Skip to main content
PMC home page
The Journal of Clinical Investigation logoLink to The Journal of Clinical Investigation
. 1998 Jan 15;101(2):413–422. doi: 10.1172/JCI119880

Bone morphogenetic protein-6 production in human osteoblastic cell lines. Selective regulation by estrogen.

D J Rickard 1, L C Hofbauer 1, S K Bonde 1, F Gori 1, T C Spelsberg 1, B L Riggs 1
PMCID: PMC508581  PMID: 9435314

Abstract

Bone morphogenetic proteins (BMPs) induce differentiation of osteoblast and chondroblast lineage cells from uncommitted mesenchymal precursors. Because estrogen has potent osteochondrogenic actions, we investigated its effect on BMP production in two estrogen-responsive, human immortalized cell lines (hFOB/ER3 and hFOB/ER9) that display the mature osteoblast phenotype. These cell lines were produced by stable transfection of the estrogen receptor (ER) gene into immortalized fetal osteoblasts at low ( approximately 800 ER/ nucleus) and at high ( approximately 3, 900 ER/nucleus) levels, respectively. As assessed by reverse transcriptase PCR, treatment with 17beta-estradiol (10(-)10 - 10(-)7 M) increased steady-state levels of BMP-6 mRNA dose dependently by twofold in the hFOB/ER3 cells and by over threefold in the hFOB/ER9 cells. Messenger RNA levels for transforming growth factors-beta1 and -beta2 and BMPs-1 through -5 and -7 levels were unchanged. The results were confirmed by sequence determination of the PCR product and by Northern blot analysis for total RNA. 17beta-estradiol increased BMP-6 protein production sixfold by Western analysis. Cotreatment with antiestrogens (ICI 182,780 or 4-hydroxytamoxifen) antagonized the effects of 17beta-estradiol. These data suggest that some of the skeletal effects of estrogen on bone and cartilage may be mediated by increased production of BMP-6 by osteoblasts.

Full Text

The Full Text of this article is available as a PDF (475.9 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ahrens M., Ankenbauer T., Schröder D., Hollnagel A., Mayer H., Gross G. Expression of human bone morphogenetic proteins-2 or -4 in murine mesenchymal progenitor C3H10T1/2 cells induces differentiation into distinct mesenchymal cell lineages. DNA Cell Biol. 1993 Dec;12(10):871–880. doi: 10.1089/dna.1993.12.871. [DOI] [PubMed] [Google Scholar]
  2. Amédée J., Bareille R., Rouais F., Cunningham N., Reddi H., Harmand M. F. Osteogenin (bone morphogenic protein 3) inhibits proliferation and stimulates differentiation of osteoprogenitors in human bone marrow. Differentiation. 1994 Dec;58(2):157–164. [PubMed] [Google Scholar]
  3. Ben-Hur H., Mor G., Blickstein I., Likhman I., Kohen F., Dgani R., Insler V., Yaffe P., Ornoy A. Localization of estrogen receptors in long bones and vertebrae of human fetuses. Calcif Tissue Int. 1993 Aug;53(2):91–96. doi: 10.1007/BF01321885. [DOI] [PubMed] [Google Scholar]
  4. Boden S. D., Hair G., Titus L., Racine M., McCuaig K., Wozney J. M., Nanes M. S. Glucocorticoid-induced differentiation of fetal rat calvarial osteoblasts is mediated by bone morphogenetic protein-6. Endocrinology. 1997 Jul;138(7):2820–2828. doi: 10.1210/endo.138.7.5125. [DOI] [PubMed] [Google Scholar]
  5. Boden S. D., McCuaig K., Hair G., Racine M., Titus L., Wozney J. M., Nanes M. S. Differential effects and glucocorticoid potentiation of bone morphogenetic protein action during rat osteoblast differentiation in vitro. Endocrinology. 1996 Aug;137(8):3401–3407. doi: 10.1210/endo.137.8.8754767. [DOI] [PubMed] [Google Scholar]
  6. Carey D. E., Liu X. Expression of bone morphogenetic protein-6 messenger RNA in bovine growth plate chondrocytes of different size. J Bone Miner Res. 1995 Mar;10(3):401–405. doi: 10.1002/jbmr.5650100310. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Chang S. C., Hoang B., Thomas J. T., Vukicevic S., Luyten F. P., Ryba N. J., Kozak C. A., Reddi A. H., Moos M., Jr Cartilage-derived morphogenetic proteins. New members of the transforming growth factor-beta superfamily predominantly expressed in long bones during human embryonic development. J Biol Chem. 1994 Nov 11;269(45):28227–28234. [PubMed] [Google Scholar]
  9. 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]
  10. Colvard D. S., Jankus W. R., Berg N. J., Graham M. L., 2nd, Jiang N. S., Ingle J. N., Spelsberg T. C. Microassay for nuclear binding of steroid receptors with use of intact cells from small samples of avian and human tissue. Clin Chem. 1988 Feb;34(2):363–369. [PubMed] [Google Scholar]
  11. Corvol M. T., Carrascosa A., Tsagris L., Blanchard O., Rappaport R. Evidence for a direct in vitro action of sex steroids on rabbit cartilage cells during skeletal growth: influence of age and sex. Endocrinology. 1987 Apr;120(4):1422–1429. doi: 10.1210/endo-120-4-1422. [DOI] [PubMed] [Google Scholar]
  12. Eriksen E. F., Colvard D. S., Berg N. J., Graham M. L., Mann K. G., Spelsberg T. C., Riggs B. L. Evidence of estrogen receptors in normal human osteoblast-like cells. Science. 1988 Jul 1;241(4861):84–86. doi: 10.1126/science.3388021. [DOI] [PubMed] [Google Scholar]
  13. Ernst M., Heath J. K., Rodan G. A. Estradiol effects on proliferation, messenger ribonucleic acid for collagen and insulin-like growth factor-I, and parathyroid hormone-stimulated adenylate cyclase activity in osteoblastic cells from calvariae and long bones. Endocrinology. 1989 Aug;125(2):825–833. doi: 10.1210/endo-125-2-825. [DOI] [PubMed] [Google Scholar]
  14. Gitelman S. E., Kirk M., Ye J. Q., Filvaroff E. H., Kahn A. J., Derynck R. Vgr-1/BMP-6 induces osteoblastic differentiation of pluripotential mesenchymal cells. Cell Growth Differ. 1995 Jul;6(7):827–836. [PubMed] [Google Scholar]
  15. Gitelman S. E., Kobrin M. S., Ye J. Q., Lopez A. R., Lee A., Derynck R. Recombinant Vgr-1/BMP-6-expressing tumors induce fibrosis and endochondral bone formation in vivo. J Cell Biol. 1994 Sep;126(6):1595–1609. doi: 10.1083/jcb.126.6.1595. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Harris S. A., Enger R. J., Riggs B. L., Spelsberg T. C. Development and characterization of a conditionally immortalized human fetal osteoblastic cell line. J Bone Miner Res. 1995 Feb;10(2):178–186. doi: 10.1002/jbmr.5650100203. [DOI] [PubMed] [Google Scholar]
  17. Harris S. A., Tau K. R., Enger R. J., Toft D. O., Riggs B. L., Spelsberg T. C. Estrogen response in the hFOB 1.19 human fetal osteoblastic cell line stably transfected with the human estrogen receptor gene. J Cell Biochem. 1995 Oct;59(2):193–201. doi: 10.1002/jcb.240590209. [DOI] [PubMed] [Google Scholar]
  18. Hughes F. J., Collyer J., Stanfield M., Goodman S. A. The effects of bone morphogenetic protein-2, -4, and -6 on differentiation of rat osteoblast cells in vitro. Endocrinology. 1995 Jun;136(6):2671–2677. doi: 10.1210/endo.136.6.7750491. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Kassem M., Okazaki R., De León D., Harris S. A., Robinson J. A., Spelsberg T. C., Conover C. A., Riggs B. L. Potential mechanism of estrogen-mediated decrease in bone formation: estrogen increases production of inhibitory insulin-like growth factor-binding protein-4. Proc Assoc Am Physicians. 1996 Mar;108(2):155–164. [PubMed] [Google Scholar]
  21. Katzenellenbogen J. A., O'Malley B. W., Katzenellenbogen B. S. Tripartite steroid hormone receptor pharmacology: interaction with multiple effector sites as a basis for the cell- and promoter-specific action of these hormones. Mol Endocrinol. 1996 Feb;10(2):119–131. doi: 10.1210/mend.10.2.8825552. [DOI] [PubMed] [Google Scholar]
  22. Kessler E., Takahara K., Biniaminov L., Brusel M., Greenspan D. S. Bone morphogenetic protein-1: the type I procollagen C-proteinase. Science. 1996 Jan 19;271(5247):360–362. doi: 10.1126/science.271.5247.360. [DOI] [PubMed] [Google Scholar]
  23. Kimble R. B., Matayoshi A. B., Vannice J. L., Kung V. T., Williams C., Pacifici R. Simultaneous block of interleukin-1 and tumor necrosis factor is required to completely prevent bone loss in the early postovariectomy period. Endocrinology. 1995 Jul;136(7):3054–3061. doi: 10.1210/endo.136.7.7789332. [DOI] [PubMed] [Google Scholar]
  24. Kingsley D. M. What do BMPs do in mammals? Clues from the mouse short-ear mutation. Trends Genet. 1994 Jan;10(1):16–21. doi: 10.1016/0168-9525(94)90014-0. [DOI] [PubMed] [Google Scholar]
  25. Komm B. S., Terpening C. M., Benz D. J., Graeme K. A., Gallegos A., Korc M., Greene G. L., O'Malley B. W., Haussler M. R. Estrogen binding, receptor mRNA, and biologic response in osteoblast-like osteosarcoma cells. Science. 1988 Jul 1;241(4861):81–84. doi: 10.1126/science.3164526. [DOI] [PubMed] [Google Scholar]
  26. Korach K. S. Insights from the study of animals lacking functional estrogen receptor. Science. 1994 Dec 2;266(5190):1524–1527. doi: 10.1126/science.7985022. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Levenson A. S., Jordan V. C. Transfection of human estrogen receptor (ER) cDNA into ER-negative mammalian cell lines. J Steroid Biochem Mol Biol. 1994 Dec;51(5-6):229–239. doi: 10.1016/0960-0760(94)90035-3. [DOI] [PubMed] [Google Scholar]
  29. Lyons K. M., Pelton R. W., Hogan B. L. Organogenesis and pattern formation in the mouse: RNA distribution patterns suggest a role for bone morphogenetic protein-2A (BMP-2A). Development. 1990 Aug;109(4):833–844. doi: 10.1242/dev.109.4.833. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Manolagas S. C., Jilka R. L. Bone marrow, cytokines, and bone remodeling. Emerging insights into the pathophysiology of osteoporosis. N Engl J Med. 1995 Feb 2;332(5):305–311. doi: 10.1056/NEJM199502023320506. [DOI] [PubMed] [Google Scholar]
  32. Oursler M. J., Cortese C., Keeting P., Anderson M. A., Bonde S. K., Riggs B. L., Spelsberg T. C. Modulation of transforming growth factor-beta production in normal human osteoblast-like cells by 17 beta-estradiol and parathyroid hormone. Endocrinology. 1991 Dec;129(6):3313–3320. doi: 10.1210/endo-129-6-3313. [DOI] [PubMed] [Google Scholar]
  33. Oursler M. J., Osdoby P., Pyfferoen J., Riggs B. L., Spelsberg T. C. Avian osteoclasts as estrogen target cells. Proc Natl Acad Sci U S A. 1991 Aug 1;88(15):6613–6617. doi: 10.1073/pnas.88.15.6613. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Oursler M. J., Pederson L., Fitzpatrick L., Riggs B. L., Spelsberg T. Human giant cell tumors of the bone (osteoclastomas) are estrogen target cells. Proc Natl Acad Sci U S A. 1994 Jun 7;91(12):5227–5231. doi: 10.1073/pnas.91.12.5227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Parker M. G. Action of "pure" antiestrogens in inhibiting estrogen receptor action. Breast Cancer Res Treat. 1993;26(2):131–137. doi: 10.1007/BF00689686. [DOI] [PubMed] [Google Scholar]
  36. Pinus H., Ornoy A., Patlas N., Yaffe P., Schwartz Z. Specific beta estradiol binding in cartilage and serum from young mice and rats is age dependent. Connect Tissue Res. 1993;30(2):85–98. doi: 10.3109/03008209309041325. [DOI] [PubMed] [Google Scholar]
  37. Reddi A. H., Cunningham N. S. Initiation and promotion of bone differentiation by bone morphogenetic proteins. J Bone Miner Res. 1993 Dec;8 (Suppl 2):S499–S502. doi: 10.1002/jbmr.5650081313. [DOI] [PubMed] [Google Scholar]
  38. Robinson J. A., Harris S. A., Riggs B. L., Spelsberg T. C. Estrogen regulation of human osteoblastic cell proliferation and differentiation. Endocrinology. 1997 Jul;138(7):2919–2927. doi: 10.1210/endo.138.7.5277. [DOI] [PubMed] [Google Scholar]
  39. Rosen V., Nove J., Song J. J., Thies R. S., Cox K., Wozney J. M. Responsiveness of clonal limb bud cell lines to bone morphogenetic protein 2 reveals a sequential relationship between cartilage and bone cell phenotypes. J Bone Miner Res. 1994 Nov;9(11):1759–1768. doi: 10.1002/jbmr.5650091113. [DOI] [PubMed] [Google Scholar]
  40. Schuchard M., Landers J. P., Sandhu N. P., Spelsberg T. C. Steroid hormone regulation of nuclear proto-oncogenes. Endocr Rev. 1993 Dec;14(6):659–669. doi: 10.1210/edrv-14-6-659. [DOI] [PubMed] [Google Scholar]
  41. Smith E. P., Boyd J., Frank G. R., Takahashi H., Cohen R. M., Specker B., Williams T. C., Lubahn D. B., Korach K. S. Estrogen resistance caused by a mutation in the estrogen-receptor gene in a man. N Engl J Med. 1994 Oct 20;331(16):1056–1061. doi: 10.1056/NEJM199410203311604. [DOI] [PubMed] [Google Scholar]
  42. Spelsberg T. C., Graham M. L., 2nd, Berg N. J., Umehara T., Riehl E., Coulam C. B., Ingle J. N. A nuclear binding assay to assess the biological activity of steroid receptors in isolated animal and human tissues. Endocrinology. 1987 Aug;121(2):631–644. doi: 10.1210/endo-121-2-631. [DOI] [PubMed] [Google Scholar]
  43. Thies R. S., Bauduy M., Ashton B. A., Kurtzberg L., Wozney J. M., Rosen V. Recombinant human bone morphogenetic protein-2 induces osteoblastic differentiation in W-20-17 stromal cells. Endocrinology. 1992 Mar;130(3):1318–1324. doi: 10.1210/endo.130.3.1311236. [DOI] [PubMed] [Google Scholar]
  44. Turner R. T., Evans G. L., Wakley G. K. Mechanism of action of estrogen on cancellous bone balance in tibiae of ovariectomized growing rats: inhibition of indices of formation and resorption. J Bone Miner Res. 1993 Mar;8(3):359–366. doi: 10.1002/jbmr.5650080313. [DOI] [PubMed] [Google Scholar]
  45. Vukicevic S., Latin V., Chen P., Batorsky R., Reddi A. H., Sampath T. K. Localization of osteogenic protein-1 (bone morphogenetic protein-7) during human embryonic development: high affinity binding to basement membranes. Biochem Biophys Res Commun. 1994 Jan 28;198(2):693–700. doi: 10.1006/bbrc.1994.1100. [DOI] [PubMed] [Google Scholar]
  46. 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]
  47. 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]
  48. Wang E. A., Israel D. I., Kelly S., Luxenberg D. P. Bone morphogenetic protein-2 causes commitment and differentiation in C3H10T1/2 and 3T3 cells. Growth Factors. 1993;9(1):57–71. doi: 10.3109/08977199308991582. [DOI] [PubMed] [Google Scholar]
  49. 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. Recombinant human bone morphogenetic protein induces bone formation. Proc Natl Acad Sci U S A. 1990 Mar;87(6):2220–2224. doi: 10.1073/pnas.87.6.2220. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Weisz A., Bresciani F. Estrogen induces expression of c-fos and c-myc protooncogenes in rat uterus. Mol Endocrinol. 1988 Sep;2(9):816–824. doi: 10.1210/mend-2-9-816. [DOI] [PubMed] [Google Scholar]
  51. 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]

Articles from Journal of Clinical Investigation are provided here courtesy of American Society for Clinical Investigation

RESOURCES