MAL/SRF complex is involved in platelet formation and megakaryocyte migration by regulating MYL9 (MLC2) and MMP9

Abstract

Megakaryoblastic leukemia 1 (MAL) is a transcriptional coactivator of serum response factor (SRF). In acute megakaryoblastic leukemia, the MAL gene is translocated and fused with the gene encoding one twenty-two (OTT). Herein, we show that MAL expression increases during the late differentiation steps of neonate and adult human megakaryopoiesis and localized into the nucleus after Rho GTPase activation by adhesion on collagen I or convulxin. MAL knockdown in megakaryocyte progenitors reduced the percentage of cells forming filopodia, lamellipodia, and stress fibers after adhesion on the same substrates, and reduced proplatelet formation. MAL repression led to dysmorphic megakaryocytes with disorganized demarcation membranes and ␣ granules heterogeneously scattered in the cytoplasm. Gene expression profiling revealed a marked decrease in metalloproteinase 9 (MMP-9) and MYL9 expression after MAL inhibition. Luciferase assays in HEK293T cells and chromatin immunoprecipitation in primary megakaryocytes showed that the MAL/SRF complex directly regulates MYL9 and MMP9 in vitro. Megakaryocyte migration in response to stromal cell-derived factor 1, through Matrigel was considerably decreased after MAL knockdown, implicating MMP9 in migration. Finally, the use of a shRNA to decrease MYL9 expression showed that MYL9 was involved in proplatelet formation. MAL/SRF complex is thus involved in platelet formation and megakaryocyte migration by regulating MYL9 and MMP9. IntroductionSerum response factor (SRF) is a widely expressed transcription factor required for the expression of immediate early, musclespecific, and cytoskeletal genes. 1-4 SRF contains a MADS domain that mediates homodimerization and DNA binding, and that allows recruitment of transcriptional cofactors. SRF binds to a CArG box present in promoter/enhancer regions of SRF-regulated genes. 5 Depending on cell lines, different extracellular stimuli activate SRF through 2 main signaling pathways: the MAP-kinase pathway through members of the ternary complex factor (TCF) 6,7 and the small GTPases pathway through the Rho family 8 members regulating the myocardin-related transcription factors (MRTFs). The Rho-actin signaling pathway 9-12 stimulates SRF by 2 ubiquitous MRTFs, megakaryoblastic leukemia 1 (MAL; MKL1, MRTF-A, BSAC) and MAL16 (MKL2, MRTF-B).MAL was initially identified in acute megakaryoblastic leukemia (AMKL, M7) as a chromosome 22 encoded protein fused in 3Ј with RNA-binding motif protein 15 (RBM15; OTT) located on chromosome 1. [13][14][15] The translocation t(1,22)(p13;q13) leads to the in-frame fusion of the quasi-totality of OTT/RBM15 to the MAL gene. The OTT-MAL fusion protein is restricted to AMKL occurring de novo in infancy, 16,17 in children older than 1 year or, occasionally, in Down syndrome patients. 18,19 The subcellular localization of MAL is regulated through its association with globular actin by its RPEL motifs in the Nterminal region. The modification of the actin treadmilling by the Rho pathway results in the nuclear accumulation of ...

SRF selectively controls tip cell invasive behavior in angiogenesis

“…6G,H). In cancer cells and megakaryocytes, it has recently been shown that SRF controls expression of myosin heavy chains and Myl9 (Gilles et al, 2009;Medjkane et al, 2009), genes known to be important for actomyosin filament contraction (Vicente-Manzanares et al, 2009). Immunostaining showed that Srf deletion in ECs led to a decrease in the expression of myosin heavy chain 10 (MYH10) (Fig.…”

The serum response factor (SRF)/megakaryocytic acute leukemia (MAL) network participates in megakaryocyte development

“…17 A contribution of MMP9 activity in the stromal-derived factor 1-induced migration of human Mks has been reported. 5,18 Taken together, changes in actin dynamics or in the expression of cell motility effectors (Tpm1, Mmp9, Myl9) may profoundly affect the migration of Mks and/or of their progenitors in the BM, leading to their accumulation in situ and defective platelets release in the bloodstream.…”

“…1,2 The human MAL gene is fused to the OTT/ RBM15 gene in acute megakaryoblastic leukemia (AMKL) 3,4 and a contribution of Mal activity to the late steps of murine and human megakaryopoiesis has been reported. 5,6 Although the lack of Srf results in major cellular defects and embryonic lethality (reviewed in Miano et al 7 ), Mal germ line deficiency results in a limited myoepithelial cells phenotype 8,9 probably because of the redundancies of Mrtf proteins.…”

Transcriptional Regulation of Platelet Formation: Harnessing the Complexity for Efficient Platelet Production In Vitro

“…Matrix metalloproteinase 9 (MMP9) and MYL9 are both directly regulated by MKL1, and through dysregulation of the expression of these two proteins, MKL1 deficiency leads to abnormalities of cell migration and proplatelet formation, respectively [161]. MKL1 has also an effect on MK polyploidisation, through dysregulation of RhoA.…”

“…Upon actin polymerisation and formation of F-actin, the pool of G-actin is depleted which promotes MKL1 nuclear localisation [159]. MKL1 expression increases during MK differentiation [160], and its nuclear localisation increases in response to TPO or upon stimulation of the RhoGTPase activation [161,162].…”