Session: 502. Hematopoiesis: Regulation of Gene Transcription, Cytokines, Signal Transduction, Apoptosis, and Cell Cycle Regulation: Poster I
Hematology Disease Topics & Pathways:
AML, Diseases, Myeloid Malignancies
To study the expression of RSK isoforms in hematopoiesis, we cultured human cord blood CD34+ (CBCD34+) hematopoietic stem and progenitor cells (HSPCs) in differentiating media containing SCF, Flt3L, TOP, IL-3, IL-6, GM-CSF, and EPO and performed qPCR with mRNA of the RSK isoforms at different stages of maturation up to 14 days. RPS6KA1 (RSK1) was expressed throughout hematopoiesis, but RPS6KA3 (RSK2) and RPS6KA2 (RSK3) showed higher expression by 2-fold (p<0.05) in the earlier stages of normal hematopoiesis. Moreover, RPS6KA3 is highly expressed in CD38-CD34+ HSCs compared with other progenitor populations (LMPP, MPP, CMP, GMP, and MEP) isolated from human cord blood. RPS6KA2 expression was higher in LMPP by 7-fold (p<0.05) and CMP by 3-fold (p<0.05) compared with HSCs but was barely detectable in MEPs. RPS6KA1 was ubiquitously expressed in all progenitor populations. These results suggest that RSK2 and RSK3 have distinct functions during early myelopoiesis.
To study the requirement of RSK isoforms during hematopoiesis, we knocked down RSK expression by transducing lentivirus expressing isoform-specific shRNAs or scramble controls into human CBCD34+ cells. On day 5 following transduction, cells were sorted and plated in methylcellulose media and assessed for the colony-forming activity of RSK knockdown in HSPCs. While RSK3 knockdown increased BFU-E colonies by 1.5-fold (p<0.05) compared with control cells, RSK1 knockdown decreased BFU-E and CFU-E erythroid colonies by 2-fold (p<0.05) but increased CFU-GM colonies by 2-fold (p<0.05) compared with control cells. However, RSK2 knockdown did not show any significant effects on colony-forming activity of HSPCs. We confirmed the isoform-specific effect of RSKs in normal hematopoiesis using liquid culture assays. We collected transduced cells at day 8, 11, and 14 after transduction and analyzed cell populations by flow cytometry. Similar to colony formation assay results, RSK3 knockdown increased the CD71+ erythroid population by 1.5-fold (p<0.001), but suppressed production of the CD14+ monocyte population. RSK1 knockdown enhanced production of the CD11b+/CD14+ myeloid population, but inhibited the CD71+ erythroid production by 1.5-fold (p<0.001) compared with control HSPCs. There was no significant change in blood cell differentiation in RSK2 knockdown cells.
These data demonstrate that RSK1 and RSK3 exert opposite functions during erythroid and myeloid differentiation of HSPCs, suggesting a novel role for RSK isoforms as a determinant of early fate decisions of HSCs.
Disclosures: No relevant conflicts of interest to declare.
See more of: Oral and Poster Abstracts