Prion diseases have been observed to deregulate the transcription of erythroid genes, and prion protein knockout mice have demonstrated a diminished response to experimental anemia. To investigate the role of the cellular prion protein (PrP(C)) in erythropoiesis, we studied the protein's expression on mouse erythroid precursors in vivo and utilized an in vitro model of the erythroid differentiation of murine erythroleukemia cells (MEL) to evaluate the effect of silencing PrP(C) through RNA interference.The expression of PrP(C) and selected differentiation markers was analyzed by quantitative multicolor flow cytometry, western blot analysis and quantitative RT-PCR. The silencing of PrP(C) expression in MEL cells was achieved by expression of shRNAmir from an integrated retroviral vector genome. The initial upregulation of PrP(C) expression in differentiating erythroid precursors was detected both in vivo and in vitro, suggesting PrP(C)'s importance to the early stages of differentiation. The upregulation was highest on early erythroblasts (16200±3700 PrP(C) / cell) and was followed by the gradual decrease of PrP(C) level with the precursor's maturation reaching 470±230 PrP(C) / cell on most mature CD71(-)Ter119(+) small precursors. Interestingly, the downregulation of PrP(C) protein with maturation of MEL cells was not accompanied by the decrease of PrP mRNA. The stable expression of anti-Prnp shRNAmir in MEL cells led to the efficient (>80%) silencing of PrP(C) levels. Cell growth, viability, hemoglobin production and the transcription of selected differentiation markers were not affected by the downregulation of PrP(C).In conclusion, the regulation of PrP(C) expression in differentiating MEL cells mimics the pattern detected on mouse erythroid precursors in vivo. Decrease of PrP(C) protein expression during MEL cell maturation is not regulated on transcriptional level. The efficient silencing of PrP(C) levels, despite not affecting MEL cell differentiation, enables created MEL lines to be used for studies of PrP(C) cellular function.

Institute of Immunology and Microbiology 1st Faculty of Medicine Charles University Prague Prague Czech Republic

Zobrazit více v PubMed

Linden R, Martins VR, Prado MA, Cammarota M, Izquierdo I, et al. Physiology of the prion protein. Physiol Rev. 2008;88:673–728. PubMed

Roucou X, Gains M, LeBlanc AC. Neuroprotective functions of prion protein. J Neurosci Res. 2004;75:153–161. PubMed

Singh A, Kong Q, Luo X, Petersen RB, Meyerson H, et al. Prion protein (PrP) knock-out mice show altered iron metabolism: a functional role for PrP in iron uptake and transport. PLoS One. 2009;4:e6115. PubMed PMC

Nicolas O, Gavin R, del Rio JA. New insights into cellular prion protein (PrPc) functions: the “ying and yang” of a relevant protein. Brain Res Rev. 2009;61:170–184. PubMed

Miele G, Manson J, Clinton M. A novel erythroid-specific marker of transmissible spongiform encephalopathies. Nat Med. 2001;7:361–364. PubMed

Brown AR, Blanco AR, Miele G, Hawkins SA, Hopkins J, et al. Differential expression of erythroid genes in prion disease. Biochem Biophys Res Commun. 2007;364:366–371. PubMed

Zivny JH, Gelderman MP, Xu F, Piper J, Holada K, et al. Reduced erythroid cell and erythropoietin production in response to acute anemia in prion protein-deficient (Prnp-/-) mice. Blood Cells Mol Dis. 2008;40:302–307. PubMed

Zhang CC, Steele AD, Lindquist S, Lodish HF. Prion protein is expressed on long-term repopulating hematopoietic stem cells and is important for their self-renewal. Proc Natl Acad Sci U S A. 2006;103:2184–2189. PubMed PMC

Dodelet VC, Cashman NR. Prion protein expression in human leukocyte differentiation. Blood. 1998;91:1556–1561. PubMed

Risitano AM, Holada K, Chen G, Simak J, Vostal JG, et al. CD34+ cells from paroxysmal nocturnal hemoglobinuria (PNH) patients are deficient in surface expression of cellular prion protein (PrPc). Exp Hematol. 2003;31:65–72. PubMed

Holada K, Vostal JG. Different levels of prion protein (PrPc) expression on hamster, mouse and human blood cells. Br J Haematol. 2000;110:472–480. PubMed

Panigaj M, Brouckova A, Glierova H, Dvorakova E, Simak J, et al. Underestimation of the expression of cellular prion protein on human red blood cells. Transfusion. 2010;51:1012–1021. PubMed

Marks PA, Rifkind RA. Induced differentiation of erythroleukemia cells by hexamethylene bisacetamide: a model for cytodifferentiation of transformed cells. Environ Health Perspect. 1989;80:181–188. PubMed PMC

Gougoumas DD, Vizirianakis IS, Tsiftsoglou AS. Transcriptional activation of prion protein gene in growth-arrested and differentiated mouse erythroleukemia and human neoplastic cells. Exp Cell Res. 2001;264:408–417. PubMed

Holada K, Simak J, Brown P, Vostal JG. Divergent expression of cellular prion protein on blood cells of human and nonhuman primates. Transfusion. 2007;47:2223–2232. PubMed

Griffiths RE, Heesom KJ, Anstee DJ. Normal prion protein trafficking in cultured human erythroblasts. Blood. 2007;110:4518–4525. PubMed

Hyman T, Rothmann C, Heller A, Malik Z, Salzberg S. Structural characterization of erythroid and megakaryocytic differentiation in Friend erythroleukemia cells. Exp Hematol. 2001;29:563–571. PubMed

Fibach E, Reuben RC, Rifkind RA, Marks PA. Effect of hexamethylene bisacetamide on the commitment to differentiation of murine erythroleukemia cells. Cancer Res. 1977;37:440–444. PubMed

Schroder B, Nickodemus R, Jurgens T, Bodemer W. Upstream AUGs modulate prion protein translation in vitro. Acta Virol. 2002;46:159–167. PubMed

Ford MJ, Burton LJ, Li H, Graham CH, Frobert Y, et al. A marked disparity between the expression of prion protein and its message by neurones of the CNS. Neuroscience. 2002;111:533–551. PubMed

Otsuka Y, Ito D, Katsuoka K, Arashiki N, Komatsu T, et al. Expression of alpha-hemoglobin stabilizing protein and cellular prion protein in a subclone of murine erythroleukemia cell line MEL. Jpn J Vet Res. 2008;56:75–84. PubMed

Chute JP, Ross JR, McDonnell DP. Minireview: Nuclear receptors, hematopoiesis, and stem cells. Mol Endocrinol. 2010;24:1–10. PubMed PMC

Kaneda T, Murate T, Sheffery M, Brown K, Rifkind RA, et al. Gene expression during terminal differentiation: dexamethasone suppression of inducer-mediated alpha 1- and beta maj-globin gene expression. Proc Natl Acad Sci U S A. 1985;82:5020–5024. PubMed PMC

Mattern J, Buchler MW, Herr I. Cell cycle arrest by glucocorticoids may protect normal tissue and solid tumors from cancer therapy. Cancer Biol Ther. 2007;6:1345–1354. PubMed

Osborne HB, Bakke AC, Yu J. Effect of dexamethasone on hexamethylene bisacetamide-induced Friend cell erythrodifferentiation. Cancer Res. 1982;42:513–518. PubMed

Tilly G, Chapuis J, Vilette D, Laude H, Vilotte JL. Efficient and specific down-regulation of prion protein expression by RNAi. Biochem Biophys Res Commun. 2003;305:548–551. PubMed

Pfeifer A, Eigenbrod S, Al-Khadra S, Hofmann A, Mitteregger G, et al. Lentivector-mediated RNAi efficiently suppresses prion protein and prolongs survival of scrapie-infected mice. J Clin Invest. 2006;116:3204–3210. PubMed PMC

Singh A, Mohan ML, Isaac AO, Luo X, Petrak J, et al. Prion protein modulates cellular iron uptake: a novel function with implications for prion disease pathogenesis. PLoS One. 2009;4:e4468. PubMed PMC

Singh A, Beveridge AJ, Singh N. Decreased CSF Transferrin in sCJD: A Potential Pre-Mortem Diagnostic Test for Prion Disorders. PLoS One. 2011;6:e16804. PubMed PMC

Clarke MF, Kukowska-Latallo JF, Westin E, Smith M, Prochownik EV. Constitutive expression of a c-myb cDNA blocks Friend murine erythroleukemia cell differentiation. Mol Cell Biol. 1988;8:884–892. PubMed PMC

Chen J, Kremer CS, Bender TP. A Myb dependent pathway maintains Friend murine erythroleukemia cells in an immature and proliferating state. Oncogene. 2002;21:1859–1869. PubMed

Roucou X, LeBlanc AC. Cellular prion protein neuroprotective function: implications in prion diseases. J Mol Med. 2005;83:3–11. PubMed

Gougoumas DD, Vizirianakis IS, Triviai IN, Tsiftsoglou AS. Activation of Prn-p gene and stable transfection of Prn-p cDNA in leukemia MEL and neuroblastoma N2a cells increased production of PrP(C) but not prevented DNA fragmentation initiated by serum deprivation. J Cell Physiol. 2007;211:551–559. PubMed

Christensen HM, Harris DA. Prion protein lacks robust cytoprotective activity in cultured cells. Mol Neurodegener. 2008;3:11. PubMed PMC

Gauczynski S, Peyrin JM, Haik S, Leucht C, Hundt C, et al. The 37-kDa/67-kDa laminin receptor acts as the cell-surface receptor for the cellular prion protein. EMBO J. 2001;20:5863–5875. PubMed PMC

Graner E, Mercadante AF, Zanata SM, Forlenza OV, Cabral AL, et al. Cellular prion protein binds laminin and mediates neuritogenesis. Brain Res Mol Brain Res. 2000;76:85–92. PubMed

Liu Y, Pop R, Sadegh C, Brugnara C, Haase VH, et al. Suppression of Fas-FasL coexpression by erythropoietin mediates erythroblast expansion during the erythropoietic stress response in vivo. Blood. 2006;108:123–133. PubMed PMC

Chang K, Elledge SJ, Hannon GJ. Lessons from Nature: microRNA-based shRNA libraries. Nat Methods. 2006;3:707–714. PubMed

Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25:402–408. PubMed

Petrak J, Myslivcova D, Man P, Cmejlova J, Cmejla R, et al. Proteomic analysis of erythroid differentiation induced by hexamethylene bisacetamide in murine erythroleukemia cells. Exp Hematol. 2007;35:193–202. PubMed

Prion Strains Differ in Susceptibility to Photodynamic Oxidation

Large Platelet and Endothelial Extracellular Vesicles in Cord Blood of Preterm Newborns: Correlation with the Presence of Hemolysis

Detection of Prions in Brain Homogenates and CSF Samples Using a Second-Generation RT-QuIC Assay: A Useful Tool for Retrospective Analysis of Archived Samples