Differential expression of NADPH oxidases in megakaryocytes and their role in polyploidy

Abstract

IntroductionMegakaryocytes (MKs) are platelet precursors in the bone marrow (BM) that undergo a complex maturation process called megakaryopoiesis. A hallmark of MK development occurs after their commitment to the MK lineage, in which MKs go through iterative rounds of endomitosis to become large polyploid cells. These cells can contain DNA content of up to and greater than 128N, with 2N being normal diploid DNA content. 1 The primary stimulus for progression of megakaryopoiesis is known to stem from thrombopoietin (TPO) signaling through the c-Mpl receptor 2 ; however, despite this being the focus of many studies, much of TPO signaling in MKs remains unclear.Recently, investigations of reactive oxygen species (ROS), their generation, and downstream signaling effects have shown that low physiologic levels of ROS play a role in promoting cell proliferation in many cells types, such as fibroblast, prostate, macrophage, endothelial, and vascular smooth muscle cells (VSMCs). 3-7 ROSproducing enzymes, such as a member of the NADPH oxidase (Nox) family, Nox1, have been described to be activated by mitogenic stimuli 8 and to produce ROS that have been shown to induce and maintain G 1 phase cyclin D1 expression in mouse lung epithelial cells. 9 Similarly, Havens et al 10 showed that ROSs increase in a cell cycle-dependent manner and oscillate with every cell division in human T98G glioblastoma cells, human leukemic T cells, and NIH 3T3 cells. Although, these investigators did not identify the source of ROS in these cells, they did show that on treatment with various antioxidants, cells undergo transient arrest and fail to make a timely G 1 -S phase progression. [11][12][13] In addition, Havens et al 10 described redox control over the activity of the anaphase promoting complex (APC) in association with Cdh1, mediating cyclin A degradation, a regulator of the G 1 /S transition. In the MK endomitotic cell cycle, cyclin D3 has been described as the predominant D-type cyclin that is up-regulated by TPO stimulation and induces high ploidy levels in cyclin D3 transgenic mice. 14-16 Ablation of cyclin D3 in cultured MKs results in a notable decrease in MK ploidy level, 16 although in vivo studies show a less dramatic effect on ploidy possibly because of compensation by other D cyclins. 17 Cyclin E is another G 1 phase cell-cycle regulator that has been suggested to play a role in MK ploidy promotion, notably inducing nonendomitotic megakaryoblastic K562 cells to undergo re-replication cycles. 18 The importance of cyclin E in promotion of ploidy is also supported by evidence from the cyclin E1 Ϫ/Ϫ E2 Ϫ/Ϫ double knockout (KO) mice produced with the use of the tetraploid complementation rescue method. 19 Interestingly, these mice developed normally with the exception of ploidy promotion in trophoblasts and MKs, showing a marked reduction in polyploid MKs even when cultured with TPO.To date, investigation of ROS in MKs has been limited to studies that used human megakaryocytic leukemic cell lines, such as MO7e and B1647 cells. 11,...

p22phox-dependent NADPH oxidase activity is required for megakaryocytic differentiation

“…In addition, supplementary analyses showed that DPI treatment was also hindering differentiation: (1) the compound prevented the increase in cell size ( Figure 3c) and acquisition of the megakaryocytic morphological features ( Figure 3d); (2) the increase in the megakaryocytic markers (CD41 and CD61) and the decrease in the erythrocytic marker GpA were partially inhibited by DPI (Figure 3e 21 This would imply that ROS production might also be involved in cell-cycle progression, thus being crucial for endomitosis. In this sense, while this paper was in preparation, a report appeared describing the potential role of NADPH oxidases in megakaryocyte polyploidisation, 22 in agreement with our results (Figure 3f). Under our experimental condition, DPI did not affect cell viability (Supplementary Figure 4c) or mitochondrial potential (Supplementary Figure 4d).…”

New Roles for Cyclin E in Megakaryocytic Polyploidization

“…Previous in vivo studies using cyclin E1, E2 double knock-out mouse model showed that cyclin E is necessary for MK polyploidy (31). Recently, we reported that up-regulation of cyclin E partially restores the ploidy levels in NADPH oxidase-inhibited MKs, further supporting a role for cyclin E as an inducer of polyploidy (47). Here, we show that TPO stimulation of MKs highly up-regulates cyclin E. This finding suggests a role for cyclin E in TPO-mediated MK polyploidization.…”

The critical roles of platelet activation and reduced NO bioavailability in fatal pulmonary arterial hypertension in a murine hemolysis model

“…At the histologic level, we also observed more extensive P-selectin staining, indicative of platelet activation/aggregation, 34 and integrin ␣IIb, a specific marker of platelet activation, 30,35 in the small perialveolar pulmonary vessels in the ThCD59 RBC mice treated with ILY 140 FI than in the ThCD59 RBC mice treated with ILY 140 SI ( Figure 5 and supplemental Figure 3). Moreover, platelet aggregates and thrombi specifically occurred in the pulmonary microvasculature, but not the vasculatures of other tissues such as kidney, liver, and heart of ThCD59 RBC mice treated with ILY 140 FI ( Figure 5; supplemental Figure 4).…”

“…Lung, liver, kidney, and heart sections were processed and stained with an anti-P-selectin (C20; Santa Cruz Biotechnology), rabbit anti-integrin ␣IIb 30 (sc-15 328; Santa Cruz Biotechnology), or rabbit anti-von Willebrand factor (VWF) 26 (Dako) antibody, 1:50 dilution, followed by the goat avidin-biotin complex (ABC) staining system (sc-2023 or sc-2018; Santa Cruz Biotechnology). Heart sections were stained with mouse anti-hCD59 monoclonal antibody Bric 229 (International Blood Group Reference Laboratory) and fluorescein isothiocyanate-conjugated horse anti-mouse secondary antibody as previously described.…”