Induction of megakaryocyte differentiation drives nuclear accumulation and transcriptional function of MKL1 via actin polymerization and RhoA activation

Abstract

• RhoA-induced actin polymerization promotes nuclear accumulation of MKL1 and transcriptional activation.• Thrombopoietin activates nuclear accumulation of MKL1 and transcriptional activation in primary megakarocytes.How components of the cytoskeleton regulate complex cellular responses is fundamental to understanding cellular function. Megakaryoblast leukemia 1 (MKL1), an activator of serum response factor (SRF) transcriptional activity, promotes muscle, neuron, and megakaryocyte differentiation. In muscle cells, where MKL1 subcellular localization is one mechanism by which cells control SRF activity, MKL1 translocation from the cytoplasm to the nucleus in response to actin polymerization is critical for its function as a transcriptional regulator. MKL1 localization is cell-type specific; it is predominantly cytoplasmic in unstimulated fibroblasts and some muscle cell types and is constitutively nuclear in neuronal cells. In the present study, we report that in megakaryocytes, subcellular localization and regulation of MKL1 is dependent on RhoA activity and actin organization. Induction of megakaryocytic differentiation of human erythroleukemia cells by 12-O-tetradecanoylphorbol-13-acetate and primary megakaryocytes by thrombopoietin promotes MKL1 nuclear localization. This MKL1 localization is blocked by drugs inhibiting RhoA activity or actin polymerization. We also show that nuclear-localized MKL1 activates the transcription of SRF target genes. This report broadens our knowledge of the molecular mechanisms regulating megakaryocyte differentiation. (Blood. 2013;121(7):1094-1101) IntroductionAlthough megakaryoblastic leukemia 1 (MKL1, also known as MRTF-A, MAL, or BSAC) plays a role in normal megakaryocytopoiesis, 1-3 much of what is known about this transcriptional coactivator of serum response factor (SRF) has been defined in fibroblasts and muscle cells. MKL1 promotes musclespecific gene expression, maintains mammary myoepithelial cell differentiation, and contributes to myocardial infarction-induced fibrosis and myofibroblast activation. [4][5][6][7] Other members of the MKL1 family include MKL2 and Myocardin. All 3 genes have been implicated in muscle cell differentiation, but have different patterns of cellular and developmental expression, which likely explains some of the differences in their knockout (KO) phenotypes. Although Mkl2-and Myocardin-KO mice are embryonic lethal with severe cardiac abnormalities, Mkl1-KO mice are viable with a less severe phenotype. Female Mkl1-KO mice have premature mammary gland involution that prevents lactation. 6,8 In addition, Mkl1-KO mice have impaired megakaryocytopoiesis defined by increased numbers of megakaryocytes in the BM, decreased ploidy of BM megakaryocytes, and low peripheral blood platelet counts. 1,3 In fibroblast cell lines, MKL1 activity is regulated posttranslationally by its subcellular localization, which is dependent on the actin cytoskeleton. 9-11 When MKL1 is bound to monomeric (G)-actin via its N-terminal RPEL domains, it is predominantly local...

Immunodeficiency and severe susceptibility to bacterial infection associated with a loss-of-function homozygous mutation of MKL1

“…15 Subsequent investigation of MKL1 function in hematopoietic cells has focused on megakaryocytes, and MKL1 deficiency in mice has been shown to result in cytoskeletal defects in this cell type disrupting differentiation, migration, and proplatelet formation. 9,10,16 Our study is the first to describe human MKL1 deficiency, and it demonstrates that although mild thrombocytopenia is associated with the condition, the major phenotype appears to relate to immunodeficiency.…”

Neonatal expression of RNA-binding protein IGF2BP3 regulates the human fetal-adult megakaryocyte transition

“…The results shown in Figure 9 indicate that several of the promegakaryocytic effects of BET inhibition occur through IGF2BP3 megakaryocyte morphogenesis. MKL1 undergoes upregulation, nuclear translocation, and transcriptional activation during megakaryopoiesis (46). Mkl1-deficient murine megakaryocytes display defects in polyploidization, enlargement, proliferation control, and proplatelet production (14,47,48), thus phenocopying normal human neonatal megakaryocytes.…”

Ott1 (Rbm15) regulates thrombopoietin response in hematopoietic stem cells through alternative splicing of c-Mpl

“…27,28 The MPL-TR isoform, generated by exclusion of exons 9 and 10, is the sole isoform shared by humans and mice, and interspecies conservation of isoforms often predicts physiologic importance. 22 Mpl-FL is insufficient to rescue Mpl 2/2 HSC engraftment when expressed by a transgene using the c-Mpl promoter, thus supporting a necessary physiologic role for alternatively spliced Mpl isoforms.…”