Inhibition of TPO-induced MEK or mTOR activity induces opposite effects on the ploidy of human differentiating megakaryocytes

Guerriero, Raffaella; Parolini, Isabella; Testa, Ugo; Samoggia, Paola; Petrucci, Eleonora; Sargiacomo, Massimo; Chelucci, Cristiana; Gabbianelli, Marco; Peschle, Cesare

doi:10.1242/jcs.02784

Cited by 62 publications

(58 citation statements)

References 48 publications

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“…Previous reports have suggested that the PI3K/AKT pathway is not necessary for the differentiation of K562 cells, 29 although other authors have related this pathway to endomitosis in CD34 þ cells. 27 In this study, we show that whereas PI3K had no effect on differentiation, AKT inhibition strongly inhibited it, and that these features were consistent in all types of cells studied. Therefore, it is likely that AKT would be activated through a mechanism not dependent on PI3K.…”

Section: Discussionsupporting

confidence: 74%

“…A representative experiment is shown. TPO, probably the most important cytokine for megakaryocytopoiesis, 27 activates different signalling pathways (such as JAK2/STAT, PI3K/AKT, MEK/ERK and JNK pathways). 28 It has previously been proposed that the activation of ERK1/2 and JNK, together with the inhibition of p38 MAPK, would be crucial for K562 cell megakaryocytic differentiation.…”

Section: Discussionmentioning

confidence: 99%

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p22phox-dependent NADPH oxidase activity is required for megakaryocytic differentiation

et al. 2010

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Transient reactive oxygen species (ROS) production is currently proving to be an important mechanism in the regulation of intracellular signalling, but reports showing the involvement of ROS in important biological processes, such as cell differentiation, are scarce. In this study, we show for the first time that ROS production is required for megakaryocytic differentiation in K562 and HEL cell lines and also in human CD34 þ cells. ROS production is transiently activated during megakaryocytic differentiation, and such production is abolished by the addition of different antioxidants (such as N-acetyl cysteine, trolox, quercetin) or the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor diphenylene iodonium. The inhibition of ROS formation hinders differentiation. RNA interference experiments have shown that a p22 phoxdependent NADPH oxidase activity is responsible for ROS production. In addition, the activation of ERK, AKT and JAK2 is required for differentiation, but the activation of phosphatidylinositol 3-kinase and c-Jun N-terminal kinase seems to be less important. When ROS production is prevented, the activation of these signalling pathways is partly inhibited. Taken together, these results show that NADPH oxidase ROS production is essential for complete activation of the main signalling pathways involved in megakaryocytopoiesis to occur. We suggest that this might also be important for in vivo megakaryocytopoiesis.

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Section: Discussionsupporting

confidence: 74%

Section: Discussionmentioning

confidence: 99%

p22phox-dependent NADPH oxidase activity is required for megakaryocytic differentiation

et al. 2010

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“…26,28 CD34 ϩ cells were incubated for 3 to 7 days in the presence of TPO or BMP4 used alone or with the indicated inhibitor. Results presented in Figure 3A reveal that if AG490, Ly294002, and rapamycin decreased cell proliferation, only Ly294002 significantly altered the number of viable CD34 ϩ cells treated with either TPO (62% vs 82% with TPO alone) or BMP4 (57% vs 76% with BMP4 alone).…”

Section: Tpo and Bmp4 Use The Same Signaling Pathways To Regulate Hummentioning

confidence: 99%

BMP4 regulation of human megakaryocytic differentiation is involved in thrombopoietin signaling

Jeanpierre

Nicolini

Kaniewski

et al. 2008

Blood

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Activin A, BMP2, and BMP4, 3 members of the transforming growth factor-␤ family, are involved in the regulation of hematopoiesis. Here, we explored the role of these molecules in human megakaryopoiesis using an in vitro serum-free assay. Our results highlight for the first time that, in the absence of thrombopoietin, BMP4 is able to induce CD34 ؉ progenitor differentiation into megakaryocytes through all stages. Although we have previously shown that activin A and BMP2 are involved in erythropoietic commitment, these molecules have no effect on human megakaryopoietic engagement and differentiation. Using signaling pathway-specific inhibitors, we show that BMP4, like thrombopoietin, exerts its effects on human megakaryopoiesis through the JAK/STAT and mTor path-

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“…36 Recently, two reports have shown that the mTOR pathway plays a critical role in both growth and polyploidization of megakaryocytes, likely in part by regulating expression and localization of cyclin D3. 37,38 Thus, it is likely that one function of the D-type cyclins in megakaryocytes is to promote cell cycle progression in a manner analogous to its role in dividing cells. However, since megakaryocytes bypass the late stages of mitosis, the end product is a cell with increased DNA content as opposed to an increase in the numbers of descendants.…”

Section: Cyclin D and Cdk4 Regulate Size And Polyploidization In Diffmentioning

confidence: 99%

Cyclin D–Cdk4 is regulated by GATA-1 and required for megakaryocyte growth and polyploidization

Muntean

Pang

Poncz

et al. 2007

Blood

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Endomitosis is a unique form of cell cycle used by megakaryocytes, in which the latter stages of mitosis are bypassed so that the cell can increase its DNA content and size. Although several transcription factors, including GATA-1 and RUNX-1, have been implicated in this process, the link between transcription factors and polyploidization remains undefined. Here we show that GATA-1-deficient megakaryocytes, which display reduced size and polyploidization, express nearly 10-fold less cyclin D1 and 10-fold increased levels of p16 compared with their wild-type counterparts. We further demonstrate that cyclin D1 is a direct GATA-1 target in megakaryocytes, but not erythroid cells. Restoration of cyclin D1 expression, when accompanied by ectopic overexpression of its partner Cdk4, resulted in a dramatic increase in megakaryocyte size and DNA content. However, terminal differentiation was not rescued. Of note, polyploidization was only modestly reduced in cyclin D1-deficient mice, likely due to compensa- IntroductionCyclin D1 is one of a battery of cell-cycle regulatory proteins, including cyclins, cyclin-dependent kinases (Cdks), and Cdk inhibitors, that is required for proper regulation of cell cycle and cell differentiation. 1 G 1 -specific cyclin-Cdk complexes, including the D-type cyclins and Cdk4/6, promote progression into S phase through the phosphorylation and inhibition of pocket proteins (pRb, p107, and p130). 2 Hypophosphorylated pocket proteins bind and inhibit the E2F family of transcription factors that control genes involved in proliferation and DNA replication, including cyclin E. 3 Phosphorylation of pocket proteins by cyclin D1-Cdk4/6 causes their dissociation from E2F proteins, leading to transcriptional activation and a commitment to cell-cycle progression. 4 However, recent data have described a more intricate balance between different phosphorylation states and function of pRb. Ezhevsky et al have shown that cyclin D-Cdk4/6 hypophosphorylates pRb in early G 1 , activating pRb as a transcriptional repressor and allowing it to bind E2F and E1A proteins. 5 In late G 1 , cyclin E-Cdk2 complexes hyperphosphorylate pRb, thereby inactivating it and allowing for progression into S phase. These findings lend credence to the model that cyclin D and cyclin E activities are not equivalent. Of note, cellular proliferation is not affected when individual D-type cyclins or Cdk4 or Cdk6 are disrupted in mice, although individual knockout lines display specific growth defects. For example, cyclin D1-deficient animals are smaller than their littermates and show hypoplasia of the retina and mammary epithelium. [6][7][8][9] Most tissues express multiple D-type cyclins, however, which allows for compensation when one is knocked out.Notably, mice that are deficient for all 3 D-type cyclins show gross defects in multiple cell types, including hematopoietic progenitors. 10 Cdk inhibitors offer another means of regulating cell-cycle progression. For example, p16 ink4A (p16), a member of the INK4 family of cell-cycle in...

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Inhibition of TPO-induced MEK or mTOR activity induces opposite effects on the ploidy of human differentiating megakaryocytes

Cited by 62 publications

References 48 publications

p22phox-dependent NADPH oxidase activity is required for megakaryocytic differentiation

p22phox-dependent NADPH oxidase activity is required for megakaryocytic differentiation

BMP4 regulation of human megakaryocytic differentiation is involved in thrombopoietin signaling

Cyclin D–Cdk4 is regulated by GATA-1 and required for megakaryocyte growth and polyploidization

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