Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1997 May;17(5):2448–2457. doi: 10.1128/mcb.17.5.2448

A-myb is expressed in bovine vascular smooth muscle cells during the late G1-to-S phase transition and cooperates with c-myc to mediate progression to S phase.

D J Marhamati 1, R E Bellas 1, M Arsura 1, K E Kypreos 1, G E Sonenshein 1
PMCID: PMC232093  PMID: 9111313

Abstract

The Myb family of transcription factors is defined by homology within the DNA binding domain and includes c-Myb, A-Myb, and B-Myb. The protein products of the myb genes all bind the Myb-binding site (MBS) [YG(A/G)C(A/C/G)GTT(G/A)]. A-myb has been found to display a limited pattern of expression. Here we report that bovine aortic smooth muscle cells (SMCs) express A-myb. Sequence analysis of isolated bovine A-myb cDNA clones spanning the entire coding region indicated extensive homology with the human gene, including the putative transactivation domain. Expression of A-myb was cell cycle dependent; levels of A-myb RNA increased in the late G1-to-S phase transition following serum stimulation of serum-deprived quiescent SMC cultures and peaked in S phase. Nuclear run-on analysis revealed that an increased rate of transcription can account for most of the increase in A-myb RNA levels. Treatment of SMC cultures with 5,6-dichlorobenzimidazole riboside, a selective inhibitor of RNA polymerase II, indicated an approximate 4-h half-life for A-myb mRNA during the S phase of the cell cycle. Expression of A-myb by SMCs was stimulated by basic fibroblast growth factor, in a cell density-dependent fashion. Cotransfection of a human A-myb expression vector activated a multimerized MBS element-driven reporter construct approximately 30-fold in SMCs. The activity of c-myb and c-myc promoters, which both contain multiple MBS elements, were similarly transactivated, approximately 30- and 50-fold, respectively, upon cotransfection with human A-myb. Lastly, A-myb RNA levels could be increased by a combination of phorbol ester plus insulin-like growth factor 1. To test the role of myb family members in progression through the cell cycle, we comicroinjected c-myc and myb expression vectors into serum-deprived quiescent SMCs. The combination of c-myc and either A-myb or c-myb but not B-myb synergistically led to entry into S phase, whereas microinjection of any vector alone had little effect on S phase entry. Thus, these results suggest that A-myb is a potent transactivator in bovine SMCs and that its expression induces progression into S phase of the cell cycle.

Full Text

The Full Text of this article is available as a PDF (2.0 MB).

Selected References

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

  1. Alitalo K., Winqvist R., Lin C. C., de la Chapelle A., Schwab M., Bishop J. M. Aberrant expression of an amplified c-myb oncogene in two cell lines from a colon carcinoma. Proc Natl Acad Sci U S A. 1984 Jul;81(14):4534–4538. doi: 10.1073/pnas.81.14.4534. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Alterman R. B., Ganguly S., Schulze D. H., Marzluff W. F., Schildkraut C. L., Skoultchi A. I. Cell cycle regulation of mouse H3 histone mRNA metabolism. Mol Cell Biol. 1984 Jan;4(1):123–132. doi: 10.1128/mcb.4.1.123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Armelin H. A., Armelin M. C., Kelly K., Stewart T., Leder P., Cochran B. H., Stiles C. D. Functional role for c-myc in mitogenic response to platelet-derived growth factor. Nature. 1984 Aug 23;310(5979):655–660. doi: 10.1038/310655a0. [DOI] [PubMed] [Google Scholar]
  4. Badiani P., Corbella P., Kioussis D., Marvel J., Weston K. Dominant interfering alleles define a role for c-Myb in T-cell development. Genes Dev. 1994 Apr 1;8(7):770–782. doi: 10.1101/gad.8.7.770. [DOI] [PubMed] [Google Scholar]
  5. Beldekas J. C., Gerstenfeld L., Sonenshein G. E., Franzblau C. Cell density and estradiol modulation of procollagen type III in cultured calf smooth muscle cells. J Biol Chem. 1982 Oct 25;257(20):12252–12256. [PubMed] [Google Scholar]
  6. Biedenkapp H., Borgmeyer U., Sippel A. E., Klempnauer K. H. Viral myb oncogene encodes a sequence-specific DNA-binding activity. Nature. 1988 Oct 27;335(6193):835–837. doi: 10.1038/335835a0. [DOI] [PubMed] [Google Scholar]
  7. Brown K. E., Kindy M. S., Sonenshein G. E. Expression of the c-myb proto-oncogene in bovine vascular smooth muscle cells. J Biol Chem. 1992 Mar 5;267(7):4625–4630. [PubMed] [Google Scholar]
  8. Brown K. E., Lawrence R., Sonenshein G. E. Concerted modulation of alpha 1(XI) and alpha 2(V) collagen mRNAs in bovine vascular smooth muscle cells. J Biol Chem. 1991 Dec 5;266(34):23268–23273. [PubMed] [Google Scholar]
  9. Burk O., Mink S., Ringwald M., Klempnauer K. H. Synergistic activation of the chicken mim-1 gene by v-myb and C/EBP transcription factors. EMBO J. 1993 May;12(5):2027–2038. doi: 10.1002/j.1460-2075.1993.tb05852.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chamley-Campbell J., Campbell G. R., Ross R. The smooth muscle cell in culture. Physiol Rev. 1979 Jan;59(1):1–61. doi: 10.1152/physrev.1979.59.1.1. [DOI] [PubMed] [Google Scholar]
  11. Chen C., Okayama H. High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol. 1987 Aug;7(8):2745–2752. doi: 10.1128/mcb.7.8.2745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  13. Clarke M. F., Kukowska-Latallo J. F., Westin E., Smith M., Prochownik E. V. Constitutive expression of a c-myb cDNA blocks Friend murine erythroleukemia cell differentiation. Mol Cell Biol. 1988 Feb;8(2):884–892. doi: 10.1128/mcb.8.2.884. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Cogswell J. P., Cogswell P. C., Kuehl W. M., Cuddihy A. M., Bender T. M., Engelke U., Marcu K. B., Ting J. P. Mechanism of c-myc regulation by c-Myb in different cell lineages. Mol Cell Biol. 1993 May;13(5):2858–2869. doi: 10.1128/mcb.13.5.2858. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Dudek H., Tantravahi R. V., Rao V. N., Reddy E. S., Reddy E. P. Myb and Ets proteins cooperate in transcriptional activation of the mim-1 promoter. Proc Natl Acad Sci U S A. 1992 Feb 15;89(4):1291–1295. doi: 10.1073/pnas.89.4.1291. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Duyao M. P., Kessler D. J., Spicer D. B., Bartholomew C., Cleveland J. L., Siekevitz M., Sonenshein G. E. Transactivation of the c-myc promoter by human T cell leukemia virus type 1 tax is mediated by NF kappa B. J Biol Chem. 1992 Aug 15;267(23):16288–16291. [PubMed] [Google Scholar]
  17. Dyson P. J., Poirier F., Watson R. J. Expression of c-myb in embryonal carcinoma cells and embryonal stem cells. Differentiation. 1989 Oct;42(1):24–27. doi: 10.1111/j.1432-0436.1989.tb00603.x. [DOI] [PubMed] [Google Scholar]
  18. Evans J. L., Moore T. L., Kuehl W. M., Bender T., Ting J. P. Functional analysis of c-Myb protein in T-lymphocytic cell lines shows that it trans-activates the c-myc promoter. Mol Cell Biol. 1990 Nov;10(11):5747–5752. doi: 10.1128/mcb.10.11.5747. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Foos G., Grimm S., Klempnauer K. H. Functional antagonism between members of the myb family: B-myb inhibits v-myb-induced gene activation. EMBO J. 1992 Dec;11(12):4619–4629. doi: 10.1002/j.1460-2075.1992.tb05564.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Foos G., Grimm S., Klempnauer K. H. The chicken A-myb protein is a transcriptional activator. Oncogene. 1994 Sep;9(9):2481–2488. [PubMed] [Google Scholar]
  22. Foos G., Natour S., Klempnauer K. H. TATA-box dependent trans-activation of the human HSP70 promoter by Myb proteins. Oncogene. 1993 Jul;8(7):1775–1782. [PubMed] [Google Scholar]
  23. Gewirtz A. M., Anfossi G., Venturelli D., Valpreda S., Sims R., Calabretta B. G1/S transition in normal human T-lymphocytes requires the nuclear protein encoded by c-myb. Science. 1989 Jul 14;245(4914):180–183. doi: 10.1126/science.2665077. [DOI] [PubMed] [Google Scholar]
  24. Gewirtz A. M., Calabretta B. A c-myb antisense oligodeoxynucleotide inhibits normal human hematopoiesis in vitro. Science. 1988 Dec 2;242(4883):1303–1306. doi: 10.1126/science.2461588. [DOI] [PubMed] [Google Scholar]
  25. Golay J., Capucci A., Arsura M., Castellano M., Rizzo V., Introna M. Expression of c-myb and B-myb, but not A-myb, correlates with proliferation in human hematopoietic cells. Blood. 1991 Jan 1;77(1):149–158. [PubMed] [Google Scholar]
  26. Golay J., Erba E., Bernasconi S., Peri G., Introna M. The A-myb gene is preferentially expressed in tonsillar CD38+, CD39-, and sIgM- B lymphocytes and in Burkitt's lymphoma cell lines. J Immunol. 1994 Jul 15;153(2):543–553. [PubMed] [Google Scholar]
  27. Golay J., Loffarelli L., Luppi M., Castellano M., Introna M. The human A-myb protein is a strong activator of transcription. Oncogene. 1994 Sep;9(9):2469–2479. [PubMed] [Google Scholar]
  28. Gonda T. J., Metcalf D. Expression of myb, myc and fos proto-oncogenes during the differentiation of a murine myeloid leukaemia. Nature. 1984 Jul 19;310(5974):249–251. doi: 10.1038/310249a0. [DOI] [PubMed] [Google Scholar]
  29. Greenberg M. E., Ziff E. B. Stimulation of 3T3 cells induces transcription of the c-fos proto-oncogene. Nature. 1984 Oct 4;311(5985):433–438. doi: 10.1038/311433a0. [DOI] [PubMed] [Google Scholar]
  30. Howe K. M., Watson R. J. Nucleotide preferences in sequence-specific recognition of DNA by c-myb protein. Nucleic Acids Res. 1991 Jul 25;19(14):3913–3919. doi: 10.1093/nar/19.14.3913. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Ibanez C. E., Lipsick J. S. trans activation of gene expression by v-myb. Mol Cell Biol. 1990 May;10(5):2285–2293. doi: 10.1128/mcb.10.5.2285. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Introna M., Golay J., Frampton J., Nakano T., Ness S. A., Graf T. Mutations in v-myb alter the differentiation of myelomonocytic cells transformed by the oncogene. Cell. 1990 Dec 21;63(6):1289–1297. doi: 10.1016/0092-8674(90)90424-d. [DOI] [PubMed] [Google Scholar]
  33. Kaczmarek L., Hyland J. K., Watt R., Rosenberg M., Baserga R. Microinjected c-myc as a competence factor. Science. 1985 Jun 14;228(4705):1313–1315. doi: 10.1126/science.4001943. [DOI] [PubMed] [Google Scholar]
  34. Kelly K., Cochran B. H., Stiles C. D., Leder P. Cell-specific regulation of the c-myc gene by lymphocyte mitogens and platelet-derived growth factor. Cell. 1983 Dec;35(3 Pt 2):603–610. doi: 10.1016/0092-8674(83)90092-2. [DOI] [PubMed] [Google Scholar]
  35. Kenney S. C., Holley-Guthrie E., Quinlivan E. B., Gutsch D., Zhang Q., Bender T., Giot J. F., Sergeant A. The cellular oncogene c-myb can interact synergistically with the Epstein-Barr virus BZLF1 transactivator in lymphoid cells. Mol Cell Biol. 1992 Jan;12(1):136–146. doi: 10.1128/mcb.12.1.136. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Kindy M. S., Sonenshein G. E. Regulation of oncogene expression in cultured aortic smooth muscle cells. Post-transcriptional control of c-myc mRNA. J Biol Chem. 1986 Sep 25;261(27):12865–12868. [PubMed] [Google Scholar]
  37. Klagsbrun M., Edelman E. R. Biological and biochemical properties of fibroblast growth factors. Implications for the pathogenesis of atherosclerosis. Arteriosclerosis. 1989 May-Jun;9(3):269–278. doi: 10.1161/01.atv.9.3.269. [DOI] [PubMed] [Google Scholar]
  38. Ku D. H., Wen S. C., Engelhard A., Nicolaides N. C., Lipson K. E., Marino T. A., Calabretta B. c-myb transactivates cdc2 expression via Myb binding sites in the 5'-flanking region of the human cdc2 gene. J Biol Chem. 1993 Jan 25;268(3):2255–2259. [PubMed] [Google Scholar]
  39. Lam E. W., Watson R. J. An E2F-binding site mediates cell-cycle regulated repression of mouse B-myb transcription. EMBO J. 1993 Jul;12(7):2705–2713. doi: 10.1002/j.1460-2075.1993.tb05932.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Lawrence R., Chang L. J., Siebenlist U., Bressler P., Sonenshein G. E. Vascular smooth muscle cells express a constitutive NF-kappa B-like activity. J Biol Chem. 1994 Nov 18;269(46):28913–28918. [PubMed] [Google Scholar]
  41. Lindner V., Reidy M. A. Proliferation of smooth muscle cells after vascular injury is inhibited by an antibody against basic fibroblast growth factor. Proc Natl Acad Sci U S A. 1991 May 1;88(9):3739–3743. doi: 10.1073/pnas.88.9.3739. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Lipsick J. S., Boyle W. J. c-myb protein expression is a late event during T-lymphocyte activation. Mol Cell Biol. 1987 Sep;7(9):3358–3360. doi: 10.1128/mcb.7.9.3358. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Marhamati D. J., Sonenshein G. E. B-Myb expression in vascular smooth muscle cells occurs in a cell cycle-dependent fashion and down-regulates promoter activity of type I collagen genes. J Biol Chem. 1996 Feb 16;271(7):3359–3365. doi: 10.1074/jbc.271.7.3359. [DOI] [PubMed] [Google Scholar]
  44. McClinton D., Stafford J., Brents L., Bender T. P., Kuehl W. M. Differentiation of mouse erythroleukemia cells is blocked by late up-regulation of a c-myb transgene. Mol Cell Biol. 1990 Feb;10(2):705–710. doi: 10.1128/mcb.10.2.705. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. McConlogue L., Gupta M., Wu L., Coffino P. Molecular cloning and expression of the mouse ornithine decarboxylase gene. Proc Natl Acad Sci U S A. 1984 Jan;81(2):540–544. doi: 10.1073/pnas.81.2.540. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Mettus R. V., Litvin J., Wali A., Toscani A., Latham K., Hatton K., Reddy E. P. Murine A-myb: evidence for differential splicing and tissue-specific expression. Oncogene. 1994 Oct;9(10):3077–3086. [PubMed] [Google Scholar]
  47. Moscovici C. Leukemic transformation with avian myeloblastosis virus: present status. Curr Top Microbiol Immunol. 1975;71:79–101. doi: 10.1007/978-3-642-66193-8_2. [DOI] [PubMed] [Google Scholar]
  48. Mucenski M. L., McLain K., Kier A. B., Swerdlow S. H., Schreiner C. M., Miller T. A., Pietryga D. W., Scott W. J., Jr, Potter S. S. A functional c-myb gene is required for normal murine fetal hepatic hematopoiesis. Cell. 1991 May 17;65(4):677–689. doi: 10.1016/0092-8674(91)90099-k. [DOI] [PubMed] [Google Scholar]
  49. Nakagoshi H., Kanei-Ishii C., Sawazaki T., Mizuguchi G., Ishii S. Transcriptional activation of the c-myc gene by the c-myb and B-myb gene products. Oncogene. 1992 Jun;7(6):1233–1240. [PubMed] [Google Scholar]
  50. Ness S. A., Marknell A., Graf T. The v-myb oncogene product binds to and activates the promyelocyte-specific mim-1 gene. Cell. 1989 Dec 22;59(6):1115–1125. doi: 10.1016/0092-8674(89)90767-8. [DOI] [PubMed] [Google Scholar]
  51. Newby A. C., George S. J. Proposed roles for growth factors in mediating smooth muscle proliferation in vascular pathologies. Cardiovasc Res. 1993 Jul;27(7):1173–1183. doi: 10.1093/cvr/27.7.1173. [DOI] [PubMed] [Google Scholar]
  52. Nicolaides N. C., Gualdi R., Casadevall C., Manzella L., Calabretta B. Positive autoregulation of c-myb expression via Myb binding sites in the 5' flanking region of the human c-myb gene. Mol Cell Biol. 1991 Dec;11(12):6166–6176. doi: 10.1128/mcb.11.12.6166. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Nomura N., Takahashi M., Matsui M., Ishii S., Date T., Sasamoto S., Ishizaki R. Isolation of human cDNA clones of myb-related genes, A-myb and B-myb. Nucleic Acids Res. 1988 Dec 9;16(23):11075–11089. doi: 10.1093/nar/16.23.11075. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Pardee A. B. G1 events and regulation of cell proliferation. Science. 1989 Nov 3;246(4930):603–608. doi: 10.1126/science.2683075. [DOI] [PubMed] [Google Scholar]
  55. Poole J. C., Cromwell S. B., Benditt E. P. Behavior of smooth muscle cells and formation of extracellular structures in the reaction of arterial walls to injury. Am J Pathol. 1971 Mar;62(3):391–414. [PMC free article] [PubMed] [Google Scholar]
  56. Reilly C. F., Kindy M. S., Brown K. E., Rosenberg R. D., Sonenshein G. E. Heparin prevents vascular smooth muscle cell progression through the G1 phase of the cell cycle. J Biol Chem. 1989 Apr 25;264(12):6990–6995. [PubMed] [Google Scholar]
  57. Reiss K., Ferber A., Travali S., Porcu P., Phillips P. D., Baserga R. The protooncogene c-myb increases the expression of insulin-like growth factor 1 and insulin-like growth factor 1 receptor messenger RNAs by a transcriptional mechanism. Cancer Res. 1991 Nov 1;51(21):5997–6000. [PubMed] [Google Scholar]
  58. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature. 1993 Apr 29;362(6423):801–809. doi: 10.1038/362801a0. [DOI] [PubMed] [Google Scholar]
  59. Sakura H., Kanei-Ishii C., Nagase T., Nakagoshi H., Gonda T. J., Ishii S. Delineation of three functional domains of the transcriptional activator encoded by the c-myb protooncogene. Proc Natl Acad Sci U S A. 1989 Aug;86(15):5758–5762. doi: 10.1073/pnas.86.15.5758. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Schwartz S. M., Reidy M. R., Clowes A. Kinetics of atherosclerosis: a stem cell model. Ann N Y Acad Sci. 1985;454:292–304. doi: 10.1111/j.1749-6632.1985.tb11869.x. [DOI] [PubMed] [Google Scholar]
  61. Simons M., Rosenberg R. D. Antisense nonmuscle myosin heavy chain and c-myb oligonucleotides suppress smooth muscle cell proliferation in vitro. Circ Res. 1992 Apr;70(4):835–843. doi: 10.1161/01.res.70.4.835. [DOI] [PubMed] [Google Scholar]
  62. Sleeman J. P. Xenopus A-myb is expressed during early spermatogenesis. Oncogene. 1993 Jul;8(7):1931–1941. [PubMed] [Google Scholar]
  63. Stern J. B., Smith K. A. Interleukin-2 induction of T-cell G1 progression and c-myb expression. Science. 1986 Jul 11;233(4760):203–206. doi: 10.1126/science.3523754. [DOI] [PubMed] [Google Scholar]
  64. Strauss B. H., Chisholm R. J., Keeley F. W., Gotlieb A. I., Logan R. A., Armstrong P. W. Extracellular matrix remodeling after balloon angioplasty injury in a rabbit model of restenosis. Circ Res. 1994 Oct;75(4):650–658. doi: 10.1161/01.res.75.4.650. [DOI] [PubMed] [Google Scholar]
  65. Takahashi T., Nakagoshi H., Sarai A., Nomura N., Yamamoto T., Ishii S. Human A-myb gene encodes a transcriptional activator containing the negative regulatory domains. FEBS Lett. 1995 Jan 16;358(1):89–96. doi: 10.1016/0014-5793(94)01402-m. [DOI] [PubMed] [Google Scholar]
  66. Thiele C. J., Cohen P. S., Israel M. A. Regulation of c-myb expression in human neuroblastoma cells during retinoic acid-induced differentiation. Mol Cell Biol. 1988 Apr;8(4):1677–1683. doi: 10.1128/mcb.8.4.1677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. Trauth K., Mutschler B., Jenkins N. A., Gilbert D. J., Copeland N. G., Klempnauer K. H. Mouse A-myb encodes a trans-activator and is expressed in mitotically active cells of the developing central nervous system, adult testis and B lymphocytes. EMBO J. 1994 Dec 15;13(24):5994–6005. doi: 10.1002/j.1460-2075.1994.tb06945.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. Watson R. J., Robinson C., Lam E. W. Transcription regulation by murine B-myb is distinct from that by c-myb. Nucleic Acids Res. 1993 Jan 25;21(2):267–272. doi: 10.1093/nar/21.2.267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  69. Weston K., Bishop J. M. Transcriptional activation by the v-myb oncogene and its cellular progenitor, c-myb. Cell. 1989 Jul 14;58(1):85–93. doi: 10.1016/0092-8674(89)90405-4. [DOI] [PubMed] [Google Scholar]
  70. Zobel A., Kalkbrenner F., Guehmann S., Nawrath M., Vorbrueggen G., Moelling K. Interaction of the v-and c-Myb proteins with regulatory sequences of the human c-myc gene. Oncogene. 1991 Aug;6(8):1397–1407. [PubMed] [Google Scholar]

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

RESOURCES