Characterization of Structural Domains of Human Osteoclastogenesis Inhibitory Factor*

Kyoji YamaguchiDagger §, Masahiko KinosakiDagger , Masaaki Goto, Fumie Kobayashi, Eisuke Tsuda, Tomonori Morinaga, and Kanji Higashio

From the Research Institute of Life Science, Snow Brand Milk Products Co., Ltd., 519 Ishibashi-machi, Shimotsuga-gun, Tochigi 329-0512, Japan

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

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.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

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.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Bacterial Strain, Cell Lines, and Culture-- Escherichia coli DH5alpha (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-alpha 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 Delta D1, Delta D2, and Delta D3 by XhoI/NdeI; Delta D4 by XhoI/SphI; Delta D5, C195S, C202S and C277S by NdeI/SphI; Delta D6 and C319S by NdeI/BstEII; Delta CL and C400S by SphI/BstEII; Delta D7 by SphI/BamHI; and Delta D67 and Delta D567 by NdeI/BamHI) and were substituted for the corresponding region of OCIF cDNA in pCEP4-OCIF. The plasmids thus constructed were designated pCEP4-Delta D1, pCEP4-Delta D2, pCEP4-Delta D3, pCEP4-Delta D4, pCEP4-Delta D5, pCEP4-C195S, pCEP4-C202S, pCEP4-C277S, pCEP4-Delta D6, pCEP4-C319S, pCEP4-Delta CL, pCEP4-C400S pCEP4-Delta D7, pCEP4-Delta D67, and pCEP4-Delta 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-OCIFDelta 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 Delta D1, Delta D2, Delta D3, Delta D4, Delta D5, Delta D6, Delta D7, Delta D67, Delta D567, Delta 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-Delta 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 Delta D567-- Mutant Delta 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 Delta 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 Delta 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-OCIFDelta D567 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 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-OCIFDelta D567, together with 0.2 µg of pCH110, an expression plasmid for beta -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.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

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, Delta D1, Delta D2, Delta D3, Delta D4, Delta D5, Delta D6, and Delta D7. We also constructed expression vector for C-terminal truncation mutants, Delta D67 and Delta 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, Delta CL, which lacks two C-terminal amino acid residues, Cys-400 and Leu-401, were prepared. All but two mutants (Delta D1 and Delta 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. Delta D1 and Delta 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 Delta D6 was impossible due to poor productivity (~50 ng/ml).


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Fig. 1.   Schematic representation of OCIF mutants. SP, signal peptide; C, cysteine residues in domains 5-7; S, positions of Cys to Ser substitutions. Numbers on the top indicate the domains. Amino acid residues deleted in each mutant are indicated in parentheses.

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), Delta D3 and Delta D4 failed to inhibit the osteoclast-like cell formation at concentrations 35 and 40 ng/ml, respectively. In contrast, Delta D5 retained the inhibitory activity with an ID50 of ~15 ng/ml. Furthermore, Delta D7 had a specific activity comparable to that of wild-type OCIF. A C-terminal truncation mutant Delta 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 Delta D567, which lacks DDHs entirely, we purified it using an anti-OCIF-antibody-immobilized affinity column. The purified Delta 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 Delta 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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Fig. 2.   Osteoclastogenesis inhibitory activity of OCIF deletion mutants. Inhibition by OCIF mutants of osteoclast-like cell formation in the co-cultures was evaluated by measuring TRAP activity. Co-cultures of ST2 and mouse spleen cells were incubated with various concentrations of the OCIF mutants. Data are expressed as mean ± SD of triplicate experiments. A, OCIF antigen in the supernatant of cells transiently expressing each mutant was quantified by an ELISA employing rabbit anti-OCIF polyclonal antibody. bullet , OCIF; open circle , Delta D3; black-square, Delta D4; square , Delta D5; black-triangle, Delta D7; and triangle , Delta D67. B, mutant Delta D567 was purified to homogeneity, and the purified protein was used for the assay. bullet , OCIF; open circle , Delta D567.

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 Delta D7, Delta D67, and Delta 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). Delta 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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Fig. 3.   Heparin-affinity HPLC profiles of the OCIF mutants with C-terminal truncation. Conditioned medium (1 ml) of cells transiently expressing each mutant was applied to a heparin-affinity FPLC column and each mutant was eluted from the column with a linear gradient of NaCl (0-1 M). Fractions were assayed for OCIF using ELISA.

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 Delta D3, Delta D4, Delta D5, and Delta D6 as a major band. In contrast, Delta D7 is present almost exclusively as a ~55-kDa protein. Thus, Delta D7 is present mainly as a monomer, while Delta D3, Delta D4, Delta D5, and Delta 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. Delta D67 and Delta D567, both lacking domain 7, migrated as monomers as expected (Fig. 4A). Difference in size between Delta D3 and Delta 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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Fig. 4.   Western blot analysis of OCIF deletion mutants. Conditined medium (10 µl) containing each mutant was subjected to SDS-PAGE (10 or 13% acrylamide). The separated proteins were transferred to a polyvinylidene difluoride membrane. The membrane was incubated with HRP-labeled anti-OCIF polyclonal antibody. The molecular mass standards are myosin (220 kDa), phosphorylase b (97.4 kDa), bovine serum albumin (66 kDa), ovalbumin (46 kDa), carbonic anhydrase (30 kDa), and lysozyme (14.3 kDa). A, analysis of OCIF deletion mutants; B, analysis of mutants C195S, C202S, C277S, C319S, C400S, and Delta CL.

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 (Delta 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 Delta 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.

C400S and Delta CL, which are present almost exclusively as a monomer in the conditioned medium, were as potent as wild-type OCIF in the inhibition of the in vitro osteoclast formation (Fig. 5). These results provide further evidence that formation of the dimer is not essential for exerting the in vitro osteoclastogenesis inhibitory activity. C195S, C202S, C277S, and C319S, which are present mainly as multimers as shown in Fig. 4B, maintained the biological activity (data not shown).


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Fig. 5.   Osteoclastogenesis inhibitory activity of OCIF mutants, C400S, and Delta CL. Inhibition by OCIF mutants of osteoclast-like cell formation was determined as described under "Experimental Procedures" and in the legend to Fig. 2. OCIF antigen in the supernatant of cells transiently expressing each mutant was quantified by an ELISA employing rabbit anti-OCIF polyclonal antibody.

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 beta -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-OCIFDelta 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-OCIFDelta 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-OCIFDelta 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-OCIFDelta 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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Fig. 6.   Expression of chimeric receptors. A, schematic representation of chimeric receptors. Hatched boxes, cysteine-rich regions of OCIF; dotted boxes, domains 5-7 of OCIF; open boxes, extracellular and intracellular domains of Fas; closed boxes, transmembrane domain of Fas; SP, signal sequence. B, detection of mRNA for the OCIF/Fas chimeric protein in 293-EBNA cells transfected with each expression plasmid. First strand cDNA was prepared from total RNA extracted from 293-EBNA cells transfected with each plasmid and was subjected to PCR with the primers described under "Experimental Procedures." The PCR products were separated on an 1% agarose gel. cDNA synthesis reactions were performed without (lanes 1-5) or with (lanes 6-10) reverse transcriptase. PCR products derived from cells transfected with pCEP4 (lanes 1 and 6), with pCEP4-Fas (lanes 2 and 7), with pCEP4-OCIF-Fas (lanes 3 and 8), with pCEP4-TM-OCIF (lanes 4 and 9), or with pCEP4-TM-OCIFDelta D567 (lanes 5 and 10) are shown. C, detection of OCIF antigen on the surface of 293-EBNA cells transfected with the plasmid that expresses OCIF/Fas chimeric protein. 293-EBNA cells (~2 × 104/well) were seeded on a 96-well plate. The cells in each well were transfected on the following day with 250 ng of either the empty vector or each expression plasmid using LipofectAMINE. Twenty hours after transfection the cells were fixed with glutaraldehyde and washed twice with PBS. The OCIF antigen on the cells was detected by HRP-conjugated anti-OCIF polyclonal antibody as described under "Experimental Procedures." Data are expressed as mean ± S.D. of triplicate experiments.


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Fig. 7.   Induction of cytotoxic signal by overexpression of TM-OCIF on 293-EBNA cells. A, morphology of 293-EBNA cells transfected with expression vectors. Cells (~4 × 104 cells/well) on a 24-well plate were transfected with the indicated expression vector (1 µg) plus pCH110 (0.2 µg). After 20 h, the cells were fixed, stained with X-gal, and photographed under a phase contrast microscope. B, lactate dehydrogenase (LDH) released in the medium. Cells were transfected as in A, and 50 µl of the conditioned medium were subjected to the assay for lactate dehydrogenase activity. Data are expressed as mean ± S.D. of triplicate experiments. C, DNA fragmentation in transfected 293-EBNA cells. Cells (~6 × 105 cells/well of a 6-well plate) were transfected with the indicated expression vectors. Twenty hours after transfection, cellular DNA was isolated and the whole DNA preparation was applied on a 2% agarose gel as described in Hartmann et al. (20). Lane 1, DNA from pCEP4-OCIF-Fas-transfected cells; lane 2, DNA from pCEP4-TM-OCIF-transfected cells; lane 3, DNA from pCEP4-transfected cells; lane 4, size markers.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

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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Fig. 8.   Representation of functional domains in OCIF. Closed box, signal sequence (SP); open boxes with vertical bars, cysteine-rich domains with cysteine residues; hatched boxes, death domain homologous regions; open square box, domain 7; C, cysteine residues in domains 5-7.

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-alpha and TNF-beta (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 Delta 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 alpha -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-alpha 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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* 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.

Dagger 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 beta -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.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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