Skip to main content
NCBI home page
The EMBO Journal logoLink to The EMBO Journal
. 1998 Aug 3;17(15):4456–4468. doi: 10.1093/emboj/17.15.4456

PU.1 regulates both cytokine-dependent proliferation and differentiation of granulocyte/macrophage progenitors.

R P DeKoter 1, J C Walsh 1, H Singh 1
PMCID: PMC1170777  PMID: 9687512

Abstract

PU.1 is a unique regulatory protein required for the generation of both the innate and the adaptive immune system. It functions exclusively in a cell-intrinsic manner to control the development of granulocytes, macrophages, and B and T lymphocytes. We demonstrate that mutation of the PU.1 gene causes a severe reduction in myeloid (granulocyte/macrophage) progenitors. PU.1 -/- myeloid progenitors can proliferate in vitro in response to the multilineage cytokines interleukin-3 (IL-3), IL-6 and stem cell factor but are unresponsive to the myeloid-specific cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF and M-CSF. The failure of PU.1 -/- progenitors to respond to G-CSF is bypassed by transient signaling with IL-3. In the presence of IL-3 and G-CSF, PU.1 -/- progenitors can differentiate into granulocytic precursors containing myeloperoxidase-positive granules. Thus PU.1 is not essential for specification of granulocytic precursors, but is required for their further differentiation. The failure of PU.1 -/- progenitors to respond to M-CSF is due to lack of c-fms gene transcription. Transduction of c-fms into PU.1 -/- myeloid progenitors bypasses the block to M-CSF-dependent proliferation but does not induce detectable macrophage differentiation. Therefore, PU. 1 appears to be essential for specification of monocytic precursors. Importantly, retroviral transduction of PU.1 into mutant progenitors restores responsiveness to myeloid-specific cytokines and development of mature granulocytes and macrophages. Thus PU.1 controls myelopoiesis by regulating both proliferation and differentiation pathways.

Full Text

The Full Text of this article is available as a PDF (687.5 KB).

Selected References

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

  1. Beck-Engeser G., Stocking C., Just U., Albritton L., Dexter M., Spooncer E., Ostertag W. Retroviral vectors related to the myeloproliferative sarcoma virus allow efficient expression in hematopoietic stem and precursor cell lines, but retroviral infection is reduced in more primitive cells. Hum Gene Ther. 1991 Spring;2(1):61–70. doi: 10.1089/hum.1991.2.1-61. [DOI] [PubMed] [Google Scholar]
  2. Bodine D. M., Orlic D., Birkett N. C., Seidel N. E., Zsebo K. M. Stem cell factor increases colony-forming unit-spleen number in vitro in synergy with interleukin-6, and in vivo in Sl/Sld mice as a single factor. Blood. 1992 Feb 15;79(4):913–919. [PubMed] [Google Scholar]
  3. Bourette R. P., Myles G. M., Choi J. L., Rohrschneider L. R. Sequential activation of phoshatidylinositol 3-kinase and phospholipase C-gamma2 by the M-CSF receptor is necessary for differentiation signaling. EMBO J. 1997 Oct 1;16(19):5880–5893. doi: 10.1093/emboj/16.19.5880. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brass A. L., Kehrli E., Eisenbeis C. F., Storb U., Singh H. Pip, a lymphoid-restricted IRF, contains a regulatory domain that is important for autoinhibition and ternary complex formation with the Ets factor PU.1. Genes Dev. 1996 Sep 15;10(18):2335–2347. doi: 10.1101/gad.10.18.2335. [DOI] [PubMed] [Google Scholar]
  5. Cormack B. P., Valdivia R. H., Falkow S. FACS-optimized mutants of the green fluorescent protein (GFP). Gene. 1996;173(1 Spec No):33–38. doi: 10.1016/0378-1119(95)00685-0. [DOI] [PubMed] [Google Scholar]
  6. Dranoff G., Crawford A. D., Sadelain M., Ream B., Rashid A., Bronson R. T., Dickersin G. R., Bachurski C. J., Mark E. L., Whitsett J. A. Involvement of granulocyte-macrophage colony-stimulating factor in pulmonary homeostasis. Science. 1994 Apr 29;264(5159):713–716. doi: 10.1126/science.8171324. [DOI] [PubMed] [Google Scholar]
  7. Eichbaum Q. G., Iyer R., Raveh D. P., Mathieu C., Ezekowitz R. A. Restriction of interferon gamma responsiveness and basal expression of the myeloid human Fc gamma R1b gene is mediated by a functional PU.1 site and a transcription initiator consensus. J Exp Med. 1994 Jun 1;179(6):1985–1996. doi: 10.1084/jem.179.6.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Feinman R., Qiu W. Q., Pearse R. N., Nikolajczyk B. S., Sen R., Sheffery M., Ravetch J. V. PU.1 and an HLH family member contribute to the myeloid-specific transcription of the Fc gamma RIIIA promoter. EMBO J. 1994 Aug 15;13(16):3852–3860. doi: 10.1002/j.1460-2075.1994.tb06696.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fukunaga R., Ishizaka-Ikeda E., Seto Y., Nagata S. Expression cloning of a receptor for murine granulocyte colony-stimulating factor. Cell. 1990 Apr 20;61(2):341–350. doi: 10.1016/0092-8674(90)90814-u. [DOI] [PubMed] [Google Scholar]
  10. Gorman D. M., Itoh N., Kitamura T., Schreurs J., Yonehara S., Yahara I., Arai K., Miyajima A. Cloning and expression of a gene encoding an interleukin 3 receptor-like protein: identification of another member of the cytokine receptor gene family. Proc Natl Acad Sci U S A. 1990 Jul;87(14):5459–5463. doi: 10.1073/pnas.87.14.5459. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hawley R. G., Lieu F. H., Fong A. Z., Hawley T. S. Versatile retroviral vectors for potential use in gene therapy. Gene Ther. 1994 Mar;1(2):136–138. [PubMed] [Google Scholar]
  12. Hestdal K., Ruscetti F. W., Ihle J. N., Jacobsen S. E., Dubois C. M., Kopp W. C., Longo D. L., Keller J. R. Characterization and regulation of RB6-8C5 antigen expression on murine bone marrow cells. J Immunol. 1991 Jul 1;147(1):22–28. [PubMed] [Google Scholar]
  13. Hogarth P. M., Hibbs M. L., Bonadonna L., Scott B. M., Witort E., Pietersz G. A., McKenzie I. F. The mouse Fc receptor for IgG (Ly-17): molecular cloning and specificity. Immunogenetics. 1987;26(3):161–168. doi: 10.1007/BF00365906. [DOI] [PubMed] [Google Scholar]
  14. Hohaus S., Petrovick M. S., Voso M. T., Sun Z., Zhang D. E., Tenen D. G. PU.1 (Spi-1) and C/EBP alpha regulate expression of the granulocyte-macrophage colony-stimulating factor receptor alpha gene. Mol Cell Biol. 1995 Oct;15(10):5830–5845. doi: 10.1128/mcb.15.10.5830. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hromas R., Orazi A., Neiman R. S., Maki R., Van Beveran C., Moore J., Klemsz M. Hematopoietic lineage- and stage-restricted expression of the ETS oncogene family member PU.1. Blood. 1993 Nov 15;82(10):2998–3004. [PubMed] [Google Scholar]
  16. Kaufmann Y., Tseng E., Springer T. A. Cloning of the murine lymphocyte function-associated molecule-1 alpha-subunit and its expression in COS cells. J Immunol. 1991 Jul 1;147(1):369–374. [PubMed] [Google Scholar]
  17. Kinsella T. M., Nolan G. P. Episomal vectors rapidly and stably produce high-titer recombinant retrovirus. Hum Gene Ther. 1996 Aug 1;7(12):1405–1413. doi: 10.1089/hum.1996.7.12-1405. [DOI] [PubMed] [Google Scholar]
  18. Kishimoto T. The biology of interleukin-6. Blood. 1989 Jul;74(1):1–10. [PubMed] [Google Scholar]
  19. 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]
  20. Lantz C. S., Boesiger J., Song C. H., Mach N., Kobayashi T., Mulligan R. C., Nawa Y., Dranoff G., Galli S. J. Role for interleukin-3 in mast-cell and basophil development and in immunity to parasites. Nature. 1998 Mar 5;392(6671):90–93. doi: 10.1038/32190. [DOI] [PubMed] [Google Scholar]
  21. Lieschke G. J., Grail D., Hodgson G., Metcalf D., Stanley E., Cheers C., Fowler K. J., Basu S., Zhan Y. F., Dunn A. R. Mice lacking granulocyte colony-stimulating factor have chronic neutropenia, granulocyte and macrophage progenitor cell deficiency, and impaired neutrophil mobilization. Blood. 1994 Sep 15;84(6):1737–1746. [PubMed] [Google Scholar]
  22. Liu F., Poursine-Laurent J., Wu H. Y., Link D. C. Interleukin-6 and the granulocyte colony-stimulating factor receptor are major independent regulators of granulopoiesis in vivo but are not required for lineage commitment or terminal differentiation. Blood. 1997 Oct 1;90(7):2583–2590. [PubMed] [Google Scholar]
  23. Liu F., Wu H. Y., Wesselschmidt R., Kornaga T., Link D. C. Impaired production and increased apoptosis of neutrophils in granulocyte colony-stimulating factor receptor-deficient mice. Immunity. 1996 Nov;5(5):491–501. doi: 10.1016/s1074-7613(00)80504-x. [DOI] [PubMed] [Google Scholar]
  24. Luskey B. D., Rosenblatt M., Zsebo K., Williams D. A. Stem cell factor, interleukin-3, and interleukin-6 promote retroviral-mediated gene transfer into murine hematopoietic stem cells. Blood. 1992 Jul 15;80(2):396–402. [PubMed] [Google Scholar]
  25. Lyman S. D., Jacobsen S. E. c-kit ligand and Flt3 ligand: stem/progenitor cell factors with overlapping yet distinct activities. Blood. 1998 Feb 15;91(4):1101–1134. [PubMed] [Google Scholar]
  26. Lübbert M., Herrmann F., Koeffler H. P. Expression and regulation of myeloid-specific genes in normal and leukemic myeloid cells. Blood. 1991 Mar 1;77(5):909–924. [PubMed] [Google Scholar]
  27. McKercher S. R., Torbett B. E., Anderson K. L., Henkel G. W., Vestal D. J., Baribault H., Klemsz M., Feeney A. J., Wu G. E., Paige C. J. Targeted disruption of the PU.1 gene results in multiple hematopoietic abnormalities. EMBO J. 1996 Oct 15;15(20):5647–5658. [PMC free article] [PubMed] [Google Scholar]
  28. Metcalf D., Burgess A. W., Johnson G. R., Nicola N. A., Nice E. C., DeLamarter J., Thatcher D. R., Mermod J. J. In vitro actions on hemopoietic cells of recombinant murine GM-CSF purified after production in Escherichia coli: comparison with purified native GM-CSF. J Cell Physiol. 1986 Sep;128(3):421–431. doi: 10.1002/jcp.1041280311. [DOI] [PubMed] [Google Scholar]
  29. Metcalf D. The granulocyte-macrophage regulators: reappraisal by gene inactivation. Exp Hematol. 1995 Jul;23(7):569–572. [PubMed] [Google Scholar]
  30. Miura N., Okada S., Zsebo K. M., Miura Y., Suda T. Rat stem cell factor and IL-6 preferentially support the proliferation of c-kit-positive murine hemopoietic cells rather than their differentiation. Exp Hematol. 1993 Jan;21(1):143–149. [PubMed] [Google Scholar]
  31. Moreau-Gachelin F. Spi-1/PU.1: an oncogene of the Ets family. Biochim Biophys Acta. 1994 Dec 30;1198(2-3):149–163. doi: 10.1016/0304-419x(94)90011-6. [DOI] [PubMed] [Google Scholar]
  32. Moreau-Gachelin F., Tavitian A., Tambourin P. Spi-1 is a putative oncogene in virally induced murine erythroleukaemias. Nature. 1988 Jan 21;331(6153):277–280. doi: 10.1038/331277a0. [DOI] [PubMed] [Google Scholar]
  33. Moulton K. S., Semple K., Wu H., Glass C. K. Cell-specific expression of the macrophage scavenger receptor gene is dependent on PU.1 and a composite AP-1/ets motif. Mol Cell Biol. 1994 Jul;14(7):4408–4418. doi: 10.1128/mcb.14.7.4408-4418.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Nagata S., Fukunaga R. Granulocyte colony-stimulating factor receptor and its related receptors. Growth Factors. 1993;8(2):99–107. doi: 10.3109/08977199309046930. [DOI] [PubMed] [Google Scholar]
  35. Nishinakamura R., Miyajima A., Mee P. J., Tybulewicz V. L., Murray R. Hematopoiesis in mice lacking the entire granulocyte-macrophage colony-stimulating factor/interleukin-3/interleukin-5 functions. Blood. 1996 Oct 1;88(7):2458–2464. [PubMed] [Google Scholar]
  36. Nishinakamura R., Nakayama N., Hirabayashi Y., Inoue T., Aud D., McNeil T., Azuma S., Yoshida S., Toyoda Y., Arai K. Mice deficient for the IL-3/GM-CSF/IL-5 beta c receptor exhibit lung pathology and impaired immune response, while beta IL3 receptor-deficient mice are normal. Immunity. 1995 Mar;2(3):211–222. doi: 10.1016/1074-7613(95)90046-2. [DOI] [PubMed] [Google Scholar]
  37. Nuchprayoon I., Meyers S., Scott L. M., Suzow J., Hiebert S., Friedman A. D. PEBP2/CBF, the murine homolog of the human myeloid AML1 and PEBP2 beta/CBF beta proto-oncoproteins, regulates the murine myeloperoxidase and neutrophil elastase genes in immature myeloid cells. Mol Cell Biol. 1994 Aug;14(8):5558–5568. doi: 10.1128/mcb.14.8.5558-5568.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Olson M. C., Scott E. W., Hack A. A., Su G. H., Tenen D. G., Singh H., Simon M. C. PU. 1 is not essential for early myeloid gene expression but is required for terminal myeloid differentiation. Immunity. 1995 Dec;3(6):703–714. doi: 10.1016/1074-7613(95)90060-8. [DOI] [PubMed] [Google Scholar]
  39. Pahl H. L., Scheibe R. J., Zhang D. E., Chen H. M., Galson D. L., Maki R. A., Tenen D. G. The proto-oncogene PU.1 regulates expression of the myeloid-specific CD11b promoter. J Biol Chem. 1993 Mar 5;268(7):5014–5020. [PubMed] [Google Scholar]
  40. Park L. S., Martin U., Sorensen R., Luhr S., Morrissey P. J., Cosman D., Larsen A. Cloning of the low-affinity murine granulocyte-macrophage colony-stimulating factor receptor and reconstitution of a high-affinity receptor complex. Proc Natl Acad Sci U S A. 1992 May 15;89(10):4295–4299. doi: 10.1073/pnas.89.10.4295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Pear W. S., Nolan G. P., Scott M. L., Baltimore D. Production of high-titer helper-free retroviruses by transient transfection. Proc Natl Acad Sci U S A. 1993 Sep 15;90(18):8392–8396. doi: 10.1073/pnas.90.18.8392. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Perez C., Coeffier E., Moreau-Gachelin F., Wietzerbin J., Benech P. D. Involvement of the transcription factor PU.1/Spi-1 in myeloid cell-restricted expression of an interferon-inducible gene encoding the human high-affinity Fc gamma receptor. Mol Cell Biol. 1994 Aug;14(8):5023–5031. doi: 10.1128/mcb.14.8.5023-5031.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Pytela R. Amino acid sequence of the murine Mac-1 alpha chain reveals homology with the integrin family and an additional domain related to von Willebrand factor. EMBO J. 1988 May;7(5):1371–1378. doi: 10.1002/j.1460-2075.1988.tb02953.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Ravetch J. V., Luster A. D., Weinshank R., Kochan J., Pavlovec A., Portnoy D. A., Hulmes J., Pan Y. C., Unkeless J. C. Structural heterogeneity and functional domains of murine immunoglobulin G Fc receptors. Science. 1986 Nov 7;234(4777):718–725. doi: 10.1126/science.2946078. [DOI] [PubMed] [Google Scholar]
  45. Reddy M. A., Yang B. S., Yue X., Barnett C. J., Ross I. L., Sweet M. J., Hume D. A., Ostrowski M. C. Opposing actions of c-ets/PU.1 and c-myb protooncogene products in regulating the macrophage-specific promoters of the human and mouse colony-stimulating factor-1 receptor (c-fms) genes. J Exp Med. 1994 Dec 1;180(6):2309–2319. doi: 10.1084/jem.180.6.2309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Rohrschneider L. R., Rothwell V. M., Nicola N. A. Transformation of murine fibroblasts by a retrovirus encoding the murine c-fms proto-oncogene. Oncogene. 1989 Aug;4(8):1015–1022. [PubMed] [Google Scholar]
  47. Scott E. W., Fisher R. C., Olson M. C., Kehrli E. W., Simon M. C., Singh H. PU.1 functions in a cell-autonomous manner to control the differentiation of multipotential lymphoid-myeloid progenitors. Immunity. 1997 Apr;6(4):437–447. doi: 10.1016/s1074-7613(00)80287-3. [DOI] [PubMed] [Google Scholar]
  48. Scott E. W., Simon M. C., Anastasi J., Singh H. Requirement of transcription factor PU.1 in the development of multiple hematopoietic lineages. Science. 1994 Sep 9;265(5178):1573–1577. doi: 10.1126/science.8079170. [DOI] [PubMed] [Google Scholar]
  49. Sears D. W., Osman N., Tate B., McKenzie I. F., Hogarth P. M. Molecular cloning and expression of the mouse high affinity Fc receptor for IgG. J Immunol. 1990 Jan 1;144(1):371–378. [PubMed] [Google Scholar]
  50. Sherr C. J. Colony-stimulating factor-1 receptor. Blood. 1990 Jan 1;75(1):1–12. [PubMed] [Google Scholar]
  51. Sherr C. J., Rettenmier C. W., Sacca R., Roussel M. F., Look A. T., Stanley E. R. The c-fms proto-oncogene product is related to the receptor for the mononuclear phagocyte growth factor, CSF-1. Cell. 1985 Jul;41(3):665–676. doi: 10.1016/s0092-8674(85)80047-7. [DOI] [PubMed] [Google Scholar]
  52. Shivdasani R. A., Orkin S. H. The transcriptional control of hematopoiesis. Blood. 1996 May 15;87(10):4025–4039. [PubMed] [Google Scholar]
  53. Singh H. Gene targeting reveals a hierarchy of transcription factors regulating specification of lymphoid cell fates. Curr Opin Immunol. 1996 Apr;8(2):160–165. doi: 10.1016/s0952-7915(96)80053-7. [DOI] [PubMed] [Google Scholar]
  54. Smith L. T., Hohaus S., Gonzalez D. A., Dziennis S. E., Tenen D. G. PU.1 (Spi-1) and C/EBP alpha regulate the granulocyte colony-stimulating factor receptor promoter in myeloid cells. Blood. 1996 Aug 15;88(4):1234–1247. [PubMed] [Google Scholar]
  55. Stanley E. R. The macrophage colony-stimulating factor, CSF-1. Methods Enzymol. 1985;116:564–587. doi: 10.1016/s0076-6879(85)16044-1. [DOI] [PubMed] [Google Scholar]
  56. Stanley E., Lieschke G. J., Grail D., Metcalf D., Hodgson G., Gall J. A., Maher D. W., Cebon J., Sinickas V., Dunn A. R. Granulocyte/macrophage colony-stimulating factor-deficient mice show no major perturbation of hematopoiesis but develop a characteristic pulmonary pathology. Proc Natl Acad Sci U S A. 1994 Jun 7;91(12):5592–5596. doi: 10.1073/pnas.91.12.5592. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Suda T., Suda J., Kajigaya S., Nagata S., Asano S., Saito M., Miura Y. Effects of recombinant murine granulocyte colony-stimulating factor on granulocyte-macrophage and blast colony formation. Exp Hematol. 1987 Oct;15(9):958–965. [PubMed] [Google Scholar]
  58. Suzuki H., Kurihara Y., Takeya M., Kamada N., Kataoka M., Jishage K., Ueda O., Sakaguchi H., Higashi T., Suzuki T. A role for macrophage scavenger receptors in atherosclerosis and susceptibility to infection. Nature. 1997 Mar 20;386(6622):292–296. doi: 10.1038/386292a0. [DOI] [PubMed] [Google Scholar]
  59. Taga T., Hibi M., Hirata Y., Yamasaki K., Yasukawa K., Matsuda T., Hirano T., Kishimoto T. Interleukin-6 triggers the association of its receptor with a possible signal transducer, gp130. Cell. 1989 Aug 11;58(3):573–581. doi: 10.1016/0092-8674(89)90438-8. [DOI] [PubMed] [Google Scholar]
  60. Tenen D. G., Hromas R., Licht J. D., Zhang D. E. Transcription factors, normal myeloid development, and leukemia. Blood. 1997 Jul 15;90(2):489–519. [PubMed] [Google Scholar]
  61. Watowich S. S., Wu H., Socolovsky M., Klingmuller U., Constantinescu S. N., Lodish H. F. Cytokine receptor signal transduction and the control of hematopoietic cell development. Annu Rev Cell Dev Biol. 1996;12:91–128. doi: 10.1146/annurev.cellbio.12.1.91. [DOI] [PubMed] [Google Scholar]
  62. Williams D. E., Eisenman J., Baird A., Rauch C., Van Ness K., March C. J., Park L. S., Martin U., Mochizuki D. Y., Boswell H. S. Identification of a ligand for the c-kit proto-oncogene. Cell. 1990 Oct 5;63(1):167–174. doi: 10.1016/0092-8674(90)90297-r. [DOI] [PubMed] [Google Scholar]
  63. Witte O. N. Steel locus defines new multipotent growth factor. Cell. 1990 Oct 5;63(1):5–6. doi: 10.1016/0092-8674(90)90280-r. [DOI] [PubMed] [Google Scholar]
  64. Yam L. T., Li C. Y., Crosby W. H. Cytochemical identification of monocytes and granulocytes. Am J Clin Pathol. 1971 Mar;55(3):283–290. doi: 10.1093/ajcp/55.3.283. [DOI] [PubMed] [Google Scholar]
  65. Yoshida H., Hayashi S., Kunisada T., Ogawa M., Nishikawa S., Okamura H., Sudo T., Shultz L. D., Nishikawa S. The murine mutation osteopetrosis is in the coding region of the macrophage colony stimulating factor gene. Nature. 1990 May 31;345(6274):442–444. doi: 10.1038/345442a0. [DOI] [PubMed] [Google Scholar]
  66. Zhang D. E., Hetherington C. J., Chen H. M., Tenen D. G. The macrophage transcription factor PU.1 directs tissue-specific expression of the macrophage colony-stimulating factor receptor. Mol Cell Biol. 1994 Jan;14(1):373–381. doi: 10.1128/mcb.14.1.373-381.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES