Use of monoclonal antibody to detect bone morphogenetic protein-4 (BMP-4)

Bone. Vol. 16. No.1 January 1995:91-96

Use of Monoclonal Antibody to Detect Bone Morphogenetic Protein-4 (BMP-4) K. MASUHARA,I T. NAKASE,I S. SUZUKI,I K. TAKAOKA,I M. MATSUI? and H. CLARKE ANDERSON 3 I

2 3

Department of Orthopaedic Surgery. Osaka Unil-ersity Medical School. 2·2 Yamada·oka. Suita 565. Japan Suntory Institute for Biomedical Research, 1-1-1 lVakayamadai Shimamoto-cho Mishima-gun, Osaka 618, Japan Department of Pathology, The University of Kansas Medical Center, 3901 Rainbow Boulemrd, Kansas City, KS 66/03, USA

the TGF-~ superfamily of molecules, which share a high degree of homology within the cysteine-rich, carboxy-terminal domains (Celeste et ai. 1990). Of these ~MPs, BMP-4, which is closely related to BMP-2, appears to be particularly important because recombinant BMP-4 by itself has been shown to elicit in vivo bone formation as is seen with purified, bone-derived BMP (Hammonds et ai. 1990; Takaoka et ai. 1993). In addition, recombinant BMP-4 has been shown to stimulate the phenotypic expression of chondrocytes (Chen et ai. 1991, 1992; Luyten et ai. 1992) and osteoblast-like cells in vitro (Chen et ai. 1991). Thus, a monoclonal antibody specific for BMP-4 might be used as one of the major biochemical markers of bone metabolism as well as in cell and tissue localization of BMP-4. We previously purified BMP-4 from murine osteosarcoma (Takaoka et ai. 1993), cloned cDNA encoding murine BMP-4 and expressed recombinant BMP-4 in Escherichia coli (E. coli) and Chinese hamster ovary (CHO) cells (Takaoka et ai. 1993). The present report describes the production and characterization of a monoclonal antibody against recombinant BMP-4 and demonstrates that an anti-BMP-4 monoclonal antibody could be used for the immunolocalization of human and murine BMP-4.

A monoclonal antibody that reacts with murine and human bone morphogenetic protein-4 (BMP·4) has been de\"eloped using recombinant BMP·4 as an immunogen. The antibody that bound most tightly to recombinant murine (rm)BMP·4 was selected, subcloned, and characterized. The specificity of the antibody was confirmed using Western blot analysis and enzyme·linked immunosorbent assay (ELISA). The antibody reacts with murine and human BMP·4 in both the reduced and nonreduced condition; howe\"er, this antibody shows cross·reactivity with neither human BI\IP·2 nor TGF·(U. Thus, the produced antibody could recognize the disulfide· linked dime ric structure of bioacti\"e BMP·4, regardless of the species. Immunocytochemical study using this antibody successfully shows the cytosolic localization of BMP·4 in os· teoinductive cells; i.e., BFO and Saos·2 in which the lenl of mRNA for BMp·4 was pro\'ed to be constituth'ely high by Northern blot analysis. In addition, the antibody could demo onstrate the presence of BMP·4 in de\'elopmenlal bone for· mation in the alveolar bone of rat embryo by immunohisto· chemistry. The antibody could be used for a more sensith'e approach for quantitative analysis of BMP·4. (Bone 16:9196; 1995)

Materials and Methods

Key Words: Monoclonal antibody; BMP-4; Western blot analysis; Northern blot analysis; Immunohistochemistry.

Preparation of Recombinalll Human BMP-4

Recombinant human (rh)BMP-4 was expressed in a baculovirus expression system. Complementary DNA (cDNA) for hBMP-4 was prepared by reverse transcription polymerase chain reaction (RT-PCR) utilizing total RNA prepared from human osteosarcoma H-5, a cell line derived from human H-OS-6 (Takaoka et ai. 1989) and specific primers for hBMP-4. The primers for the RT-PCR were designed according to the sequences reported previously. The cDNA products encoding hBMP-4 were changed to obtain a restriction enzyme-cleavable site and inserted into an Nru I restriction enzyme recognition site in a vector pBm4. The transfer vector, pBm4-hBMP-4, and genomic DNA of wild-type virus BmNPV (strain P6E) was cotransfected into NISES BoMoISAIIc, a Bombyx mori cell line, and the recombinant virus was obtained in the medium after lysis of the infected cells. A total of 0.5 x lOs plaque-forming units (PFU) of the recombinant virus was injected into the body of the fifth inster larvae of the silkworm. Four or 5 days after the injection, hemolymph was recovered and purification was accomplished by heparin- and phenyl-sepharose column chromatographies. The details of expression and purification procedures for hBMPs are described elsewhere.

Introduction Observations of the bone-inducing activity of a demineralized bone matrix prompted research into the factors that promote bone morphogenesis (Reddi & Huggins 1972; Urist 1965). Recently, a group of proteins known as bone morphogenetic proteins (BMPs), which includes osteogenin (BMP-3) and osteogenic protein (OP-l or BMP-7), has been isolated in sufficient quantity and purity to provide amino acid sequence il)formation (Ozkaynak et ai. 1990; Sampath et ai. 1987; Wang et ai. 1988). Several genes have been subsequently cloned and designated BMP-l, BMP-2 (BMP-2A),.BMP-3 (osteogenin), BMP-4 (BMP-2B), BMP-5, BMP-6 and BMP-7 (OP-I) (Celeste et ai. 1990; Wozney et ai. 1988). The predicted amino acid sequences of the proteins indicated that BMP-2 through BMP-7 are all in

Addressfor correspondence and reprints: Dr. K. Masuhara. Department of Orthopaedic Surgery, Osaka University Medical School, 2-2 Yamadaoka, Suita 565, Japan. © 1995 by Elsevier Science Inc.

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The final products of rhBMP-4 showed several stained bands in SDS-polyacrylamide gel electrophoresis between 19 kDa and 17 kDa in a reduced condition. In a nonreduced condition, a diffused band was observed at 32 kDa. The minimum required quantity of recombinant BMP-4 for bone-inducing activity ill vivo was approximately 100 ng protein per milligram carrier coIlagen, i.e., as potenf as highly purified mBMP-4 from Dunn osteosarcoma. A structural analysis of the heterogeneity of the hBMP-4 protein core portions and glycosylation is under investigation.

bound antibody was subsequently detected by the addition of alkaline phosphatase-conjugated anti-mouse immunoglobulin antibody (Bio-Rad, Richmond, CA), followed by the addition of p-nitrophenyl phosphate. The amount of p-nitrophenol released by alkaline phosphate was determined at 405 nm with a multiscan apparatus (Nippon InterMed K. K., Japan). The positive hybrids were expanded and were selected by the limiting dilution method. After cloning, the monoclonal antibodies were isolated from BALBfc mice ascites using chromatography on protein A sepharose (Pierce, Rockford, IL).

Expressioll of mBMP-4 in E. coli

Characterization of Monoclonal Antibody

To prepare mBMP-4 for immunization, the recombinant mBMP-4 was expressed in an E. coli expression system. The expression vector used in this study was the pUCPL-cI which contains a PL promoter, cIts857, a Shine-Dalgamo sequence, multiple cloning sites and Trp terminator (Kuroki et al. 1992). The mature region of mBMP-4 (from Ser293 to Arg408) (Takaoka et al. 1993) was amplified by PCR under standard conditions using a DNA Thermal Cycler (Perkin-Elmer Cetus USA). The resultant PCR product (370 bp) which contained an EcoR I site and a translation initiator ATG codon at the 5'-end and AUG, translation stop codon and Sal I site at the 3' -end was digested with EcoR I and Sal I, then cloned into the EcoR I and Sal I sites of pUCPL-cI to yield pUC-mBMP-4 (mature). The expression vector was transfected into E. coli W3 I IO and mBMP-4 protein was synthesized by culturing the transformants at 42°C for 2 h. The ceIls were then disrupted using a French Pressure Cell Press (SLM Instruments, Inc., USA). After centrifugation for 10 min at 4000 g, the pellets that contained inclusion bodies were dissolved in 4 M urea and applied to a preparative 15% SDS-gel. After electrophoresis, gel slices containing an approximate molecular weight of 13 kDa, were collected and submitted to an Extraphore Electronic Concentrator (Pharmacia, LKB). The eluent was dialyzed against 10 mM TrisHCI (pH 8.2) and lyophilized. The mBMP-4 polypeptide was used as an antigen to produce monoclonal antibodies in mice.

The reactivity of anti-BMP-4 monoclonal antibody with highly purified murine osteosarcoma-derived BMP-4 (Takaoka et al. 1993), rhBMP-2 (Yamanouchi Pharmaceutical Co., Japan), rhBMP-4 and TGF-131 (R&D Systems, Minneapolis, MN) was studied by Western immunoblot analysis and ELISA. For the Western blot analysis, all the samples (purified mBMP-4, rhBMP-2 and rhBMP-4) were dissolved in a Laemmli sample buffer heated in boiling water for 3 min with or without dithiothreitol (100 mM) prior to electrophoresis. Samples subjected to 15% SDS-PAGE were transferred to a nitrocellulose filter. After blocking with 3% gelatin, the filter was incubated with an antiBMP-4 monoclonal antibody, and then with horseradish peroxidase-conjugated, goat anti-mouse immunoglobulin antibody for 2 h each at 3rC. Immunoblotting was visualized by the addition of NTB (Nitrotetrazolium blue) using a POD Immunostain set (Wako Pure Chemical Industries, Ltd., Japan). The isotype (classes, subclasses and light chains) of the anti-BMP-4 monoclonal antibody was determined using a mouse monoclonalantibody isotyping kit (Amersham International pIc, UK).

Production of Monoclonal Antibody Against BMP-4 Four female BALB/c mice (6 weeks old) were immunized by subcutaneous injection with 50 J.Lg of rmBMP-4 in Freund's complete adjuvant. A booster injection of the same amount of antigen in Freund's incomplete adjuvant was given after 2 weeks and a final intraperitoneal injection of 10 J.Lg antigen was given 2 weeks later. Two sets of fusions were performed by the technique developed by Kohler and Milstein (1975). For each fusion, 2 mice were kiIIed 3 days after the final booster injection and their spleens removed. Spleen cells were isolated and 2.8 x 108 ceIls were fused with 3.2 x 107 murine plasmacytoma cells (P3X63 Ag8, 653) in 40% polyethylene glycol .(Boehringer Mannheim); then, 2.0 x 108 viable hybrid cells were distributed into six microtiter trays (96 wells/tray). Hybrids were cultured in Iscove's modified Dulbccco's medium (Gibco) supplemented with hypoxanthine, aminopterin, thymidine and 20% calf serum (Gibco). An enzyme-linked immunosorbent assay (ELISA) was used to test the culture fluids for antibodies to rmBMP-4. Briefly, microtiter plates were coated with rmBMP-4 (50 ng/well) for I h at 37°C. The plates were blocked with TBS containing 20% FBS for 30 min, washed thoroughly with a 0.2% Tween-20 TBS solution and incubated for I h at 37°C with hybridoma culture fluid. The wells were washed again with TBS Tween and the

Immunofluorescent Staining of Osteoinductive Cells with Anti-BMP-4 Monoclonal Antibody BFO and Saos-2 ceIls, known as the BMP-producing murine (Tsuda et al. 1989) and human (Anderson et al. 1992; Raval et al. 1994) osteosarcoma cell lines, were used for immunohistochemistry. NIH3T3 fibroblasts were also studied. BFO, Saos and NIH3T3 cells were cultured in Eagle's MEM containing 10% fetal bovine serum, McCoy 5A containing 15% fetal bovine serum and D-MEM containing 10% calf serum, respectively. Briefly, cultured monolayer cells were rinsed with PBS, then fixed in 60% cold acetone for 5 min and then air dried at room temperature (RT). The cells were blocked with normal goat serum, for 20 min at 3rC, and then incubated with an anti-BMP-4 antibody for 2 h at 37°C. After thorough washing in PBS, the cells were incubated with a 1:200 dilution of fluorescein isothiocyanate (FITC)-conjugated, goat anti-mouse immunoglobulin, as a secondary antibody, for I h at RT. The cells were then washed thoroughly, mounted in 90% glycerol and 10% PBS solution, and viewed with a universal microscope spectrophotometer (Carl Zeiss, Germany). As controls, cells were treated with normal mouse serum at the first incubating solution, or with the secondary antibody alone. Northern Blo/ Analysis of Osteoinductive Cells Northern hybridization was carried out in the BFO, Saos-2 and NIH3T3 cells to detect BMP-4 mRNA. RNAs from the cultured cells were prepared using the guanidine thiocyanate procedure, as previously described (Chirgwin et al. 1979). Polyadenylated RNA was separated under denaturing conditions on 1.2% (w/v)

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agarose gels containing 2.2 M formaldehyde, and transferred to a nitrocellulose membrane. The membrane was hybridized with O.67-kb Xbal-Pstl eDNA fragment of BMP-4, labeled with 32p (Suzuki et al. 1993). After hybridization, the filter was washed with 0.1 x SSC containing 0.1 % (w/v) SDS at 65°C for 20 min. Autoradiograms were obtained by exposing the filter to Kodak X-Omat film for 24 h at -70°C.

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Anti-BMP-4 Monoclonal Antibody A total of 12 positive hybridomas were obtained from two sets of cell fusion. The antibody that bound most tightly to rmBMP-4 was selected from these anti-BMP-4 antibodies, and was then subcloned and named 3H2.3. A Western immunoblot analysis revealed that this monoclonal antibody reacted with purified bioactive mBMP-4 and rhBMP-4 in both reduced and nonreduced conditions (Figure 1). The 3H2.3 monoclonal antibody detected

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Jaws of 18-day-old rat embryos were fixed in 4% paraformaldehyde in phosphate buffer (pH 7.4), dehydrated and embedded in paraffin. Sections of 4-lJ..m thickness were prepared and were used for immunohistochemical analysis as described hereafter. The sections were dewaxed, rehydrated and washed in PBS. They were treated for 3 h at 37°C with a monoclonal antibody against BMP-4 (3H2.3) and rinsed with PBS. They were then incubated for 1 h with fluorescein isothiocyanate-conjugated goat anti-mouse IgG (Tago, CA). After washing with PBS, they were mounted in a gel mount (Biomeda, Foster City, CA). Control sections were exposed to an identical staining sequence, except that the anti-BMP-4 antibody was replaced by mouse IgG. These sections were viewed with a fluorescence photomicroscope (UMSP 80, Carl Zeiss, Germany).

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Immunolocalization of BMP-4 in the Alveolar Bone of Rat Embryo

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immunoreactive components in the 30--32-kDa range under nonreduced conditions and in the 14-19-kDa range under reduced conditions, while the rest of positive hybridomas reacted with purified mBMP-4 and rhBMP-4 only in the reduced condition. The 3H2.3 antibody showed no reactivity with rhBMP-2 in either condition. An ELISA revealed no cross-reactivity of this monoclonal antibody to recombinant hBMP-2 and TGF-I)l (Figure 2). The 3H2.3 antibody was subtyped as IgG-'Y (2B) and its light chain was identified as K (Figure 3). Expressions of BMP-4 Protein and mRNA in Osteoinductive Cells No nonspecific staining was detected in the controls treated with nonimmune serum, but BMP-4 was intensely stained in murine (BFO) and human (Saos-2) osteoinductive cells. Both the BFO and the Saos-2 cells were labeled mainly in the cytosolic region (Figures 4A and B) whereas the NIH3T3 cells showed no positive BMP-4 identification in any region (Figure 4C). The level of mRNA for BMP-4 was found to be constitutively high in the BFO and Saos-2 cells. Two mRNAs (1.9 and 1.7 kb) for BMP-4 were identified in both the BFO and the Saos cells. However, no BMP-4 mRNA expression was detected in the NIH3T3 cells (Figure 5).

27.5 immunolocalization of BMP-4 in the Embryonic Development of the Rat

8.5 Positive staining was observed in osteogenic cells surrounding calcified bone matrix in the alveolar bone of upper jaw of 18-

Figure 1. Western blol analysis of BMPs against anti-BMP-4 monoclonal antibody. Reactivity of the antibody with 10 iJ.g of purified mBMP-4 (lane l) and rhBMP-4 (lane 2) is shown under reduced (left lanes) and nonreduced (right lanes) conditions. No reactivity of the antibody with 10 ILg of rhBMP-2 (lane 3) is observed under either condition.

Figure 3. lsotype of anti-BMP-4 monoclonal The isotype of the antibody is indicated by the two lines on the one of which corresponds to the antibody subclass (IgG2b), and one that corresponds to the antibody light chain (K).

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BMP-4

ture healing (;-!akase et a!. 1994). B:MP-4 might have a mesoderm-inducing ability (Koster et a1. 1991) which could act as a ventralizing factor in early Xenopus development (Dale et al. 1992; Jones et a!. 1992). Recent studies have indicated that B.\1P-4 could have appreciable effects on the process of endochondral ossification. Proteoglycan production and DNA synthesis by chick limb bud mesodenn have been shown to increase significantly with 10 ng/ml rhBMP-4 (Chen et al. 1992). Similarly, proteoglycan synthesis by bovine cartilage explants has been shown to increase in dose-dcpendem manner with up to 30 ng/ml rhBMP-4 (Luytcn et a!. 1992). The rhBMP-4 (0.1-10 ng/m!) is also capable of stimulating DNA and collagen synthesis, as well as alkaline phosphatase activity in rat osteoblastenriched cultures (Chen et a1. 1991). Yloreovcr, a striking in vivo osteogenic activity was observed with as little as 100 ng rhBMP-4 (Hammonds ct a!. ] 990). These reports suggest that rccombinant BMP-4 may be an equal or greater stimulator of chondrogenesis and osteogenesis than recombinant BMP-2. Thus, the immunological detection of BMP-4 is potentially of great value. The present study demonstrates that the anti-BMP-4 monoclonal antibody (3H2.3) reacts with 30-32 kDa unreduced mBMP-4, highly purified from the Dunn osteosarcoma from which the clone encoding mBMP-4 was isolated. Furthermore, the antibody 3H2.3 which was raised against a monomer chain of recombinant mBMP-4 has been shown to detect bioactive rhBMP-4 with a dimeric structure. Recent reports have demonstrated a 98% identity of the mature protein region betwecn mBYlP-4 and hB!\lP-4 (both differ at only two residues), and appear to confinn a high degree of homology between mouse and human BYlP-4s (Takaoka et al. 1993). Although BMP-2 and BMP-4 exhibit 92% sequence identity in thc carboxy-terminal 101 residues, both are quite different in the amino-tenninal 10% of the mature protein. Both differ by over 50% in the first 15 residues (Celeste et al. 1990). In addition, BMP-4 and TGF-f) I showed much less homology (only 31 % sequence identity). Thesc structural differences could account for the finding that anti-BMP-4 monoclonal antibody reacts with neither recombinant human BMP-2 nor TFG-l3l. Thus, it is important to note that the produced monoclonal antibody, 3H2.3, could be specific for the disulfide-linked dimeric structure of bioactive BYlP-4, regardless of the species. The search for patterns of BMP distribution within cells has long been hampered by a number of problems. The small number of cell lines that express sufficient bone-inducing activity and the incomplete purification of BMPs necessary to raise specific an-

1 Figure 4. Immunofluorescence of BMP-4 in BFO (A), Saos·2 (B) and )'I;lH3T3 (C) cells. The cytoso!ic regions of BFO and Sa05·2 cells are intensely stained for BMP-4. No positive staining is observed in .\ilH3T3 cells.

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day-old rat embryo (Figure 6B). Control sections exposed to nonimIIlUne IgG exhibited no evidence of positive staining (Figure 6C). Discussion Based on in situ hybridization, BMP-4 has been suggested to playa key role in the initial stages of neurogenesis and organogenesis during murine development (Jones et a!. ! 991) and frac-

Hgure 5. )'I;orrhem blot analysis of mvlP-4 mRNA. Total RNA samples (20 ~g per lane) from NlH3T3 (lane I), BFO (lane 2) and Saos (lane 3) cells are subject~d to electrophoresis and hybridized with BMPA cD:-IA labeled with 3cp. Two mR:-JAs (1.9 and 1.7 kb) for BMP-4 are identified in both the BFO and the Saos cells.

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The results are also supported by our unpublished observation with in situ hybridization andRT-PCR that BMP-4 is highly expressed in the alveolar bone of murine embryo. Although the precise molecular structure of BMP-4 protein in various tissues or in serum remains uncertain, the monoclonal antibody against BMP-4 described here could be a useful tool for various purposes, such as in vivo imaging via coupling with a radioactive tag, or for the mass screening of serum specimens to detect BMP-4 circulating in the serum. If BMP-4 can be shown to exist in serum, it may be useful clinically as a biocherrrical marker of bone metabolism, since it can be measured by an immunoassay such as for plasma osteocalcin. The production and characterization of the anti-BMP-4 monoclonal antibody described in this fu'iicle represents a requisite first step toward realizing some of these uses for BMP-4,

References

Figure 6. Immunohistochemical staining with anti-BMP-4 antibody in 18-day-old rat embryo. Ali ate sections of upper jaw of 18-day-old rat fetus. (A) A section stained with hematoxyiin and eosin followed by von Kassa. Dark area shows the calcified bone matrix. (B) A continuous section from (A) stained with the antibody against BMP-4. Positive staining is noted in cells surrounding the calcified malrix. (C) A control section stained with a nonimmune mouse IgG [continuous from (A)]. Almost no staining is observed. lA-C ate same magnification; bar = 50 fLmJ

tibodies have limited the success of antibody development and immunolocalization of BMPs. Figure 4 shows that a cytosolic localization of BMP-4 has now been confirmed in osteoinductive cells using the new anti-B~P-4 monoclonal antibody (3H2.3). These immunocytochemical findings seem to be compatible with the resu;ts obtained from the Northern blot analysis showing significant BNIP-4 mR:\A expression in BFO and Saos-2 ce!ls. Furthermore, the immunohistochemical study lIsing the antibody (3H2.3) clearly demonstrates [he presence of BMP-4 in osteogenic cells of fetal rat alveolar bone, which is consistent with more recent reports (Harris et a1. 1994; Schildhauer et al. 993).

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Nakase T.; Nomura, S.; Yoshikawa, H.; Hashimoto, J.; Hirota, S.; Kitamura, Y.; Oikawa, S.; Ono, K.; Takaoka, K. Transient and localized expression of bone morphogenetic protein 4 messenger RNA during fracture healing. J Bone Min Res 9:651-659; 1994. Ozkaynak, E.; Rueger, D. C.; Drier, E. A.; Corbett, C.; Ridge, R. J.; Sampath, T. K.; Oppermann, H. OP-I eDNA encodes an osteogenic protein in the TGF-j3 family. EMBO J ,9:2085-2093; 1990. Raval, P.; Schneider, D.; Bonewald, L. F.; Anderson, H. C. Relationship between expression of bone morphogenetic proteins and osteoinductive activity in osteosarcoma cells. Trans Orthop Res Soc 19:272; 1994. Reddi, A. H.; Huggins, C. B. Biochemical sequences in the transformation of normal fibroblasts in adolescent rats. Proc Natl Acad Sci USA 69:1601-1605; 1972. Sampath, T. K.; Muthukumaran, N.; Reddi, A. H. Isolation of osteogenin, an extracellular matrix-associated bone-inductive protein, by heparin affinity chromatography. Proc Nat! Acad Sci USA 84:7109-7113; 1987. Schildhauer, T. A.; Bostrom, M. P. G.; Lane, J. M.; Berberian, W. S.; Tomin, E.; Missri, A. A. E.; Weiland, A.; Rosen, V. M.; Doty, S. Immunolocalization of bone morphogenetic proteins 214 in the embryological development of the rat. J Bone Min Res 8:S317; 1993. Suzuki, S.; Koga, M.; Takaoka, K.; Ono, K.; Sato, B. Effects of retinoic acid on steroid and vitamin DJ receptors in cultured mouse osteosarcoma cells. Bone 14:7-12; 1993. Takaoka, K.; Yoshikawa, H.; Masuhara, K.; Sugamoto, K.; Tsuda, T.; Aoki, Y.; Ono, K.; Sakamoto, Y. Establishment of a cell line producing bone morpho-

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Bone, Vol. 16, No. I January 1995:91-96 genetic protein from a human osteosarcoma. Clin Orthop ReI Res 244:258264; 1989. Takaoka, K.; Yoshikawa, H.; Hashimoto, J.; Miyamoto, S.; Masuhara, K.; Nakahara, H.; Matsui, M.; Ono, K. Purification and characterization of a boneinducing protein from a murine osteosarcoma (Dunn type). Clin Orthop ReI Res 292:329-336; 1993. Takaoka, K.; Yoshikawa, H.; Hashimoto, J.; Masuhara, K.; Miyamoto, S.; Suzuki, S.; Ono, K.; Matsui, M.; Oikawa, S.; Tsuruoka, N.; Tawaragi, Y.; Inuzuka, C.; Katayama, T.; Sugiyama, M.; Tsujimoto, M.; Nakanishi, T.; Nakazato, H. Gene cloning and expression of a bone morphogenetic protein derived from a murine osteosarcoma. Clin Orthop ReI Res 294:344-352; 1993. Tsuda, T.; Masuhara, K.; Yoshikawa, H.; Shimizu, N.; Takaoka, K. Establishment of an osteoinductive murine osteosarcoma clonal cell line showing osteoblastic traits. Bone 10:195-200; 1989. , Urist, M. R. Bone formation by autoinduction. Science 150:893-899; 1965. Wang, E. A.; Rosen, V.; Cordes, P.; Hewick, R. M.; Kriz, M. J.; Luxenberg, D. P.; Sibley, B. S.; Womey, J. M. Purification and characterization of other distinct bone-inducing factors. Proc Nat! Acad Sci USA 85:9484-9488; 1988. Womey, J. M.; Rosen, V.; Celeste, A. J.; Mitsock, L. M.; Whitters, M. J.; Kriz, R. W.; Hewick, R. M.; Wang, E. A. Novel regulato~ of bone formation: molecular clones and activities. Science 242:1528-1534; 1988.

Date Received: September 14, 1993 Date Re"ised: October 26, 1993 Date Accepted: August 17, 1994