Expression and Characterization of Purinergic Receptor, $P2Y_{10}$ in Hematopoietic Stem Cells

  • Lee Eun-Jong (Cell and Gene Therapy Research Institue, Graduate School of Life Science and Biotechnology, Pochon CHA University) ;
  • Kim Dong-Ku (Cell and Gene Therapy Research Institue, Graduate School of Life Science and Biotechnology, Pochon CHA University)
  • Published : 2005.06.01

Abstract

Hematopoietic stem cells (HSC) are multipotent cells that reside in the bone marrow and replenish all adult hematopoietic lineages throughoutthe lifetime. In this study, we analyzed the expression of receptors of $P2Y_{10}$, purinergic receptor families in murine hematopoietic stem cells, hematopoietic progenitor cells. In addition, the biological activity of $P2Y_{10}$ was investigated with B lymphocyte cell line, Ba/F3 in effect to cell growth and cell cycle. From the analysis of expression in hematopoieticstem cell. and progenitor with RT-PCR, $P2Y_{10}$ was strongly expressed in murine hematopoieticstem cells (c-kit+ Sca-l+ Lin-) and progenitor cell population, such as c-kit- Sca-l+ Lin-, c-kit+ Sca-l- Lin- and c-kit- Sca-l- Lin-. To investigate the biological effects by $P2Y_{10}$, retroviral vector from subcloned murine $P2Y_{10}$ cDNA was used fur gene introduction into Ba/F3 cells, and stable transfectant cells were obtained by flow cytometry sorting. In cell proliferation assay, the proliferation ability of $P2Y_{10}$ receptor gene­transfected cells was strongly inhibited, and the cell cycle was arrested at G1 phase. These result suggest that the $P2Y_{10}$ may be involved the biological activity in hematopoietic stem cells and immature B lymphocytes.

Keywords

References

  1. Anderson DJ, Gage FH, Weissman JM (2001): Can stem cells cross lineage boundaries? Nat Med 7:393-395 https://doi.org/10.1038/86439
  2. Blau HM, Brazelton TR, Weimann JM (2001): The evolving concept of a stem cell: entity of function? Cell 105:829-841 https://doi.org/10.1016/S0092-8674(01)00409-3
  3. Di Virgilio F, Chiozzi P, Ferrari D, Falzoni S, Sanz JM, Morelli A, Torboli M, Bolognesi G, Baricordi OR (2001): Nucelotide receptors: an emerging family of regulatory molecules in blood cells. Blood 97:587-600 https://doi.org/10.1182/blood.V97.3.587
  4. Ema H, Nakauchi H (2000): Expansion of hematopoietic stem cells in the developing liver of a mouse embryo. Blood 95:2284-2288
  5. Falzoni S, Munerati M, Ferrari D, Spisani S, Moretti S, Di virgilio F (1995): The purinergic P2Z receptor of human macrophage cells: Characterization and possible physiological role. J Clin Invest 95:1207-1216 https://doi.org/10.1172/JCI117770
  6. Dubyak GR, El-Moatassim C (1993): Signal transduction via P2-purinergic receptors for extracellular ATP and other nucleotides. Am J Physiol 265:577-606 https://doi.org/10.1152/ajpcell.1993.265.3.C577
  7. Ferrari D, La Sala A, Chiozzi, Morelli A, Falzoni S, Girolomoni G, Idzko M, Dichmann S, Norgauer J, Di Virgilio F (2001): The P2 purinergic receptors of human dendritic cells: identification and coupling to cytokine release. FASEB J 14:2466-2476 https://doi.org/10.1096/fj.00-0031com
  8. Honda S, Sasaki Y, Ohsawa K, Imai Y, Nakamura Y, Inoue K, Kohsaka S (2001): Extracellular ATP or ADP induce chemotaxis of cultured microglia through G1/0-coupled P2Y receptors. J Neurosc 21:1975-1982
  9. Hogquist KA, Unanue ER, Chapline DD (1991): Release of IL-1 from mononuclear phagocytes. J Immunol 147: 2181-2186
  10. Idzko M, Dichmann S, Ferrari D, Di Virgilio F, la Sala A, Girolomoni G, Panter E, Norgauer J (2003): Neucleotides induce chemotaxis and actin polymerization in immature but not mature human dendritic cells via activation of pertussis toxin-sensitive P2Y receptors. Blood 100:925-932 https://doi.org/10.1182/blood.V100.3.925
  11. Idzko M, Panther E, Bremer He, Schrichter S, Luttmann W, Virchow CJ Jr, Di Virgilio F, Herouy Y, Norgauer J, Ferrari D (2003): Stimulation of P2 purinergic receptors induces the release of eosinophil cationic protein and interleukin-8 from human eosinophils, Br J Pharmacol 138:1244-1250 https://doi.org/10.1038/sj.bjp.0705145
  12. Lagasse E, Shizuru JA, Uchida N, Tsukamoto A, Weissman IL (2001): Toward regenerative medicine. Immunity 14:425-436 https://doi.org/10.1016/S1074-7613(01)00123-6
  13. Lemoli RM, Ferrari D, Fogli M, Rossi L, Pizzirani, Forchap S, Chiozzi P, Vaselli D, Bertolini F, Foutz T, Aluigi M, Baccarani M, Virgilio FD (2004): Extracellular nucleotides are potent stimulators of human hematopoietic stem cells in vitro and in vivo. Blood 15: 1662-1670
  14. McCloskey MA, Fan Y, Luther S (1999): Chemotaxis of rat mast cells toward adenine nucleotides. J Immunol 163:970-977
  15. Morrison SJ, Shah NM, Anderson DJ (1997): Regulatory mechanism in stem cell biology. Cell 88: 287-298 https://doi.org/10.1016/S0092-8674(00)81867-X
  16. Moore KA, Ema H, Lemischka IR (1997): In vitro maintenance of highly purified, transplatable hematopoietic stem cells. Blood 89:4337-4347
  17. North A, Surprenant AM (2000): Pharmacology of cloned P2X receptors. Annu Rev Pharmacol Toxicol 40:563-580 https://doi.org/10.1146/annurev.pharmtox.40.1.563
  18. Ross EA, Anderson N, Micklem HS (1982): Serial depletion and regeneration of the murine hematopoietic system. Implications for hematopoietic organization and the study of cellular aging. J Exp Med 155:432-444 https://doi.org/10.1084/jem.155.2.432
  19. Ryten M, Dunn PM, Neary JT, Burnstock G (2002): ATP regulates the differentiation of mammalian skeletal muscle by activation of a $P2X_{5}$ receptor on satellite cells. J Cell Biol 158:345-355 https://doi.org/10.1083/jcb.200202025
  20. Ryu JK, Choi HB, Hatori K, Heisel RL, Pelech SL, Mclarnon JG, Kim SU (2003): Adenosine triphosphate induces proliferation of human neural stem cells:role of calcium and P70 ribosomal protein 56 kinase. J Neurosci Res 72:352-362 https://doi.org/10.1002/jnr.10507
  21. Ralevic V, Burnstock G (1998): Receptors for purines and pyrimidines. Pharmacol Rev 50:413-492
  22. Sak K, Boeynaems JM, Everaus H (2003): Involvement of P2Y receptors in the differentiation of heamatopoietic cells. J leukoc Biol 73:442-447 https://doi.org/10.1189/jlb.1102561
  23. Suh BC, kim JS, Namgung U, Ha H, Kim KT (2001): $P2X_{7}$ nucleotide receptor mediation of membrane pore formation and superoxide generation in human promyelocytes and neutrophils. J Immunol 166:6754-6763 https://doi.org/10.4049/jimmunol.166.11.6754
  24. Von Kugelgen I, Wetter A (2000): Molecular pharmacology of P2Y-receptors. Naunyn Schmiedebergs Arch Phamacol 62:310-323