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. 1993 Mar 15;90(6):2300–2304. doi: 10.1073/pnas.90.6.2300

Constitutive transcription of the osteocalcin gene in osteosarcoma cells is reflected by altered protein-DNA interactions at promoter regulatory elements.

R Bortell 1, T A Owen 1, V Shalhoub 1, A Heinrichs 1, M A Aronow 1, C Rochette-Egly 1, Y Lutz 1, J L Stein 1, J B Lian 1, G S Stein 1
PMCID: PMC46074  PMID: 8460137

Abstract

The bone-specific osteocalcin (OC) gene is transcribed only after completion of proliferation in normal diploid calvarial-derived osteoblasts during extracellular matrix mineralization. In contrast, the OC gene is expressed constitutively in both proliferating and nonproliferating ROS 17/2.8 osteosarcoma cells. To address molecular mechanisms associated with these tumor-related modifications in transcriptional control, we examined sequence-specific interactions of transactivation factors at key basal and hormone-responsive elements in the OC gene promoter. In ROS 17/2.8 cells compared to normal diploid osteoblasts, the absence of a stringent requirement for cessation of proliferation to support both induction of OC transcription and steroid hormone-mediated transcriptional modulation is reflected by modifications in transcription factor binding at (i) the two primary basal regulatory elements, the OC box (which contains a CCAAT motif as a central core) and the TATA/glucocorticoid-responsive element domain, and (ii) the vitamin D-responsive element. Particularly striking are two forms of the vitamin D receptor complex that are present in proliferating osteoblasts and osteosarcoma cells. Both forms of the complex are sensitive to vitamin D receptor antibody and retinoic X receptor antibody. After the down-regulation of proliferation, only the lower molecular weight complex is found in normal diploid osteoblasts. Both forms of the complex are present in nonproliferating ROS 17/2.8 cells with increased representation of the complex exhibiting reduced electrophoretic mobility that is phosphorylation-dependent.

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Selected References

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

  1. Aronow M. A., Gerstenfeld L. C., Owen T. A., Tassinari M. S., Stein G. S., Lian J. B. Factors that promote progressive development of the osteoblast phenotype in cultured fetal rat calvaria cells. J Cell Physiol. 1990 May;143(2):213–221. doi: 10.1002/jcp.1041430203. [DOI] [PubMed] [Google Scholar]
  2. Baumbach L. L., Marashi F., Plumb M., Stein G., Stein J. Inhibition of DNA replication coordinately reduces cellular levels of core and H1 histone mRNAs: requirement for protein synthesis. Biochemistry. 1984 Apr 10;23(8):1618–1625. doi: 10.1021/bi00303a006. [DOI] [PubMed] [Google Scholar]
  3. Bellows C. G., Aubin J. E., Heersche J. N., Antosz M. E. Mineralized bone nodules formed in vitro from enzymatically released rat calvaria cell populations. Calcif Tissue Int. 1986 Mar;38(3):143–154. doi: 10.1007/BF02556874. [DOI] [PubMed] [Google Scholar]
  4. Bhargava U., Bar-Lev M., Bellows C. G., Aubin J. E. Ultrastructural analysis of bone nodules formed in vitro by isolated fetal rat calvaria cells. Bone. 1988;9(3):155–163. doi: 10.1016/8756-3282(88)90005-1. [DOI] [PubMed] [Google Scholar]
  5. Bortell R., Owen T. A., Bidwell J. P., Gavazzo P., Breen E., van Wijnen A. J., DeLuca H. F., Stein J. L., Lian J. B., Stein G. S. Vitamin D-responsive protein-DNA interactions at multiple promoter regulatory elements that contribute to the level of rat osteocalcin gene expression. Proc Natl Acad Sci U S A. 1992 Jul 1;89(13):6119–6123. doi: 10.1073/pnas.89.13.6119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bortell R., van Wijnen A. J., Ramsey-Ewing A. L., Stein G. S., Stein J. L. Differential regulation of H4 histone gene expression in 3T3-L1 pre-adipocytes during arrest of proliferation following contact inhibition or differentiation and its modulation by TGF beta 1. J Cell Biochem. 1992 Sep;50(1):62–72. doi: 10.1002/jcb.240500111. [DOI] [PubMed] [Google Scholar]
  7. Brown T. A., DeLuca H. F. Phosphorylation of the 1,25-dihydroxyvitamin D3 receptor. A primary event in 1,25-dihydroxyvitamin D3 action. J Biol Chem. 1990 Jun 15;265(17):10025–10029. [PubMed] [Google Scholar]
  8. Chen T. L., Hauschka P. V., Cabrales S., Feldman D. The effects of 1,25-dihydroxyvitamin D3 and dexamethasone on rat osteoblast-like primary cell cultures: receptor occupancy and functional expression patterns for three different bioresponses. Endocrinology. 1986 Jan;118(1):250–259. doi: 10.1210/endo-118-1-250. [DOI] [PubMed] [Google Scholar]
  9. Dame M. C., Pierce E. A., Prahl J. M., Hayes C. E., DeLuca H. F. Monoclonal antibodies to the porcine intestinal receptor for 1,25-dihydroxyvitamin D3: interaction with distinct receptor domains. Biochemistry. 1986 Aug 12;25(16):4523–4534. doi: 10.1021/bi00364a011. [DOI] [PubMed] [Google Scholar]
  10. Demay M. B., Gerardi J. M., DeLuca H. F., Kronenberg H. M. DNA sequences in the rat osteocalcin gene that bind the 1,25-dihydroxyvitamin D3 receptor and confer responsiveness to 1,25-dihydroxyvitamin D3. Proc Natl Acad Sci U S A. 1990 Jan;87(1):369–373. doi: 10.1073/pnas.87.1.369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dignam J. D., Lebovitz R. M., Roeder R. G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 1983 Mar 11;11(5):1475–1489. doi: 10.1093/nar/11.5.1475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Ecarot-Charrier B., Glorieux F. H., van der Rest M., Pereira G. Osteoblasts isolated from mouse calvaria initiate matrix mineralization in culture. J Cell Biol. 1983 Mar;96(3):639–643. doi: 10.1083/jcb.96.3.639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Ecarot-Charrier B., Shepard N., Charette G., Grynpas M., Glorieux F. H. Mineralization in osteoblast cultures: a light and electron microscopic study. Bone. 1988;9(3):147–154. doi: 10.1016/8756-3282(88)90004-x. [DOI] [PubMed] [Google Scholar]
  14. Eilam Y., Silbermann M., Szydel N. Growth inhibition of clonal osteosarcoma cell-lines by low concentrations of glucocorticoid hormones. Biochem Biophys Res Commun. 1980 Sep 16;96(1):299–305. doi: 10.1016/0006-291x(80)91214-0. [DOI] [PubMed] [Google Scholar]
  15. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  16. Grimes S., Weisz-Carrington P., Daum H., 3rd, Smith J., Green L., Wright K., Stein G., Stein J. A rat histone H4 gene closely associated with the testis-specific H1t gene. Exp Cell Res. 1987 Dec;173(2):534–545. doi: 10.1016/0014-4827(87)90293-x. [DOI] [PubMed] [Google Scholar]
  17. Gundberg C. M., Hauschka P. V., Lian J. B., Gallop P. M. Osteocalcin: isolation, characterization, and detection. Methods Enzymol. 1984;107:516–544. doi: 10.1016/0076-6879(84)07036-1. [DOI] [PubMed] [Google Scholar]
  18. Holthuis J., Owen T. A., van Wijnen A. J., Wright K. L., Ramsey-Ewing A., Kennedy M. B., Carter R., Cosenza S. C., Soprano K. J., Lian J. B. Tumor cells exhibit deregulation of the cell cycle histone gene promoter factor HiNF-D. Science. 1990 Mar 23;247(4949 Pt 1):1454–1457. doi: 10.1126/science.247.4949.1454. [DOI] [PubMed] [Google Scholar]
  19. Hsieh J. C., Jurutka P. W., Galligan M. A., Terpening C. M., Haussler C. A., Samuels D. S., Shimizu Y., Shimizu N., Haussler M. R. Human vitamin D receptor is selectively phosphorylated by protein kinase C on serine 51, a residue crucial to its trans-activation function. Proc Natl Acad Sci U S A. 1991 Oct 15;88(20):9315–9319. doi: 10.1073/pnas.88.20.9315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kerner S. A., Scott R. A., Pike J. W. Sequence elements in the human osteocalcin gene confer basal activation and inducible response to hormonal vitamin D3. Proc Natl Acad Sci U S A. 1989 Jun;86(12):4455–4459. doi: 10.1073/pnas.86.12.4455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Leid M., Kastner P., Lyons R., Nakshatri H., Saunders M., Zacharewski T., Chen J. Y., Staub A., Garnier J. M., Mader S. Purification, cloning, and RXR identity of the HeLa cell factor with which RAR or TR heterodimerizes to bind target sequences efficiently. Cell. 1992 Jan 24;68(2):377–395. doi: 10.1016/0092-8674(92)90478-u. [DOI] [PubMed] [Google Scholar]
  22. Lian J., Stewart C., Puchacz E., Mackowiak S., Shalhoub V., Collart D., Zambetti G., Stein G. Structure of the rat osteocalcin gene and regulation of vitamin D-dependent expression. Proc Natl Acad Sci U S A. 1989 Feb;86(4):1143–1147. doi: 10.1073/pnas.86.4.1143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Liao J., Ozono K., Sone T., McDonnell D. P., Pike J. W. Vitamin D receptor interaction with specific DNA requires a nuclear protein and 1,25-dihydroxyvitamin D3. Proc Natl Acad Sci U S A. 1990 Dec;87(24):9751–9755. doi: 10.1073/pnas.87.24.9751. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. MacDonald P. N., Haussler C. A., Terpening C. M., Galligan M. A., Reeder M. C., Whitfield G. K., Haussler M. R. Baculovirus-mediated expression of the human vitamin D receptor. Functional characterization, vitamin D response element interactions, and evidence for a receptor auxiliary factor. J Biol Chem. 1991 Oct 5;266(28):18808–18813. [PubMed] [Google Scholar]
  25. Majeska R. J., Rodan S. B., Rodan G. A. Parathyroid hormone-responsive clonal cell lines from rat osteosarcoma. Endocrinology. 1980 Nov;107(5):1494–1503. doi: 10.1210/endo-107-5-1494. [DOI] [PubMed] [Google Scholar]
  26. Markose E. R., Stein J. L., Stein G. S., Lian J. B. Vitamin D-mediated modifications in protein-DNA interactions at two promoter elements of the osteocalcin gene. Proc Natl Acad Sci U S A. 1990 Mar;87(5):1701–1705. doi: 10.1073/pnas.87.5.1701. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Morrison N. A., Shine J., Fragonas J. C., Verkest V., McMenemy M. L., Eisman J. A. 1,25-dihydroxyvitamin D-responsive element and glucocorticoid repression in the osteocalcin gene. Science. 1989 Dec 1;246(4934):1158–1161. doi: 10.1126/science.2588000. [DOI] [PubMed] [Google Scholar]
  28. Owen T. A., Aronow M. S., Barone L. M., Bettencourt B., Stein G. S., Lian J. B. Pleiotropic effects of vitamin D on osteoblast gene expression are related to the proliferative and differentiated state of the bone cell phenotype: dependency upon basal levels of gene expression, duration of exposure, and bone matrix competency in normal rat osteoblast cultures. Endocrinology. 1991 Mar;128(3):1496–1504. doi: 10.1210/endo-128-3-1496. [DOI] [PubMed] [Google Scholar]
  29. Owen T. A., Aronow M., Shalhoub V., Barone L. M., Wilming L., Tassinari M. S., Kennedy M. B., Pockwinse S., Lian J. B., Stein G. S. Progressive development of the rat osteoblast phenotype in vitro: reciprocal relationships in expression of genes associated with osteoblast proliferation and differentiation during formation of the bone extracellular matrix. J Cell Physiol. 1990 Jun;143(3):420–430. doi: 10.1002/jcp.1041430304. [DOI] [PubMed] [Google Scholar]
  30. Owen T. A., Bortell R., Yocum S. A., Smock S. L., Zhang M., Abate C., Shalhoub V., Aronin N., Wright K. L., van Wijnen A. J. Coordinate occupancy of AP-1 sites in the vitamin D-responsive and CCAAT box elements by Fos-Jun in the osteocalcin gene: model for phenotype suppression of transcription. Proc Natl Acad Sci U S A. 1990 Dec;87(24):9990–9994. doi: 10.1073/pnas.87.24.9990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Owen T. A., Holthuis J., Markose E., van Wijnen A. J., Wolfe S. A., Grimes S. R., Lian J. B., Stein G. S. Modifications of protein-DNA interactions in the proximal promoter of a cell-growth-regulated histone gene during onset and progression of osteoblast differentiation. Proc Natl Acad Sci U S A. 1990 Jul;87(13):5129–5133. doi: 10.1073/pnas.87.13.5129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Ozono K., Liao J., Kerner S. A., Scott R. A., Pike J. W. The vitamin D-responsive element in the human osteocalcin gene. Association with a nuclear proto-oncogene enhancer. J Biol Chem. 1990 Dec 15;265(35):21881–21888. [PubMed] [Google Scholar]
  33. Rodan G. A., Noda M. Gene expression in osteoblastic cells. Crit Rev Eukaryot Gene Expr. 1991;1(2):85–98. [PubMed] [Google Scholar]
  34. Schepmoes G., Breen E., Owen T. A., Aronow M. A., Stein G. S., Lian J. B. Influence of dexamethasone on the vitamin D-mediated regulation of osteocalcin gene expression. J Cell Biochem. 1991 Oct;47(2):184–196. doi: 10.1002/jcb.240470212. [DOI] [PubMed] [Google Scholar]
  35. Sone T., Ozono K., Pike J. W. A 55-kilodalton accessory factor facilitates vitamin D receptor DNA binding. Mol Endocrinol. 1991 Nov;5(11):1578–1586. doi: 10.1210/mend-5-11-1578. [DOI] [PubMed] [Google Scholar]
  36. Stein G. S., Lian J. B., Owen T. A. Bone cell differentiation: a functionally coupled relationship between expression of cell-growth- and tissue-specific genes. Curr Opin Cell Biol. 1990 Dec;2(6):1018–1027. doi: 10.1016/0955-0674(90)90151-4. [DOI] [PubMed] [Google Scholar]
  37. Stein G., Lian J., Stein J., Briggs R., Shalhoub V., Wright K., Pauli U., van Wijnen A. Altered binding of human histone gene transcription factors during the shutdown of proliferation and onset of differentiation in HL-60 cells. Proc Natl Acad Sci U S A. 1989 Mar;86(6):1865–1869. doi: 10.1073/pnas.86.6.1865. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Strömstedt P. E., Poellinger L., Gustafsson J. A., Carlstedt-Duke J. The glucocorticoid receptor binds to a sequence overlapping the TATA box of the human osteocalcin promoter: a potential mechanism for negative regulation. Mol Cell Biol. 1991 Jun;11(6):3379–3383. doi: 10.1128/mcb.11.6.3379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Terpening C. M., Haussler C. A., Jurutka P. W., Galligan M. A., Komm B. S., Haussler M. R. The vitamin D-responsive element in the rat bone Gla protein gene is an imperfect direct repeat that cooperates with other cis-elements in 1,25-dihydroxyvitamin D3- mediated transcriptional activation. Mol Endocrinol. 1991 Mar;5(3):373–385. doi: 10.1210/mend-5-3-373. [DOI] [PubMed] [Google Scholar]
  40. Wong M. M., Rao L. G., Ly H., Hamilton L., Tong J., Sturtridge W., McBroom R., Aubin J. E., Murray T. M. Long-term effects of physiologic concentrations of dexamethasone on human bone-derived cells. J Bone Miner Res. 1990 Aug;5(8):803–813. doi: 10.1002/jbmr.5650050803. [DOI] [PubMed] [Google Scholar]
  41. Yu V. C., Delsert C., Andersen B., Holloway J. M., Devary O. V., När A. M., Kim S. Y., Boutin J. M., Glass C. K., Rosenfeld M. G. RXR beta: a coregulator that enhances binding of retinoic acid, thyroid hormone, and vitamin D receptors to their cognate response elements. Cell. 1991 Dec 20;67(6):1251–1266. doi: 10.1016/0092-8674(91)90301-e. [DOI] [PubMed] [Google Scholar]
  42. van Wijnen A. J., Ramsey-Ewing A. L., Bortell R., Owen T. A., Lian J. B., Stein J. L., Stein G. S. Transcriptional element H4-site II of cell cycle regulated human H4 histone genes is a multipartite protein/DNA interaction site for factors HiNF-D, HiNF-M, and HiNF-P: involvement of phosphorylation. J Cell Biochem. 1991 Jun;46(2):174–189. doi: 10.1002/jcb.240460211. [DOI] [PubMed] [Google Scholar]
  43. van Wijnen A. J., Wright K. L., Lian J. B., Stein J. L., Stein G. S. Human H4 histone gene transcription requires the proliferation-specific nuclear factor HiNF-D. Auxiliary roles for HiNF-C (Sp1-like) and HiNF-A (high mobility group-like). J Biol Chem. 1989 Sep 5;264(25):15034–15042. [PubMed] [Google Scholar]
  44. van der Houven van Oordt C. W., van Wijnen A. J., Carter R., Soprano K., Lian J. B., Stein G. S., Stein J. L. Protein-DNA interactions at the H4-site III upstream transcriptional element of a cell cycle regulated histone H4 gene: differences in normal versus tumor cells. J Cell Biochem. 1992 May;49(1):93–110. doi: 10.1002/jcb.240490115. [DOI] [PubMed] [Google Scholar]

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