From the Research Institute of Life Science, Snow Brand Milk Products Co., Ltd., 519 Ishibashi-machi, Shimotsuga-gun, Tochigi 329-0512, Japan
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ABSTRACT |
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Osteoclastogenesis inhibitory factor (OCIF) is a heparin-binding secretory glycoprotein that belongs to the tumor necrosis factor receptor (TNFR) family. OCIF is present both as a ~60-kDa monomer and a disulfide-linked homodimer. We attempted to characterize the seven structural domains of OCIF by determining the capabilities of various OCIF mutants to inhibit osteoclastogenesis, to interact with heparin, and to form dimers. We also examined a potential of domains 5 and 6, death domain homologous regions (DDHs), for inducing cell death by expressing OCIF/Fas fusion proteins. Our results show that: (i) the N-terminal portion of OCIF containing domains 1-4, which have structural similarity to the extracellular domains of the TNFR family proteins, is sufficient to inhibit osteoclastogenesis; (ii) a heparin-binding site is located in domain 7, and affinity for heparin does not correlate with the inhibitory activity; (iii) Cys-400 in domain 7 is the residue responsible for dimer formation; and (iv) the C-terminal portion containing domains 5 and 6, DDHs, has a high potential for mediating a cytotoxic signal when it is expressed in cells as an OCIF/Fas fusion protein in which the transmembrane region of Fas is inserted in front of DDHs.
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INTRODUCTION |
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In the vertebrate, homeostasis and remodeling of bone are by strictly controlled by mostly unrevealed mechanisms. Much effort has been made to clarify the mechanisms, and several protein factors were found to participate in bone homeostasis (1-3). Recently, we have isolated one such factor termed osteoclastogenesis inhibitory factor (OCIF)1 from the conditioned medium of human embryonic lung fibroblasts, IMR-90 (4). Both a ~60-kDa monomer and a disulfide-linked homodimer are present in the conditioned medium, and the two forms have similar specific activity in inhibition of osteoclast formation in vitro (4). However, the mechanism by which OCIF inhibits osteoclastogenesis is not yet known.
Based on the partial amino acid sequence, cDNA for human OCIF was molecularly cloned. The amino acid sequence deduced from the nucleotide sequence of OCIF cDNA predicted that it consists of 401 amino acid residues, including a putative 21-amino acid residue signal sequence (5). The nucleotide sequence analysis has revealed that OCIF is identical to osteoprotegerin (6). OCIF has seven major domains (domains 1-7) and has overall similarity to proteins of the tumor necrosis factor receptor (TNFR) family, although OCIF lacks an apparent transmembrane region (5, 6). Domains 1-4 are cysteine-rich structures with a characteristic of extracellular domains of the TNFR family proteins. Domains 5 and 6 share structural features with "death domains" of TNFR 1, Fas, DR 3 (also designated as Apo 3, Wsl 1, and TRAMP), the TRAIL receptor, and the several recently identified cytoplasmic proteins mediating apoptosis (5, 7-17). However, unlike previously characterized death domains, two death domain homologous regions (DDHs), domains 5 and 6 of OCIF, exist in extracellular environments, because OCIF is secreted into conditioned medium. Domain 7, which does not resemble any protein motifs characterized thus far, consists of 50 amino acid residues and has a relatively high net positive charge; it contains eight basic amino acid residues (Lys and Arg) and only one acidic residue (Glu).
To determine which residue(s) or domain(s) is/are involved in the in vitro biological activity, binding to heparin, and dimer formation, we generated and characterized various mutants of OCIF. We also examined the potential of domains 5 and 6 for mediating cell death by overexpressing chimeric proteins in which portions containing the transmembrane domain derived from Fas were inserted into OCIF.
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EXPERIMENTAL PROCEDURES |
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Bacterial Strain, Cell Lines, and Culture--
Escherichia
coli DH5 (Life Technologies, Inc.) was used to propagate and
amplify plasmids. 293-EBNA (CLONTECH), a human
fetal kidney cell line, was grown in Iscove's modified Dulbecco's
medium (IMDM) containing 10% fetal bovine serum (FBS) and 250 µg/ml
geneticin (Sigma). A mouse bone marrow-derived stromal cell line, ST2
(Riken Cell Bank RCB0224, Japan) was grown in minimum essential
medium-
containing 10% FBS.
Construction of Plasmids and Expression of Mutants of
OCIF--
Mammalian expression plasmid pCEP4
(CLONTECH) was used for expression of OCIF mutants,
Fas, and OCIF-Fas chimeric proteins. Full-length OCIF cDNA was
subcloned into the XhoI and BamHI sites of pCEP4
to yield pCEP4-OCIF in which the cDNA is expressed under the
control of the cytomegalovirus promoter. Human Fas cDNA (18) was
amplified by PCR using Ex Taq polymerase (Takara Shuzo) and primers 5'-TCTTTCACTTCGGAGGATTG-3' (sense) and
5'-TCTAGACCAAGCTTTGGATTTC-3' (antisense). A human activated T cell
cDNA library (CLONTECH) was used as a template
for the PCR. Mutagenesis and genetic fusions were performed according
to a method called "recombinant polymerase chain reaction" (19). To
generate the expression vector for OCIF mutants, DNA fragments
amplified by PCR were digested with appropriate restriction enzymes
(DNA fragments for D1,
D2, and
D3 by
XhoI/NdeI;
D4 by
XhoI/SphI;
D5, C195S, C202S and C277S by
NdeI/SphI;
D6 and C319S by
NdeI/BstEII;
CL and C400S by
SphI/BstEII;
D7 by
SphI/BamHI; and
D67 and
D567 by
NdeI/BamHI) and were substituted for the
corresponding region of OCIF cDNA in pCEP4-OCIF. The plasmids thus
constructed were designated pCEP4-
D1, pCEP4-
D2, pCEP4-
D3,
pCEP4-
D4, pCEP4-
D5, pCEP4-C195S, pCEP4-C202S, pCEP4-C277S, pCEP4-
D6, pCEP4-C319S, pCEP4-
CL, pCEP4-C400S pCEP4-
D7,
pCEP4-
D67, and pCEP4-
D567, respectively. To construct a vector
expressing OCIF-Fas, DNA fragment coding for domains 1-4 of
OCIF (amplified using primers 5'-TGACAAATGTCCTCCTGGTA-3' and
5'-AGATCTCGATCCATCTATTCCACATTTTTGAGTTG-3') and that for the
transmembrane domain through the intracellular region of Fas
(amplified using primers 5'-TGTGGAATAGATGGATCGAGATCTAACTTGGGGTGGCTT-3' and 5'-CCGGATCCTCTAGACCAAGCTTTGGATTTC-3') were fused as described previously (19). The fused fragment was digested with NdeI
and BamHI, and the resultant fragment was substituted for
the NdeI/BamHI fragment of OCIF cDNA in
pCEP4-OCIF to generate pCEP4-OCIF-Fas. To generate pCEP4-TM-OCIF,
first, a DNA fragment encoding the transmembrane region of Fas was
amplified by PCR with primers 5'-GGCTCGAGTCTTTCACTTCGGAGGATTG-3'
(sense) and 5'-CCTCCGGAACCTTGGTTTTCCTTTCTGTG-3' (antisense), and
the human activated T cell cDNA library as a template. Next, the
amplified fragment was digested with BspEI, and the
~700-bp fragment produced was ligated with the 0.4-kb BspEI/SphI fragment of OCIF cDNA. Then, the
ligated DNA fragment was digested with BglII and
SphI, and a ~550-bp fragment was isolated. Finally, a
~700-bp BglII/XhoI fragment from
pCEP4-OCIF-Fas, the ~550-bp fragment, and an ~11-kb
XhoI/SphI fragment from pCEP4-OCIF were ligated.
To construct pCEP4-TM-OCIF
D567, a DNA sequence encoding domains 5 to
7 was deleted from pCEP4-TM-OCIF by recombinant PCR (19). Mutations
were confirmed by DNA sequencing. For the production of
D1,
D2,
D3,
D4,
D5,
D6,
D7,
D67,
D567,
CL, C195S,
C202S, C277S, C319S, and C400S, 293-EBNA cells were seeded on 24-well
plates at a cell density of ~2 × 105/well, and the
cells in each well were transfected with 1 µg of the respective
expression vector on the following day using LipofectAMINE (Life
Technologies, Inc.). Five hours after transfection, the DNA precipitate
was removed, and the cells were cultured in serum-free medium for
40 h. Cells transfected with pCEP4-
D567 were cultured in IMDM
containing 10% FBS and 200 µg/ml hygromycin B (Wako) to establish
stable transformants.
Establishing ELISA to Quantify OCIF and OCIF Mutants-- An ELISA employing rabbit anti-OCIF polyclonal antibody was used to quantify OCIF mutants in the conditioned medium. The polyclonal antibody was prepared as follows. OCIF produced in 293-EBNA harboring pCEP4-OCIF was purified to homogeneity as described previously (4). JW rabbits were immunized with the purified OCIF. Anti-OCIF antibody was purified from serum of the rabbits using protein G-Sepharose (Pharmacia Biotech Inc.). Horseradish peroxidase-labeled anti-OCIF antibody was prepared using a maleimide-activated peroxidase kit (Pierce).
Assay for Biological Activity of OCIF Mutants-- Osteoclastogenesis inhibitory activity was determined by observing the suppression of osteoclast-like cell formation. Osteoclast-like cell formation was evaluated by measuring tartaric acid-resistant acid phosphatase (TRAP) activity in co-culture of mouse spleen cells and ST2 cells after cultivating for 1 week in the presence of 10 nM 1,25-dihydroxyvitamin D3 and 100 nM dexamethasone concomitant with various concentrations of OCIF mutants as described previously (4). TRAP activity is expressed in absorbance at 405 nm as described previously (4).
Purification of D567--
Mutant
D567 was purified from
conditioned medium of a stable 293-EBNA transfectant using rabbit
anti-OCIF polyclonal antibody-immobilized HiTrap NHS-activated column
(Pharmacia). The conditioned medium (~100 ml) was applied to the
column at a flow rate of 0.5 ml/min. After washing the column with 21.5 ml of 50 mM Tris-HCl buffer (pH 7.5) containing 1 M NaCl and 0.1% CHAPS (Sigma) and subsequently with 8 ml
of 0.15 M NaCl containing 0.1% CHAPS, proteins were eluted
from the column with 0.2 M acetic acid (pH 2.5) containing 0.15 M NaCl and 0.1% CHAPS at a flow rate of 0.5 ml/min.
Fractions containing
D567 were determined by an ELISA employing
anti-OCIF polyclonal antibody, collected, and applied to a HiTrap Blue
(Pharmacia) column equilibrated with 10 mM phosphate buffer
(pH 6.0) containing 0.1% CHAPS. After washing the column with the same
buffer, proteins bound to the column were eluted with a linear gradient
from 0 to 2 M NaCl at a flow rate of 0.5 ml/min. Fractions
containing
D567 (~10 µg) were concentrated and desalted using
Centricon 10 (Amicon).
Heparin-Sepharose Chromatography-- The affinity of wild-type OCIF and OCIF mutants for heparin was determined by FPLC on HiTrap heparin column (Pharmacia). Conditioned medium (~1 ml) containing each OCIF mutant was applied to the column equilibrated with 50 mM Tris-HCl (pH 7.0) containing 0.1% CHAPS. The column was developed with a 40-min linear gradient of 0 to 1 M NaCl in equilibration buffer at a flow rate of 1.0 ml/min, and fractions (1.0 ml) were collected. The concentration of the mutant in each fraction was determined by ELISA employing rabbit anti-OCIF polyclonal antibody.
Western Blotting-- Proteins were separated on SDS-polyacrylamide gel (10 or 13%) electrophoresis. Rainbow-colored molecular weight markers (Bio-Rad) were used as standards. Proteins were blotted onto ProBlott (Perkin-Elmer Corp.) using a semidry-type electroblotter (Bio-Rad). OCIF or OCIF mutants were detected using horseradish peroxidase (HRP)-conjugated rabbit anti-OCIF polyclonal antibody, and the membrane was exposed to x-ray film using enhanced chemiluminescence system (ECL, Amersham Corp.).
Reverse Transcriptase PCR-- Total RNA was isolated from cells 24 h posttransfection using Trizol (Life Technologies, Inc.). Reverse transcriptase-PCR was performed with Super Script preamplification system (Life Technologies, Inc.) using 1 µg of the total RNA. Primers for the reverse transcriptase-PCR were 5'-ATGAACAACTTGCTGTGCTGCGCGCT-3' (sense) and 5'-CAAACTGTATTTCGCTCTGG-3' (antisense). The primers were designed to amplify a 424-bp fragment corresponding to the putative signal peptide and domains 1-3 of OCIF. Size of the amplified products was determined by 1% agarose gel electrophoresis.
Detection of OCIF Antigen Expressed on Cell
Surface--
293-EBNA cells were inoculated into each well in a
96-well plate at a cell density of ~2 × 104
cells/200 µl/well. The cells in each well were transfected on the
following day with 250 ng of empty vector, pCEP4-Fas, pCEP4-OCIF-Fas, pCEP4-TM-OCIF, or pCEP4-TM-OCIFD567 using LipofectAMINE. Twenty hours after transfection, cells were fixed with 1% glutaraldehyde (Wako) for 10 min at room temperature. After washing the cells twice
with 200 µl of phosphate buffered saline (PBS), HRP-conjugated anti-OCIF polyclonal antibody was added to the wells, and the plate was
incubated for 2 h at room temperature. After washing the cells
five times with 200 µl of PBS, 100 µl of substrate solution (0.4 mg/ml o-phenylenediamine dihydrochloride (Sigma) and 0.006% H2O2 in 0.1 M citrate-phosphate
buffer, pH 4.5) was added to the cells, and the incubation was
continued for additional 3 min at room temperature. The reaction was
stopped by adding 50 µl of 6 N
H2SO4 to each well, and the absorbance at 490 nm was measured.
Cell Death Assays--
293-EBNA cells were inoculated into each
well in a 24-well plate at a cell density of ~5 × 104 cells/2 ml/well. The cells in each well were
transfected on the following day with 1 µg of empty vector,
pCEP4-Fas, pCEP4-OCIF-Fas, pCEP4-TM-OCIF, or pCEP4-TM-OCIFD567,
together with 0.2 µg of pCH110, an expression plasmid for
-galactosidase (Pharmacia), using LipofectAMINE. Five hours after
transfection, the DNA precipitate was removed, and IMDM containing 10%
FBS (500 µl/well) was added to each well. After 15 h of
cultivation, cells were fixed by treating with 1% glutaraldehyde for 5 min at room temperature, washed twice with 1 ml of PBS, and stained
with X-gal (Wako). Cell morphology was examined under a phase-contrast
microscope, and the percentage of round-shaped blue cells
versus total blue cells was calculated. For the assay of
lactate dehydrogenase activity in the medium, aliquots of conditioned
medium were removed before fixing the cells. Lactate dehydrogenase
activity was measured using a colorimetric kit (Shino-test, Tokyo,
Japan). DNA fragmentation assay was performed as described previously
(20) using cells cultivated for 20 h after transfection.
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RESULTS |
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Construction of Vectors and Expression of OCIF Mutants--
A
schematic representation of OCIF and mutants of OCIF used in this work
is shown in Fig. 1. We constructed a
series of expression vectors and transfected them into 293-EBNA cells
to produce deletion mutants, D1,
D2,
D3,
D4,
D5,
D6,
and
D7. We also constructed expression vector for C-terminal
truncation mutants,
D67 and
D567. Furthermore, to identify a
residue responsible for dimer formation of OCIF, a series of Cys to Ser
mutants, C195S, C202S, C277S, C319S, and C400S, in which each Cys
residue in domains 5, 6, and 7 was replaced with Ser, and a deletion
mutant,
CL, which lacks two C-terminal amino acid residues, Cys-400
and Leu-401, were prepared. All but two mutants (
D1 and
D2) were
detected in the conditioned medium by Western blot analysis (see below) employing anti-OCIF polyclonal antibody. OCIF mutants were quantified by ELISA using anti-OCIF polyclonal antibody.
D1 and
D2 were not
secreted at levels detectable by Western blotting or ELISA. The
concentration of most of the mutants in their conditioned media ranged
from 200 ng/ml to 2 µg/ml. Accurate determination of inhibitory
activity of
D6 was impossible due to poor productivity (~50
ng/ml).
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The N-terminal Portion of OCIF Containing Domains 1-4 Is
Sufficient to Inhibit Osteoclastogenesis--
Fig.
2A shows the
osteoclastogenesis inhibitory activity of the deletion mutants measured
based on the inhibition of TRAP-positive osteoclast-like cell formation
in the cocultures. Although wild-type OCIF inhibited the
osteoclast-like cell formation in a dose range of 5 to 40 ng/ml
(half-maximal inhibitory dose (ID50) of ~6 ng/ml), D3
and
D4 failed to inhibit the osteoclast-like cell formation at
concentrations 35 and 40 ng/ml, respectively. In contrast,
D5
retained the inhibitory activity with an ID50 of ~15
ng/ml. Furthermore,
D7 had a specific activity comparable to that of wild-type OCIF. A C-terminal truncation mutant
D67, which lacks domains 6 and 7, possessed the osteoclastogenesis inhibitory activity, although potency in the inhibitory activity was considerably lower than
that of wild-type OCIF (an ID50 of 10 ng/ml) (Fig.
2A). For the accurate determination of the inhibitory
activity of
D567, which lacks DDHs entirely, we purified it using an
anti-OCIF-antibody-immobilized affinity column. The purified
D567
inhibited osteoclastogenesis (Fig. 2B), indicating that
truncation of domains 5-7 (consisting of the C-terminal 204 residues)
does not abolish the biological activity. However, the potency of
D567 was approximately 10% of that of wild-type OCIF (Fig.
2B) as estimated from their ID50. These results
indicate that the N-terminal portion containing the first four domains
is sufficient for exerting the osteoclastogenesis inhibitory activity
in vitro.
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Deletion of Domain 7 Decreases the Affinity of OCIF for
Heparin--
To examine the significance of the binding of OCIF to
heparin in the inhibition of osteoclastogenesis, we analyzed the
affinity of D7,
D67, and
D567 for heparin by FPLC on HiTrap
heparin column. Conditioned medium of the cells transiently expressing each OCIF mutant was loaded on the column and each mutant was eluted
from the column with NaCl-containing buffer. Wild-type OCIF was eluted
at NaCl concentrations of 0.55 M and 0.74 M,
which correspond to those at which the monomer and the dimer form of OCIF are eluted, respectively (Fig. 3).
D7 was eluted as a single peak at an NaCl concentration of
0.24M. Further truncation had only marginal effects on the
binding of OCIF to heparin (Fig. 3). These three mutant proteins were
eluted as single peaks, probably because they are present as monomers
(see below). These results strongly suggest that domain 7, which
occupies the C-terminal 50 amino acid residues, contains a
heparin-binding site. The fact that deletion of domain 7 did not affect
the inhibition of osteoclastogenesis (Fig. 2A) but
significantly decreased the binding of OCIF to heparin (Fig. 3)
indicates that binding ability of OCIF to heparin does not correlate
with its osteoclastogenesis inhibitory activity in
vitro.
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Domain 7 Is Involved in the Dimerization of OCIF--
OCIF from
IMR-90-conditioned medium is present as two forms, a monomer with an
approximate molecular mass of 60 kDa and a disulfide-linked dimer with
an approximate mass of 120 kDa (4). To identify (a)
domain(s) responsible for the dimer formation, the capability of the
domain deletion mutants to form dimers was analyzed using
immunoblotting as shown in Fig.
4A. A protein with a mass of
80-100 kDa was detected for D3,
D4,
D5, and
D6 as a major
band. In contrast,
D7 is present almost exclusively as a ~55-kDa
protein. Thus,
D7 is present mainly as a monomer, while
D3,
D4,
D5, and
D6 are present in two forms, a monomer and a dimer
(or a multimer) in the conditioned medium, suggesting that domain 7 is
involved in the dimer formation.
D67 and
D567, both lacking
domain 7, migrated as monomers as expected (Fig. 4A).
Difference in size between
D3 and
D4 (Fig. 4A) is
probably due to a different degree of glycosylation. Indeed, there are three potential N-glycosylation sites
(Asn-X-Ser/Thr) in domain 3, whereas there is no such site
in domain 4 (5, 6).
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Cys-400 Is Responsible for the Dimer Formation--
Since there is
only one Cys residue (Cys-400) in domain 7 (5, 6), participation of the
residue in the intermolecular disulfide-linkage was suspected. To
confirm this, two mutants, one with substitution of a Ser residue for
Cys-400 (C400S) and the other with a deletion of the two C-terminal
amino acid residues Cys-400 and Leu-401 (CL) were produced. As a
control, a series of Cys to Ser mutants, C195S, C202S, C277S, and
C319S, in which each Cys residue in domains 5 and 6 was replaced with a
Ser residue, were generated. The mutants were transiently expressed in
293-EBNA cells, and the structure of the mutants was analyzed by
Western blotting as shown in Fig. 4B. The results indicate
that both C400S and
CL exist almost exclusively as a monomer with a
mass of ~60 kDa (Fig. 4B). No monomer-form OCIF with a
mass of ~60 kDa was detected in the conditioned medium of C195S,
C202S, C277S, or C319S (Fig. 4B). These four mutants
migrated even slower than the dimer form OCIF with a mass of ~120
kDa. The slower migrating bands may represent higher order multimers
derived from unusual disulfide bonding. These results demonstrate that
Cys-400 is responsible for the dimer formation of OCIF. Thus, we
conclude that the 120-kDa protein detected in the conditioned medium of
OCIF-producing cells is a homodimer consisting of two 60-kDa monomers
linked together by an intermolecular disulfide bond between two Cys-400
residues.
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Overexpression in 293-EBNA Cells of Chimeric Proteins Consisting of
OCIF and the Fas Transmembrane Domain Causes Apoptosis--
We next
asked whether domains 5 and 6 (DDHs) have a potential for mediating
cytotoxic signals. For this purpose, we transfected plasmids encoding
various OCIF/Fas fusion proteins together with pCH110, an expression
plasmid for -galactosidase, in 293-EBNA cells. The structure of
OCIF, Fas, and their fusion proteins used in this experiment is
schematically illustrated in Fig.
6A. The presence of mRNA
derived from each chimeric construct in the transfected cells was
confirmed by reverse transcriptase-PCR. Primers were designed to
amplify a 424-bp fragment corresponding to the 5' portion of OCIF
mRNA. As shown in Fig. 6B, reverse transcriptase-PCR using RNA from the cells transfected with pCEP4-OCIF-Fas,
pCEP4-TM-OCIF, or pCEP4-TM-OCIF
D567 generated the 424-bp fragment,
but not with pCEP4 or pCEP4-Fas. HRP-labeled anti-OCIF polyclonal
antibody specifically bound to the cells transfected with
pCEP4-OCIF-Fas, pCEP4-TM-OCIF, or pCEP4-TM-OCIF
D567, but not with
the empty vector or pCEP4-Fas (Fig. 6C). These results
indicate that each chimeric cDNA was efficiently expressed, and the
fusion products were translocated to the surface of the transfected
cells. These cells were then stained with X-gal to examine the size and
the shape of the cells harboring each expression plasmid. Microscopic
examination of the cells transfected with pCEP4-Fas, pCEP4-OCIF-Fas, or
pCEP4-TM-OCIF revealed that 30-60% of the blue cells were round and
shrunken, showing signs of cell death (Fig.
7A). In contrast, when
transfected with the empty vector, pCEP4-OCIF or pCEP4-TM-OCIF
D567,
more than 90% of the blue cells retained the flat and adherent
appearance (Fig. 7A). Lactate dehydrogenase activity in the
conditioned medium of the cells transfected with pCEP4-OCIF-Fas or
pCEP4-TM-OCIF was significantly higher than that transfected with the
empty vector or pCEP4-TM-OCIF
D567 (Fig. 7B), showing that
overexpression of OCIF-Fas or TM-OCIF induced cell death. The
transfection efficiency was almost the same (approximately 40%) in all
transfection experiments. Cytotoxic signal induced by OCIF-Fas was
apparently stronger than that induced by Fas for a currently unknown
reason. To examine whether the cell death was caused by
apoptosis, we next analyzed the integrity of DNA in the cells
transfected with the empty vector, pCEP4-OCIF-Fas or pCEP4-TM-OCIF.
Cells transfected with pCEP4-OCIF-Fas or pCEP4-TM-OCIF showed severe
fragmentation of DNA, a clear symptom of apoptosis, as compared with
controls (Fig. 7C). These results demonstrate that OCIF is
capable of triggering apoptosis when the Fas transmembrane region is
inserted between cysteine-rich regions and DDHs and that domains 5-7
have a potential comparable to the death domain of Fas in the induction
of apoptosis.
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DISCUSSION |
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OCIF belongs to the TNFR family, containing four cysteine-rich domains and two DDHs followed by a domain with a high net positive charge (5, 6). In addition, OCIF has some characteristics that not all of the TNFR family proteins possess: (i) it is a secretory protein with no apparent transmembrane region; (ii) it is present in two forms, a monomer and a dimer; and (iii) it interacts with heparin. In the present study, we examined which structural domains are involved in the inhibition of osteoclastogenesis, the binding to heparin, and the formation of the dimer. We also examined whether the two DDHs have potential for mediating the cytotoxic signal when a chimeric protein in which the Fas transmembrane region is inserted in front of DDHs is produced in cells. The results are summarized in Fig. 8.
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By analyzing the osteoclastogenesis inhibitory activity of deletion and
C-terminal truncation mutants, we found that the N-terminal portion
containing domains 1-4 is sufficient to inhibit osteoclastogenesis in vitro. Domains 1-4 correspond to the extracellular
cysteine-rich regions of the TNFR family proteins. For the TNFR family
proteins, these regions protrude outside the cells and are involved in
the interaction with their ligands. Even for the soluble form
receptors, the cysteine-rich regions still have the ability to
associate with their ligands. A naturally occurring secreted form of
Fas lacking the transmembrane domain is present in patients with
systemic lupus erythematosus, suggesting that the molecule works as an antagonist against Fas (22). A soluble form of TNFR consisting of the
cysteine-rich region, which is generated by a proteolytic cleavage,
inhibits the activity of both TNF- and TNF-
(23-26). Thus, most
of the soluble variants of the TNFR family proteins act as antagonists
against the membrane-bound signaling receptors. This fact raises the
possibility that OCIF competes with a currently unidentified receptor
capable of triggering osteoclast formation, by binding to a ligand with
a structural similarity to TNF (4).
Recently, Simonet et al. (6) have reported the isolation of
a cDNA coding for osteoprotegerin, a protein identical to OCIF. They showed that the first four domains alone can exert its inhibitory activity in vitro by analyzing the biological activity of
C-terminal truncation mutants (6). However, the biological activity of the mutants was not accurately determined in their study. We first found that conditioned medium of cells producing D567 was capable of
inhibiting osteoclastogenesis in a dose-dependent manner
(data not shown). Then, the mutant was purified to homogeneity to
determine the potency. By analyzing the in vitro biological
activity of the mutant, we concluded that the N-terminal portion of
OCIF is sufficient to inhibit osteoclastogenesis, although the potency is approximately one tenth of that of wild-type OCIF (Fig.
2B).
Analysis of heparin binding capability of the mutants revealed that
domain 7 is involved in heparin binding. Binding to heparin or
heparin-like molecules is known to be important for such growth factors
as basic fibroblast growth factor to function in vitro and
in vivo (21). The affinity of OCIF for heparin, however, did
not correlate with the in vitro biological activity. For
some proteins, changes in heparin-binding capability affect stability, rate of clearance, and target cell specificity in vivo. For
example, a point mutation in superoxide dismutase that caused reduction of affinity for heparin results in a 10-fold increase in its plasma concentration without affecting the specific enzymatic activity in vitro (27). Therefore, it is worth examining the
possibility that affinity of OCIF for heparin may be of some
physiological importance in vivo. Most of heparin binding
sites of known growth factors consist of a cluster of basic amino acid
residues (28). Although no apparent cluster of positively charged amino
acid residues is present in domain 7, application of Edmundson's wheel model to residues 361-378 shows an -helix in which Lys-361,
Lys-368, Lys-369, Arg-372, and His-375 exist on one side of the helix
and hydrophobic residues such as Leu, Val, Ile, or Phe on the opposite side (data not shown). Such localized basic residues may contribute to
the binding of OCIF to heparin. This was supported by the results that
the mutants with alanine substitutions for Lys-368, Lys-369, and
Arg-372 in OCIF had a marked decrease in affinity for
heparin.2
Domain 7 is also responsible for the dimerization of OCIF. This finding
was derived from Western blot analysis of the mutants (Fig.
4A). Subsequent study identified Cys-400 as the residue essential for the dimer formation (Fig. 4B). Substitution or
deletion of Cys-400 did not affect the activity of OCIF (Fig. 5),
confirming the previous observation that both the monomer and the dimer
form OCIF have similar specific activity in inhibition of in
vitro osteoclastogenesis (4). The dimerization by disulfide
bridges is extremely important for other TNF receptor homologs to exert inhibitory activity. Myxoma virus T2 protein, a TNFR homolog, is
secreted as both a monomer and a dimer, and they bind to rabbit TNF-
with a similar affinity. Interestingly, the dimer is a more potent TNF
inhibitor (29). The significance of dimer formation of OCIF remains
open for the further investigation.
Domains 5 and 6 have a homology to death domains that are involved in transmitting apoptotic signals in many cells. Domains 5 and 6 are not essential for the inhibitory activity (Fig. 2, A and B), heparin binding (Fig. 3), or the dimerization (Fig. 4A), although deletion of both domains resulted in a marked decrease in in vitro biological activity (Fig. 2B). Therefore, we examined whether the two domains have a potential for mediating "death" signals when expressed in the cytoplasm of 293-EBNA cells. It has been reported that ectopic expression of death domain-containing receptor proteins including TNFR 1, Fas, DR3 (also known as Apo-3, Wsl-1, or TRAMP) and the TRAIL receptor lead to cell death via apoptosis (11-14). Although overexpression of OCIF does not cause cell death, a receptor type OCIF (TM-OCIF), which contains the transmembrane region of Fas between domains 4 and 5 of OCIF, possessed an ability to induce apoptosis in 293-EBNA cells (Fig. 7, A-C). Truncation of the DDHs prevented the cell death, strongly suggesting that domains 5 and/or 6 have a high potential for mediating the apoptosis. Analysis of the OCIF gene failed to identify any sequences encoding a potential membrane-spanning domain, suggesting that OCIF exists only in soluble form.3 Therefore, it is unlikely that OCIF triggers apoptosis in a manner similar to TNFR 1 or Fas. Instead, OCIF may induce apoptosis in an unknown fashion under certain conditions.
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ACKNOWLEDGEMENTS |
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We thank Dr. N. Washida, T. Satake, and A. Tomoyasu for preparing anti-OCIF antibody and an affinity column employing anti-OCIF antibody. We also thank Dr A. Murakami, Dr. N. Shima, and H. Yasuda for helpful discussions.
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FOOTNOTES |
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* 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.
These authors contributed equally to this work.
§ To whom correspondence should be addressed: Tel.: 81-285-52-1332; Fax: 81-285-53-1314; E-mail: fvbd7042{at}mb.infoweb.or.jp.
1
The abbreviations used are: OCIF,
osteoclastogenesis inhibitory factor; TNF, tumor necrosis factor; TNFR,
tumor necrosis factor receptor; DDH, death domain homologous region;
TRAP, tartaric-resistant acid phosphatase; CHAPS,
3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate; PCR,
polymerase chain reaction; ELISA, enzyme-linked immunosorbent assay;
HRP, horseradish peroxidase; FBS, fetal bovine serum; PBS, phosphate-buffered saline; IMDM, Iscove's modified Dulbecco's medium;
FPLC, fast protein liquid chromatography; X-gal,
5-bromo-4-chloro-3-indolyl -D-galactopyranoside; bp,
base pair(s); kb, kilobase pair(s).
2 K. Yamaguchi and M. Kinosaki, unpublished observations.
3 T. Morinaga, N. Nakagawa, H. Yasuda, E. Tsuda, and K. Higashio, unpublished observations.
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REFERENCES |
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