive
osteoclasts were formed after 7 days. Treatment with 5 µM
OCIL antisense oligonucleotide complementary to sequences of the
extracellular domain resulted in a 7-fold increase in the number of
multinucleate osteoclasts (Fig. 3B). Both mononuclear and
multinucleate TRAP-positive cells were further characterized as
osteoclasts by the presence of calcitonin receptors demonstrated by
immunostaining, using a rabbit polyclonal antibody specific for the
C-terminal intracellular domain of the mouse and rat Cla calcitonin
receptor (30) (data not shown). Co-cultures treated with sense or
scrambled oligonucleotides did not alter osteoclast formation, and
osteoclast numbers were equivalent to similar co-cultures in which
oligonucleotides were not added.
Effect of Recombinant mOCIL on Osteoclast Formation--
The
increase in osteoclast formation when OCIL expression was inhibited by
antisense oligonucleotides suggested that OCIL acts to inhibit
osteoclast formation. This was tested by assessing osteoclast formation
in the presence of the extracellular domain of mOCIL expressed as a
soluble recombinant protein.
The effect of MBP-OCIL fusion protein on osteoclast formation by
adherent adult mouse spleen cells treated with 50 ng/ml soluble RANKL
and 25 ng/ml M-CSF for 7 days is shown in Fig.
4. Using this osteoclast generation
system, potential effects of OCIL on stromal cells were eliminated, and
since the cells were adherent cells (monocytes and macrophages),
potential effects of OCIL on lymphocytic cells were also eliminated.
Any effect of MBP on osteoclast formation was determined by the
addition of MBP vector control alone. MBP-OCIL fusion protein
dose-dependently inhibited the formation of osteoclasts,
but MBP alone did not affect osteoclast formation.
Time Course of Effects of Recombinant mOCIL on Osteoclast
Formation--
In vitro osteoclast formation can be divided
into two distinct phases: proliferation (days 0-3) and differentiation
(days 4-7) (42). The effects of MBP-OCIL fusion protein on the two phases of osteoclast development were examined with adult murine adherent spleen cells. MBP-OCIL fusion protein was effective in inhibiting osteoclast formation predominantly during the proliferative phase (days 0-3), with a smaller effect (70% inhibition) during the
differentiation phase (days 4-7) (Fig.
5).
Expression of OCIL and OPG mRNAs during Osteoclast Formation in
Mouse Bone Marrow Culture--
In this system, multinucleate
osteoclast formation was observed after day 5 and was associated with
an increase in expression of mRNA for the IL-11 Regulation of OCIL mRNA Expression in Rat
Osteoblasts--
OCIL mRNA levels were examined by Northern blot
analyses in primary mouse calvarial osteoblast cells and the rat
osteoblast-like cell line UMR 106 following treatment with osteotropic
factors for 24 h (Fig. 7). OCIL
mRNA expression was up-regulated by IL-1 Tissue Distribution of OCIL mRNA in Adult Mouse--
Total RNA
was extracted from adult (week 7) mice to determine the expression of
OCIL mRNA in extraskeletal tissues by Northern blot analysis. OCIL
mRNA was expressed by all tissues examined, with the highest
expression in kidney, liver, gut, heart, and skeletal muscle (Fig.
8).
In Situ Hybridization and Immunohistochemistry--
Skeletal and
extraskeletal tissues were obtained from C57BL/6J male mice to study
the spatial distribution of OCIL mRNA and protein by in
situ hybridization and immunohistochemistry, respectively. Long
bones from the femurs and tibiae were obtained from embryonic, newborn,
and adult mice, and calvarial bones were obtained from newborn and
adult mice. In long bones, immunohistochemistry showed strong
expression of OCIL protein in osteoblasts, chondrocytes in the growth
plate, and skeletal muscle overlying the bone (Fig. 9A). In situ
hybridization similarly showed strong expression of OCIL mRNA in
osteoblasts (Fig. 9C).
For the study of extraskeletal tissue distribution, the following
tissues were obtained from embryonic (15 days of gestation), newborn
(day 1), and adult (week 8) mice: brain, kidney, lung, heart, liver,
small intestine, skin, skeletal muscle, and spleen (week 5 adult only).
The results of in situ hybridization and immunohistochemistry for OCIL expression are tabulated in Table I.
We report the cloning and characterization of an inhibitor of
osteoclast formation (mOCIL) that belongs to the C-type lectin family.
The structure of mOCIL is reminiscent of a type II transmembrane protein, with a carboxyl-terminal extracellular domain and a short amino-terminal cytoplasmic domain.
Lectins are nonenzymatic sugar-binding proteins that bind with
considerable specificity to complex carbohydrate structures found on
cell surfaces and in the extracellular matrix and secreted glycoproteins. They are involved in numerous cellular processes such as
host-pathogen interactions, targeting of proteins within cells, and
cell-cell interactions (40, 44). The calcium-dependent (C-type) lectin family includes cell adhesion molecules like selectins, which target leukocytes to lymphoid tissues and sites of inflammation (45, 46); mannose-binding proteins that function in
antibody-independent host defense against pathogens (47, 48); and
lecticans, a family of chondroitin sulfate proteoglycans including
aggrecan, versican, neurocan, and brevican (49, 50). These proteins contain a C-type carbohydrate recognition domain attached to other domains responsible for the physiological functions of the molecule (51). Protein sequences containing C-type lectin domains are classified
into seven different categories according to sequence homology and/or
the overall modular architecture of the protein (52). The C-type lectin
domain of mOCIL has a 36% homology to the C-lectin domain of human
CD69. CD69 is the earliest leukocyte activation antigen and is
expressed mainly by activated T, B, and natural killer cells. Studies
in mice deficient in CD69 through targeted gene deletion suggested that
CD69 plays a role in B cell development, with otherwise normal
hematopoietic cell development as well as normal T cell subpopulations
and functions (53). CD69 belongs to the group V category of C-type
lectins, which includes natural killer dimeric cell-surface receptors
CD94/NKG2, Ly-49, and NKR-P1 (48). There is little information
available regarding the structure of these receptors, how they
recognize their ligands, and how they form dimers.
Recombinant OCIL inhibited osteoclast formation predominantly in the
proliferative phase of osteoclastogenesis, with a smaller effect during
the differentiation phase. Osteoclast inhibitors such as IL-18 and
GM-CSF (21) elicit their actions only during the proliferative phase of
osteoclast formation, whereas legumain appears to block the later
stages of osteoclast formation (22). We assessed OCIL action with
adherent spleen cells to eliminate the effects of lymphocytes because
IL-18 inhibits osteoclast formation via the production of GM-CSF by T
cells, suggesting that the immune system may have a regulatory role in
osteoclastogenesis. Identical results were obtained with T
cell-depleted spleen cells (data not shown), thus suggesting that OCIL
acts on osteoclast precursors independently of T cells and other lymphocytes.
Osteoblasts express RANKL and OPG as well as OCIL. Like RANKL, OCIL is
a transmembrane protein that may be dependent on cell-cell interactions
for its effects. Our results suggest that OCIL is capable of acting
independently in regulating osteoclast formation, although
cooperativity with OPG cannot be excluded. The ability of OCIL to act
independently of OPG was demonstrated with adherent spleen cultures
treated with RANKL and M-CSF. Such cultures lack osteoblasts; and as a
consequence, OPG levels were very low (data not shown). In the mouse
bone marrow culture, OCIL and OPG were constitutively expressed during
the proliferative phase (days 0-4), permitting both to act in the
early phase of osteoclast formation to inhibit osteoclast formation. In
the later phase of osteoclast formation (days 5-8), OCIL mRNA
expression was substantially suppressed, enabling the formation of
multinucleate osteoclasts. In contrast, OPG mRNA not only was
maintained, but was slightly increased during this phase of osteoclast
formation. Although this would appear to be contrary to the actions of
OPG in osteoclast formation, we hypothesize that during this later
phase, OPG may be acting to inhibit the resorptive action of mature
osteoclasts (19). The marked down-regulation of OCIL mRNA
expression during the later phase implies that OCIL does not share this
secondary function with OPG, highlighting a major difference in action
between these two molecules.
The regulation of OCIL mRNA expression was also different from that
of OPG. OCIL mRNA expression was increased by a variety of
osteotropic factors such as retinoic acid, 1,25-dihydroxyvitamin D3, parathyroid hormone, IL-1 One of the more intriguing aspects of recent knowledge on RANKL and OPG
has been the demonstration that both molecules are widely distributed
in tissues other than bone, thus raising questions about their possible
functions in extraskeletal tissues. Apart from bone, RANKL is expressed
in lymphoid tissues, heart, skeletal muscle, lung, stomach, placenta,
and thyroid tissue (5-8, 60) and has been shown to play an important
role in the immune response (57) as well as the formation of
lobulo-alveolar mammary structures during pregnancy (56). OPG is even
more widely distributed than RANKL, having been detected in lung,
heart, kidney, liver, gastrointestinal system, skin, brain, spinal
cord, endothelial cells, aortic smooth muscle cells, dendritic and B
lymphocytic cell lines, thyroid, and bone (16, 18, 58, 59). OCIL
expression was found to be very similar to our previously described
distribution for RANKL (60), being present also in brain, heart,
skeletal muscle, skin, lung, and kidney, and the expression of OCIL in
these tissues was maintained in newborn as well as adult tissues. The
function of OCIL in tissues other than bone is unknown. The widespread distribution of RANKL, OPG, and OCIL suggests that each molecule probably has a multifunctional role with the possibility of different effects depending on tissue-specific microenvironments.
Recent discoveries of RANKL and OPG as promoter and inhibitor,
respectively, of osteoclast formation highlight the importance of local
control in this process. Studies showing that the induction of
osteoclastogenesis is dependent upon the relative abundance of RANKL
compared with the levels of OPG (7, 11, 12, 54, 55, 61) have led to the
suggestion that RANKL and OPG may be the final two effectors
determining osteoclast formation (62). Nevertheless, the results
presented in this study provide good evidence that OCIL, in conjunction
with RANKL, may also play an important role in the regulation of
osteoclast formation. The proximity of OCIL to RANKL on the osteoblast
cell membrane, together with in vitro evidence that OCIL
inhibits RANKL- and M-CSF-induced osteoclast formation, is strongly
suggestive of an interaction between these molecules in skeleton and
perhaps in other tissues as well.
and -11, and retinoic acid. In rodent tissues, Northern blotting, in situ hybridization, and
immunohistochemistry demonstrated OCIL expression in osteoblasts and
chondrocytes as well as in a variety of extraskeletal tissues. The
overlapping tissue distribution of OCIL mRNA and protein with that
of RANKL strongly suggests an interaction between these molecules in
the skeleton and in extraskeletal tissues.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B ligand), osteoprotegerin
(OPG),1 and RANK, the
cognate receptor for RANKL, along with M-CSF are crucial to the
formation of osteoclasts. Osteoclasts, multinucleate cells responsible
for bone resorption, are derived from hematopoietic stem cells that
differentiate along the macrophage/monocyte lineage (1, 2). Direct
contact between osteoblasts or stromal cells with mononuclear
precursors of osteoclasts is required for their differentiation into
mature, functional, multinucleate osteoclasts (3). Osteoblasts/stromal
cells express a membrane-bound protein termed RANKL, which stimulates
the differentiation and formation of multinucleate osteoclasts from
mononuclear precursors when it binds to its receptor, RANK (4). RANKL
is also known as TRANCE (5, 6) ODF (7), or OPGL (8, 9). Recombinant protein corresponding to the extracellular domain of RANKL stimulates the formation of active, bone-resorbing osteoclasts from hematopoietic cells derived from spleen even in the absence of stromal cells (10).
RANKL expression is stimulated by bone-resorbing factors such as
parathyroid hormone, PGE2, 1,25-dihydroxyvitamin
D3, and interleukin-1
and -11 (7, 11, 12). In addition
to the stimulation of osteoclast differentiation, RANKL also enhances the activity of mature osteoclasts (13), inhibits osteoclast apoptosis
(14), and enhances osteoclast survival (15).
as well as RANKL (14, 17-19). Other
inhibitors of osteoclast formation have been identified in recent
years. They include IL-4, IL-10, IL-18, interferon-
, and GM-CSF (20,
21) as well as legumain and Sca, which were cloned and isolated from a
human osteoclast cDNA expression library. Legumain (22) is a member of the mammalian cysteine protease family, the asparaginyl
endopeptidases (23), whereas Sca is a
glycosylphosphatidylinositol-linked osteoclast inhibitory factor
(24).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-minimal
essential medium containing 10% fetal bovine serum. Incubation was
carried out at 37 °C in a humidified atmosphere equilibrated with
5% CO2 in air. Dr. S. Rodan (Merck Research Laboratories,
West Point, PA) generously provided a rat ROS 17/2.8 cDNA library.
M-CSF was a gift from Genetics Institute (Cambridge, MA). Recombinant
murine RANKL was obtained from Preprotech Inc. (Canton, MA).
[
-32P]dCTP was purchased from PerkinElmer Life
Sciences. All other reagents were of analytic grade and were obtained
from standard suppliers. The mouse cDNA library was purchased from
Stratagene (La Jolla, CA).
gt11 arms. A 25-bp antisense primer that was complementary to a
sequence in the 321-bp fragment, 5'-TGA GTG TTG TCT GTC CAC TTC CAA
G-3', was used with either the
gt11 forward primer (5'-GGT GGC
GAC GAC TCC TGG AGC C-3') or the
gt11 reverse primer (5'-GAC ACC
AGA CCA ACT GGT AAT G-3') (CLONTECH) to
amplify further nucleotide sequences. Cycling parameters were 94 °C
for 5 min and then 80 cycles of 94 °C for 30 s, 60 °C for
30 s, and 72 °C for 2 min, followed by a final extension step
of 72 °C for 10 min. A 402-bp fragment, designated rOCIL402,
was obtained with the
gt11 reverse primer as the anchored primer.
The 402-bp fragment was labeled with [
-32P]dCTP using
a random primer labeling kit (Roche Molecular Biochemicals) to screen a
mouse liver cDNA library at 65 °C in hybridization buffer
containing 4× SSPE (SSPE = 0.15 M NaCl, 0.01 M NaH2PO4, and 0.001 M
EDTA), 5× Denhardt's solution, and 0.5% SDS for 24 h. The
filters were then washed sequentially in 2× SSC at 65 °C for 15 min, 2× SSC with 0.1% SDS at 65 °C for 30 min, and finally 0.1×
SSC at 65 °C for 10 min. A 1206-bp cDNA termed mOCIL was obtained.
-minimal essential medium containing 10% fetal
bovine serum. Coverslips were then placed on fresh 10-mm diameter wells
containing 200 µl of
-minimal essential medium and 10% fetal
bovine serum. Treatments were added on day 0 and with the medium change
on day 3.
-D-galactopyranoside for 3 h at
37 °C. The MBP-OCIL fusion protein was isolated from the soluble
bacterial fraction by affinity chromatography as outlined in the
manufacturer's instructions. The eluant fractions were subjected to SDS-polyacrylamide gel electrophoresis and transferred to
polyvinylidene difluoride membranes (Roche Molecular Biochemicals). Western blot analyses were performed with a rabbit anti-MBP serum (New
England Biolabs Inc.) and a Boehringer Mannheim
chemiluminescence blotting substrate peroxidase detection
system (Roche Molecular Biochemicals). Fractions containing the
MBP-OCIL fusion protein were pooled and concentrated using an Amicon
ultrafiltration YM-10 membrane (Millipore, Bedford, MA). The protein
concentration was measured in a BCA protein assay (Pierce).
, IL-1
,
IL-11, dexamethasone, and 1,25-dihydroxyvitamin D3. Total
RNA was isolated, separated on a formaldehyde-containing 1.5% agarose
gel, and transferred to nylon filters. Filters (GeneScreen, PerkinElmer
Life Sciences) were hybridized with a digoxigenin-labeled mOCIL
riboprobe overnight, and specifically bound probe was visualized by
autoradiography after detection with anti-digoxigenin antibody as
described previously (31). Relative mRNA levels were normalized for
loading variability by comparison with 18 S rRNA levels in the same filters.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Deduced amino acid sequence of mOCIL.
The deduced amino acid sequence of mOCIL is shown, segmented into the
cytoplasmic, transmembrane, and extracellular domains. The
extracellular domain predicts a neck region of 15 amino acids, a
113-amino acid C-type lectin motif from positions 80 to 192 featuring
the carbohydrate-binding domain, and a 15-amino acid C-terminal
extension. Asterisks indicate cysteine residues, and
arrowheads indicate putative N-linked
glycosylation sites.
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Fig. 2.
Structural homology of deduced amino acid
sequences of the extracellular domains, including the C-type lectin
motifs, of mOCIL, rat OCIL (rOCIL), human CD69
(hCD69), and murine CD69
(mCD69). Gaps (indicated by
dots) have been introduced to maximize the
alignments.
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Fig. 3.
Effect of mOCIL antisense oligonucleotides on
osteoclast formation. A, murine bone marrow
cells and primary calvarial osteoblasts were co-cultured for 7 days as
described under "Experimental Procedures." Antisense
oligonucleotide for the intracellular (dotted bar) or
extracellular (hatched bar) domain of OCIL was added on days
0 and 3. Scrambled oligonucleotides with the same base composition as
the antisense oligonucleotides were used as controls (C,
white bar) for these experiments. At the end of the
incubation period, TRAP-positive mononuclear cells (TRAP+
Mono) were counted. B, murine bone marrow cells and
primary calvarial osteoblasts were co-cultured for 7 days in the
presence of 10 nM 1,25-dihydroxyvitamin D3
(1,25 (OH)2D3) and 10 nM PGE2. Multinucleate TRAP-positive
osteoclasts (TRAP+ MNC) were formed after 7 days.
Mononucleate and multinucleate TRAP-positive cells express calcitonin
receptors (data not shown). Data are representative of experiments
performed three times. Bars represent means ± S.E.
(n = 3 for each treatment). *, p < 0.001 versus control. A/S, antisense.
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Fig. 4.
Effect of recombinant mOCIL on osteoclast
formation. Adherent adult murine spleen cultures were treated with
soluble RANKL (50 ng/ml) and M-CSF (25 ng/ml) for 7 days in the
presence of increasing concentrations of MBP-OCIL fusion protein
(black bars) or MBP vector control alone (hatched
bars). The white bar (C, control) represents
wells to which no MBP or MBP-OCIL was added. TRAP-positive
multinucleate osteoclasts (TRAP+ MNC) were counted at the
end of the incubation period. Data are representative of three
independent experiments. Bars represent means ± S.E.
(n = 3 for each treatment). *, p < 0.001 versus MBP alone.
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Fig. 5.
Time course of effects of recombinant mOCIL
on osteoclast formation. Adherent adult murine spleen cultures
were treated with soluble RANKL (50 ng/ml) and M-CSF (25 ng/ml) for 7 days. In A, the control (white bar) is compared
with 500 ng/ml MBP alone (hatched bar) and 500 ng/ml
MBP-OCIL fusion protein (black bar). Increasing
concentrations of MBP-OCIL fusion protein (black bars) were
added to the cultures, either during the proliferative phase
(B; days 0-3) or the differentiation phase (C;
days 4-7). Data are representative of three independent experiments.
TRAP+ MNC, TRAP-positive multinucleate osteoclasts.
receptor as well
as the calcitonin receptor (43). OCIL mRNA was constitutively
expressed in fresh bone marrow cells. Upon stimulation by
1,25-dihydroxyvitamin D3, a time-dependent decrease in OCIL mRNA relative to GAPDH mRNA occurred. By day 3, OCIL mRNA was 13% of control levels, decreasing even further by
day 8. Like OCIL, OPG mRNA was constitutively expressed in fresh
bone marrow cells. However, unlike OCIL, OPG mRNA expression increased 2-fold by day 4 in cultures treated with
1,25-dihydroxyvitamin D3; and by day 8, it had increased
4-fold (Fig. 6).
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Fig. 6.
Expression of OCIL and OPG mRNAs during
osteoclast formation in murine bone marrow culture. Murine
bone marrow cells were cultured for 10 days in the presence of 10 nM 1,25-dihydroxyvitamin D3. Total RNA was
isolated at each 24-h time point. Reverse transcriptase-PCR was carried
out to determine the relative expression of OCIL, OPG, and GAPDH
mRNAs as described under "Experimental Procedures." The
OCIL/GAPDH ratio is plotted against time as percent control levels
( ), and the OPG/GAPDH ratio is plotted against time as -fold
induction (
). CTR+, calcitonin receptor; TRAP+
MNC, TRAP-positive multinucleate osteoclasts.
, IL-11, and
1,25-dihydroxyvitamin D3 in primary mouse calvarial cells
and by retinoic acid as well as parathyroid hormone-(1-34) in UMR 106 cells.
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Fig. 7.
Northern blot analyses of OCIL mRNA
expression in osteoblasts. Primary mouse calvarial osteoblast
cells and rat osteoblast-like cells (UMR 106) were treated in culture
for 24 h with osteotropic factors at the concentrations listed.
Total RNA was isolated and probed for the expression of OCIL mRNA
with a digoxigenin-labeled mOCIL riboprobe. Relative mRNA levels
were normalized for loading variability by comparison with 18 S rRNA
levels in the same filters. The OCIL/18 S RNA ratios, normalized to
control levels (1.0), are indicated below the Northern blots.
Dex, dexamethasone;
1,25(OH)2D3,
1,25-dihydroxyvitamin D3; RA, retinoic acid;
PTH, parathyroid hormone.
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Fig. 8.
Tissue distribution of mOCIL. Tissues
obtained from 7-week-old adult mice were subjected to Northern blot
analysis using a digoxigenin-labeled mOCIL riboprobe as described under
"Experimental Procedures." To ensure equal starting quantities of
RNA in each sample, relative mRNA levels were normalized for
loading variability by comparison with 18 S rRNA levels in the same
filters. S. Muscle, skeletal muscle.
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Fig. 9.
Immunohistochemistry and in situ
hybridization for mOCIL in mouse long bone.
Immunohistochemistry (A and B) and in
situ hybridization (C and D) were carried
out on a section of long bone from neonatal (day 1) mouse as described
under "Experimental Procedures." A, OCIL protein
expression was observed in proliferating chondrocytes (PC),
hypertrophic chondrocytes (HC), and overlying skeletal
muscle (SM). B, shown is a nonimmune negative
control of long bone. C, OCIL mRNA expression was
demonstrated in osteoblasts as indicated by the arrowheads.
D, shown is a RNase-treated section of bone included as a
negative control. Original magnification × 50 (A and
B) and × 200 (C and D).
In situ hybridization and immunohistochemistry for OCIL mRNA and
protein, respectively, in skeletal and extraskeletal tissues from
embryonic (day 15), newborn (day 1), and adult (weeks 5-8) mice
, absence of or negligible signal/staining; NA, not
applicable; ND, not determined; mk, megakaryoblast of fetal liver; ISH,
in situ hybridization; IHC, immunohistochemistry.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, and IL-11. Similar
osteotropic factors up-regulate RANKL mRNA expression (7, 12). In
contrast, OPG mRNA expression is decreased by 1,25-dihydroxyvitamin
D3, glucocorticoids, and PGE2 (11, 54, 55).
Unlike the osteoclast inhibitors IL-18, GM-CSF, interferon-
, IL-4,
and IL-10 (20), which are largely refractile to regulation by
osteotropic agents, or the case with OPG, whose regulation opposes that
of RANKL, regulation of OCIL and RANKL appears concordant, suggesting
that OCIL may be a tonic inhibitor of the actions of RANKL under
physiological conditions in bone. Under pathological conditions of
increased bone turnover, in which osteoclast formation is increased
through the actions of cytokines, the concurrent up-regulation of OCIL might act as a self-regulatory mechanism to limit osteoclast formation.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Drs. T. J. Martin and M. Parker for constructive comments and to Dr. M. Ikegame for providing access to RNA from mouse marrow cultures.
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FOOTNOTES |
---|
* This work was supported by Program Grant 003211 (to H. Z., V. K., J. M. W. Q., M. T. G., and K. W. N.) from the National Health and Medical Research Council of Australia. Portions of this work were presented at the 21st Annual Meeting of the American Society of Bone and Mineral Research, September 30 to October 4, 1999, St. Louis, MO.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF321552 and AF321553.
To whom correspondence should be addressed. Tel.: 613-9288-2574;
Fax: 613-9288-2581; E-mail: k.ng@medicine.unimelb.edu.au.
Published, JBC Papers in Press, February 13, 2001, DOI 10.1074/jbc.M011554200
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ABBREVIATIONS |
---|
The abbreviations used are: OPG, osteoprotegerin; M-CSF, macrophage colony-stimulating factor; GM-CSF, granulocyte/macrophage colony-stimulating factor; PGE2, prostaglandin E2; IL, interleukin; OCIL, osteoclast inhibitory lectin; mOCIL, murine OCIL; PCR, polymerase chain reaction; bp, base pair(s); TRAP, tartrate-resistant acid phosphatase; MBP, maltose-binding protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
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