Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1995 Jan;15(1):476–487. doi: 10.1128/mcb.15.1.476

Identification of a ras-activated enhancer in the mouse osteopontin promoter and its interaction with a putative ETS-related transcription factor whose activity correlates with the metastatic potential of the cell.

X Guo 1, Y P Zhang 1, D A Mitchell 1, D T Denhardt 1, A F Chambers 1
PMCID: PMC231995  PMID: 7799957

Abstract

The role of RAS in transducing signals from an activated receptor into altered gene expression is becoming clear, though some links in the chain are still missing. Cells possessing activated RAS express higher levels of osteopontin (OPN), an alpha v beta 3 integrin-binding secreted phosphoprotein implicated in a number of developmental, physiological, and pathological processes. We report that in T24 H-ras-transformed NIH 3T3 cells enhanced transcription contributes to the increased expression of OPN. Transient transfection studies, DNA-protein binding assays, and methylation protection experiments have identified a novel ras-activated enhancer, distinct from known ras response elements, that appears responsible for part of the increase in OPN transcription in cells with an activated RAS. In electrophoretic mobility shift assays, the protein-binding motif GGAGGCAGG was found to be essential for the formation of several complexes, one of which (complex A) was generated at elevated levels by cell lines that are metastatic. Southwestern blotting and UV light cross-linking studies indicated the presence of several proteins able to interact with this sequence. The proteins that form these complexes have molecular masses estimated at approximately 16, 28, 32, 45, 80, and 100 kDa. Because the approximately 16-kDa protein was responsible for complex A formation, we have designated it MATF for metastasis-associated transcription factor. The GGANNNAGG motif is also found in some other promoters, suggesting that they may be similarly controlled by MATF.

Full Text

The Full Text of this article is available as a PDF (794.7 KB).

Selected References

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

  1. Augereau P., Chambon P. The mouse immunoglobulin heavy-chain enhancer: effect on transcription in vitro and binding of proteins present in HeLa and lymphoid B cell extracts. EMBO J. 1986 Aug;5(8):1791–1797. doi: 10.1002/j.1460-2075.1986.tb04428.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Beato M., Arnemann J., Chalepakis G., Slater E., Willmann T. Gene regulation by steroid hormones. J Steroid Biochem. 1987;27(1-3):9–14. doi: 10.1016/0022-4731(87)90288-3. [DOI] [PubMed] [Google Scholar]
  3. Behrend E. I., Chambers A. F., Wilson S. M., Denhardt D. T. Comparative analysis of two alternative first exons reported for the mouse osteopontin gene. J Biol Chem. 1993 May 25;268(15):11172–11175. [PubMed] [Google Scholar]
  4. Blenis J. Signal transduction via the MAP kinases: proceed at your own RSK. Proc Natl Acad Sci U S A. 1993 Jul 1;90(13):5889–5892. doi: 10.1073/pnas.90.13.5889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bokoch G. M., Der C. J. Emerging concepts in the Ras superfamily of GTP-binding proteins. FASEB J. 1993 Jun;7(9):750–759. doi: 10.1096/fasebj.7.9.8330683. [DOI] [PubMed] [Google Scholar]
  6. Bollag G., McCormick F. Regulators and effectors of ras proteins. Annu Rev Cell Biol. 1991;7:601–632. doi: 10.1146/annurev.cb.07.110191.003125. [DOI] [PubMed] [Google Scholar]
  7. Bortner D. M., Langer S. J., Ostrowski M. C. Non-nuclear oncogenes and the regulation of gene expression in transformed cells. Crit Rev Oncog. 1993;4(2):137–160. [PubMed] [Google Scholar]
  8. Bruder J. T., Heidecker G., Rapp U. R. Serum-, TPA-, and Ras-induced expression from Ap-1/Ets-driven promoters requires Raf-1 kinase. Genes Dev. 1992 Apr;6(4):545–556. doi: 10.1101/gad.6.4.545. [DOI] [PubMed] [Google Scholar]
  9. Cantley L. C., Auger K. R., Carpenter C., Duckworth B., Graziani A., Kapeller R., Soltoff S. Oncogenes and signal transduction. Cell. 1991 Jan 25;64(2):281–302. doi: 10.1016/0092-8674(91)90639-g. [DOI] [PubMed] [Google Scholar]
  10. Chambers A. F., Behrend E. I., Wilson S. M., Denhardt D. T. Induction of expression of osteopontin (OPN; secreted phosphoprotein) in metastatic, ras-transformed NIH 3T3 cells. Anticancer Res. 1992 Jan-Feb;12(1):43–47. [PubMed] [Google Scholar]
  11. Chambers A. F., Denhardt G. H., Wilson S. M. ras-transformed NIH 3T3 cell lines, selected for metastatic ability in chick embryos, have increased proportions of p21-expressing cells and are metastatic in nude mice. Invasion Metastasis. 1990;10(4):225–240. [PubMed] [Google Scholar]
  12. Chambers A. F., Tuck A. B. Ras-responsive genes and tumor metastasis. Crit Rev Oncog. 1993;4(2):95–114. [PubMed] [Google Scholar]
  13. Coffer P., de Jonge M., Mettouchi A., Binetruy B., Ghysdael J., Kruijer W. junB promoter regulation: Ras mediated transactivation by c-Ets-1 and c-Ets-2. Oncogene. 1994 Mar;9(3):911–921. [PubMed] [Google Scholar]
  14. Craig A. M., Bowden G. T., Chambers A. F., Spearman M. A., Greenberg A. H., Wright J. A., McLeod M., Denhardt D. T. Secreted phosphoprotein mRNA is induced during multi-stage carcinogenesis in mouse skin and correlates with the metastatic potential of murine fibroblasts. Int J Cancer. 1990 Jul 15;46(1):133–137. doi: 10.1002/ijc.2910460124. [DOI] [PubMed] [Google Scholar]
  15. Craig A. M., Denhardt D. T. The murine gene encoding secreted phosphoprotein 1 (osteopontin): promoter structure, activity, and induction in vivo by estrogen and progesterone. Gene. 1991 Apr;100:163–171. doi: 10.1016/0378-1119(91)90362-f. [DOI] [PubMed] [Google Scholar]
  16. Craig A. M., Smith J. H., Denhardt D. T. Osteopontin, a transformation-associated cell adhesion phosphoprotein, is induced by 12-O-tetradecanoylphorbol 13-acetate in mouse epidermis. J Biol Chem. 1989 Jun 5;264(16):9682–9689. [PubMed] [Google Scholar]
  17. Crews C. M., Erikson R. L. Extracellular signals and reversible protein phosphorylation: what to Mek of it all. Cell. 1993 Jul 30;74(2):215–217. doi: 10.1016/0092-8674(93)90411-i. [DOI] [PubMed] [Google Scholar]
  18. DeLucia A. L., Lewton B. A., Tjian R., Tegtmeyer P. Topography of simian virus 40 A protein-DNA complexes: arrangement of pentanucleotide interaction sites at the origin of replication. J Virol. 1983 Apr;46(1):143–150. doi: 10.1128/jvi.46.1.143-150.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Denhardt D. T., Guo X. Osteopontin: a protein with diverse functions. FASEB J. 1993 Dec;7(15):1475–1482. [PubMed] [Google Scholar]
  20. Fisher R. J., Koizumi S., Kondoh A., Mariano J. M., Mavrothalassitis G., Bhat N. K., Papas T. S. Human ETS1 oncoprotein. Purification, isoforms, -SH modification, and DNA sequence-specific binding. J Biol Chem. 1992 Sep 5;267(25):17957–17965. [PubMed] [Google Scholar]
  21. Foa C., Aubert C. Ultrastructural comparison between cultured and tumor cells of human malignant melanoma. Cancer Res. 1977 Nov;37(11):3957–3963. [PubMed] [Google Scholar]
  22. Hall A. A biochemical function for ras--at last. Science. 1994 Jun 3;264(5164):1413–1414. doi: 10.1126/science.8197454. [DOI] [PubMed] [Google Scholar]
  23. Hijiya N., Setoguchi M., Matsuura K., Higuchi Y., Akizuki S., Yamamoto S. Cloning and characterization of the human osteopontin gene and its promoter. Biochem J. 1994 Oct 1;303(Pt 1):255–262. doi: 10.1042/bj3030255. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hill S. A., Wilson S., Chambers A. F. Clonal heterogeneity, experimental metastatic ability, and p21 expression in H-ras-transformed NIH 3T3 cells. J Natl Cancer Inst. 1988 Jun 1;80(7):484–490. doi: 10.1093/jnci/80.7.484. [DOI] [PubMed] [Google Scholar]
  25. Imler J. L., Schatz C., Wasylyk C., Chatton B., Wasylyk B. A Harvey-ras responsive transcription element is also responsive to a tumour-promoter and to serum. Nature. 1988 Mar 17;332(6161):275–278. doi: 10.1038/332275a0. [DOI] [PubMed] [Google Scholar]
  26. Kamachi Y., Ogawa E., Asano M., Ishida S., Murakami Y., Satake M., Ito Y., Shigesada K. Purification of a mouse nuclear factor that binds to both the A and B cores of the polyomavirus enhancer. J Virol. 1990 Oct;64(10):4808–4819. doi: 10.1128/jvi.64.10.4808-4819.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kedar P. S., Lowy D. R., Widen S. G., Wilson S. H. Transfected human beta-polymerase promoter contains a ras-responsive element. Mol Cell Biol. 1990 Jul;10(7):3852–3856. doi: 10.1128/mcb.10.7.3852. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Klemsz M. J., McKercher S. R., Celada A., Van Beveren C., Maki R. A. The macrophage and B cell-specific transcription factor PU.1 is related to the ets oncogene. Cell. 1990 Apr 6;61(1):113–124. doi: 10.1016/0092-8674(90)90219-5. [DOI] [PubMed] [Google Scholar]
  29. Kothary R., Clapoff S., Darling S., Perry M. D., Moran L. A., Rossant J. Inducible expression of an hsp68-lacZ hybrid gene in transgenic mice. Development. 1989 Apr;105(4):707–714. doi: 10.1242/dev.105.4.707. [DOI] [PubMed] [Google Scholar]
  30. Krook A., Rapoport M. J., Anderson S., Pross H., Zhou Y. C., Denhardt D. T., Delovitch T. L., Haliotis T. p21ras and protein kinase C function in distinct and interdependent signaling pathways in C3H 10T1/2 fibroblasts. Mol Cell Biol. 1993 Mar;13(3):1471–1479. doi: 10.1128/mcb.13.3.1471. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lee K. A., Bindereif A., Green M. R. A small-scale procedure for preparation of nuclear extracts that support efficient transcription and pre-mRNA splicing. Gene Anal Tech. 1988 Mar-Apr;5(2):22–31. doi: 10.1016/0735-0651(88)90023-4. [DOI] [PubMed] [Google Scholar]
  32. Maroteaux L., Heilig R., Dupret D., Mandel J. L. Repetitive satellite-like sequences are present within or upstream from 3 avian protein-coding genes. Nucleic Acids Res. 1983 Mar 11;11(5):1227–1243. doi: 10.1093/nar/11.5.1227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. McCormick F. Signal transduction. How receptors turn Ras on. Nature. 1993 May 6;363(6424):15–16. doi: 10.1038/363015a0. [DOI] [PubMed] [Google Scholar]
  34. Miyazaki Y., Setoguchi M., Yoshida S., Higuchi Y., Akizuki S., Yamamoto S. The mouse osteopontin gene. Expression in monocytic lineages and complete nucleotide sequence. J Biol Chem. 1990 Aug 25;265(24):14432–14438. [PubMed] [Google Scholar]
  35. Morris V. L., Tuck A. B., Wilson S. M., Percy D., Chambers A. F. Tumor progression and metastasis in murine D2 hyperplastic alveolar nodule mammary tumor cell lines. Clin Exp Metastasis. 1993 Jan;11(1):103–112. doi: 10.1007/BF00880071. [DOI] [PubMed] [Google Scholar]
  36. Nielsen D. A., Chou J., MacKrell A. J., Casadaban M. J., Steiner D. F. Expression of a preproinsulin-beta-galactosidase gene fusion in mammalian cells. Proc Natl Acad Sci U S A. 1983 Sep;80(17):5198–5202. doi: 10.1073/pnas.80.17.5198. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Noda M., Vogel R. L., Craig A. M., Prahl J., DeLuca H. F., Denhardt D. T. Identification of a DNA sequence responsible for binding of the 1,25-dihydroxyvitamin D3 receptor and 1,25-dihydroxyvitamin D3 enhancement of mouse secreted phosphoprotein 1 (SPP-1 or osteopontin) gene expression. Proc Natl Acad Sci U S A. 1990 Dec;87(24):9995–9999. doi: 10.1073/pnas.87.24.9995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Nye J. A., Petersen J. M., Gunther C. V., Jonsen M. D., Graves B. J. Interaction of murine ets-1 with GGA-binding sites establishes the ETS domain as a new DNA-binding motif. Genes Dev. 1992 Jun;6(6):975–990. doi: 10.1101/gad.6.6.975. [DOI] [PubMed] [Google Scholar]
  39. Owen R. D., Ostrowski M. C. A nuclear factor that binds to ras-responsive enhancer elements is present in human tumor cells. Cell Growth Differ. 1990 Dec;1(12):601–606. [PubMed] [Google Scholar]
  40. Patarca R., Saavedra R. A., Cantor H. Molecular and cellular basis of genetic resistance to bacterial infection: the role of the early T-lymphocyte activation-1/osteopontin gene. Crit Rev Immunol. 1993;13(3-4):225–246. [PubMed] [Google Scholar]
  41. Redondo J. M., Hata S., Brocklehurst C., Krangel M. S. A T cell-specific transcriptional enhancer within the human T cell receptor delta locus. Science. 1990 Mar 9;247(4947):1225–1229. doi: 10.1126/science.2156339. [DOI] [PubMed] [Google Scholar]
  42. Schlokat U., Bohmann D., Schöler H., Gruss P. Nuclear factors binding specific sequences within the immunoglobulin enhancer interact differentially with other enhancer elements. EMBO J. 1986 Dec 1;5(12):3251–3258. doi: 10.1002/j.1460-2075.1986.tb04636.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Senger D. R., Perruzzi C. A., Papadopoulos A. Elevated expression of secreted phosphoprotein I (osteopontin, 2ar) as a consequence of neoplastic transformation. Anticancer Res. 1989 Sep-Oct;9(5):1291–1299. [PubMed] [Google Scholar]
  44. Seth A., Ascione R., Fisher R. J., Mavrothalassitis G. J., Bhat N. K., Papas T. S. The ets gene family. Cell Growth Differ. 1992 May;3(5):327–334. [PubMed] [Google Scholar]
  45. Thompson C. C., Brown T. A., McKnight S. L. Convergence of Ets- and notch-related structural motifs in a heteromeric DNA binding complex. Science. 1991 Aug 16;253(5021):762–768. doi: 10.1126/science.1876833. [DOI] [PubMed] [Google Scholar]
  46. Timmers H. T., Pronk G. J., Bos J. L., van der Eb A. J. Analysis of the rat JE gene promoter identifies an AP-1 binding site essential for basal expression but not for TPA induction. Nucleic Acids Res. 1990 Jan 11;18(1):23–34. doi: 10.1093/nar/18.1.23. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Trimble W. S., Johnson P. W., Hozumi N., Roder J. C. Inducible cellular transformation by a metallothionein-ras hybrid oncogene leads to natural killer cell susceptibility. Nature. 1986 Jun 19;321(6072):782–784. doi: 10.1038/321782a0. [DOI] [PubMed] [Google Scholar]
  48. Tuck A. B., Wilson S. M., Khokha R., Chambers A. F. Different patterns of gene expression in ras-resistant and ras-sensitive cells. J Natl Cancer Inst. 1991 Apr 3;83(7):485–491. doi: 10.1093/jnci/83.7.485. [DOI] [PubMed] [Google Scholar]
  49. Xin J. H., Cowie A., Lachance P., Hassell J. A. Molecular cloning and characterization of PEA3, a new member of the Ets oncogene family that is differentially expressed in mouse embryonic cells. Genes Dev. 1992 Mar;6(3):481–496. doi: 10.1101/gad.6.3.481. [DOI] [PubMed] [Google Scholar]
  50. Ye Z. S., Samuels H. H. Cell- and sequence-specific binding of nuclear proteins to 5'-flanking DNA of the rat growth hormone gene. J Biol Chem. 1987 May 5;262(13):6313–6317. [PubMed] [Google Scholar]
  51. de Wet J. R., Wood K. V., DeLuca M., Helinski D. R., Subramani S. Firefly luciferase gene: structure and expression in mammalian cells. Mol Cell Biol. 1987 Feb;7(2):725–737. doi: 10.1128/mcb.7.2.725. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES