The May-Hegglin anomaly gene MYH9 is a negative regulator of platelet biogenesis modulated by the Rho-ROCK pathway.
ABSTRACT The gene implicated in the May-Hegglin anomaly and related macrothrombocytopenias, MYH9, encodes myosin-IIA, a protein that enables morphogenesis in diverse cell types. Defective myosin-IIA complexes are presumed to perturb megakaryocyte (MK) differentiation or generation of proplatelets. We observed that Myh9(-/-) mouse embryonic stem (ES) cells differentiate into MKs that are fully capable of proplatelet formation (PPF). In contrast, elevation of myosin-IIA activity, by exogenous expression or by mimicking constitutive phosphorylation of its regulatory myosin light chain (MLC), significantly attenuates PPF. This effect occurs only in the presence of myosin-IIA and implies that myosin-IIA influences thrombopoiesis negatively. MLC phosphorylation in MKs is regulated by Rho-associated kinase (ROCK), and consistent with our model, ROCK inhibition enhances PPF. Conversely, expression of AV14, a constitutive form of the ROCK activator Rho, blocks PPF, and this effect is rescued by simultaneous expression of a dominant inhibitory MLC form. Hematopoietic transplantation studies in mice confirm that interference with the putative Rho-ROCK-myosin-IIA pathway selectively decreases the number of circulating platelets. Our studies unveil a key regulatory pathway for platelet biogenesis and hint at Sdf-1/CXCL12 as one possible extracellular mediator. The unexpected mechanism for Myh9-associated thrombocytopenia may lead to new molecular approaches to manipulate thrombopoiesis.
SourceAvailable from: Alessandro Malara[Show abstract] [Hide abstract]
ABSTRACT: Megakaryocytes are rare cells found in the bone marrow, responsible for the everyday production and release of millions of platelets into the bloodstream. Since the discovery and cloning, in 1994, of their principal humoral factor, thrombopoietin, and its receptor c-Mpl, many efforts have been directed to define the mechanisms underlying an efficient platelet production. However, more recently different studies have pointed out new roles for megakaryocytes as regulators of bone marrow homeostasis and physiology. In this review we discuss the interaction and the reciprocal regulation of megakaryocytes with the different cellular and extracellular components of the bone marrow environment. Finally, we provide evidence that these processes may concur to the reconstitution of the bone marrow environment after injury and their deregulation may lead to the development of a series of inherited or acquired pathologies.Cellular and Molecular Life Sciences CMLS 01/2015; 72(8). DOI:10.1007/s00018-014-1813-y · 5.86 Impact Factor
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ABSTRACT: Anagrelide represents a treatment option for essential thrombocythemia patients. It lowers platelet counts through inhibition of megakaryocyte maturation and polyploidization, although the basis for this effect remains unclear. Based on its rapid onset of action, we assessed whether, besides blocking megakaryopoiesis, anagrelide represses proplatelet formation (PPF) and aimed to clarify the underlying mechanisms. Exposure of cord blood-derived megakaryocytes to anagrelide during late stages of culture led to a dose- and time-dependent inhibition in PPF and reduced proplatelet complexity, which were independent of anagrelide-induced effect on megakaryocyte maturation. Whereas anagrelide was shown to phosphorylate cAMP-substrate VASP, two pharmacologic inhibitors of the cAMP pathway were completely unable to revert anagrelide-induced repression in megakaryopoiesis and PPF, suggesting these effects are unrelated to its ability to inhibit phosphodiesterase (PDE) 3. The reduction in thrombopoiesis was not the result of downregulation of transcription factors which coordinate PPF, while the myosin pathway was identified as a candidate target, as anagrelide was shown to phosphorylate myosin light chain and the PPF phenotype was partially rescued after inhibition of myosin activity with blebbistatin. The platelet-lowering effect of anagrelide results from impaired megakaryocyte maturation and reduced PPF, both of which are deregulated in essential thrombocythemia. These effects seem unrelated to PDE3 inhibition, which is responsible for anagrelide's cardiovascular side effects and antiplatelet activity. Further work on this field may lead to the potential development of drugs to treat thrombocytosis in myeloproliferative disorders with improved pharmacologic profile. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.Journal of Thrombosis and Haemostasis 01/2015; 13(4). DOI:10.1111/jth.12850 · 5.55 Impact Factor
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ABSTRACT: Megakaryocytes (MKs) are the highly specialized precursor cells that produce platelets via cytoplasmic extensions called proplatelets. Proplatelet formation (PPF) requires profound changes in microtubule and actin organization. In this work, we demonstrated that DIAPH1 (mDia1), a mammalian homolog of Drosophila diaphanous that works as an effector of the small GTPase Rho, negatively regulates PPF by controlling the dynamics of the actin and microtubule cytoskeletons. Moreover, we showed that inhibition of both DIAPH1 and the Rock/myosin pathway increased PPF via coordination of both cytoskeletons. We provide evidence that two major effectors of the Rho GTPase pathway (DIAPH1 and Rock/myosin II) are involved not only in Rho-mediated stress fibers assembly but also in the regulation of microtubule stability and dynamics during PPF.Blood 10/2014; 124(26). DOI:10.1182/blood-2013-12-544924 · 9.78 Impact Factor