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

Abstract

IntroductionMYH9 encodes myosin-IIA, a nonmuscle myosin heavy chain that assembles within actomyosin complexes and facilitates shape changes in diverse cell types. [1][2][3] Patients with autosomal dominant inherited MYH9-related disorders, 4-6 including the May-Hegglin anomaly, 7,8 exhibit macrothrombocytopenia and variable degrees of hearing loss, nephritis, and cataracts. These individuals experience mild bleeding symptoms as a result of reduced numbers of misshapen blood platelets that can be 2 to 5 times larger than normal. 9,10 In the May-Hegglin anomaly, macrothrombocytopenia is due to a defect in platelet release by megakaryocytes (MKs) in the bone marrow, but platelets that do form seem to circulate and function normally. [9][10][11] The MYH9-associated syndromes are thus regarded as disorders of thrombopoiesis, 12 and defective myosin-IIA complexes are presumed to perturb some aspect of MK differentiation, likely late in the course of cell maturation.Polyploid MKs accumulate an enormous and complex cytoplasm before they assemble and release blood platelets. Two key processes are thought to govern the timing and execution of platelet release. Mature MKs travel within the bone marrow and come to lie close to sinusoidal vessels 13 ; this process responds to cellular and humoral interactions, mediated in part by the chemokine stromal cell-derived factor 1 (Sdf-1 or CXCL12). [14][15][16] In their final stages, MKs extend long cytoplasmic projections called proplatelets and actively assemble nascent platelets at the tips of these structures. [17][18][19] Proplatelet formation (PPF) is preceded and accompanied by characteristic changes in cell shape, reorganization of the actin and microtubule cytoskeletons, and generation of intracellular force. 20 As an abundant motor protein within MKs and the only representative of its family, 21 myosin-IIA is a good candidate mediator of proplatelet extension or platelet release. Biochemical studies suggest that certain N-terminal mutations found in patients with MYH9-related disorders reduce intrinsic ATPase activity, 22 whereas other common C-terminal mutations may compromise myosin filament formation. 23 Although both classes of mutations may interfere with myosin-IIA function, it is unclear whether monoallelic MYH9 mutations produce clinical phenotypes as a result of haploinsufficiency or dominant-negative effects. The role of myosin-IIA in platelet assembly and release is hence uncertain.Conventional myosin-II complexes contain a dimer of heavy chains, each associated with the myosin regulatory light chain (MLC). Phosphorylated MLC enhances actin-dependent myosin motor activity, 24 and 3 kinases can phosphorylate MLC: Rhoassociated kinase (ROCK), myosin light chain kinase (MLCK), and p21-activated kinase. 24 Both ROCK and MLCK participate in platelet activation, 25-27 but their roles in MK maturation or platelet Submitted February 1, 2007; accepted March 21, 2007. Prepublished online as Blood First Edition paper, March 28, 2007; DOI 10.1182 DOI 10. /blood-2007 An In...

Myosin-II inhibition and soft 2D matrix maximize multinucleation and cellular projections typical of platelet-producing megakaryocytes

“…S3E) (25). The morphology effects are reminiscent of blebbistatin, causing an increased number of MKs with proplatelet extensions when cells are grown on plastic (13,14), and we indeed find an approximately threefold increase in mean length of proplatelet extensions under such conditions (Fig. 2E).…”

“…In mouse, deletion of MYH9 in MKs also produces MayHegglin-like defects (12,13). Paradoxically, pharmacological inhibition of NMM-II ATPase by blebbistatin in mouse adult (13) and mouse embryonic (14) systems is reported to produce a two-to ∼threefold increase in proplatelet extensions but not affect MK ploidy or size, at least for the doses or times tested. Whether human MK-poiesis is regulated by myosin-II has remained unclear, but possible pathways include direct phosphorylation of NMM-II (i.e., heavy-chain regulation that decreases myosin activity in other cells) (15).…”

“…Whether human MK-poiesis is regulated by myosin-II has remained unclear, but possible pathways include direct phosphorylation of NMM-II (i.e., heavy-chain regulation that decreases myosin activity in other cells) (15). Rho-associated protein kinase can affect myosin-II and is known to increase MK ploidy by approximately twofold (14). Here, NMM-IIA's role in MK maturation and subsequent platelet production is examined directly starting with human bone marrow (BM)-derived CD34 + cells cultured with just two cytokines to differentiate into MK progenitors followed by sustained reversible inhibition of myosin-II for 3 d (Fig.…”

Differential expression of wild-type and mutant NMMHC-IIA polypeptides in blood cells suggests cell-specific regulation mechanisms in MYH9 disorders

“…31,32 Recent findings have shown that NMMHC-IIA attenuates proplatelet formation, 33,34 which occurs in terminally mature megakaryocytes where most of the cytoplasm is converted into lengthy beaded extensions. 35 Thus, NMMHC-IIA might restrain thrombopoiesis until megakaryocytes accumulate sufficient quantities of the materials required for optimal platelet assembly; loss of myosin-IIA function could trigger precocious proplatelet formation, whereas gain-of-function mutations might limit platelet production.…”

Historical hematology: May–Hegglin anomaly

“…Whether or not a dominant negative effect of mutant allele or haploinsufficiency is responsible for hematological abnormalities [23][24][25] is still controversial. The role of NMMHC-IIA in megakaryocyte differentiation and platelets release also needs to be investigated [26]. …”