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• W2513847293 abstract "Abstract Abstract 3650 Poster Board III-586 Background Megakaryocytes and erythroblasts differentiate from MEPs, but the mechanism of lineage shift of MEPs to either megakaryocyte or erythroblast still remains unknown. Transcription factors, GATA DNA-binding genes, ETS family genes, EKLF and RUNX1, play a key role in megakaryocytic and erythroid differentiation. Among them, FLI1, one of ETS family, is considered to play the main role for determination of megakaryocytic differentiation. We previously reported a unique leukemia cell line, JAS-R, being a good model to study the megakaryo-erythroid divergence mechanisms (Leukemia Res. 2007 G 31:1537-43). These cells acquire megakaryocytic phenotypes by adhesion to extracellular matrices. We examined how cell attachment augments FLI1 expression. Materials and Methods JAS-R cells, a megakaryo-erythroid cell line, were used. Gene transfection was done by electroporation. FLI1 promoter activity was measured by luciferase reporter gene assay. Knock-down of a specific gene function was obtained by RNA-interference using a short-hairpin expression vector. Messenger RNAs were studied by Northern blot or RT-PCR. Protein was studied by Immunoblot. Integrins were analyzed by flow cytometry. Results As previously reported, JAS-R cells were segregated into two populations depending on adhesion. One is adherent megakaryocytic cells (JAS-RAD), and the other is floating erythroid cells (JAS-REN). JAS-RAD expressed higher amount of FLI1 message. Knock-down of FLI1 gene by short-hairpin RNA (shFLI1) reduced the adhesion (80% of JAS-RAD cells transfected with control vector adhered compared to 45% of shFLI1-transfected). Changes of CD41 and CD61 were studied by flow cytometry. The mean fluorescence of both CD41 and CD61 was significantly decreased in FLI1 knock-downed cells. While FLI1 was high in JAS-RAD cells, the expression of NFE2 did not differ. Thus, transcriptional activity of two genes was examined in JAS-RAD and JAS-REN cells. Luciferase reporter gene experiments revealed that the promoter activity of FLI1 gene (-835 to -36 from translation initiation site) was dramatically high in JAS-RAD, while the NFE2 promoter was equal in both lineages. These data demonstrated that the adhesion increases the FLI1 promoter activity in JAS-RAD cells, leading megakaryocytic differentiation. Next, we searched the region of FLI1 promoter responsive for the activation in JAS-RAD. A series of deletion vectors disclosed that -596 to -417 contained the critical region responsible for the difference between JAS-RAD and JAS-REN cells. Luciferase assay carried out with mutations in each transcription factor binding site demonstrated that GATA- and ETS-binding sites were responsible for the transcription in JAS-RAD, but NFkB, AP1, or STAT binding sites were not. Indeed, the FLI1 promoter activity decreased to the half by introduction of shFLI1 indicates the existence of auto-augmentation mechanisms of FLI1 gene. Next, we studied whether the interference of adhesion affected on the FLI1 promoter activity. JAS-RAD cells did not attach on the substratum of Ultra Low Attachment surface culture dishes, instead they aggregated and grew as floating cells. The promoter activity decreased by 20% in Ultra Low dishes, and this reduction was significant. Knock-down of either ITGB1 or ITGB3 gene by shRNA failed to reduce the FLI1 promoter activity, but the concomitant knock-down of two integrin genes reduced the promoter activity by 50%. Conclusion Several lines of evidences support the important function of cell adhesion for maintenance and differentiation of hematopoietic cells. Our data support that once cell-attachment occurs in JAS-R cells, FLI1 is induced, and adhesion molecules increase profoundly. Adherent cells further augment FLI1 itself and are critically destined to differentiate into megakaryocytes. Disclosures: No relevant conflicts of interest to declare." @default.
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• W2513847293 date "2009-11-20" @default.
• W2513847293 modified "2023-10-01" @default.
• W2513847293 title "Mutual-Activation of FLI1 and Integrins Functions as a Key Event During Megakaryocytic Differentiation of Megakaryo-Erythroid JAS-R Cells." @default.
• W2513847293 doi "https://doi.org/10.1182/blood.v114.22.3650.3650" @default.
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