Down-regulation of stathmin expression is required for megakaryocyte maturation and platelet production

Abstract

The final stages of of megakaryocyte (MK) maturation involve a series of steps, including polyploidization and proplatelet formation. Although these processes are highly dependent on dynamic changes in the microtubule (MT) cytoskeleton, the mechanisms responsible for regulation of MTs in MKs remain poorly defined. Stathmin is a highly conserved MTregulatory protein that has been suggested to play a role in MK differentiation of human leukemic cell lines. However, previous studies defining this relationship have reached contradictory conclusions. In this study, we addressed this controversy and investigated the role of stathmin in primary human MKs. To explore the importance of stathmin down-regulation during megakaryocytopoiesis, we used a lentiviral-mediated gene delivery system to prevent physiologic down-regulation of stathmin in primary MKs. We demonstrated that sustained expression of constitutively active stathmin delayed cytoplasmic maturation (ie, glycoprotein GPIb and platelet factor 4 expression) and reduced the ability of MKs to achieve high levels of ploidy. Moreover, platelet production was impaired in MKs in which downregulation of stathmin expression was prevented. These studies indicate that suppression of stathmin is biologically important for MK maturation and platelet production and support the importance of MT regulation during the final stages of thrombopoiesis. (Blood. 2011; 117(17):4580-4589) IntroductionMegakaryocytopoiesis is a complex process in which hematopoietic stem cells proliferate, differentiate, undergo terminal maturation, and give rise to circulating platelets. 1 During the early stages of megakaryocytopoiesis, megakaryocyte (MK) progenitors are diploid, proliferative and express early markers of the MK lineage. In the later stages, MKs cease to divide but continue to replicate their DNA and become polyploid (ie, DNA content Ͼ 2N). This is accompanied by expression of more MK-specific markers and an increase in nuclear complexity and cell size. Lastly, mature MKs develop long cytoplasmic extensions, known as proplatelets, that release large numbers of platelets into the circulation. 2 The processes of polyploidization and proplatelet formation are unique to the MK lineage. Both processes are characterized by dramatic changes in the organization of the microtubule (MT) cytoskeleton. After multiple rounds of normal cell divisions, MK progenitors switch from a normal to a form of abortive mitosis, known as endomitosis, in which MKs enter and progress normally through mitosis but fail to undergo cytokinesis and divide. [3][4][5] This process is mediated by an atypical organization of MTs that forms a complex, spherical mitotic spindle. 4,6 Recent studies have suggested that the failure of MKs to complete cytokinesis is associated, in part, with defective elongation of spindle MTs. 5 On the other hand, studies by Patel et al have demonstrated that the cytoplasm of mature MKs contains bundles of polymerized MTs that extend into the proplatelets and provide their structural scaffold ...

Neonatal expression of RNA-binding protein IGF2BP3 regulates the human fetal-adult megakaryocyte transition

“…For the human progenitor retroviral transduction experiments enforcing IGF2BP3 expression, analyses were conducted on viable GFP + gated cells. Assessment of platelet release was conducted as previously described (38,39). Studies on transplanted mice were conducted 6 weeks after transplant.…”

“…These loss-of-function studies thus reveal an influence of IGF2BP3 levels on P-TEFb activation and megakaryocytic morphogenesis, consistent with a role as a critical ontogenic determinant. To address effects of IGF2BP3 knockdown on platelet production, a recently described ex vivo platelet-release assay (38,39) was applied to the culture system. Interpretation was complicated by the accelerated growth arrest and maturation associated with loss of IGF2BP3, but the studies supported an association between knockdown and enhanced platelet production on a per megakaryocyte basis (Supplemental Figure 10).…”

Sympathetic stimulation facilitates thrombopoiesis by promoting megakaryocyte adhesion, migration, and proplatelet formation

“…7,[21][22][23] Briefly, CD34 1 cell-derived MKs were collected by centrifugation at 1200 rpm for 5 minutes and mixed with the supernatants that were centrifuged a second time at 3000 rpm for 10 minutes. The pellets containing platelets were labeled with the FITC-CD41, APC-CD42b antibodies and 10 mg/mL PI (Sigma-Aldrich).…”

hGH promotes megakaryocyte differentiation and exerts a complementary effect with c-Mpl ligands on thrombopoiesis

“…Peripheral blood-derived human platelets were analyzed as the control for primary megakaryocyte-derived platelets. The operation was performed according to previous reports, [29][30][31] and the details of the assay procedure are shown in supplemental Figure 1 (available on the Blood Web site).…”