The May-Hegglin anomaly gene MYH9 is a negative regulator of platelet biogenesis modulated by the Rho-ROCK pathway

Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA.
Blood (Impact Factor: 10.45). 08/2007; 110(1):171-9. DOI: 10.1182/blood-2007-02-071589
Source: PubMed


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.

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    • "Furthermore, ROCK inhibitors were reported to induce embryonic stem cell differentiation into neuro-ectodermal and mesodermal lineages [5]. On the other hand, suppression of ROCK in ES cell-derived Flk positive mesodermal precursor cells was reported to promote endothelial cell differentiation and expansion [6], while activation of ROCK in megakaryocytes reduces proplatelet formation [7] [8]. These results suggest that ROCKs are important in maintaining pluripotency and controlling lineage differentiation of ES and ES cell-derived progenitor cells. "
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    ABSTRACT: Rho-associated kinase (ROCK) plays an important role in maintaining embryonic stem (ES) cell pluripotency. To determine whether ROCK is involved in ES cell differentiation into cardiac and hematopoietic lineages, we evaluated the effect of ROCK inhibitors, Y-27632 and fasudil on murine ES and induced pluripotent stem (iPS) cell differentiation. Gene expression levels were determined by real-time PCR, Western blot analysis and immunofluorescent confocal microscopy. Cell transplantation of induced differentiated cells were assessed in vivo in a mouse model (three groups, n=8/group) of acute myocardial infarction (MI). The cell engraftment was examined by immunohistochemical staining and the outcome was analyzed by echocardiography. Cells were cultured in hematopoietic differentiation medium in the presence or absence of ROCK inhibitor and colony formation as well as markers of ES, hematopoietic stem cells (HSC) and cells of cardiac lineages were analyzed. ROCK inhibition resulted in a drastic change in colony morphology accompanied by loss of hematopoietic markers (GATA-1, CD41 and β-Major) and expressed markers of cardiac lineages (GATA-4, Isl-1, Tbx-5, Tbx-20, MLC-2a, MLC-2v, α-MHC, cTnI and cTnT) in murine ES and iPS cells. Fasudil-induced cardiac progenitor (Mesp-1 expressing) cells were infused into a murine MI model. They engrafted into the peri-infarct and infarct regions and preserved left ventricular function. These findings provide new insights into the signaling required for ES cell differentiation into hematopoietic as well as cardiac lineages and suggest that ROCK inhibitors are useful in directing iPS cell differentiation into cardiac progenitor cells for cell therapy of cardiovascular diseases. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.
    International journal of cardiology 08/2015; 201:441-448. DOI:10.1016/j.ijcard.2015.08.118 · 4.04 Impact Factor
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    • "Interaction of megakaryocyte with collagen type I through GPIa-IIa activates the small GTPase Rho and the Rho-kinase ROCK, which in turn phosphorylates the regulatory light chains of myosin IIA resulting in the inhibition of proplatelet formation. Type I collagen is abundant in the stem cell niche of BM and, thus, this pathway serves to prevent megakaryocytes from a premature release of platelets in this space before they reach the bone marrow sinusoids (Chen et al, 2007). This inhibitory mechanism is affected in MYH9-related disease due to defective myosin IIA. "
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    ABSTRACT: Our knowledge of the cellular and molecular mechanisms of platelet production has greatly expanded in recent years due to the opportunity to culture in vitro megakaryocytes and to create transgenic animals with specific genetic defects that interfere with platelet biogenesis. However, in vitro models do not reproduce the complexity of the bone marrow microenvironment where megakaryopoiesis takes place, and experience shows that what is seen in animals does not always happen in humans. So, these experimental models tell us what might happen in humans, but does not assure us that these events really occur. In contrast, inherited thrombocytopenias offer the unique opportunity to verify in humans the actual effects of abnormalities in specific molecules on platelet production. There are currently 20 genes whose defects are known to result in thrombocytopenia and, on this basis, this review tries to outline a model of megakaryopoiesis based on firm evidence. Inherited thrombocytopenias have not yet yielded all the information they can provide, because nearly half of patients have forms that do not fit with any known disorder. So, further investigation of inherited thrombocytopenias will advance not only the knowledge of human illnesses, but also our understanding of human platelet production.
    British Journal of Haematology 01/2014; 165(2). DOI:10.1111/bjh.12752 · 4.71 Impact Factor
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    • "Coordinated cellular processes that alter cell and tissue morphology, such as apical constriction, are often driven by the assembly and activation of contractile networks of F-actin and non-muscle myosin II (reviewed in [1]). This contractility and the resulting changes in cell shape are required for the proper development of numerous tissues including the vasculature, heart, central nervous system, kidney, and gut [2]–[14]. The signaling and mechanistic aspects of apical constriction have been widely studied and have recently been placed in a cellular framework. "
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    ABSTRACT: Rho-associated coiled coil containing protein kinase (Rho-kinase or Rock) is a well-defined determinant of actin organization and dynamics in most animal cells characterized to date. One of the primary effectors of Rock is non-muscle myosin II. Activation of Rock results in increased contractility of myosin II and subsequent changes in actin architecture and cell morphology. The regulation of Rock is thought to occur via autoinhibition of the kinase domain via intramolecular interactions between the N-terminus and the C-terminus of the kinase. This autoinhibited state can be relieved via proteolytic cleavage, binding of lipids to a Pleckstrin Homology domain near the C-terminus, or binding of GTP-bound RhoA to the central coiled-coil region of Rock. Recent work has identified the Shroom family of proteins as an additional regulator of Rock either at the level of cellular distribution or catalytic activity or both. The Shroom-Rock complex is conserved in most animals and is essential for the formation of the neural tube, eye, and gut in vertebrates. To address the mechanism by which Shroom and Rock interact, we have solved the structure of the coiled-coil region of Rock that binds to Shroom proteins. Consistent with other observations, the Shroom binding domain is a parallel coiled-coil dimer. Using biochemical approaches, we have identified a large patch of residues that contribute to Shrm binding. Their orientation suggests that there may be two independent Shrm binding sites on opposing faces of the coiled-coil region of Rock. Finally, we show that the binding surface is essential for Rock colocalization with Shroom and for Shroom-mediated changes in cell morphology.
    PLoS ONE 12/2013; 8(12):e81075. DOI:10.1371/journal.pone.0081075 · 3.23 Impact Factor
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