title: Expression of prion protein in mouse erythroid progenitors and differentiating murine erythroleukemia cells.
abstract: 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.
authors.affiliation: Institute of Immunology and Microbiology, First Faculty of Medicine, Charles University in Prague, Prague, Czech Republic.
authors.forename: Martin
authors.initials: M
authors.lastname: Panigaj
authors.forename: Hana
authors.initials: H
authors.lastname: Glier
authors.forename: Marcela
authors.initials: M
authors.lastname: Wildova
authors.forename: Karel
authors.initials: K
authors.lastname: Holada
mesh_terms: D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology; D002454:Cell Differentiation / Q000235:genetics*; D045744:Cell Line, Tumor; D015536:Down-Regulation; D015672:Erythroid Precursor Cells / Q000166:cytology / Q000378:metabolism*; D015972:Gene Expression Regulation, Neoplastic; D005822:Genetic Vectors / Q000235:genetics; D006454:Hemoglobins / Q000096:biosynthesis; D004915:Leukemia, Erythroblastic, Acute / Q000235:genetics / Q000473:pathology*; D051379:Mice; D011328:Prions / Q000235:genetics*; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D012190:Retroviridae / Q000235:genetics; D013154:Spleen / Q000166:cytology
chemical_list: D006454:Hemoglobins; D011328:Prions; D012333:RNA, Messenger; D034741:RNA, Small Interfering