HDAC6 promotes PMA-induced megakaryocyte differentiation of K562 cells by regulating ROS levels via NOX4 and repressing Glycophorin A

Abstract

AbstractThe human erythroleukemia (K562) cells are considered as bipotent megakaryocyte-erythroid progenitor cells and the differentiation of these cells to megakaryocytes (MK) in the presence of phorbol 12-myristate 13-acetate (PMA) mimics in vivo differentiation of MEP (megakaryocyte-erythroid progenitor) cells in the bone marrow. Histone deacetylases (HDACs) are involved in gene suppression and their roles during the MK differentiation remains largely undefined. In the present study, we have studied the expression levels of class I and class II HDACs during phorbol 12-myristate 13-acetate (PMA)-induced differentiation of K562 cells to MK. Class IIb HDACs (HDAC6 & HDAC10) were significantly up regulated time dependently upto 4 days of PMA-induced MK differentiation along with decreased acetylation levels of H3K9 and H3K56. Pharmacological inhibition and knockdown studies of HDAC6 using tubastatin A (TubA) and shRNA-HDAC6 respectively, during MK differentiation resulted in down regulation of MK lineage marker CD61 and up regulation of erythroid lineage gene glycophorin A (GYPA). HDAC6 over expression in K562 cells showed significant up regulation of CD61, MK transcription factors (FOG1 and GATA2) and down regulation of GYPA. ChIP-PCR studies showed enrichment of HDAC6 protein on GYPA promoter during differentiation indicating GYPA gene repression by HDAC6. Further studies on elucidating the role of HDAC6 in MK differentiation clearly indicated that HDAC6 is required for the production of sustainable levels of reactive oxygen species (ROS), an important regulator of MK differentiation, via NOX4.- ROS-HDAC6 circuit. In this study, we provide the first evidence that during PMA-induced megakaryocyte differentiation of K562 cells, HDAC6 represses erythroid lineage marker gene, GYPA, and promotes the sustainable levels of ROS via NOX4 required for MK differentiation.Key pointsHDAC6 upregulated during MK differentiation is involved in sustainable production of ROS via the circuit - HDAC6-NOX4-ROS-HDAC6.HDAC6 inhibits erythroid lineage gene, GYPA, by forming a repressor complex over the promoter region.