Role of a serine/threonine kinase,Mst1, in megakaryocyte differentiation

Platelets, which play a central role in thrombosis and hemostasis, develop from megakaryocytes. Signal transduction originated from the megakaryocyte growth and development factor, the Mpl ligand, which leads to megakaryocyte differentiation, polyploidization, and maturation, has been gradually characterized. In this study, we report the inducibility of Mst1, a recently described serine/threonine kinase, by Mpl ligand and the effect of its induced expression on megakaryocyte differentiation. The steady‐state level of mst1 message and Mst1‐associated kinase activity increased in response to Mpl ligand. Ectopic expression of human mst1 in a mouse megakaryocytic cell line resulted in a drastic increase in DNA content per cell. Elevated expression of megakaryocyte differentiation markers, such as acetylcholine esterase, PF4, and GPIIb was also observed in hmst1‐expressing cells. Activation of p38 MAPK, a known downstream effector of Mst1, was shown to be required for polyploidization, but not for enhanced expression of differentiation markers. Our study thus designates Mst1 as a Mpl ligand‐responsive signaling molecule that promotes induction of lineage‐specific cellular programming. J. Cell. Biochem. 76:44–60, 1999. © 1999 Wiley‐Liss, Inc.     

Signaling by the Mpl receptor involves IKK and NF-kappaB

Binding of tumor necrosis factor-alpha (TNF-alpha) to its receptor activates IKK complex, which leads to inducement of NF-kappaB activity. Here we report that activation of Mpl ligand is also linked to IKK and NF-kappaB activity. Mpl ligand, also known as thrombopoietin (TPO) or megakaryocyte growth and development factor (MGDF), induces megakaryocyte differentiation and inhibition of mitotic proliferation, followed by induction of polyploidization and fragmentation into platelets. The latter process is often observed in megakaryocytes undergoing apoptosis. Treatment of a Mpl ligand-responding megakaryocytic cell line with this cytokine led to an immediate, transient increase in IKK activity followed by a profound decrease in this kinase activity over time. This decrease was not due to an effect on the levels of the IKK regulatory components IKKalpha and IKKbeta. Proliferating megakaryocytes displayed a constitutive DNA-binding activity of NF-kappaB p50 homodimers and of NF-kappaB p50-p65 heterodimers. As expected, reduced IKK activity in Mpl ligand-treated cells was associated with a significant reduction in NF-kappaB DNA binding activity and in the activity of a NF-kappaB-dependent promoter. Our study is thus the first to identify a constitutive NF-kappaB activity in proliferating megakaryocytes as well as to describe a link between Mpl receptor signaling and IKK and NF-kappaB activities. Since a variety of proliferation-promoting genes and anti-apoptotic mechanisms are activated by NF-kappaB, retaining its low levels would be one potential mechanism by which inhibition of mitotic proliferation is maintained and apoptosis is promoted during late megakaryopoiesis.     

Interference RNA (RNAi)-based silencing of endogenous thrombopoietin receptor (Mpl) in Dami cells resulted in decreased hNUDC-mediated megakaryocyte proliferation and differentiation

Recently our laboratory reported evidence showing that hNUDC acts as an additional cytokine for thrombopoietin receptor (Mpl). Previously known as the human homolog of a fungal nuclear migration protein, hNUDC plays a critical role in megakaryocyte differentiation and maturation. Here we sought to further clarify the hNUDC-Mpl ligand-receptor relationship by utilizing interference RNA (RNAi) to knockdown Mpl expression in a megakaryocyte cell line. We created U6 promoter driven constructs to express short hairpin RNAs (shRNA) with affinity for different sites on Mpl mRNA. By including Mpl-EGFP fusion protein in these constructs, we were able to effectively screen the shRNA that was most efficient in inhibiting Mpl mRNA expression. This shRNA was subsequently transferred into a lentivirus vector and transduced into Dami cells, a cell line which constitutively expresses endogenous Mpl. This lentiviral vector was also designed to simultaneously express EGFP to monitor transfection efficiency. Our results show that lentivirus can be used to effectively deliver shRNAs into Dami cells and cause specific inhibition of Mpl protein expression after transduction. Furthermore, we show the functional effects of shRNA-mediated Mpl silencing by demonstrating reduced hNUDC stimulated megakaryocyte proliferation and differentiation. Thus, the use of a RNAi knockdown strategy has allowed us to pinpoint the connection of hNUDC with Mpl in the regulation of megakaryocyte maturation.     

Rat NAP1: cDNA cloning and upregulation by Mpl ligand

The Mpl ligand is a hematopoietic cytokine which exerts its effects through association with the c-Mpl receptor. It regulates the proliferation, polyploidization and maturation of platelet precursors, the megakaryocytes. Using a differential display polymerase chain reaction (PCR) approach, we have identified an mRNA, belonging to a family of nucleosome assembly proteins, whose expression is upregulated in response to Mpl ligand. Multiple size classes of this mRNA (1.7, 2.5 and 4.3 kb) are readily detected in rat primary bone marrow cells and hematopoietic tissues. The size classes are also expressed to different extents in cell lines of all hematopoietic lineages. We isolated the full-length cDNA encoding the rat megakaryocyte 1.7 kb mRNA, referred to as rNAP1. Bacterially expressed recombinant protein encoded by the 1.7 kb cDNA facilitates the formation of nucleosomes on relaxed circular DNA in vitro. Our data indicate that rNAPs, which may facilitate chromatin reorganization, are upregulated by Mpl ligand. It is possible that NAPs contribute to Mpl ligand's induced effects on hematopoietic cells.     

Oncoprotein 18 levels and phosphorylation mediate megakaryocyte polyploidization in human erythroleukemia cells

Megakaryocytes undergo an unusual cell cycle during differentiation that results in polyploidy through largely unknown mechanism(s). It has been shown that serine phosphorylation of oncoprotein 18 (Op18) is required for cell cycle progression specifically at the G2/M transition. Moreover, mutant forms of Op18 that are defective in one or more of the four serine residues induce G2/M arrest and subsequent polyploidization. Op18 phosphorylation is rapidly induced with phorbol myristate acetate (PMA) treatment in a wide range of human cells. In this study, we investigated the role of Op18 in PMA induced polyploidization during megakaryocyte differentiation of the human erythroleukemia cell line. Crucial to the molecular analysis of megakaryocyte differentiation, is the ability to fractionate cell populations with different ploidy levels. We have utilized cell elutriation as a fractionation strategy to analyze Op18 expression in synchronized cell subpopulations in different phases of the cell cycle or with progressive megakaryocyte polyploidization. In the absence of PMA, increased phosphorylation of Op18 was observed in HEL cells during cell cycle progression, as for other proliferating cells. However, in contrast to Jurkat leukemia cells chosen as control, HEL cells exhibited a lack of Op18 phosphorylation in response to PMA, which was accompanied by polyploidization and differentiation along the megakaryocytic lineage. To further determine the role of Op18 in polyploidization, HEL cells were transfected with different Op18 expression constructs. Differences in cell survival and polyploidization were observed between high and low Op18 expressors. An increased Op18 level reduced cell survival during the early stage of PMA induced megakaryocyte differentiation, but enhanced polyploidization efficiency. Our findings suggest that maintenance of a high level of unphosphorylated Op18 is required for efficient polyploidization during the differentiation program of megakaryocytes.     

Multiple upstream signals converge on the adaptor protein Mst50 in Magnaporthe grisea

Rice blast fungus (Magnaporthe grisea) forms a highly specialized infection structure for plant penetration, the appressorium, the formation and growth of which are regulated by the Mst11-Mst7-Pmk1 mitogen-activated protein kinase cascade. We characterized the MST50 gene that directly interacts with both MST11 and MST7. Similar to the mst11 mutant, the mst50 mutant was defective in appressorium formation, sensitive to osmotic stresses, and nonpathogenic. Expressing a dominant active MST7 allele in mst50 complemented its defects in appressorium but not lesion formation. The sterile alpha-motif (SAM) domain of Mst50 was essential for its interaction with Mst11 and for appressorium formation. Although the SAM and Ras-association domain (RAD) of Mst50 were dispensable for its interaction with Mst7, deletion of RAD reduced appressorium formation and virulence on rice (Oryza sativa) seedlings. The interaction between Mst50 and Mst7 or Mst11 was detected by coimmunoprecipitation assays in developing appressoria. Mst50 also interacts with Ras1, Ras2, Cdc42, and Mgb1 in yeast two-hybrid assays. Expressing a dominant active RAS2 allele in the wild-type strain but not in mst50 stimulated abnormal appressorium formation. These results indicate that MST50 functions as an adaptor protein interacting with multiple upstream components and plays critical roles in activating the Pmk1 cascade for appressorium formation and plant infection in M. grisea.     

Repression of A TAF(II)32 isoform as part of a program of genes regulated during mpl ligand-induced megakaryocyte differentiation.

Circulating platelets, essential for thrombosis and hemostasis, originate from megakaryocytes. Megakaryocyte growth, differentiation and survival processes are regulated by the c-Mpl receptor ligand. In the current study we used differential display to identify part of the program of genes regulated during Mpl ligand-induced murine megakaryocyte differentiation. Several of the genes, including the retinoblastoma binding protein p84, were found to be induced, while others were repressed. One such repressed gene was identified as a TATA-binding protein (TBP)-Associated Factor (TAF) family member, TAF(II)32, previously reported to be upregulated during apoptosis. Our analysis of various cell types suggested that the previously identified species homologs, human TAF(II)32 and murine TAF(II)32, are in fact different isoforms, which we propose to re-name TAF(II)32alpha and TAF(II)32beta, respectively. Only the TAF(II)32beta isoform is regulated during Mpl ligand-induced megakaryocyte differentiation, which suggests individual roles for the two forms.     

Identification of Regulators of Polyploidization Presents Therapeutic Targets for Treatment of AMKL

The mechanism by which cells decide to skip mitosis to become polyploid is largely undefined. Here we used a high-content image-based screen to identify small-molecule probes that induce polyploidization of megakaryocytic leukemia cells and serve as perturbagens to help understand this process. Our?study implicates five networks of kinases that?regulate the switch to polyploidy. Moreover, we find that dimethylfasudil (diMF, H-1152P) selectively increased polyploidization, mature cell-surface marker expression, and apoptosis of malignant megakaryocytes. An integrated target identification approach employing proteomic and shRNA screening revealed that a major target of diMF is Aurora kinase A (AURKA). We further find that MLN8237 (Alisertib), a selective inhibitor of AURKA, induced polyploidization and expression of mature megakaryocyte markers in acute megakaryocytic leukemia (AMKL) blasts and displayed potent anti-AMKL activity in?vivo. Our findings provide a rationale to support clinical trials of MLN8237 and other inducers of polyploidization and differentiation in AMKL.     

Phosphatidylinositol 3-kinase is necessary but not sufficient for thrombopoietin-induced proliferation in engineered Mpl-bearing cell lines as well as in primary megakaryocytic progenitors.

Thrombopoietin and its receptor (Mpl) support survival and proliferation in megakaryocyte progenitors and in BaF3 cells engineered to stably express Mpl (BaF3/Mpl). The binding of thrombopoietin to Mpl activates multiple kinase pathways, including the Jak/STAT, Ras/Raf/MAPK, and phosphatidylinositol 3-kinase pathways, but it is not clear how these kinases promote cell cycling. Here, we show that thrombopoietin induces phosphatidylinositol 3-kinase and that phosphatidylinositol 3-kinase is required for thrombopoietin-induced cell cycling in BaF3/Mpl cells and in primary megakaryocyte progenitors. Treatment of BaF3/Mpl cells and megakaryocytes with the phosphatidylinositol 3-kinase inhibitor LY294002 inhibited mitotic and endomitotic cell cycl-ing. BaF3/Mpl cells treated with thrombopoietin and LY294002 were blocked in G(1), whereas megakaryocyte progenitors treated with thrombopoietin and LY294002 showed both a G(1) and a G(2) cell cycle block. Expression of constitutively active Akt in BaF3/Mpl cells restored the ability of thrombopoietin to promote cell cycling in the presence of LY294002. Constitutively active Akt was not sufficient to drive proliferation of BaF3/Mpl cells in the absence of thrombopoietin. We conclude that in BaF3/Mpl cells and megakaryocyte progenitors, thrombopoietin-induced phosphatidylinositol 3-kinase activity is necessary but not sufficient for thrombopoietin-induced cell cycle progression. Phosphatidylinositol 3-kinase activity is likely to be involved in regulating the G(1)/S transition.     

Thrombopoietin (TPO) induces c-myc expression through a PI3K- and MAPK-dependent pathway that is not mediated by Akt, PKCzeta or mTOR in TPO-dependent cell lines and primary megakaryocytes

Thrombopoietin (TPO) and its receptor (c-Mpl) are the major regulators of megakaryocyte and platelet production and serve a critical and non-redundant role in hematopoietic stem cell (HSC) biology. TPO signals through the Jak-STAT, Ras-Raf-MAPK, and PI3K pathways, and promotes survival, proliferation, and polyploidization in megakaryocytes. The proto-oncogene c-myc also plays an important role in many of these same processes. In this work we studied the regulated expression of c-myc in megakaryocytic cell lines and primary cells by quantitative real-time RT-PCR. We found that TPO induced expression of c-myc in 1 h in both hematopoietic cell lines (UT-7 and BaF3/Mpl) and mature murine megakaryocytes. The TPO-induced expression of c-myc was blocked by a phosphatidylinositol 3-kinase (PI3K) inhibitor, suggesting that TPO stimulated c-myc expression through a PI3K-dependent pathway. Of interest, our study showed that overexpression of active Akt did not rescue the effect of PI3K blockade on c-myc expression, rather, enhanced it. In addition, inhibitors of protein kinase C (PKC)zeta and the target of rapamycin (mTOR) also failed to affect c-myc mRNA expression, while c-myc mRNA expression was reduced by inhibition of the mitogen activated protein kinase (MAPK) pathway. Therefore, we conclude that TPO stimulates c-myc expression in primary megakaryocytes through a PI3K- and MAPK-dependent pathway that is not mediated by Akt, PKCzeta or mTOR.     

Thrombopoietin stimulates proliferation and megakaryocytic differentiation of mouse pro-B cell line BF-TE22

We have isolated and characterized a thrombopoietin (TPO)-dependent BF-TE22 cell line endogenously expressing murine Mpl, which is a subclone of murine pro-B Ba/F3 cells. TPO stimulated the proliferation of BF-TE22 cells in a dose-dependent manner, and also induced the expression of megakaryocyte lineage-specific AP-51 and CD61 cell surface antigens. The results indicate that the murine Mpl on BF-TE22 cells can transmit both proliferation and megakaryocyte lineage-specific differentiation signals to cells. Furthermore, it was shown that IL-3 inhibits the TPO-induced differentiation signals of BF-TE22 cells. These results suggest that the signals mediated by IL-3 predominate over those of TPO in BF-TE22 cells. Thus, BF-TE22 cells will be useful for the biological and biochemical studies of the TPO-Mpl signal transduction mechanism. This revised version was published online in June 2006 with corrections to the Cover Date.     

Regulation of cell differentiation by hNUDC via a Mpl-dependent mechanism in NIH 3T3 cells

Thrombopoietin receptor (Mpl) belongs to the cytokine receptor surperfamily with a large extracellular N-terminal portion responsible for cytokine recognition and binding. Thrombopoietin (TPO) has so far been the only widely studied cytokine for Mpl. However we have recently identified human NUDC (hNUDC), previously described as a human homolog of a fungal nuclear migration protein, as another putative binding partner of Mpl. The purpose of this study is to test the extent of the functioning of hNUDC by identifying protein-protein interactions with Mpl in mammalian cells. The full-length cDNAs encoding Mpl and hNUDC were cloned into pEGFP-N1 and pDsRed2-N1 respectively which were subsequently expressed as Mpl-EGFP (green) and hNUDC-DsRed (red) fusion proteins. Using ELISA and immunofluorescence studies, we have demonstrated the direct binding of hNUDC to cell surface-captured Mpl. We also observed that hNUDC induced significant changes in cellular morphology in NIH 3T3 cells stably transfected with pMpl-EGFP. Interestingly, these morphological changes were characteristic of cells undergoing megakaryocyte differentiation. Extracellular-signal-regulated protein kinases 1 and 2 (ERK1/2) have been shown to mediate such megakaryocyte-like differentiation. In addition, co-expression of Mpl-EGFP and hNUDC-DsRed led to the release of hNUDC-DsRed into the culture medium.     

Genetic Deletion of Mst1 Alters T Cell Function and Protects against Autoimmunity

Mammalian sterile 20-like kinase 1 (Mst1) is a MAPK kinase kinase kinase which is involved in a wide range of cellular responses, including apoptosis, lymphocyte adhesion and trafficking. The contribution of Mst1 to Ag-specific immune responses and autoimmunity has not been well defined. In this study, we provide evidence for the essential role of Mst1 in T cell differentiation and autoimmunity, using both genetic and pharmacologic approaches. Absence of Mst1 in mice reduced T cell proliferation and IL-2 production in vitro, blocked cell cycle progression, and elevated activation-induced cell death in Th1 cells. Mst1 deficiency led to a CD4⁺ T cell development path that was biased toward Th2 and immunoregulatory cytokine production with suppressed Th1 responses. In addition, Mst1^−/− B cells showed decreased stimulation to B cell mitogens in vitro and deficient Ag-specific Ig production in vivo. Consistent with altered lymphocyte function, deletion of Mst1 reduced the severity of experimental autoimmune encephalomyelitis (EAE) and protected against collagen-induced arthritis development. Mst1^−/− CD4⁺ T cells displayed an intrinsic defect in their ability to respond to encephalitogenic antigens and deletion of Mst1 in the CD4⁺ T cell compartment was sufficient to alleviate CNS inflammation during EAE. These findings have prompted the discovery of novel compounds that are potent inhibitors of Mst1 and exhibit desirable pharmacokinetic properties. In conclusion, this report implicates Mst1 as a critical regulator of adaptive immune responses, Th1/Th2-dependent cytokine production, and as a potential therapeutic target for immune disorders.     

Schizosaccharomyces pombe histone acetyltransferase Mst1 (KAT5) is an essential protein required for damage response and chromosome segregation

Schizosaccharomyces pombe Mst1 is a member of the MYST family of histone acetyltransferases and is the likely ortholog of Saccharomyces cerevisiae Esa1 and human Tip60 (KAT5). We have isolated a temperature-sensitive allele of this essential gene. mst1 cells show a pleiotropic phenotype at the restrictive temperature. They are sensitive to a variety of DNA-damaging agents and to the spindle poison thiabendazole. mst1 has an increased frequency of Rad22 repair foci, suggesting endogenous damage. Two-hybrid results show that Mst1 interacts with a number of proteins involved in chromosome integrity and centromere function, including the methyltransferase Skb1, the recombination mediator Rad22 (Sc Rad52), the chromatin assembly factor Hip1 (Sc Hir1), and the Msc1 protein related to a family of histone demethylases. mst1 mutant sensitivity to hydroxyurea suggests a defect in recovery following HU arrest. We conclude that Mst1 plays essential roles in maintenance of genome stability and recovery from DNA damage.     

Physical and Functional Interactions between Mitotic Kinases during Polyploidization and Megakaryocytic Differentiation

Human Polo-like kinase 3 (Plk3), a protein serine/threonine kinase, is involved in the regulation of cell cycle progression at multiple stages. Our previous studies revealed that Plk3 is closely associated with centrosomes and plays an important role in the regulation of microtubule dynamics. Here we describe the physical interaction of Plk3 with Aurora A and BubR1 kinases, and the significance of this interaction during terminal differentiation and polyploidization of megakaryocytes. Specifically, double immunofluorescence staining confirms that Plk3 and Aurora A co-localize to centrosomes or spindle poles during essentially all phases of the cell cycle and that BubR1 also exhibits spindle pole localization during metaphase. Pull-down assays show that Plk3 physically interacts with Aurora A as well as BubR1. Upon treatment with phorbol 12-myristate 13-acetate (PMA), human erythroleukemic cells (K562) underwent megakaryocytic differentiation characterized by polyploidization and expression of mature megakaryocyte surface markers such as CD41. Plk3 protein levels were seen to be increased during PMA-induced megakaryocytic differentiation of K562 cells, correlating well with the ploidy level in these cells. Similarly, Aurora A and its phosphorylated form also increased after PMA treatment. In contrast, BubR1 levels were markedly reduced. Taken together, our study suggests that Plk3 and Aurora A kinases may lie in the same regulatory pathway and that Plk3 and Aurora A as well as BubR1 may play an important role in polyploidization and megakaryocytic differentiation.     

Thrombopoietin signal transduction: studies from cell lines and primary cells

Thrombopoietin (TPO) and its receptor Mpl support all of the developmental step necessary for megakaryocytopoiesis. In the past few years, the signaling pathways utilized by this member of the cytokine receptor family have been extensively studied, especially JAK/STAT, Ras/MAP kinase, Shc, and other adapter molecules. Many if not most of the secondary signaling pathways activated by thrombopoietin have also been identified upon binding of other hematopoietic growth factors to their cognate receptors, making the study of Mpl signaling representative of the field in general. However, identifying unique molecules or combinations of signals that direct megakaryocyte development has been an elusive goal and has led some investigators to conclude that there is little specificity during Mpl signal transduction. In this article we review the data regarding Mpl signaling with particular attention to the methods employed and critical interpretation of the data generated. Future studies will have to focus on primary bone marrow cells and intact animal models rather than transformed cell lines. Furthermore, it is likely that a comprehensive, integrative analysis of the many pathways activated by ligand binding will be necessary to understand the physiology of cytokine signaling.     

Control of thrombopoietin-induced megakaryocytic differentiation by the mitogen-activated protein kinase pathway.

Thrombopoietin (TPO) is the major regulator of both growth and differentiation of megakaryocytes. We previously showed that both functions can be generated by TPO in the megakaryoblastic cell line UT7, in which murine Mpl was introduced, and are independently controlled by distinct regions of the cytoplasmic domain of Mpl. Particularly, residues 71 to 94 of this domain (deleted in the mutant mpl delta3) were found to be required for megakaryocytic maturation but dispensable for proliferation. We show here that TPO-induced differentiation in UT7 cells is tightly dependent on a strong, long-lasting activation of the mitogen-activated protein kinase (MAPK) pathway. Indeed, (i) in UT7-mpl cells, TPO induced a strong activation of extracellular signal-regulated kinases (ERK) which was persistent until at least 4 days in TPO-containing medium; (ii) a specific MAPK kinase (MEK) inhibitor inhibited TPO-induced megakaryocytic gene expression; (iii) the Mpl mutant mpl delta3, which displayed no maturation activity, transduced only a weak and transient ERK activation in UT7 cells; and (iv) TPO-induced megakaryocytic differentiation in UT7-mpl delta3 cells was partially restored by expression of a constitutively activated mutant of MEK. The capacity of TPO to trigger a strong and prolonged MAPK signal depended on the cell in which Mpl was introduced. In BAF3-mpl cells, TPO triggered a weak and transient ERK activation, similar to that induced in UT7-mpl delta3 cells. In these cells, no difference in MAPK activation was found between normal Mpl and mpl delta3. Thus, depending on the cellular context, several distinct regions of the cytoplasmic domain of Mpl and signaling pathways may contribute to generate quantitative variations in MAPK activation.