3'-Half of the Thrombopoietin cDNA Confers Higher Expression of Erythropoietin at the RNA Level but Not at the Protein Level

  • Kim, Tae-Won (Department of Biology, Dongguk University) ;
  • Ji, Jin Woo (Department of Biology, Dongguk University) ;
  • Chang, Ho Gun (Department of Biology, Dongguk University) ;
  • Kim, Myoung Ok (Department of Biology, Dongguk University) ;
  • Ryoo, Zae Young (Laboratory Animal Center, Catholic Medical College) ;
  • Park, In Kook (Department of Biology, Dongguk University) ;
  • Kim, Sun Jung (Department of Biology, Dongguk University)
  • Received : 2004.10.09
  • Accepted : 2004.12.21
  • Published : 2005.04.30

Abstract

Both erythropoietin (EPO) and the short-form thrombopoietin (TPO) were expressed at low levels whereas the long-form TPO was expressed at high levels in transgenic animals. To elucidate the role of carboxy-terminal half of the long-form TPO which is absent in the short-form, we generated recombinant TPO or EPO expression vectors which contain or lack the carboxy-terminal half of TPO and examined their expression in the HC11 and 293 cells. The long-form TPO was expressed higher than the short-form regardless of the cell types, transfection modes, and promoters. When 3'-half of the long-form TPO cDNA was placed downstream of the EPO cDNA to act as a 3'-untranslated region, expression of EPO was moderately increased at the RNA level, however, no remarkable increase was observed at the protein level. These results suggest that the low expression of EPO, as like as the short-form TPO, is due to absence of the 3'-half in the full-length TPO that confers stability both at the RNA and protein levels.

Keywords

Acknowledgement

Supported by : Korea Science and Engineering Foundation

References

  1. Aguirre, A., Castro-Palomino, N., De la Fuente, J., and Ovidio Castro, F. O. (1998) Expression of human erythropoietin transgenes and of the endogenous WAP gene in the mammary gland of transgenic rabbits during gestation and lactation. Transgenic Res. 7, 311-317 https://doi.org/10.1023/A:1008882332133
  2. Alexander, W. S. and Hyland, C. (2004) Thrombopoietin bioassay. Methods Mol. Biol. 272, 347-360
  3. Apostel, F., Dammann, R., Pfeifer, G. P., and Greeve, J. (2002) Reduced expression and increased CpG dinucleotide methylation of the rat APOBEC-1 promoter in transgenic rabbits. Biochim. Biophys. Acta 27, 384-394
  4. Bartley, T. D., Bogenberger, J., Hunt, P., Li, Y. S., Lu, H. S., et al. (1994) Identification and cloning of a megakaryocyte growth and development factor that is a ligand for the cytokine receptor Mpl. Cell 77, 1117-1124 https://doi.org/10.1016/0092-8674(94)90450-2
  5. de Sauvage, F. J., Hass, P. E., Spencer, S. D., Malloy, B. E., Gurney, A. L., et al. (1994) Stimulation of megakaryocytopoiesis and thrombopoiesis by the c-Mpl ligand. Nature 369, 533-538 https://doi.org/10.1038/369533a0
  6. Fisher, J. W. (2003) Erythropoietin: physiology and pharmacology update. Exp. Biol. Med. 228, 1-14
  7. Foster, D. and Lok, S. (1996) Biological roles for the second domain of thrombopoietin. Stem Cells 1, 102-107
  8. Ghezzi, P. and Brines, M. (2004) Erythropoietin as an antiapoptotic, tissue-protective cytokine. Cell Death Differ. 11, S37- 44 https://doi.org/10.1038/sj.cdd.4401450
  9. Gurney, A. L., Kuang, W. J., Xie, M. H., Malloy, B. E., Eaton, D. L., et al. (1995) Genomic structure, chromosomal localization, and conserved alternative splice forms of thrombopoietin. Blood 85, 981-988
  10. Haas, M. J., Dragan, Y. P., Hikita, H., Shimel, R., Takimoto, K., et al. (1999) Transgene expression and repression in transgenic rats bearing the phosphoenolpyruvate carboxykinase-simian virus 40 T antigen or the phosphoenolpyruvate carboxykinasetransforming growth factor-alpha constructs. Am. J. Pathol. 155, 183-192 https://doi.org/10.1016/S0002-9440(10)65112-7
  11. Hoffman, R. C., Andersen, H., Walker, K., Krakover, J. D., Patel, S., et al. (1996) Peptide, disulfide, and glycosylation mapping of recombinant human thrombopoietin from ser1 to Arg246. Biochemistry 35, 14849-14861 https://doi.org/10.1021/bi961075b
  12. Jacobs, K., Shoemaker, C., Rudersdorf, R., Neill, S. D., Kaufman, R. J., et al. (1985) Isolation and characterization of genomic and cDNA clones of human erythropoietin. Nature 313, 806-810 https://doi.org/10.1038/313806a0
  13. Kim, T. W., Moon, H. B., and Kim, S. J. (2003) Interleukin-10 is up-regulated by prolactin and serum-starvation in cultured mammary epithelial cells. Mol. Cells 16, 168-172 https://doi.org/10.4313/JKEM.2003.16.2.168
  14. Kirito, K., Fox, N., and Kaushansky, K. (2004) Thrombopoietin induces HOXA9 nuclear transport in immature hematopoietic cells: potential mechanism by which the hormone favorably affects hematopoietic stem cells. Mol. Cell Biol. 24, 6751- 6762 https://doi.org/10.1128/MCB.24.15.6751-6762.2004
  15. Korhonen, V. P., Tolvanen, M., Hyttinen, J. M., Uusi-Oukari, M., Sinervirta, R., et al. (1997) Expression of bovine betalactoglobulin/ human erythropoietin fusion protein in the milk of transgenic mice and rabbits. Eur. J. Biochem. 245, 482- 489 https://doi.org/10.1111/j.1432-1033.1997.00482.x
  16. Linden, H. M. and Kaushansky, K. (2000) The glycan domain of thrombopoietin enhances its secretion. Biochemistry 39, 3044-3051 https://doi.org/10.1021/bi991756h
  17. Linden, H. M. and Kaushansky, K. (2002) The glycan domain of thrombopoietin (TPO) acts in trans to enhance secretion of the hormone and other cytokines. J. Biol. Chem. 277, 35240- 35247 https://doi.org/10.1074/jbc.M201297200
  18. Lok, S., Kaushansky, K., Holly, R. D., Kuijper, J. L., Lofton-Day, C. E., et al. (1994) Cloning and expression of murine thrombopoietin cDNA and stimulation of platelet production in vivo. Nature 369, 565-568 https://doi.org/10.1038/369565a0
  19. Massoud, M., Attal, J., Thepot, D., Pointu, H., Stinnakre, M. G., et al. (1996) The deleterious effects of human erythropoietin gene driven by the rabbit whey acidic protein gene promoter in transgenic rabbits. Reprod. Nutr. Dev. 36, 555-563 https://doi.org/10.1051/rnd:19960511
  20. Mikus, T., Maly, P., Poplstein, M., Landa, V., Trefil, P., et al. (2001) Expression of human erythropoietin gene in the mammary gland of a transgenic mouse. Folia Biol. 47, 187-195
  21. Narhi, L. O., Arakawa, T., Aoki, K. H., Elmore, R., Rohde, M. F., et al. (1991) The effect of carbohydrate on the structure and stability of erythropoietin. J. Biol. Chem. 266, 23022- 23026
  22. Rodriguez, A., Castro, F. O., Aguilar, A., Ramos, B., Del Barco, D. G., et al. (1995) Expression of active human erythropoietin in the mammary gland of lactating transgenic mice and rabbits. Biol. Res. 28, 141-153
  23. Sohn, B. H., Kim, S. J., Park, H., Park, S. K., Lee, S. C., et al. (1999) Expression and characterization of bioactive human thrombopoietin in the milk of transgenic mice. DNA Cell Biol. 18, 845-852 https://doi.org/10.1089/104454999314845
  24. Spivak, J. L. and Hogans, B. B. (1989) The in vivo metabolism of recombinant human erythropoietin in the rat. Blood 73, 90-99
  25. Takeuchi, M., Takasaki, S., Shimada, M., and Kobata, A. (1990) Role of sugar chains in the in vitro biological activity of human erythropoietin produced in recombinant Chinese hamster ovary cells. J. Biol. Chem. 265, 12127-12130
  26. Tibbles, H. E., Navara, C. S., Hupke, M. A., Vassilev, A. O., and Uckun, F. M. (2002) Thrombopoietin induces p-selectin expression on platelets and subsequent platelet/leukocyte interactions. Biochem. Biophys. Res. Commun. 292, 987-991 https://doi.org/10.1006/bbrc.2002.6629
  27. Ulich, T. R., del Castillo, J., Senaldi, G., Cheung, E., Roskos, L., et al. (1999) The prolonged hematologic effects of a single injection of PEG-rHuMGDF in normal and thrombocytopenic mice. Exp. Hematol. 27, 117-130 https://doi.org/10.1016/S0301-472X(98)00012-5
  28. Wagner, K. F., Katschinski, D. M., Hasegawa, J., Schumacher, D., Meller, B., et al. (2001) Chronic inborn erythrocytosis leads to cardiac dysfunction and premature death in mice overexpressing erythropoietin. Blood 97, 536-542 https://doi.org/10.1182/blood.V97.1.198