Influence of Prion Gene Downregulation by RNAi on Erythroid Differentiation in Vitro. First Insight.

Cellular prion protein (PrPc) has significant medical importance but its physiological role remains unclear. Some reports indicate that PrPc may play role in the cell survival and/or differentiation. Connection between prion pathogenesis and erythropoiesis was suggested by downregulation of alpha hemoglobin stabilizing protein (AHSP) during prion disease. In addition we recently demonstrated the reduction of erythroid cell and erythropoietin production in PrP-null mice in response to acute anemia (Zivny et al. BCMD 40(2008)302–7). Supporting data for possible involvement of PrPc in hematopoiesis come from the study showing that long-term hematopoietic stem cells of PrP-null bone marrow exhibited impaired self-renewal in serial transplantation of lethally irradiated mouse recipients. Pilot studies in cell culture model, represented by murine erythroleukemic (MEL) cells, indicated upregulation of PrPc expression during erythroid differentiation suggesting its importance in the process. In order to study the role of PrPc during the erythroid differentiation we created 3 stable transfected MEL lines infected with retroviruses coding the short interfering RNA in context of miRNA (1 nonsilencing control - LN and 2 RNAs - LP1 and LP2 targeting murine prion gene (Prnp)). Anti Prnp sequences (HP_285770 and HP_288208) were chosen from publicly available database (RNAi codex). As a vector we used MSCV/LTRmiR30-PIG (Open Biosystems) retroviral plasmid which was transfected to packaging cell line HEK293GP2 and medium containing the viruses was used to infect the MEL cells. Upon selection with 0.5 μg/mL of Puromycin we obtained ~95% of positive cells (estimated by eGFP expression). Downregulation of PrPc was verified by western blot and qRT-PCR. Significant inhibition of PrPc extending from ~70 to more than 90 % compared to LN - MEL cells was observable during the 6 day course of the differentiation (Fig.). We analyzed the expression of 3 genes by qRT-PCR: c-myb, AHSP and hemoglobin alpha (HBA). C-myb is required for early cellular expansion and its downregulation allows the terminal differentiation of cells. Interestingly on the start of differentiation we found ~70% upregulation of c-myb in lines with silenced PrPc. After entering of cells to differentiation, all lines downregulated the c-myb, but the decrease in c-myb expression was more pronounced in LP1 and LP2 cells (15% of initial expression after 24 hours) in comparison with LN cells (30%). Analysis of AHSP and HBA, showed similar upregulation in all cell lines. Since many reports suggest cytoprotective role of PrPc in prevention of apoptosis we analyzed potential influence of PrPc on MEL cell survival. Previous study showed that neither physiological upregulation nor enhanced expression by exogenous delivered Prnp cDNA altered percentage of apoptotic MEL cells during differentiation. We wondered if opposite approach- downregulation of PrPc will affect cell survival. Flow cytometry analysis upon cell staining with 7-AAD and Hoechst 33342 showed that inhibition of translation of PrPc during differentiation probably does not play role in sensitization of cells to apoptosis under physiological conditions. We hypothesized that if under normal conditions cell lines do not differ in level of apoptosis, then they could react differentially after exposure to external stress. We promoted the disturbing conditions by several different treatments: oxidative stress by presence of H₂O₂ (500 μM), Staurosporin (125 nM) and elevated incubation temperature (40°C). We also treated uninduced cells with increasing concentration of Cu²⁺ or exposed them to serum deprivation. Contrary to belief that absence of PrPc sensitizes the cells against damage, we found that inhibition of PrPc expression did not altered level of apoptosis/necrosis in MEL cells. Finally, we have introduced the new cell culture model for study of PrPc involvement in erythroid differentiation. Our initial observations suggest that direct cytoprotection is not mode of PrPc action in studied process, but it may play role in cell cycle which is a subject of undergoing research.

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