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. 1994 Sep 2;126(6):1595–1609. doi: 10.1083/jcb.126.6.1595

Recombinant Vgr-1/BMP-6-expressing tumors induce fibrosis and endochondral bone formation in vivo

PMCID: PMC2290953  PMID: 8089189

Abstract

Members of the TGF-beta superfamily appear to modulate mesenchymal differentiation, including the processes of cartilage and bone formation. Nothing is yet known about the function of the TGF-beta- related factor vgr-1, also called bone morphogenetic protein-6 (BMP-6), and only limited studies have been conducted on the most closely related factors BMP-5, osteogenic protein-1 (OP-1) or BMP-7, and OP-2. Because vgr-1 mRNA has been localized in hypertrophic cartilage, this factor may play a vital role in endochondral bone formation. We developed antibodies to vgr-1, and documented that vgr-1 protein was expressed in hypertrophic cartilage of mice. To further characterize the role of this protein in bone differentiation, we generated CHO cells that overexpressed recombinant murine vgr-1 protein. Western blot analysis documented that recombinant vgr-1 protein was secreted into the media and was proteolytically processed to yield the mature vgr-1 molecule. To assess the biological activity of recombinant vgr-1 in vivo, we introduced the vgr-1-expressing CHO cells directly into the subcutaneous tissue of athymic nude mice. CHO-vgr-1 cells produced localized tumors, and the continuous secretion of vgr-1 resulted in tumors with a strikingly different gross and histological appearance as compared to the parental CHO cells. The tumors of control CHO cells were hemorrhagic, necrotic, and friable, whereas the CHO-vgr-1 tumors were dense, firm, and fibrotic. In contrast with control CHO tumors, the nests of CHO-vgr-1 tumor cells were surrounded by extensive connective tissue, which contained large regions of cartilage and bone. Further analysis indicated that secretion of vgr-1 from the transfected CHO tumor cells induced the surrounding host mesenchymal cells to develop along the endochondral bone pathway. These findings suggest that endochondral bone formation.

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Selected References

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  1. Asahina I., Sampath T. K., Nishimura I., Hauschka P. V. Human osteogenic protein-1 induces both chondroblastic and osteoblastic differentiation of osteoprogenitor cells derived from newborn rat calvaria. J Cell Biol. 1993 Nov;123(4):921–933. doi: 10.1083/jcb.123.4.921. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barcellos-Hoff M. H., Derynck R., Tsang M. L., Weatherbee J. A. Transforming growth factor-beta activation in irradiated murine mammary gland. J Clin Invest. 1994 Feb;93(2):892–899. doi: 10.1172/JCI117045. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Basler K., Edlund T., Jessell T. M., Yamada T. Control of cell pattern in the neural tube: regulation of cell differentiation by dorsalin-1, a novel TGF beta family member. Cell. 1993 May 21;73(4):687–702. doi: 10.1016/0092-8674(93)90249-p. [DOI] [PubMed] [Google Scholar]
  4. Beck L. S., Deguzman L., Lee W. P., Xu Y., McFatridge L. A., Gillett N. A., Amento E. P. Rapid publication. TGF-beta 1 induces bone closure of skull defects. J Bone Miner Res. 1991 Nov;6(11):1257–1265. doi: 10.1002/jbmr.5650061117. [DOI] [PubMed] [Google Scholar]
  5. Celeste A. J., Iannazzi J. A., Taylor R. C., Hewick R. M., Rosen V., Wang E. A., Wozney J. M. Identification of transforming growth factor beta family members present in bone-inductive protein purified from bovine bone. Proc Natl Acad Sci U S A. 1990 Dec;87(24):9843–9847. doi: 10.1073/pnas.87.24.9843. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chen T. L., Bates R. L., Dudley A., Hammonds R. G., Jr, Amento E. P. Bone morphogenetic protein-2b stimulation of growth and osteogenic phenotypes in rat osteoblast-like cells: comparison with TGF-beta 1. J Bone Miner Res. 1991 Dec;6(12):1387–1393. doi: 10.1002/jbmr.5650061216. [DOI] [PubMed] [Google Scholar]
  7. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  8. Cunningham N. S., Paralkar V., Reddi A. H. Osteogenin and recombinant bone morphogenetic protein 2B are chemotactic for human monocytes and stimulate transforming growth factor beta 1 mRNA expression. Proc Natl Acad Sci U S A. 1992 Dec 15;89(24):11740–11744. doi: 10.1073/pnas.89.24.11740. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Doctor J. S., Jackson P. D., Rashka K. E., Visalli M., Hoffmann F. M. Sequence, biochemical characterization, and developmental expression of a new member of the TGF-beta superfamily in Drosophila melanogaster. Dev Biol. 1992 Jun;151(2):491–505. doi: 10.1016/0012-1606(92)90188-m. [DOI] [PubMed] [Google Scholar]
  10. Esko J. D., Rostand K. S., Weinke J. L. Tumor formation dependent on proteoglycan biosynthesis. Science. 1988 Aug 26;241(4869):1092–1096. doi: 10.1126/science.3137658. [DOI] [PubMed] [Google Scholar]
  11. Gazit D., Ebner R., Kahn A. J., Derynck R. Modulation of expression and cell surface binding of members of the transforming growth factor-beta superfamily during retinoic acid-induced osteoblastic differentiation of multipotential mesenchymal cells. Mol Endocrinol. 1993 Feb;7(2):189–198. doi: 10.1210/mend.7.2.8385738. [DOI] [PubMed] [Google Scholar]
  12. Gorman C., Padmanabhan R., Howard B. H. High efficiency DNA-mediated transformation of primate cells. Science. 1983 Aug 5;221(4610):551–553. doi: 10.1126/science.6306768. [DOI] [PubMed] [Google Scholar]
  13. Green N., Alexander H., Olson A., Alexander S., Shinnick T. M., Sutcliffe J. G., Lerner R. A. Immunogenic structure of the influenza virus hemagglutinin. Cell. 1982 Mar;28(3):477–487. doi: 10.1016/0092-8674(82)90202-1. [DOI] [PubMed] [Google Scholar]
  14. Hammonds R. G., Jr, Schwall R., Dudley A., Berkemeier L., Lai C., Lee J., Cunningham N., Reddi A. H., Wood W. I., Mason A. J. Bone-inducing activity of mature BMP-2b produced from a hybrid BMP-2a/2b precursor. Mol Endocrinol. 1991 Jan;5(1):149–155. doi: 10.1210/mend-5-1-149. [DOI] [PubMed] [Google Scholar]
  15. Hiraki Y., Inoue H., Shigeno C., Sanma Y., Bentz H., Rosen D. M., Asada A., Suzuki F. Bone morphogenetic proteins (BMP-2 and BMP-3) promote growth and expression of the differentiated phenotype of rabbit chondrocytes and osteoblastic MC3T3-E1 cells in vitro. J Bone Miner Res. 1991 Dec;6(12):1373–1385. doi: 10.1002/jbmr.5650061215. [DOI] [PubMed] [Google Scholar]
  16. Hughes F. J., Aubin J. E., Heersche J. N. Differential chemotactic responses of different populations of fetal rat calvaria cells to platelet-derived growth factor and transforming growth factor beta. Bone Miner. 1992 Oct;19(1):63–74. doi: 10.1016/0169-6009(92)90844-4. [DOI] [PubMed] [Google Scholar]
  17. Jessell T. M., Melton D. A. Diffusible factors in vertebrate embryonic induction. Cell. 1992 Jan 24;68(2):257–270. doi: 10.1016/0092-8674(92)90469-s. [DOI] [PubMed] [Google Scholar]
  18. Jones C. M., Lyons K. M., Hogan B. L. Involvement of Bone Morphogenetic Protein-4 (BMP-4) and Vgr-1 in morphogenesis and neurogenesis in the mouse. Development. 1991 Feb;111(2):531–542. doi: 10.1242/dev.111.2.531. [DOI] [PubMed] [Google Scholar]
  19. Jones C. M., Simon-Chazottes D., Guenet J. L., Hogan B. L. Isolation of Vgr-2, a novel member of the transforming growth factor-beta-related gene family. Mol Endocrinol. 1992 Nov;6(11):1961–1968. doi: 10.1210/mend.6.11.1480182. [DOI] [PubMed] [Google Scholar]
  20. Joyce M. E., Roberts A. B., Sporn M. B., Bolander M. E. Transforming growth factor-beta and the initiation of chondrogenesis and osteogenesis in the rat femur. J Cell Biol. 1990 Jun;110(6):2195–2207. doi: 10.1083/jcb.110.6.2195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Katagiri T., Yamaguchi A., Ikeda T., Yoshiki S., Wozney J. M., Rosen V., Wang E. A., Tanaka H., Omura S., Suda T. The non-osteogenic mouse pluripotent cell line, C3H10T1/2, is induced to differentiate into osteoblastic cells by recombinant human bone morphogenetic protein-2. Biochem Biophys Res Commun. 1990 Oct 15;172(1):295–299. doi: 10.1016/s0006-291x(05)80208-6. [DOI] [PubMed] [Google Scholar]
  22. Lee S. J. Identification of a novel member (GDF-1) of the transforming growth factor-beta superfamily. Mol Endocrinol. 1990 Jul;4(7):1034–1040. doi: 10.1210/mend-4-7-1034. [DOI] [PubMed] [Google Scholar]
  23. Luyten F. P., Cunningham N. S., Ma S., Muthukumaran N., Hammonds R. G., Nevins W. B., Woods W. I., Reddi A. H. Purification and partial amino acid sequence of osteogenin, a protein initiating bone differentiation. J Biol Chem. 1989 Aug 15;264(23):13377–13380. [PubMed] [Google Scholar]
  24. Lyons K. M., Jones C. M., Hogan B. L. The DVR gene family in embryonic development. Trends Genet. 1991 Nov-Dec;7(11-12):408–412. doi: 10.1016/0168-9525(91)90265-r. [DOI] [PubMed] [Google Scholar]
  25. Lyons K. M., Pelton R. W., Hogan B. L. Patterns of expression of murine Vgr-1 and BMP-2a RNA suggest that transforming growth factor-beta-like genes coordinately regulate aspects of embryonic development. Genes Dev. 1989 Nov;3(11):1657–1668. doi: 10.1101/gad.3.11.1657. [DOI] [PubMed] [Google Scholar]
  26. Lyons K., Graycar J. L., Lee A., Hashmi S., Lindquist P. B., Chen E. Y., Hogan B. L., Derynck R. Vgr-1, a mammalian gene related to Xenopus Vg-1, is a member of the transforming growth factor beta gene superfamily. Proc Natl Acad Sci U S A. 1989 Jun;86(12):4554–4558. doi: 10.1073/pnas.86.12.4554. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Marcelli C., Yates A. J., Mundy G. R. In vivo effects of human recombinant transforming growth factor beta on bone turnover in normal mice. J Bone Miner Res. 1990 Oct;5(10):1087–1096. doi: 10.1002/jbmr.5650051013. [DOI] [PubMed] [Google Scholar]
  28. McPherron A. C., Lee S. J. GDF-3 and GDF-9: two new members of the transforming growth factor-beta superfamily containing a novel pattern of cysteines. J Biol Chem. 1993 Feb 15;268(5):3444–3449. [PubMed] [Google Scholar]
  29. Miettinen P. J., Heikinheimo K. Transforming growth factor-alpha (TGF-alpha) and insulin gene expression in human fetal pancreas. Development. 1992 Apr;114(4):833–840. doi: 10.1242/dev.114.4.833. [DOI] [PubMed] [Google Scholar]
  30. Noda M., Camilliere J. J. In vivo stimulation of bone formation by transforming growth factor-beta. Endocrinology. 1989 Jun;124(6):2991–2994. doi: 10.1210/endo-124-6-2991. [DOI] [PubMed] [Google Scholar]
  31. Ozkaynak E., Rueger D. C., Drier E. A., Corbett C., Ridge R. J., Sampath T. K., Oppermann H. OP-1 cDNA encodes an osteogenic protein in the TGF-beta family. EMBO J. 1990 Jul;9(7):2085–2093. doi: 10.1002/j.1460-2075.1990.tb07376.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Ozkaynak E., Schnegelsberg P. N., Jin D. F., Clifford G. M., Warren F. D., Drier E. A., Oppermann H. Osteogenic protein-2. A new member of the transforming growth factor-beta superfamily expressed early in embryogenesis. J Biol Chem. 1992 Dec 15;267(35):25220–25227. [PubMed] [Google Scholar]
  33. Padgett R. W., St Johnston R. D., Gelbart W. M. A transcript from a Drosophila pattern gene predicts a protein homologous to the transforming growth factor-beta family. Nature. 1987 Jan 1;325(6099):81–84. doi: 10.1038/325081a0. [DOI] [PubMed] [Google Scholar]
  34. Pfeilschifter J., Wolf O., Naumann A., Minne H. W., Mundy G. R., Ziegler R. Chemotactic response of osteoblastlike cells to transforming growth factor beta. J Bone Miner Res. 1990 Aug;5(8):825–830. doi: 10.1002/jbmr.5650050805. [DOI] [PubMed] [Google Scholar]
  35. Reddi A. H., Huggins C. Biochemical sequences in the transformation of normal fibroblasts in adolescent rats. Proc Natl Acad Sci U S A. 1972 Jun;69(6):1601–1605. doi: 10.1073/pnas.69.6.1601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Roberts A. B., Sporn M. B., Assoian R. K., Smith J. M., Roche N. S., Wakefield L. M., Heine U. I., Liotta L. A., Falanga V., Kehrl J. H. Transforming growth factor type beta: rapid induction of fibrosis and angiogenesis in vivo and stimulation of collagen formation in vitro. Proc Natl Acad Sci U S A. 1986 Jun;83(12):4167–4171. doi: 10.1073/pnas.83.12.4167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Simonsen C. C., Levinson A. D. Isolation and expression of an altered mouse dihydrofolate reductase cDNA. Proc Natl Acad Sci U S A. 1983 May;80(9):2495–2499. doi: 10.1073/pnas.80.9.2495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Urist M. R. Bone: formation by autoinduction. Science. 1965 Nov 12;150(3698):893–899. doi: 10.1126/science.150.3698.893. [DOI] [PubMed] [Google Scholar]
  40. Urlaub G., Chasin L. A. Isolation of Chinese hamster cell mutants deficient in dihydrofolate reductase activity. Proc Natl Acad Sci U S A. 1980 Jul;77(7):4216–4220. doi: 10.1073/pnas.77.7.4216. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Vukicevic S., Luyten F. P., Reddi A. H. Stimulation of the expression of osteogenic and chondrogenic phenotypes in vitro by osteogenin. Proc Natl Acad Sci U S A. 1989 Nov;86(22):8793–8797. doi: 10.1073/pnas.86.22.8793. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Wall N. A., Blessing M., Wright C. V., Hogan B. L. Biosynthesis and in vivo localization of the decapentaplegic-Vg-related protein, DVR-6 (bone morphogenetic protein-6). J Cell Biol. 1993 Jan;120(2):493–502. doi: 10.1083/jcb.120.2.493. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Wigler M., Sweet R., Sim G. K., Wold B., Pellicer A., Lacy E., Maniatis T., Silverstein S., Axel R. Transformation of mammalian cells with genes from procaryotes and eucaryotes. Cell. 1979 Apr;16(4):777–785. doi: 10.1016/0092-8674(79)90093-x. [DOI] [PubMed] [Google Scholar]
  44. Wood W. I., Cachianes G., Henzel W. J., Winslow G. A., Spencer S. A., Hellmiss R., Martin J. L., Baxter R. C. Cloning and expression of the growth hormone-dependent insulin-like growth factor-binding protein. Mol Endocrinol. 1988 Dec;2(12):1176–1185. doi: 10.1210/mend-2-12-1176. [DOI] [PubMed] [Google Scholar]
  45. Wozney J. M., Rosen V., Celeste A. J., Mitsock L. M., Whitters M. J., Kriz R. W., Hewick R. M., Wang E. A. Novel regulators of bone formation: molecular clones and activities. Science. 1988 Dec 16;242(4885):1528–1534. doi: 10.1126/science.3201241. [DOI] [PubMed] [Google Scholar]
  46. Wozney J. M. The bone morphogenetic protein family and osteogenesis. Mol Reprod Dev. 1992 Jun;32(2):160–167. doi: 10.1002/mrd.1080320212. [DOI] [PubMed] [Google Scholar]
  47. Yamaguchi A., Kahn A. J. Clonal osteogenic cell lines express myogenic and adipocytic developmental potential. Calcif Tissue Int. 1991 Sep;49(3):221–225. doi: 10.1007/BF02556122. [DOI] [PubMed] [Google Scholar]
  48. Yamaguchi A., Katagiri T., Ikeda T., Wozney J. M., Rosen V., Wang E. A., Kahn A. J., Suda T., Yoshiki S. Recombinant human bone morphogenetic protein-2 stimulates osteoblastic maturation and inhibits myogenic differentiation in vitro. J Cell Biol. 1991 May;113(3):681–687. doi: 10.1083/jcb.113.3.681. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Yang E. Y., Moses H. L. Transforming growth factor beta 1-induced changes in cell migration, proliferation, and angiogenesis in the chicken chorioallantoic membrane. J Cell Biol. 1990 Aug;111(2):731–741. doi: 10.1083/jcb.111.2.731. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Zhou X., Sasaki H., Lowe L., Hogan B. L., Kuehn M. R. Nodal is a novel TGF-beta-like gene expressed in the mouse node during gastrulation. Nature. 1993 Feb 11;361(6412):543–547. doi: 10.1038/361543a0. [DOI] [PubMed] [Google Scholar]
  51. von Heijne G. A new method for predicting signal sequence cleavage sites. Nucleic Acids Res. 1986 Jun 11;14(11):4683–4690. doi: 10.1093/nar/14.11.4683. [DOI] [PMC free article] [PubMed] [Google Scholar]

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