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. 1989 Nov;86(21):8338–8342. doi: 10.1073/pnas.86.21.8338

Isolation of an inhibitory insulin-like growth factor (IGF) binding protein from bone cell-conditioned medium: a potential local regulator of IGF action.

S Mohan 1, C M Bautista 1, J Wergedal 1, D J Baylink 1
PMCID: PMC298276  PMID: 2479022

Abstract

Inhibitory insulin-like growth factor binding protein (In-IGF-BP) has been purified to homogeneity from medium conditioned by TE89 human osteosarcoma cells by two different methods using Sephadex G-100 gel filtration, FPLC Mono Q ion-exchange, HPLC C4 reverse-phase, HPLC CN reverse-phase, and affinity chromatographies. In-IGF-BP thus purified appeared to be homogeneous and unique by the following criteria. (i) N-terminal sequence analysis yielded a unique sequence (Asp-Glu-Ala-Ile-His-Cys-Pro-Pro-Glu-Ser-Glu-Ala-Lys-Leu-Ala). (ii) Amino acid composition of In-IGF-BP revealed marked differences with the amino acid compositions of other known BPs. (iii) In-IGF-BP exhibited a single band with a molecular mass of 25 kDa under reducing conditions on sodium dodecyl sulfate/polyacrylamide gels. IGF-I and IGF-II but not insulin displaced the binding of 125I-labeled IGF-I or 125I-labeled IGF-II binding to In-IGF-BP. In-IGF-BP inhibited basal, IGF-stimulated bone cell proliferation and serum-stimulated bone cell proliferation. Forskolin increased synthesis of In-IGF-BP in TE85 human osteosarcoma cells in a dose-dependent manner. Based on these findings, we conclude that In-IGF-BP is a protein that has a unique sequence and significant biological actions on bone cells.

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Selected References

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  1. Baxter R. C. The insulin-like growth factors and their binding proteins. Comp Biochem Physiol B. 1988;91(2):229–235. doi: 10.1016/0305-0491(88)90137-x. [DOI] [PubMed] [Google Scholar]
  2. Bell S. C., Keyte J. W. N-terminal amino acid sequence of human pregnancy-associated endometrial alpha 1-globulin, an endometrial insulin-like growth factor (IGF) binding protein--evidence for two small molecular weight IGF binding proteins. Endocrinology. 1988 Aug;123(2):1202–1204. doi: 10.1210/endo-123-2-1202. [DOI] [PubMed] [Google Scholar]
  3. Brewer M. T., Stetler G. L., Squires C. H., Thompson R. C., Busby W. H., Clemmons D. R. Cloning, characterization, and expression of a human insulin-like growth factor binding protein. Biochem Biophys Res Commun. 1988 May 16;152(3):1289–1297. doi: 10.1016/s0006-291x(88)80425-x. [DOI] [PubMed] [Google Scholar]
  4. Brinkman A., Groffen C., Kortleve D. J., Geurts van Kessel A., Drop S. L. Isolation and characterization of a cDNA encoding the low molecular weight insulin-like growth factor binding protein (IBP-1). EMBO J. 1988 Aug;7(8):2417–2423. doi: 10.1002/j.1460-2075.1988.tb03087.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brown A. L., Chiariotti L., Orlowski C. C., Mehlman T., Burgess W. H., Ackerman E. J., Bruni C. B., Rechler M. M. Nucleotide sequence and expression of a cDNA clone encoding a fetal rat binding protein for insulin-like growth factors. J Biol Chem. 1989 Mar 25;264(9):5148–5154. [PubMed] [Google Scholar]
  6. Conover C. A., Liu F., Powell D., Rosenfeld R. G., Hintz R. L. Insulin-like growth factor binding proteins from cultured human fibroblasts. Characterization and hormonal regulation. J Clin Invest. 1989 Mar;83(3):852–859. doi: 10.1172/JCI113968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Daughaday W. H., Ward A. P., Goldberg A. C., Trivedi B., Kapadia M. Characterization of somatomedin binding in human serum by ultracentrifugation and gel filtration. J Clin Endocrinol Metab. 1982 Nov;55(5):916–921. doi: 10.1210/jcem-55-5-916. [DOI] [PubMed] [Google Scholar]
  8. De Leon D. D., Wilson D. M., Bakker B., Lamsom G., Hintz R. L., Rosenfeld R. G. Characterization of insulin-like growth factor binding proteins from human breast cancer cells. Mol Endocrinol. 1989 Mar;3(3):567–574. doi: 10.1210/mend-3-3-567. [DOI] [PubMed] [Google Scholar]
  9. De Mellow J. S., Baxter R. C. Growth hormone-dependent insulin-like growth factor (IGF) binding protein both inhibits and potentiates IGF-I-stimulated DNA synthesis in human skin fibroblasts. Biochem Biophys Res Commun. 1988 Oct 14;156(1):199–204. doi: 10.1016/s0006-291x(88)80824-6. [DOI] [PubMed] [Google Scholar]
  10. Elgin R. G., Busby W. H., Jr, Clemmons D. R. An insulin-like growth factor (IGF) binding protein enhances the biologic response to IGF-I. Proc Natl Acad Sci U S A. 1987 May;84(10):3254–3258. doi: 10.1073/pnas.84.10.3254. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Julkunen M., Koistinen R., Aalto-Setälä K., Seppälä M., Jänne O. A., Kontula K. Primary structure of human insulin-like growth factor-binding protein/placental protein 12 and tissue-specific expression of its mRNA. FEBS Lett. 1988 Aug 29;236(2):295–302. doi: 10.1016/0014-5793(88)80041-3. [DOI] [PubMed] [Google Scholar]
  12. Koistinen R., Kalkkinen N., Huhtala M. L., Seppälä M., Bohn H., Rutanen E. M. Placental protein 12 is a decidual protein that binds somatomedin and has an identical N-terminal amino acid sequence with somatomedin-binding protein from human amniotic fluid. Endocrinology. 1986 Apr;118(4):1375–1378. doi: 10.1210/endo-118-4-1375. [DOI] [PubMed] [Google Scholar]
  13. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  14. Lee Y. L., Hintz R. L., James P. M., Lee P. D., Shively J. E., Powell D. R. Insulin-like growth factor (IGF) binding protein complementary deoxyribonucleic acid from human HEP G2 hepatoma cells: predicted protein sequence suggests an IGF binding domain different from those of the IGF-I and IGF-II receptors. Mol Endocrinol. 1988 May;2(5):404–411. doi: 10.1210/mend-2-5-404. [DOI] [PubMed] [Google Scholar]
  15. Linkhart S., Mohan S., Linkhart T. A., Kumegawa M., Baylink D. J. Human skeletal growth factor stimulates collagen synthesis and inhibits proliferation in a clonal osteoblast cell line (MC3T3-E1). J Cell Physiol. 1986 Aug;128(2):307–312. doi: 10.1002/jcp.1041280224. [DOI] [PubMed] [Google Scholar]
  16. Martin J. L., Baxter R. C. Insulin-like growth factor-binding proteins (IGF-BPs) produced by human skin fibroblasts: immunological relationship to other human IGF-BPs. Endocrinology. 1988 Oct;123(4):1907–1915. doi: 10.1210/endo-123-4-1907. [DOI] [PubMed] [Google Scholar]
  17. McCusker R. H., Clemmons D. R. Insulin-like growth factor binding protein secretion by muscle cells: effect of cellular differentiation and proliferation. J Cell Physiol. 1988 Dec;137(3):505–512. doi: 10.1002/jcp.1041370316. [DOI] [PubMed] [Google Scholar]
  18. Mohan S., Jennings J. C., Linkhart T. A., Baylink D. J. Isolation and purification of a low-molecular-weight skeletal growth factor from human bones. Biochim Biophys Acta. 1986 Nov 19;884(2):234–242. doi: 10.1016/0304-4165(86)90168-6. [DOI] [PubMed] [Google Scholar]
  19. Mohan S., Jennings J. C., Linkhart T. A., Baylink D. J. Primary structure of human skeletal growth factor: homology with human insulin-like growth factor-II. Biochim Biophys Acta. 1988 Jul 14;966(1):44–55. doi: 10.1016/0304-4165(88)90127-4. [DOI] [PubMed] [Google Scholar]
  20. Mottola C., MacDonald R. G., Brackett J. L., Mole J. E., Anderson J. K., Czech M. P. Purification and amino-terminal sequence of an insulin-like growth factor-binding protein secreted by rat liver BRL-3A cells. J Biol Chem. 1986 Aug 25;261(24):11180–11188. [PubMed] [Google Scholar]
  21. Ocrant I., Pham H., Oh Y., Rosenfeld R. G. Characterization of insulin-like growth factor binding proteins of cultured rat astroglial and neuronal cells. Biochem Biophys Res Commun. 1989 Mar 31;159(3):1316–1322. doi: 10.1016/0006-291x(89)92254-7. [DOI] [PubMed] [Google Scholar]
  22. Ooi G. T., Herington A. C. The biological and structural characterization of specific serum binding proteins for the insulin-like growth factors. J Endocrinol. 1988 Jul;118(1):7–18. doi: 10.1677/joe.0.1180007. [DOI] [PubMed] [Google Scholar]
  23. Póvoa G., Enberg G., Jörnvall H., Hall K. Isolation and characterization of a somatomedin-binding protein from mid-term human amniotic fluid. Eur J Biochem. 1984 Oct 15;144(2):199–204. doi: 10.1111/j.1432-1033.1984.tb08449.x. [DOI] [PubMed] [Google Scholar]
  24. Póvoa G., Isaksson M., Jörnvall H., Hall K. The somatomedin-binding protein isolated from a human hepatoma cell line is identical to the human amniotic fluid somatomedin-binding protein. Biochem Biophys Res Commun. 1985 May 16;128(3):1071–1078. doi: 10.1016/0006-291x(85)91049-6. [DOI] [PubMed] [Google Scholar]
  25. Ritvos O., Ranta T., Jalkanen J., Suikkari A. M., Voutilainen R., Bohn H., Rutanen E. M. Insulin-like growth factor (IGF) binding protein from human decidua inhibits the binding and biological action of IGF-I in cultured choriocarcinoma cells. Endocrinology. 1988 May;122(5):2150–2157. doi: 10.1210/endo-122-5-2150. [DOI] [PubMed] [Google Scholar]
  26. Suikkari A. M., Jalkanen J., Koistinen R., Bützow R., Ritvos O., Ranta T., Seppälä M. Human granulosa cells synthesize low molecular weight insulin-like growth factor-binding protein. Endocrinology. 1989 Feb;124(2):1088–1090. doi: 10.1210/endo-124-2-1088. [DOI] [PubMed] [Google Scholar]
  27. Szabo L., Mottershead D. G., Ballard F. J., Wallace J. C. The bovine insulin-like growth factor (IGF) binding protein purified from conditioned medium requires the N-terminal tripeptide in IGF-1 for binding. Biochem Biophys Res Commun. 1988 Feb 29;151(1):207–214. doi: 10.1016/0006-291x(88)90580-3. [DOI] [PubMed] [Google Scholar]
  28. Wergedal J. E., Mohan S., Taylor A. K., Baylink D. J. Skeletal growth factor is produced by human osteoblast-like cells in culture. Biochim Biophys Acta. 1986 Nov 28;889(2):163–170. doi: 10.1016/0167-4889(86)90100-x. [DOI] [PubMed] [Google Scholar]
  29. 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]

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