A role for Rab27b in NF-E2-dependent pathways of platelet formation

Abstract

Megakaryocytes release platelets by reorganizing the cytoplasm into proplatelet extensions. Fundamental to this process is the need to coordinate transport of products and organelles in the appropriate abundance to nascent platelets. The importance of the Rab family of small GTPases (guanosine 5-triphosphatases) in platelet biogenesis is revealed in gunmetal (gm/gm) mice, which show deficient Rab isoprenylation and macrothrombocytopenia with few granules and abnormal megakaryocyte morphology. Although some Rab proteins are implicated in vesicle and organelle transport along microtubules or actin, the role of any Rab protein in platelet biogenesis is unknown. The limited number of Rab proteins with defective membrane association in gm/gm megakaryocytes prominently includes Rab27a and Rab27b. Normal expression of Rab27b is especially increased with terminal megakaryocyte differentiation and dependent on nuclear factor-erythroid 2 (NF-E2), a transcription factor required for thrombopoiesis. Chromatin immunoprecipitation demonstrates recruitment of NF-E2 to the putative Rab27B promoter. Inhibition of endogenous IntroductionMammalian blood platelets are released from bone marrow megakaryocytes (MKs) in a process that transforms the entire MK cytoplasm into long pseudopodia known as proplatelets. 1-3 Nascent platelets are assembled within these structures. 4 The need for dramatic cytoplasmic and cytoskeletal reorganization and concomitant assembly of anucleate platelets presents unusual challenges to differentiated MKs. The cellular and molecular response to these challenges is poorly understood.Selected mouse models of thrombocytopenia and qualitative platelet defects have proved invaluable in probing the molecular basis of platelet biogenesis. Mice lacking either of 2 erythromegakaryocytic transcription factors, GATA1, and nuclear factorerythroid 2 (NF-E2), show dramatically arrested MK maturation 5,6 and thus implicate their target genes in aspects of platelet assembly. However, the transcriptional targets of GATA1 and NF-E2 that are immediately relevant to MK fragmentation and platelet release are not known. Other insights derive from findings in animal models of the human Hermansky-Pudlak syndrome (HPS), a multigenic group of recessively inherited disorders that result from abnormal synthesis of 3 related organelles: melanosomes, platelet-dense granules, and lysosomes. 7 HPS proteins regulate intracellular vesicle traffic, including the ␦ and ␤3A subunits of the AP-3 adaptor complex, which captures organelle membrane proteins at the trans-Golgi apparatus and/or endosomes, and the pallid protein, which interacts with syntaxin-13 to mediate vesicle fusion. 8,9 These HPS proteins highlight molecular pathways that serve to assemble and transport organelles but they do not overtly influence the efficiency with which MKs release platelets. In contrast, thrombocytopenia is a cardinal feature of the gunmetal mouse. 10 The cellular morphology of gm/gm MKs closely resembles that seen in the absence of NF-E2, with reduced ...

NF-E2 domination over Nrf2 promotes ROS accumulation and megakaryocytic maturation

“…14 The expression of Thromboxane synthetase (Txas) and Rab27b, 2 direct target genes of p45, is decreased in p45-null megakaryocytes. 15,16 In addition, p45 depletion impairs proliferation of megakaryocytes, 17 suggesting that p45 also contributes to the proliferation of megakaryocytes.…”

An immunohistochemical analysis of Rab27B distribution in fetal and adult tissue

“…granules , dense granules in platelets (Barral et al, 2002 and megakaryocytes (Tiwari et al, 2003), urothelial fusiform vesicles (Chen et al, 2003), parotid and pancreatic acinar zymogen granules (Chen et al, 2004, Imai et al, 2004, mast cell secretory granules (Mizuno et al, 2007), gastric parietal tubular vesicles (Suda et al, 2011), and secretory granules in alveolar epithelial type II and Clara cells (Bolasco et al, 2011). Furthermore, Rab27B is abundantly expressed in stomach acid-secreting parietal cells and mucus cells, as shown in a transgenic mouse model expressing beta-galactosidase under the Rab27B promoter (Gomi et al, 2007).…”

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

“…Tubb1 knockout mice have thrombocytopenia with spherical platelets [149], and a mutation in TUBB1 has been identified in a patient with congenital macrothrombocytopenia [150]. Mice with deficient RAB signalling have macrothrombocytopenia with few granules and abnormal MK morphology, and RAB27B may coordinate granule transport during proplatelet formation [146]. Nfe2 null MKs fail to bind to fibrinogen in response to platelet agonists indicative of a defect in the signalling leading to activation of GPIIB/IIIA, and, similarly, Casp12 null platelets have a defect in GPIIB/IIIA which is a well-documented positive regulator of proplatelet formation [151].…”

“…The apparent role of NFE2 in proplatelet formation might be explained through the function of some of its direct transcriptional targets such as tubulin β1 (TUBB1) [145], RAB27b [146], caspase 12 (CASP12) [147] and HSD3B1 (3-betahydroxysteroid dehydrogenase) [148]. Tubb1 knockout mice have thrombocytopenia with spherical platelets [149], and a mutation in TUBB1 has been identified in a patient with congenital macrothrombocytopenia [150].…”