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Identification of Rat Bone Morphogenetic Protein-3b (BMP-3b), a New Member of BMP-3
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
219, 656–662 (1996)
0289
Identification of Rat Bone Morphogenetic Protein-3b (BMP-3b), a New Member of BMP-3 Makoto Takao,*,† Jun Hino,* Norimatsu Takeshita,† Yasuhiko Konno,† Tsutomu Nishizawa,† Hisayuki Matsuo,* and Kenji Kangawa*,1 *National Cardiovascular Center Research Institute, Fujishirodai, Suita, Osaka 565, Japan; and †HQL Research Labs, Sumitomo Metal Industries Ltd., 3-5 Hikaridai, Seika-Cho, Sohraku-Gun, Kyoto 619-02, Japan Received January 9, 1996 Rat bone morphogenetic protein-3 (BMP-3) cDNA and BMP-3b cDNA were isolated from rat femur cDNA library by the RT-PCR method using degenerate oligonucleotide primers corresponding to human BMP-3. The deduced amino acid sequence of rat BMP-3 in the mature region reveals 2 amino acid changes compared to that of human BMP-3 (98% identity). The deduced BMP-3b amino acid sequence shows 81% similarity with the mature region of rat BMP-3 and only 37% similarity with the propeptide region. By Northern blot analysis, rat BMP-3b mRNA was detected in costa, costicartilage, femur, calvaria, trachea, aorta and brain. Among these tissues, BMP-3b transcripts were predominantly expressed in cerebellum. BMP-3 mRNA was found in femur, calvaria, trachea, lung and ovary. Although BMP-3b and BMP-3 are very closely related to each other, their transcripts are distributed in different tissues except that both are found in bone. The distribution pattern of BMP-3b mRNA suggests that BMP-3b plays an important role in the central nervous system as well as in bone formation and remodeling. © 1996 Academic Press, Inc.
Bone morphogenetic proteins (BMPs) belonging to the transforming growth factor b (TGF-b) superfamily, have been identified as proteins that induce ectopic bone formation in rat. In mammals, seven different forms of BMP, termed BMP-2 to 8, have been isolated (1–4). Several recombinant BMPs (BMP-2, 4 and 7) have been shown to induce chondrogenesis and osteogenesis in vivo (5–7). Various biological activities of BMP have been demonstrated in vitro (7–12). Although it is recognized that all BMPs are active in embryogenesis and developmental pattern formation beyond osteogenesis, little is known about the function and distribution of BMP-3, compared with other BMPs. BMPs fall into three subgroups based on their sequence homology. BMP-2 and BMP-4 are very closely related (86% amino acid identity in mature region) and form the first group. BMP-5, BMP-6, BMP-7 and BMP-8 are also closely related to each other (61–79%) and form the second group. BMP-3 is quite different from other BMPs and so falls alone into a third category. We attempted to identify a novel cDNA closely related to BMP-3 by the method of degenerate reverse transcription-polymerase chain reaction (RT-PCR). We succeeded in cloning a novel member of the TGF-b superfamily, which we have designated BMP-3b. Here we report the isolation and cDNA sequence of rat BMP-3b and rat BMP-3. In addition, regional differences in the expression of the BMP-3b gene, compared with BMP-3, were examined by RNA blot analysis. MATERIALS AND METHODS RT-PCR. The degenerate oligonucleotide primers synthesized for RT-PCR, according to the sequence of human BMP-3 mature region are as follows: P1, 59-GCNTG(C,T)CA(G,A)TT(C,T)-CCNATGCC-39 [bases 1524–1543]; P2, 59GC(G,A)CAN(G,C)(T,A)(C,T)TCNACNGTCAT-39 [complementary to base 1710–1729]. Nucleotide numbering is based on the published sequence of human BMP-3 mature region (GenBank Accession No. M22491). For the primer sequences,
1
To whom correspondence should be addressed. Fax: (+81)-6-872-7485. Abbreviations: BMP, bone morphogenetic protein; TGF-b, transforming growth factor b. The nucleotide sequences of rat BMP-3b and BMP-3 are deposited at the GenBank database under accession Nos. D49494 and D63860, respectively. 656 0006-291X/96 $18.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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N denotes any nucleotide. RT-PCR was performed using Perkin Elmer RNA PCR kit. One microgram of total RNA isolated from rat femur was used as a template for single strand cDNA synthesis by reverse transcriptase with P2 primers. The PCR was performed under the following conditions: 1 min at 94°C, 2 min at 55°C and 2 min at 72°C for 25 cycles. Amplified DNA (about 200 bp) was cloned directly into the EcoRV site of Bluescript II SK+ vector by the TA cloning method (13) and transformed to E. coli JM109. Approximately 600 transformants were screened by colony hybridization using human BMP-3 probe (nucleotide No. 1555–1736) which was prepared by PCR. Hybridization was performed at 42°C in 20% formamide, 6 × SSPE, 5 × Denhardt’s solution, 0.5% SDS, and 100 mg/ml denatured salmon sperm DNA. Two classes of clones were obtained, as indicated by strong (Clone A) or weak (Clone B) hybridization signals. The cDNA inserts of these clones were sequenced using the dideoxy chain termination method (14). cDNA library construction and cloning. Total RNA was extracted from rat femur by the acid guanidium thiocyanatephenol-chloroform method (15). Poly(A)+ RNA was isolated on an oligo(dT)-latex (Nippon Roche). An oligo(dT)-primed cDNA library of rat femur was constructed in lgt10 vector using cDNA Synthesis Kit (Pharmacia). For cloning BMP-3b cDNA, approximately 1 × 106 phages were screened by plaque hybridization using 32P-labeled double strand probe of Clone B, which was prepared by RT-PCR described above. Hybridization was performed at 42°C in 40% formamide, 6 × SSPE, 5 × Denhardt’s solution, 0.5% SDS and 100 mg/ml denatured salmon sperm DNA, and washed at 55°C in 2 × SSC, 0.1% SDS. A positive clone RB45 harboring the longest cDNA insert was subcloned into the Bluescript II SK+ vector and sequenced. Next, for cloning rat BMP-3 cDNA, the same filters were screened using 32P-labeled cDNA insert of Clone A as a probe. Hybridization was performed under the same conditions as for BMP-3b. A positive clone RC7 harboring the longest cDNA insert was used for sequencing. RNA blot analysis. Tissues were obtained from Sprague-Dawley rats. Cerebellum from rat at embryo (18 days) and different stages of development were obtained as indicated in the legend to Fig. 4. All poly(A)+ RNA were denatured using glyoxal and dimetylsulfoxide, and were fractionated on a 0.8% agarose gel. After electrophoresis, RNA was transferred to a nylon membrane (Zeta Probe, Bio-Rad). The membrane was prehybridized and hybridized at 42°C in 50% formamide, 6 × SSPE, 5 × Denhardt’s solution, 0.5% SDS, and 100 mg/ml denatured salmon sperm DNA. The blot was washed at 60°C once in 2 × SSC, 0.1% SDS, once in 0.5 × SSC, 0.1% SDS, and finally once in 0.1 × SSC, 0.1% SDS. The blot was exposed to X-ray films. The same membrane was used for sequential hybridizations with probes specific for rat BMP-3b (2.4 kb Not I fragment of RB45 reported in this paper), rat BMP-3 (2.2 kb Not I fragment of RC7 reported in this paper) and rat BMP-2 (2.7 kb full length cDNA of rat BMP-2 cloned from rat placenta cDNA library, data not shown). Probes were labeled by the random-primed method.
RESULTS AND DISCUSSION Cloning of rat BMP-3 and BMP-3b cDNA. In order to obtain clones for rat BMP-3 and unknown BMP-3 related protein, degenerate oligonucleotide primers (P1 and P2) were synthesized, which included all possible sequences encoding Ala-Cys-Gln-Phe-Pro-Met-Pro and Met-Thr-Val-GluSer-Cys-Ala, corresponding to human BMP-3 mature region. These primers were used in a PCR by using cDNA from rat femur as a template. As a result, a single 200 base-pair fragment was amplified. This PCR product was subcloned to Bluescript II SK+ and colony hybridization was done at low stringency using human BMP-3 fragment (nucleotide No. 1555–1736) as a probe. Two classes of clones, one giving a strong signal and the other a weak signal, were obtained. Nucleotide sequence analysis indicated that clones (Clone B) giving weak signal have a novel cDNA (Fig. 1, indicated by an overline), while clones (Clone A) giving a strong signal have a cDNA thought to be a rat homologue of human BMP-3 (data not shown). We have designated this novel cDNA rat BMP-3b. To obtain the full length of rat BMP-3 cDNA and rat BMP-3b cDNA, rat femur library was constructed. First, approximately 1 × 106 recombinant clones were screened using a 200 bp of cDNA fragment from Clone B as a probe for BMP-3b. When hybridizations were performed at high stringency, about twenty positive clones were isolated. Next, for cloning rat BMP-3, a 200 bp of cDNA fragment obtained from Clone A as a probe for BMP-3 was used and about twenty positive clones were isolated. The cDNA inserts of BMP-3b and BMP-3 were subcloned into the Not I site of Bluescript II SK+ and complete sequences were determined. Structure of rat BMP-3b cDNA. Fig. 1 shows the complete nucleotide sequence of rat BMP-3b cDNA, which is 2411 nucleotides long. A putative initiation codon ATG is located at nucleotides 1–3, preceded by the consensus sequence A/CCCATGG for the initiation (16), while a termination 657
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FIG. 1. Nucleotide sequence and deduced amino acid sequence of rat BMP-3b cDNA. Numbering of nucleotides and of amino acids (on the left margin) starts with the first ATG of the BMP-3b coding sequence. A boxed Arg-Arg-Arg sequence precedes putative maturation sites. Potential sites of N-linked glycosylation are indicated by thick underline. Dashed underline indicates the AATAAA sequence. Amplified cDNA fragment obtained by RT-PCR using degenerate oligonucleotide primers is indicated by overline.
codon TAA is found 476 codons later at nucleotides 1429–1431. A typical polyadenylation signal, AATAAA, is found only at nucleotides 2153–2158, but no poly(A)+ tail is contained within this cDNA. The open reading frame encodes a putative precursor for rat BMP-3b, which consists of 476 amino acid residues. The N-terminal sequence of the precursor is thought to be a signal peptide, 658
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based on its characteristic hydrophobic features (17). The putative precursor contains four N-linked glycosylation sites. The TGF-b homologous sequence is located in the C-terminal containing about 100-amino acid polypeptide. Three Arg at positions 364–366 precede the mature region of the BMP-3b sequence. By analogy with members of TGF-b superfamily (18), proteolytic cleavage of BMP-3b may take place following these basic residues, resulting in the release of a polypeptide of 110 residues with one potential N-glycosylation site (at position 467). Like other members of the TGF-b superfamily, the functional BMP-3b product is probably a homodimer of the C-terminal segments of BMP-3b precursors. Structure of rat BMP-3 cDNA. We sequenced the full length of rat BMP-3 cDNA. Predicted BMP-3 precursor consists of 468 amino acids. The nucleotide sequence of rat BMP-3 is deposited at the GenBank database (No. D63860, data not shown). The deduced amino acid sequence of rat BMP-3 precursor is approximately 79% identical to that of human. In the mature region rat BMP-3 revealed 2 amino acid changes compared to that of human BMP-3 (98% identity). Comparison with other BMP subfamily. Fig. 2 shows the amino acid sequence of C-terminal region of rat BMP-3b and BMP-3 with other members of BMP subfamily, aligned by seven highly conserved cysteine residues. The mature region of rat BMP-3b shows a high degree of amino acid identity with comparable regions of rat BMP-3 (81%) and a low degree of identity (42–47%) with human BMP-4, 5, 6, 7 and 8. In the propeptides region, BMP-3b (amino acids No. 1–366 in Fig. 1) and BMP-3 are 37% identity. But the homology between propeptide region of BMP-3b and other BMPs is very poor. BMPs are divided into three subgroups. BMP-2 and BMP-4 are very closely related (86% amino acid identity in the mature region) and form one group. BMP-5, BMP-6 and BMP-7 are also closely related to one another (an average of 78%) and form a second group. BMP-3 is quite different from other BMPs and forms a third category. Because BMP-3b is most closely related to BMP-3 (81%), BMP-3b can be considered a second member of BMP-3 subgroup within the BMP family.
FIG. 2. Alignment of amino acid sequence of C-terminal region of rat BMP-3b and corresponding regions of BMP subfamily. For ease of comparison, BMPs have been separated into three subgroups. Amino acid sequences (one-letter symbols) are aligned for maximal homology. Gaps are indicated by periods. Conserved amino acids in each subgroup are boxed. Seven conserved cysteines in the TGF-b superfamily are indicated with asterisks above the figure. 659
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mRNA expression of rat BMP-3b, BMP-3 and BMP-2. To determine the expression site of BMP-3b and to compare BMP-3b expression with that of BMP-3 and BMP-2, we screened mRNA preparations from several organ tissues of adult and neonatal rat by Northern blot hybridization (Fig. 3). In bone tissues BMP-3b mRNA was observed in femur, calvaria and costa as two species of 2.8 kb (major band) and 4.1 kb (minor band). BMP-3 mRNA was also found in these bone tissues as multiple mRNA species; major species of 6.7 kb and minor species of 4.7 and 2.6 kb were detected. BMP-2 mRNA was expressed in costa as double mRNA species (2.5 kb and 3.8 kb). These results indicate that BMP-3b was expressed in bone tissues in a manner similar to that of BMP-3 and BMP-2. Recombinant BMP-2 has been demonstrated to induce ectopic bone formation in vivo. On the other hand, little is known about biological activities of BMP-3. In our preliminary experiments, recombinant BMP-3b and BMP-3 expressed in CHO cells did not induce ectopic bone formation (data not shown). However relatively high level of BMP-3 protein was observed in bone tissue (1). Thus, expression of BMP-3b and BMP-3 mRNA in bone tissue suggests that these two BMPs may be acting in bone with distinct mechanism from other BMPs. We compared the relative abundance of BMP-3 and BMP-3b transcripts in calvaria and femur of neonatal and adult rat. Similar to BMP-3, the levels of BMP-3b mRNA in neonatal femur and calvaria were higher than those of in adult femur and calvaria. This result implies that BMP-3b has physiological importance in new bone formation in embryogenesis. In costicartilage, BMP-3b was highly expressed, while BMP-3 mRNA was not detected and BMP-2 mRNA was only slightly detected. Cartilage consists of only chondrocyte cells. BMP-3b
FIG. 3. Expression of rat BMP-3b, rat BMP-3 and rat BMP-2 mRNA in different rat tissues. Poly(A)+ RNA (10 mg) was loaded onto each lane, electrophoresed on a 0.8% agarose gel, blotted on to a nylon membrane and hybridized. The same filter was used for sequential hybridizations with 32P-labeled DNA probes. Probes used for hybridization are (A) rat BMP-3b probe, (B) rat BMP-3 probe and (C) rat BMP-2 probe. RNA ladder (0.24-9.5 kb; BRL INC) was used as size standard. 660
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FIG. 4. Northern blot analysis of BMP-3b mRNA from rat cerebellum isolated at various stages of development. Ages are given as days post coitum (d.p.c), days postnatal (p.n.) and weeks adult (wks.). Equal amounts of Poly(A)+ RNA (2 mg) isolated from cerebellum were loaded onto each lane. Hybridization was done with probe for rat BMP-3b. Lanes: (1) Poly (A)+ RNA obtained from cerebellum of 18 day embryos; (2) 0 day postnatal; (3) 1 day postnatal; (4) 2 days postnatal; (5) 3 days postnatal; (6) 1 week old; (7) 2 weeks old; (8) 3 weeks old; (9) 10–20 weeks old. The same filter was rehybridized with human b-actin cDNA probe.
protein is synthesized and secreted by chondrocytes, and might participate in cartilage formation and metabolism, and maintenance of the extracellular matrix. In other tissues, BMP-3b mRNA was found at high levels in brain, trachea and aorta and at lower levels in ovary, testis and skeletal muscle. Interestingly, BMP-3b mRNA in cerebellum was more abundant than in bone tissue. When it became apparent that BMP-3b mRNA was almost exclusively expressed in cerebellum, different stages of developmental expression of BMP-3b mRNA in cerebellum were examined. In embryo cerebellum (18 days), the BMP-3b transcripts were expressed weakly (Fig. 4). Its transcripts strongly appeared in postneonate and were constantly expressed in adult. Therefore, BMP-3b may play a role in the development and/or function of the cerebellum. The action of BMP-3b in the nervous system is unknown, but several members of the TGF-b superfamily are known to be an important regulator in early stages of neural development (19,20). BMP-7 is expressed in brain (2), and has been shown to induce the neural cell adhesion molecule (N-CAM) in a neuroblastoma-glioma hybrid cell line (10). Similar to BMP-7, BMP-3b also may act in the nervous system. BMP-3 mRNA was expressed at high levels in ovary, lung and trachea, but at low levels in kidney, aorta, and spleen. BMP-2 transcripts were detected at high levels in trachea, spleen, small intestine, and ovary, and at low levels in cerebellum, costa and bone marrow. The mRNA distribution of BMP-3b is distinct from that of BMP-3, although BMP-3b and BMP-3 are very closely related to each other. For example, BMP-3b mRNA is highly expressed in cerebellum and costicartilage, while BMP-3 mRNA is not observed in these tissues. In lung, kidney and small intestine, BMP-3b mRNA is not detected but BMP-3 mRNA is found. These results may suggest that physiological functions of BMP-3b are different from those of BMP-3. In conclusion, we cloned BMP-3b cDNA, encoding a novel protein closely related to BMP-3. Expression of BMP-3b mRNA in bone tissue and cerebellum suggests that BMP-3b may be involved in bone and cartilage metabolism and in the central nervous system. 661
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ACKNOWLEDGMENTS We thank Drs. K. Fukuda and M. Miyazato for helpful discussions. This work was supported in part by Special Coordination funds for Promoting Science and Technology from the Science and Technology Agency (Encouragement System of C.O.E.), and research grants from the Ministry of Health and Welfare, and the Human Science Foundation of Japan.
REFERENCES 1. Wozney, J. M., Rosen, V., Celeste, A. J., Mitsock, L. M., Whitters, M. J., Kriz, R. W., Hewick, R. M., and Wang, E. A., (1988) Science 242, 1528–1534. 2. Özkaynak, E., Rueger, D. C., Drier, E. A., Corbett, C., Ridge, J., Sampath, T. K., and Oppermann, H., (1990) EMBO J. 9, 2085–2093. 3. Celeste, A. J., Iannazzi, J. A., Taylor, R. C., Hewick, R. M., Rosen, V., Wang, E. A., and Wozney, J. M., (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 9843–9847. 4. Özkaynak, E., Schnegelsberg, P. N. J., Jin, D. F., Clifford, G. M., Warren, F. D., Drier, E. A., and Oppermann, H., (1992) J. Biol. Chem. 267, 25220–25227. 5. Wang, E. A., Rosen, V., D’Alessandro, J. S., Bauduy, M., Cordes, P., Harada, T., Israel, D. I., Hewick, R. M., Kerns, K. M., Lapan, P., Luxenberg, D. P., McQuaid, D., Moutsatsos, I. K., Nove, J., and Wozney, J. M., (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 2220–2224. 6. Hammonds, R. G., Schwall, J. R., Dudley, A., Berkemeier, L., Lai, C., Lee, J., Cunningham, N., Reddi, A. H., Wood, W. I., and Mason, A. J., (1991) Mol. Endocrinol. 5, 149–155. 7. Sampath, T. K., Maliakal, J. C., Hauschka, P. V., Jones, W. K., Sasak, H., Tucker, R. F., White, K. F., Coughlin, J. E., Tucker, M. M., Pang, R. H. L., Corbett, C., Özkaynak, E., Oppermann, H., and Rueger, D. C., (1992) J. Biol. Chem. 267, 20352–20362. 8. Yamaguchi, A., Katagiri, T., Ikeda, T., Wozney, J. M., Rosen, V., Wang, E. A., Kahn, A. J., Suda, T., and Yoshiki, S., (1991) J. Cell. Biol. 113, 681–687. 9. Cunningham, N. S., Paralkar, V., and Reddi, A. H., (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 11740–11744. 10. Perides, G., Safran, R. M., Downing, L. A., and Charness, M. E., (1994) J. Biol. Chem. 269, 765–770. 11. Lyons, K. M., Pelton, R. W., and Hogan, B. L. M., (1989) Genes and Development 3, 1657–1668. 12. Lyons, K. M., Pelton, R. W., and Hogan, B. L. M., (1990) Development 109, 833–844. 13. Marchuk, D., Drumm, M., Saulino, A., and Collins, F. S., (1991) Nucleic Acids Res. 19, 1154. 14. Sanger, F., Nicklen, S., and Coulson, A. R., (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 5463–5467. 15. Chomczynsky, P., and Sacchi, N., (1987) Anal. Biochem. 162, 156–159. 16. Kozak, M., (1987) Nucleic Acids Res. 15, 8125–8148. 17. von Heijne, G., (1983) Eur. J. Biochem. 133, 17–21. 18. Derynck, R., Jarrett, J. A., Chen, E. Y., Eaton, D. H., Bell, J. R., Assoian, R. K., Roberts, A. B., Sporn, M. B., and Goeddel, D. V., (1985) Nature 316, 701–705. 19. Jones, C. M., Lyones, K. M., and Hogan, B. L. M., (1990) Development 111, 531–542. 20. Liem, K. F., Tremml, G., Roelink, H., and Jessell, T. M., (1995) Cell 82, 969–979.
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
219, 656–662 (1996)
0289
Identification of Rat Bone Morphogenetic Protein-3b (BMP-3b), a New Member of BMP-3 Makoto Takao,*,† Jun Hino,* Norimatsu Takeshita,† Yasuhiko Konno,† Tsutomu Nishizawa,† Hisayuki Matsuo,* and Kenji Kangawa*,1 *National Cardiovascular Center Research Institute, Fujishirodai, Suita, Osaka 565, Japan; and †HQL Research Labs, Sumitomo Metal Industries Ltd., 3-5 Hikaridai, Seika-Cho, Sohraku-Gun, Kyoto 619-02, Japan Received January 9, 1996 Rat bone morphogenetic protein-3 (BMP-3) cDNA and BMP-3b cDNA were isolated from rat femur cDNA library by the RT-PCR method using degenerate oligonucleotide primers corresponding to human BMP-3. The deduced amino acid sequence of rat BMP-3 in the mature region reveals 2 amino acid changes compared to that of human BMP-3 (98% identity). The deduced BMP-3b amino acid sequence shows 81% similarity with the mature region of rat BMP-3 and only 37% similarity with the propeptide region. By Northern blot analysis, rat BMP-3b mRNA was detected in costa, costicartilage, femur, calvaria, trachea, aorta and brain. Among these tissues, BMP-3b transcripts were predominantly expressed in cerebellum. BMP-3 mRNA was found in femur, calvaria, trachea, lung and ovary. Although BMP-3b and BMP-3 are very closely related to each other, their transcripts are distributed in different tissues except that both are found in bone. The distribution pattern of BMP-3b mRNA suggests that BMP-3b plays an important role in the central nervous system as well as in bone formation and remodeling. © 1996 Academic Press, Inc.
Bone morphogenetic proteins (BMPs) belonging to the transforming growth factor b (TGF-b) superfamily, have been identified as proteins that induce ectopic bone formation in rat. In mammals, seven different forms of BMP, termed BMP-2 to 8, have been isolated (1–4). Several recombinant BMPs (BMP-2, 4 and 7) have been shown to induce chondrogenesis and osteogenesis in vivo (5–7). Various biological activities of BMP have been demonstrated in vitro (7–12). Although it is recognized that all BMPs are active in embryogenesis and developmental pattern formation beyond osteogenesis, little is known about the function and distribution of BMP-3, compared with other BMPs. BMPs fall into three subgroups based on their sequence homology. BMP-2 and BMP-4 are very closely related (86% amino acid identity in mature region) and form the first group. BMP-5, BMP-6, BMP-7 and BMP-8 are also closely related to each other (61–79%) and form the second group. BMP-3 is quite different from other BMPs and so falls alone into a third category. We attempted to identify a novel cDNA closely related to BMP-3 by the method of degenerate reverse transcription-polymerase chain reaction (RT-PCR). We succeeded in cloning a novel member of the TGF-b superfamily, which we have designated BMP-3b. Here we report the isolation and cDNA sequence of rat BMP-3b and rat BMP-3. In addition, regional differences in the expression of the BMP-3b gene, compared with BMP-3, were examined by RNA blot analysis. MATERIALS AND METHODS RT-PCR. The degenerate oligonucleotide primers synthesized for RT-PCR, according to the sequence of human BMP-3 mature region are as follows: P1, 59-GCNTG(C,T)CA(G,A)TT(C,T)-CCNATGCC-39 [bases 1524–1543]; P2, 59GC(G,A)CAN(G,C)(T,A)(C,T)TCNACNGTCAT-39 [complementary to base 1710–1729]. Nucleotide numbering is based on the published sequence of human BMP-3 mature region (GenBank Accession No. M22491). For the primer sequences,
1
To whom correspondence should be addressed. Fax: (+81)-6-872-7485. Abbreviations: BMP, bone morphogenetic protein; TGF-b, transforming growth factor b. The nucleotide sequences of rat BMP-3b and BMP-3 are deposited at the GenBank database under accession Nos. D49494 and D63860, respectively. 656 0006-291X/96 $18.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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N denotes any nucleotide. RT-PCR was performed using Perkin Elmer RNA PCR kit. One microgram of total RNA isolated from rat femur was used as a template for single strand cDNA synthesis by reverse transcriptase with P2 primers. The PCR was performed under the following conditions: 1 min at 94°C, 2 min at 55°C and 2 min at 72°C for 25 cycles. Amplified DNA (about 200 bp) was cloned directly into the EcoRV site of Bluescript II SK+ vector by the TA cloning method (13) and transformed to E. coli JM109. Approximately 600 transformants were screened by colony hybridization using human BMP-3 probe (nucleotide No. 1555–1736) which was prepared by PCR. Hybridization was performed at 42°C in 20% formamide, 6 × SSPE, 5 × Denhardt’s solution, 0.5% SDS, and 100 mg/ml denatured salmon sperm DNA. Two classes of clones were obtained, as indicated by strong (Clone A) or weak (Clone B) hybridization signals. The cDNA inserts of these clones were sequenced using the dideoxy chain termination method (14). cDNA library construction and cloning. Total RNA was extracted from rat femur by the acid guanidium thiocyanatephenol-chloroform method (15). Poly(A)+ RNA was isolated on an oligo(dT)-latex (Nippon Roche). An oligo(dT)-primed cDNA library of rat femur was constructed in lgt10 vector using cDNA Synthesis Kit (Pharmacia). For cloning BMP-3b cDNA, approximately 1 × 106 phages were screened by plaque hybridization using 32P-labeled double strand probe of Clone B, which was prepared by RT-PCR described above. Hybridization was performed at 42°C in 40% formamide, 6 × SSPE, 5 × Denhardt’s solution, 0.5% SDS and 100 mg/ml denatured salmon sperm DNA, and washed at 55°C in 2 × SSC, 0.1% SDS. A positive clone RB45 harboring the longest cDNA insert was subcloned into the Bluescript II SK+ vector and sequenced. Next, for cloning rat BMP-3 cDNA, the same filters were screened using 32P-labeled cDNA insert of Clone A as a probe. Hybridization was performed under the same conditions as for BMP-3b. A positive clone RC7 harboring the longest cDNA insert was used for sequencing. RNA blot analysis. Tissues were obtained from Sprague-Dawley rats. Cerebellum from rat at embryo (18 days) and different stages of development were obtained as indicated in the legend to Fig. 4. All poly(A)+ RNA were denatured using glyoxal and dimetylsulfoxide, and were fractionated on a 0.8% agarose gel. After electrophoresis, RNA was transferred to a nylon membrane (Zeta Probe, Bio-Rad). The membrane was prehybridized and hybridized at 42°C in 50% formamide, 6 × SSPE, 5 × Denhardt’s solution, 0.5% SDS, and 100 mg/ml denatured salmon sperm DNA. The blot was washed at 60°C once in 2 × SSC, 0.1% SDS, once in 0.5 × SSC, 0.1% SDS, and finally once in 0.1 × SSC, 0.1% SDS. The blot was exposed to X-ray films. The same membrane was used for sequential hybridizations with probes specific for rat BMP-3b (2.4 kb Not I fragment of RB45 reported in this paper), rat BMP-3 (2.2 kb Not I fragment of RC7 reported in this paper) and rat BMP-2 (2.7 kb full length cDNA of rat BMP-2 cloned from rat placenta cDNA library, data not shown). Probes were labeled by the random-primed method.
RESULTS AND DISCUSSION Cloning of rat BMP-3 and BMP-3b cDNA. In order to obtain clones for rat BMP-3 and unknown BMP-3 related protein, degenerate oligonucleotide primers (P1 and P2) were synthesized, which included all possible sequences encoding Ala-Cys-Gln-Phe-Pro-Met-Pro and Met-Thr-Val-GluSer-Cys-Ala, corresponding to human BMP-3 mature region. These primers were used in a PCR by using cDNA from rat femur as a template. As a result, a single 200 base-pair fragment was amplified. This PCR product was subcloned to Bluescript II SK+ and colony hybridization was done at low stringency using human BMP-3 fragment (nucleotide No. 1555–1736) as a probe. Two classes of clones, one giving a strong signal and the other a weak signal, were obtained. Nucleotide sequence analysis indicated that clones (Clone B) giving weak signal have a novel cDNA (Fig. 1, indicated by an overline), while clones (Clone A) giving a strong signal have a cDNA thought to be a rat homologue of human BMP-3 (data not shown). We have designated this novel cDNA rat BMP-3b. To obtain the full length of rat BMP-3 cDNA and rat BMP-3b cDNA, rat femur library was constructed. First, approximately 1 × 106 recombinant clones were screened using a 200 bp of cDNA fragment from Clone B as a probe for BMP-3b. When hybridizations were performed at high stringency, about twenty positive clones were isolated. Next, for cloning rat BMP-3, a 200 bp of cDNA fragment obtained from Clone A as a probe for BMP-3 was used and about twenty positive clones were isolated. The cDNA inserts of BMP-3b and BMP-3 were subcloned into the Not I site of Bluescript II SK+ and complete sequences were determined. Structure of rat BMP-3b cDNA. Fig. 1 shows the complete nucleotide sequence of rat BMP-3b cDNA, which is 2411 nucleotides long. A putative initiation codon ATG is located at nucleotides 1–3, preceded by the consensus sequence A/CCCATGG for the initiation (16), while a termination 657
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FIG. 1. Nucleotide sequence and deduced amino acid sequence of rat BMP-3b cDNA. Numbering of nucleotides and of amino acids (on the left margin) starts with the first ATG of the BMP-3b coding sequence. A boxed Arg-Arg-Arg sequence precedes putative maturation sites. Potential sites of N-linked glycosylation are indicated by thick underline. Dashed underline indicates the AATAAA sequence. Amplified cDNA fragment obtained by RT-PCR using degenerate oligonucleotide primers is indicated by overline.
codon TAA is found 476 codons later at nucleotides 1429–1431. A typical polyadenylation signal, AATAAA, is found only at nucleotides 2153–2158, but no poly(A)+ tail is contained within this cDNA. The open reading frame encodes a putative precursor for rat BMP-3b, which consists of 476 amino acid residues. The N-terminal sequence of the precursor is thought to be a signal peptide, 658
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based on its characteristic hydrophobic features (17). The putative precursor contains four N-linked glycosylation sites. The TGF-b homologous sequence is located in the C-terminal containing about 100-amino acid polypeptide. Three Arg at positions 364–366 precede the mature region of the BMP-3b sequence. By analogy with members of TGF-b superfamily (18), proteolytic cleavage of BMP-3b may take place following these basic residues, resulting in the release of a polypeptide of 110 residues with one potential N-glycosylation site (at position 467). Like other members of the TGF-b superfamily, the functional BMP-3b product is probably a homodimer of the C-terminal segments of BMP-3b precursors. Structure of rat BMP-3 cDNA. We sequenced the full length of rat BMP-3 cDNA. Predicted BMP-3 precursor consists of 468 amino acids. The nucleotide sequence of rat BMP-3 is deposited at the GenBank database (No. D63860, data not shown). The deduced amino acid sequence of rat BMP-3 precursor is approximately 79% identical to that of human. In the mature region rat BMP-3 revealed 2 amino acid changes compared to that of human BMP-3 (98% identity). Comparison with other BMP subfamily. Fig. 2 shows the amino acid sequence of C-terminal region of rat BMP-3b and BMP-3 with other members of BMP subfamily, aligned by seven highly conserved cysteine residues. The mature region of rat BMP-3b shows a high degree of amino acid identity with comparable regions of rat BMP-3 (81%) and a low degree of identity (42–47%) with human BMP-4, 5, 6, 7 and 8. In the propeptides region, BMP-3b (amino acids No. 1–366 in Fig. 1) and BMP-3 are 37% identity. But the homology between propeptide region of BMP-3b and other BMPs is very poor. BMPs are divided into three subgroups. BMP-2 and BMP-4 are very closely related (86% amino acid identity in the mature region) and form one group. BMP-5, BMP-6 and BMP-7 are also closely related to one another (an average of 78%) and form a second group. BMP-3 is quite different from other BMPs and forms a third category. Because BMP-3b is most closely related to BMP-3 (81%), BMP-3b can be considered a second member of BMP-3 subgroup within the BMP family.
FIG. 2. Alignment of amino acid sequence of C-terminal region of rat BMP-3b and corresponding regions of BMP subfamily. For ease of comparison, BMPs have been separated into three subgroups. Amino acid sequences (one-letter symbols) are aligned for maximal homology. Gaps are indicated by periods. Conserved amino acids in each subgroup are boxed. Seven conserved cysteines in the TGF-b superfamily are indicated with asterisks above the figure. 659
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mRNA expression of rat BMP-3b, BMP-3 and BMP-2. To determine the expression site of BMP-3b and to compare BMP-3b expression with that of BMP-3 and BMP-2, we screened mRNA preparations from several organ tissues of adult and neonatal rat by Northern blot hybridization (Fig. 3). In bone tissues BMP-3b mRNA was observed in femur, calvaria and costa as two species of 2.8 kb (major band) and 4.1 kb (minor band). BMP-3 mRNA was also found in these bone tissues as multiple mRNA species; major species of 6.7 kb and minor species of 4.7 and 2.6 kb were detected. BMP-2 mRNA was expressed in costa as double mRNA species (2.5 kb and 3.8 kb). These results indicate that BMP-3b was expressed in bone tissues in a manner similar to that of BMP-3 and BMP-2. Recombinant BMP-2 has been demonstrated to induce ectopic bone formation in vivo. On the other hand, little is known about biological activities of BMP-3. In our preliminary experiments, recombinant BMP-3b and BMP-3 expressed in CHO cells did not induce ectopic bone formation (data not shown). However relatively high level of BMP-3 protein was observed in bone tissue (1). Thus, expression of BMP-3b and BMP-3 mRNA in bone tissue suggests that these two BMPs may be acting in bone with distinct mechanism from other BMPs. We compared the relative abundance of BMP-3 and BMP-3b transcripts in calvaria and femur of neonatal and adult rat. Similar to BMP-3, the levels of BMP-3b mRNA in neonatal femur and calvaria were higher than those of in adult femur and calvaria. This result implies that BMP-3b has physiological importance in new bone formation in embryogenesis. In costicartilage, BMP-3b was highly expressed, while BMP-3 mRNA was not detected and BMP-2 mRNA was only slightly detected. Cartilage consists of only chondrocyte cells. BMP-3b
FIG. 3. Expression of rat BMP-3b, rat BMP-3 and rat BMP-2 mRNA in different rat tissues. Poly(A)+ RNA (10 mg) was loaded onto each lane, electrophoresed on a 0.8% agarose gel, blotted on to a nylon membrane and hybridized. The same filter was used for sequential hybridizations with 32P-labeled DNA probes. Probes used for hybridization are (A) rat BMP-3b probe, (B) rat BMP-3 probe and (C) rat BMP-2 probe. RNA ladder (0.24-9.5 kb; BRL INC) was used as size standard. 660
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FIG. 4. Northern blot analysis of BMP-3b mRNA from rat cerebellum isolated at various stages of development. Ages are given as days post coitum (d.p.c), days postnatal (p.n.) and weeks adult (wks.). Equal amounts of Poly(A)+ RNA (2 mg) isolated from cerebellum were loaded onto each lane. Hybridization was done with probe for rat BMP-3b. Lanes: (1) Poly (A)+ RNA obtained from cerebellum of 18 day embryos; (2) 0 day postnatal; (3) 1 day postnatal; (4) 2 days postnatal; (5) 3 days postnatal; (6) 1 week old; (7) 2 weeks old; (8) 3 weeks old; (9) 10–20 weeks old. The same filter was rehybridized with human b-actin cDNA probe.
protein is synthesized and secreted by chondrocytes, and might participate in cartilage formation and metabolism, and maintenance of the extracellular matrix. In other tissues, BMP-3b mRNA was found at high levels in brain, trachea and aorta and at lower levels in ovary, testis and skeletal muscle. Interestingly, BMP-3b mRNA in cerebellum was more abundant than in bone tissue. When it became apparent that BMP-3b mRNA was almost exclusively expressed in cerebellum, different stages of developmental expression of BMP-3b mRNA in cerebellum were examined. In embryo cerebellum (18 days), the BMP-3b transcripts were expressed weakly (Fig. 4). Its transcripts strongly appeared in postneonate and were constantly expressed in adult. Therefore, BMP-3b may play a role in the development and/or function of the cerebellum. The action of BMP-3b in the nervous system is unknown, but several members of the TGF-b superfamily are known to be an important regulator in early stages of neural development (19,20). BMP-7 is expressed in brain (2), and has been shown to induce the neural cell adhesion molecule (N-CAM) in a neuroblastoma-glioma hybrid cell line (10). Similar to BMP-7, BMP-3b also may act in the nervous system. BMP-3 mRNA was expressed at high levels in ovary, lung and trachea, but at low levels in kidney, aorta, and spleen. BMP-2 transcripts were detected at high levels in trachea, spleen, small intestine, and ovary, and at low levels in cerebellum, costa and bone marrow. The mRNA distribution of BMP-3b is distinct from that of BMP-3, although BMP-3b and BMP-3 are very closely related to each other. For example, BMP-3b mRNA is highly expressed in cerebellum and costicartilage, while BMP-3 mRNA is not observed in these tissues. In lung, kidney and small intestine, BMP-3b mRNA is not detected but BMP-3 mRNA is found. These results may suggest that physiological functions of BMP-3b are different from those of BMP-3. In conclusion, we cloned BMP-3b cDNA, encoding a novel protein closely related to BMP-3. Expression of BMP-3b mRNA in bone tissue and cerebellum suggests that BMP-3b may be involved in bone and cartilage metabolism and in the central nervous system. 661
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ACKNOWLEDGMENTS We thank Drs. K. Fukuda and M. Miyazato for helpful discussions. This work was supported in part by Special Coordination funds for Promoting Science and Technology from the Science and Technology Agency (Encouragement System of C.O.E.), and research grants from the Ministry of Health and Welfare, and the Human Science Foundation of Japan.
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