MASL1 Induces Erythroid Differentiation In Human Erythropoietin-Dependent CD34⁺ Cells Involving Raf/MEK/ERK Signaling Pathway

Abstract 4228

Human erythropoiesis is a dynamic complex multistep process that involves differentiation of early erythroid progenitors to enucleated red blood cells. Importantly, erythroid differentiation involves lineage-specific activation and restriction of gene expression. However, the mechanisms that play a role in erythropoiesis still remain incompletely understood. We previously demonstrated that erythropoietin-stimulated clone-1 (EP1), which is selectively expressed in normal human erythroid lineage cells shares 99.5% identity with the malignant fibrous histiocytoma amplified sequence 1(MFHAS1 or MASL1). MASL1 is an important oncogene that is highly expressed in malignant fibrous histiocytomas (MFH). MASL1 protein has several domains; ras, three leucine zipper, ATP/GTP-binding site and leucine-rich tandem repeat motif which are important structural or functional elements for interactions among proteins related to the cell cycle. However, the function of the MASL1 gene in erythropoiesis has not been studied. We hypothesized that MASL1 gene plays a role in the erythroid differentiation, and used a human liquid erythroid culture system to explore this concept. MASL1 mRNA and protein expression levels were significantly increased during the erythroid differentiation of CD34⁺ cells following EPO treatment. Conversely, small interfering RNA-mediated knock down of MASL1 in CD34⁺ cells resulted in a reduction of a double positive of transferrin receptor and glycophorin A (GPA) (14.1 ± 4.7%) when compared with mock (77.9 ± 4.4%) or control vector-transfected CD34⁺ cells (76.7 ± 8.8%) at day 14. May-Grunwald-Giemsa staining confirmed a phenotype of MASL1 knockdown CD34⁺ cells that most of cells were pro- or basophilic erythroblasts. Western blotting also showed a significant decrease in hemoglobin protein levels in MASL1 knockdown CD34⁺ cells. In addition, MASL1-knockdown in CD34⁺ cells demonstrated an interruption of Raf/MEK/ERK signaling pathway. Inhibition assay of SH3 domain of Son of Sevenless (SOS) which is an upstream adapter protein in EPO-induced erythroid differentiation, confirmed that a suppression of MASL1 reduced the phosphorylation of Raf/MEK/ERK kinases and delays erythroid differentiation of Epo-induced CD34⁺ cells. Taken together, our study provides novel insights into MASL1-regulated erythropoiesis through the Raf/MEK/ERK signaling pathway.