Megakaryocytic Activity Research Articles

Sphingosine kinase 2 (Sphk2) regulates platelet biogenesis by providing intracellular sphingosine 1-phosphate (S1P)

AbstractHuman megakaryocytes (MKs) release trillions of platelets each day into the circulation to maintain normal homeostatic platelet levels. We have previously shown that extracellular sphingosine 1-phosphate (S1P) plays a key role in thrombopoiesis via its receptor S1pr1. In addition to its role as an extracellular mediator, S1P can also function as a second messenger in the intracellular compartment. Although signaling via intracellular S1P is involved in various cellular processes, a role in thrombopoiesis has not been examined. Sphingosine kinases are the key enzymes that produce intracellular S1P. Here we report that sphingosine kinase 2 (Sphk2) is the major messenger RNA species present in MKs. Sphk2 predominantly localizes to the nucleus and is the major source of intracellular S1P in MKs. Loss of Sphk2 significantly reduced intracellular S1P in MKs and downregulated the expression and activity of Src family kinases (SFKs). Loss of Sphk2 and inhibition of SFK activity resulted in defective intravascular proplatelet shedding, the final stage of thrombopoiesis. Correspondingly, mice lacking Sphk2 in the hematopoietic system display thrombocytopenia. Together, our data suggest that Sphk2 provides the source of intracellular S1P that controls thrombopoiesis, which is associated with SFK expression and activity in MKs.

Induction of insulin resistance by the adipokines resistin, leptin, plasminogen activator inhibitor-1 and retinol binding protein 4 in human megakaryocytes

In normal platelets, insulin inhibits agonist-induced Ca(2+) mobilization by raising cyclic AMP. Platelet from patients with type 2 diabetes are resistant to insulin and show increased Ca(2+) mobilization, aggregation and procoagulant activity. We searched for the cause of this insulin resistance. Platelets, the megakaryocytic cell line CHRF-288-11 and primary megakaryocytes were incubated with adipokines and with plasma from individuals with a disturbed adipokine profile. Thrombin-induced Ca(2+) mobilization and signaling through the insulin receptor and insulin receptor substrate 1 were measured. Abnormalities induced by adipokines were compared with abnormalities found in platelets from patients with type 2 diabetes. Resistin, leptin, plasminogen activator inhibitor-1 and retinol binding protein 4 left platelets unchanged but induced insulin resistance in CHRF-288-11 cells. Interleukin-6, tumor necrosis factor-α and visfatin had no effect. These results were confirmed in primary megakaryocytes. Contact with adipokines for 2 hours disturbed insulin receptor substrate 1 Ser(307)-phosphorylation, while contact for 72 hours caused insulin receptor substrate 1 degradation. Plasma with a disturbed adipokine profile also made CHRF-288-11 cells insulin-resistant. Platelets from patients with type 2 diabetes showed decreased insulin receptor substrate 1 expression. Adipokines resistin, leptin, plasminogen activator-1 and retinol binding protein 4 disturb insulin receptor substrate 1 activity and expression in megakaryocytes. This might be a cause of the insulin resistance observed in platelets from patients with type 2 diabetes.

Regulation of fas/fas ligand‐mediated apoptosis by nuclear factor of activated T cells in megakaryocytes

Signal transduction pathways in megakaryocytes, a rare population of bone marrow cells, are poorly understood. We have previously shown that the calcineurin-dependent transcription factor Nuclear Factor of Activated T cells (NFAT) is expressed in megakaryocytes and is required for the transcription of specific megakaryocytic genes. The biological role of NFAT in megakaryocytes, however, is unknown. Here we show that activation of the calcineurin/NFAT pathway in megakaryocytes forces the cells to go into apoptosis. Calcineurin/NFAT activation in megakaryocytes leads to membrane expression of Fas ligand (FASLG), a pro-apoptotic member of the tumour necrosis factor superfamily. Expression of FASLG was augmented in cells stably overexpressing NFATC2 and suppressed in cells either pretreated with the calcineurin inhibitor ciclosporin A (CsA) or expressing the specific peptide inhibitor of NFAT, VIVIT. In cocultures with Fas-expressing Jurkat T cells, the presence of activated megakaryocytic cells, but not of unstimulated cells or cells stimulated in the presence of CsA, significantly induced apoptosis in Jurkat cells in a Fas/FASLG- and NFAT-dependent manner. These results represent the first evidence for a biological function of the calcineurin/NFAT pathway in megakaryocytes, and suggest that the biological role of megakaryocytes may include the induction of apoptosis in bystander cells.

Generation of Transgenic Mice for Platelet-Specific Expression of Human GPVI From GPVI-Knockout Mice

Abstract 1139Transgenic mice with human gene knock-in (KI) in platelets can be very useful tools for the evaluation of anti-thrombotic therapeutics in vivo, as many murine thrombosis models have been established and well-characterized. Previously we have successfully generated the GPVI-knockout (KO) mice (S Lockyer et al. Thromb. Res. 2006, 118: 371–80), and used the KO mice for the development of potent anti-human GPVI (hGPVI)-specific monoclonal antibodies (mAb) (Y Matsumoto et al. Thromb. Res. 2007, 119: 319–29), one of which (OM-2) was humanized for further clinical development. As the mAbs only recognize human and monkey GPVI but not murine counterpart, there is a need to develop hGPVI-expressing murine model for the evaluation of these mAbs in vivo. In the present study, we further introduced the hGPVI gene under the control of platelet-specific promoters into the fully back-crossed GPVI-KO C57BL/6 mice, by perivitelline space microinjection with lentivirus. In order to achieve platelet-specific expression, we cloned the promoters from human GPIIb, rat platelet factor 4 and murine GPIbα, confirmed their specific promoter activity in megakaryocytes and used in lentiviral vector construction for the generation of KI mice. Full length cDNA encoding human GPVI was cloned from platelet and put downstream of these promoters in the lentivirus. One-day old fertilized eggs from GPVI-KO mice were isolated. Concentrated lentiviruses were injected into the eggs with an automated Eppendorf micro-injection apparatus. Next day, embryos at two-cell stage were implanted into the uterus of pseudo-pregnant CD-1 mice. Six weeks after delivery, blood was collected for the examination of hGPVI expression in platelets and other cells with flow cyotmetry after immunofluorescent staining with FITC-conjugated OM-2. Results: We demonstrated the expression of hGPVI on the surface of platelets from the hGPVI-KI transgenic mice by immunofluorescent staining with OM-2 and with GPVI-specific FITC-convulxin. No significant difference was observed in platelet counts and other blood cells in these mice. It was further shown that the platelets from hGPVI-KI mice aggregated in platelet-rich plasma (PRP) and whole blood with collagen stimulation concentration-dependently and was inhibited by OM-2 Fab. The platelets from hGPVI-KI mice also adhered to fibrillar collagen-coated plate under both static and flow condition, similar to wild-type platelets, and was inhibited by OM-2 Fab. Pulmonary thromboembolism induced by co-injection of collagen and epinephrine was tested with the mice. GPVI-KO mice survived the pulmonary thromboembolism challenge due to lack of GPVI in platelets, but 9 out of 10 hGPVI-KI mice died after collagen/epinephrine injection. However the death of the KI mice (10 out of 10) were prevented by OM-2 Fab (0.2 mg/kg body weight) pre-injection. These data indicate that the expressed hGPVI in the platelets of the transgenic mice is fully functional, and the platelets activate to collagen similar to the platelets from wild-type mice. Using FITC-conjugated convulxin, we found the expression levels of hGPVI in the platelets from the hGPVI-KI transgenic mice were comparable to wild-type platelets. It is therefore concluded that GPVI-mediated platelet function is rescued in these hGPVI-KI transgenic mice from the GPVI-KO mice which manifest the lack of platelet response to collagen in aggregation and adhesion studies. These hGPVI-KI transgenic mice have been further used successfully in the evaluation of the role of GPVI in the development of rheumatoid arthritis (RA) and of the therapeutic effect of OM-2 in RA, and would be very useful and serve as cheap animal models for the evaluation of other anti-hGPVI therapeutics (such as for the reduction of cardiac infarct size after ischemic injury and reperfusion and prevention of cancer cell metastasis), and for pre-clinical toxicity and dose-finding studies. To our best knowledge, this is the first time of reported success of functional replacement of murine GPVI with its human homologue in mice. Disclosures:Sun:Otsuka: Employment. Tao:Otsuka: Employment. Xu:Otsuka: Employment. Zhang:Otsuka: Consultancy. Lockyer:Otsuka: Employment. Tandon:Otsuka: Employment.

Calpain Drives Megakaryopoiesis by Targeting the 7SK snRNP Complex: A Connection to Down Syndrome Megakaryocytic Neoplasia

Abstract 552Megakaryocytic differentiation uniquely relies on a hypertrophic program involving massive cellular enlargement and nuclear polyploidization. A critical mechanism driving this program consists of global P-TEFb activation, a process involving the release of an active Cdk9-cyclin T kinase module from a larger, inactive 7SK snRNP complex. We previously implicated the protease complex calpain 2/S1 in P-TEFb activation during megakaryopoiesis and demonstrated a direct contribution of calpain activity to megakaryocytic differentiation (Elagib et al., ASH 2009). The clinical relevance of this pathway was suggested by findings in patient samples and knockin mice of defective calpain 2 upregulation in megakaryocytes bearing the leukemogenic GATA-1s mutant. Current studies address: 1) the mechanism by which calpain 2/S1 activates P-TEFb during normal megakaryopoiesis, 2) the genetic program controlled by P-TEFb during megakaryopoiesis, and 3) the relevance of calpain 2 deficiency to abnormal megakaryopoiesis associated with GATA-1s. Regarding the mechanism of P-TEFb activation by calpain, analysis of a P-TEFb interactome database identified an interaction of calpain S1 with the protein BCDIN3 (Jeronimo CD. et. al. Mol Cell 2007). BCDIN3 stabilizes the 7SK snRNA through capping activity, but also exerts enzyme-independent stabilization by forming a core complex with 7SK snRNA and the protein LARP7. This core complex provides the foundation for assembly of the 7SK snRNP involved in restraining P-TEFb activity. Using adult human CD34+ cells cultured in unilineage megakaryocytic medium, both BCDIN3 and LARP7 proteins were found to undergo dramatic downregulation during differentiation, leading to global destabilization of 7SK snRNA. By contrast, neither BCDIN3, LARP7, nor 7SK snRNA underwent downregulation during erythroid differentiation. Megakaryocytic downregulation of BCDIN3, but not LARP7, required calpain activity. Calpain inhibitors as well as shRNA knockdown of calpain 2 prevented BCDIN3 downregulation during megakaryocytic cultures. In vitro reactions with recombinant purified factors confirmed direct cleavage of BCDIN3 by calpain 2/S1. The role of BCDIN3 downregulation in megakaryopoiesis was assessed by shRNA knockdown experiments. Notably, enforcing downregulation of BCDIN3 in CD34+ cells in erythroid medium reprogrammed differentiation away from the erythroid lineage and toward the megakaryocytic lineage. Thus BCDIN3 is a critical target of calpain 2/S1 during megakaryocytic activation of P-TEFb. To identify P-TEFb target genes, a bioinformatic approach was used to screen megakaryocytic expression databases for genes coregulated with known P-TEFb targets, e.g. HEXIM1. This approach yielded a cohort of coregulated genes encoding actin associated factors: ACTN1, FLNA, TGFB1I1, and MKL1. Immunoblot analsysis validated that all four factors underwent concomitant upregulation during megakaryocytic but not erythroid differentiation. shRNA knockdowns of calpain 2 and Cdk9 confirmed that upregulation of these factors depended on calpain and P-TEFb activation. Previous studies have indicated a requirement for ACTN1 downregulation in cytokinesis. Thus, shRNA knockdowns determined whether ACTN1 upregulation contributed to megakaryocytic polyploidization. Strikingly, ACTN1 knockdown blocked the polyploidization of human CD34+ cells but allowed other features of megakaryocytic differentiation, e.g. CD41 upregulation. Thus, a key aspect of the genetic program controlled by P-TEFb in megakaryopoiesis consists of the upregulation of a cohort of actin-associated factors which contribute to a hypertrophic program of cellular enlargement and polyploidization. To address the role of calpain in the aberrant megakaryopoiesis associated with mutant GATA-1s, fetal liver (fl) cells from GATA-1s knockin (G1s ki) mice were studied. Previous studies have shown a hyperproliferative phenotype in G1s ki fl but not adult megakaryocytes. In the current study, enforced calpain 2 expression by lentiviral transduction reversed this phenotype and enhanced terminal differentiation, reflected by polyploidization and CD42 expression. Thus, the defective upregulation of calpain 2 in megakaryocytes with GATA-1s most likely plays a critical role in the abnormal megakaryopoiesis seen in Down syndrome-associated megakaryoctyic proliferative disorders. Disclosures:No relevant conflicts of interest to declare.

Prevention of Chemotherapy-Induced Anemia and Thrombocytopenia by Constant Administration of Stem Cell Factor

Chemotherapy-induced apoptosis of immature hematopoietic cells is a major cause of anemia and thrombocytopenia in cancer patients. Although hematopoietic growth factors such as erythropoietin and colony-stimulating factors cannot prevent the occurrence of drug-induced myelosuppression, stem cell factor (SCF) has been previously shown to protect immature erythroid and megakaryocytic cells in vitro from drug-induced apoptosis. However, the effect of SCF in vivo as a single myeloprotective agent has never been elucidated. The ability of SCF to prevent the occurrence of chemotherapy-induced anemia and thrombocytopenia was tested in a mouse model of cisplatin-induced myelosuppression. To highlight the importance of maintaining a continuous antiapoptotic signal in immature hematopoietic cells, we compared two treatment schedules: in the first schedule, SCF administration was interrupted during chemotherapy treatment and resumed thereafter, whereas in the second schedule, SCF was administered without interruption for 7 days, including the day of chemotherapy treatment. The administration of SCF to cisplatin-treated mice could preserve bone marrow integrity, inhibit apoptosis of erythroid and megakaryocytic precursors, prevent chemotherapy-induced anemia, and rapidly restore normal platelet production. Treatment with SCF increased the frequency of Bcl-2/Bcl-XL-positive bone marrow erythroid cells and sustained Akt activation in megakaryocytes. Myeloprotection was observed only when SCF was administered concomitantly with cisplatin and kept constantly present during the days following chemotherapy treatment. SCF treatment can prevent the occurrence of chemotherapy-induced anemia and thrombocytopenia in mice, indicating a potential use of this cytokine in the supportive therapy of cancer patients.

Increased Expression of Proteasome-Related Genes In Patients with Primary Myelofibrosis

AbstractAbstract 4117 Introduction:The proteasome is an ubiquituous enzyme complex that plays a critical role in the degradation of many proteins involved in cell cycle regulation, apoptosis, and angiogenesis. Since these pathways and functions are often deregulated in cancer cells, inhibition of the proteasome is an attractive potential anticancer therapy. Bortezomib (Velcade, formerly PS-341) is an extremely potent and selective proteasome inhibitor that shows strong activity against many solid and hematologic tumor types. Moreover, bortezomib, mainly by inhibition of the NF-kappaB pathway, has a chemosensitizing effect when administered together with other antitumoral drugs. Bortezomib is a well-established treatment in multiple myeloma and studies are focusing in the potential benefit of bortezomib in other haematological malignancies, including malignant lymphomas. Since the NF-kappaB pathway is considered to be implicated in the abnormal release of cytokines in primary myelofibrosis (PMF), the proteasome inhibitor bortezomib might be a potential therapy. In a murine model, bortezomib has been demonstrated to inhibit thrombopoietin (TPO)-induced NF-kappaB activation in megakaryocytes and to reduce myeloproliferation induced by high TPO levels. Accordingly, from in vitro studies it was concluded that bortezomib might be a promising therapy for future treatment of PMF patients. Surprisingly, however, these encouraging results have not been achieved in clinical trials testing bortezomib in patients with myelofibrosis. We have performed gene expression profiling of patients with PMF and in patients with other chronic myeloproliferative neoplasms (CMPNs) in order to describe aberrant genes in the proteasome pathway in PMF. Materials and methods:The HG-U133 Plus 2.0 microarray from Affymetrix was used to profile expression of 38500 genes in whole blood from 70 patients with CMPNs, including 9 patients with PMF and 61 patients with other CMPNs. All patients were diagnosed according to the WHO criteria of a CMPN (ET=19, PV=41, PMF=9). The patients were diagnosed and followed in two institutions. Most patients were studied on cytoreductive therapy, which for the large majority included hydroxyurea. Total RNA was purified from whole blood and amplified to biotin-labeled aRNA and hybridized to microarray chips.Differences in gene expression between the two groups were calculated for each gene in the dataset by using Welch two sample t test, and the Benjamini Hochberg method was applied to control for multiple hypothesis testing (false discovery rate (FDR) < 0.05). Data were integrated with biological pathways and networks using Gene Microarray Pathway Profiler (GenMAPP 2.1) and Cytoscape 2.6.3, respectively. Hypothesis driven discovery was used to find significantly differentially expressed genes and pathways associated with PMF. Results:Single gene analysis demonstrated significantly elevated expression of seventeen proteasomal subunit genes in patients with PMF (PSMA1, PSMA2, PSMA6, PSMA7, PSMB4, PSMB5, PSMB6, PSMB7, PSMC2, PSMC3, PSMD10, PSMD14, PSMD4, PSMD8, PSMD9, PSMG1, and PSMG3 (FDR < 0.05). Only one gene, PSMB4, was significantly downregulated (FDR < 0.05). Global pathway analysis showed a significant upregulation of the proteasome degradation pathway (adjusted P < 0.03), and the network analysis revealed a significant subnetwork only composed of upregulated genes (CDC25A, CDC6, CDT1, GMNN, ORC1L, PSMA6, PSMA7, PSMB5, PSMB6, PSMB7, PSMC3, PSMD5, PSMD8, PSMD9, PSMD14) of which 10 were proteasomal genes (Z=2.6). Conclusion:In this study, we have for the first time described the gene signature of the proteasome in peripheral blood cells from patients with myelofibrosis and patients with ET and PV. Using single gene analysis, global pathway and network analysis, we found significant upregulation of the proteasomal transcriptome in patients with PMF as compared to patients with ET and PV as a group. This study has added new important information of the genes involved in the upregulation of the proteasome degradation pathway in these patients. Disclosures:No relevant conflicts of interest to declare.

Novel Role for Host-Derived Megakaryocytes In Facilitating Stem Cell Engraftment through Enhancement of Osteoblastic Niche Restoration Following Radioablation

AbstractAbstract 558The osteoblastic niche is a critical site of engraftment following stem cell transplantation. We have previously demonstrated that the osteoblastic niche expands within the first 48 hours following marrow radioablation. This expansion is accompanied by relocalization of megakaryocytes from their homeostatic location within the central marrow space to the endosteal surface at sites of osteoblast expansion, suggesting that these relocalized megakaryocytes may play a key role in reconstitution of the niche. To determine whether megakaryocytes contribute to the radioablation-induced osteoblast expansion and consequently assist in facilitating engraftment, we have examined osteoblast expansion and stem cell engraftment in wildtype (WT) mice, thrombopoietin receptor (c-Mpl) deficient mice (mpl−/−) that have less than 20% of normal megakaryocyte numbers, and in mice treated with an anti-CD41 blocking antibody (MWREG30). BrdU incorporation and TUNEL assays demonstrated that megakaryocyte relocalization following radioablation occurs through active migration of viable megakaryocytes. mpl−/− mice or anti-CD41 treated WT mice developed less than 20% of the megakaryocyte endosteal migration seen in untreated WT mice 48 hours after radioablation (*P<0.001), and anti-CD41 treatment of irradiated mpl−/− mice reduced endosteal megakaryocytes to baseline pre-radiation numbers. Moreover, mpl−/− mice developed less than 50% of the osteoblast expansion seen in WT mice following irradiation; thus, abrogating megakaryocyte activity blocks critical signals required for osteoblast expansion. Bone marrow expression of IGF-1, a recognized osteoblast growth factor, increased 5-fold within 48 hours after radiation relative to pre-radiation levels in WT mice, but this IGF-1 spike is completely blocked by c-Mpl deficiency, suggesting that megakaryocytes may induce osteoblast expansion through a pathway in which c-Mpl signaling leads to IGF-1 expression. To test the functional significance of radiation-induced megakaryocyte migration and osteoblast expansion, we transplanted lethally irradiated WT or mpl−/− mice with or without anti-CD41 treatment using bone marrow from GFP-transgenic mice. Initial bone marrow engraftment of WT GFP+ donor cells within 24h of transplantation was significantly reduced (*P<0.01 for all groups) in anti-CD41 treated WT (51% of engraftment seen in untreated wildtype recipients), untreated mpl−/− (45%), and anti-CD41 treated mpl−/− (35%) recipients. Expansion of engrafted WT GFP+ donor cells at 3, 5 and 7 days post-transplant was also significantly reduced in untreated mpl−/− recipients (36%, 53%, 63% of untreated WT recipients at 3, 5, and 7 days, respectively, *P<0.05) and anti-CD41 treated mpl−/− recipients (25%, 33%, and 30% at 3, 5, and 7 days, respectively, *P<0.05), with prominent deficits specifically in the reconstitution of the B lymphocyte lineage. Bone marrow cellularity remained significantly reduced in anti-CD41 treated WT and untreated or anti-CD41 treated mpl−/− recipients by 35–45% relative to untreated WT recipients at least out to 3 weeks post-transplant (*P<0.01). Using competitive repopulation secondary transplantation assays performed with marrow harvested from primary recipients at 24h after primary transplantation, we showed that progenitor and short term hematopoietic stem cell (HSC) engraftment was significantly decreased in mpl−/− versus WT primary recipients (*P<0.05). Secondary transplant assays performed with marrow harvested from primary recipients 3 weeks after initial transplantation demonstrated that engraftment and expansion of long term-HSC (*P <0.05) and B cell reconstitution (*P<0.005) are significantly impaired in anti-CD41 treated mpl−/− versus untreated WT recipients. Taken together, our findings demonstrate that host megakaryocytes migrate to the endosteal surface following marrow radioablation where, through the enhancement of osteoblast niche expansion and potential osteoblast-independent effects, they play a pivotal role in facilitating efficient HSC engraftment following transplantation. Further understanding of these stem cell niche restoration pathways may reveal novel therapeutic targets to improve engraftment efficiency in the clinical setting. Disclosures:No relevant conflicts of interest to declare.

Correlation between immature platelet fraction and reticulated platelets. Usefulness in the etiology diagnosis of thrombocytopenia

Reticulated platelets (RP) are a surrogate marker for megakaryocytic activity, but the limitation of this determination is the lack of standardization of methodology. The determination of the immature platelet fraction (IPF) is performed in a simple, automated, and reproducible way between laboratories. We analyzed the correlation between IPF and RP, and usefulness of IPF in patients with thrombocytopenia. RP were determined by flow cytometry using double staining with thiazole orange and CD61 PerCP. IPF was performed with Sysmex XE2100 analyzer. We used a control group with normal platelets, and thrombocytopenic patients were classified into three groups: Group 1. Central thrombocytopenia, Group 2. Thrombocytopenia as a result of enhanced peripheral platelet destruction, and Group 3. Peripheral non-immune thrombocytopenia by abnormal distribution. Fourteen controls and 66 patients were analyzed. Group 1: 25 patients, they had mean and confidence interval 95% (95% CI) for IPF 8.67% (6.49-10.46%) and RP 4.08% (2.86-5.30%). Group 2: 20 patients, they had mean and 95%CI for IPF 16.80% (12.20-21.39%) and RP 16.14% (9.89-22.40%). Group 3: 21 patients, they had mean and 95% CI for IPF 9.04% (6.95-11.14%) and RP 5.23% (3.41-7.05%). The overall Pearson linear correlation between IPF and RP was r: 0.65. There were statistically significant differences in values of IPF and RP between Group 2 and the other two groups (P < 0.01). There is a good correlation between IPF and RP mainly in thrombocytopenia by peripheral destruction. Determination of IPF is an easy technique in their implementation, standardized and reproducible, so it could be a useful screening technique in patients with thrombocytopenia.

Lineage-specific combinatorial action of enhancers regulates mouse erythroid Gata1 expression

AbstractPrecise spatiotemporal control of Gata1 expression is required in both early hematopoietic progenitors to determine erythroid/megakaryocyte versus granulocyte/monocyte lineage output and in the subsequent differentiation of erythroid cells and megakaryocytes. An enhancer element upstream of the mouse Gata1 IE (1st exon erythroid) promoter, mHS−3.5, can direct both erythroid and megakaryocytic expression. However, loss of this element ablates only megakaryocytes, implying that an additional element has erythroid specificity. Here, we identify a double DNaseI hypersensitive site, mHS−25/6, as having erythroid but not megakaryocytic activity in primary cells. It binds an activating transcription factor complex in erythroid cells where it also makes physical contact with the Gata1 promoter. Deletion of mHS−25/6 or mHS−3.5 in embryonic stem cells has only a modest effect on in vitro erythroid differentiation, whereas loss of both elements ablates both primitive and definitive erythropoiesis with an almost complete loss of Gata1 expression. Surprisingly, Gata2 expression was also concomitantly low, suggesting a more complex interaction between these 2 factors than currently envisaged. Thus, whereas mHS−3.5 alone is sufficient for megakaryocytic development, mHS−3.5 and mHS−25/6 collectively regulate erythroid Gata1 expression, demonstrating lineage-specific differences in Gata1 cis-element use important for development of these 2 cell types.

Ectopic Expression and Role of RCAN1 in Myeloid Leukemia Cells.

Abstract 1274Poster Board I-296 IntroductionRCAN1 (also called DSCR1, calcipressin 1, MCIP1, Adapt78) is known to be a regulator of calcineurin, a Ca2+/calmodulin-dependent protein phosphatase, and has also been shown to regulate the activity of certain transcription factors, including NF-AT and NF-kB. RCAN1 expression is widely found in various tissues, including the brain, kidney, liver and heart, but the expression level of RCAN1 is extremely low in normal hematopoiesis-related organs (bone marrow and spleen). Although RCAN1 has been shown to act as a negative regulator of FceRI-induced mast cell activation and NF-AT signaling in megakaryocytes, the cellular function of RCAN1 in myeloid cells remains unknown. Recently, we found that RCAN1 was highly expressed in acute phase of chronic myeloid leukemia (CML) by DNA microarray analysis. In this study, to explore the role of RCAN1 in leukemogenesis, we compared RCAN1 expression in leukemia cells and that in normal hematopoietic cells. We also analyzed regulatory mechanisms of RCAN1 gene promoter function and searched for novel RCAN1-interacting molecules in myeloid leukemia cells. MethodsExpression of RCAN1 was examined by quantitative PCR using normal human bone marrow cells, bone marrow cells from patients with acute myeloid leukemia (AML) and patients with CML, and various human hematological tumor cell lines. Promoter activity of the human RCAN1 gene was evaluated by a luciferase assay using various deletion mutants in KCL22 cells. To identify novel RCAN1-interacting proteins, a yeast two-hybrid assay was performed using a cDNA library from K562 leukemia cells. ResultsFractions of CD34+CD38-, CD34+CD38+, CD33+CD14-CD16-, CD33+CD14+ and CD33+CD16+ cells from normal human bone marrow showed extremely low levels of RCAN1, indicating that RCAN1 is scarcely expressed in normal hematopoietic cells, including stem cells and progenitor cells. In contrast, bone marrow mononuclear cells (BMMC) from 15 of 16 patients with AML and all four patients with CML in acute phase showed substantial levels of RCAN1. Furthermore, RCAN1 was expressed in all human cell lines examined in this study that were derived from AML (THP-1, U937, HL60, KY821), CML in acute phase (KCL22,K562, KU812), acute lymphoblastic leukemia (Jarkat, Raji) and multiple myeloma (RPMI8226, U266). Interestingly, expression levels of RCAN1 were significantly decreased in BMMC from three of four AML patients when the patients were in complete remission. These results suggest that RCAN1 is extraordinarily expressed in a wide range of hematological tumor cells. To reveal the underlying mechanisms of ectopic expression of RCAN1, we next examined the promoter function of RCAN1 gene, which consists of seven exons. Although RCAN1 expresses several alternative isoforms that selectively include each of exons 1-4 and use distinct promoters, we found that only exon 1 isoform was expressed in these leukemia cells. Analysis of the function of human RCAN1 gene promoter upstream of exon 1 demonstrated that NKX2-5 binding site is important for basal activity of RCAN1 gene promoter in KCL22 cells. NKX2-5 is a homeobox-containing transcription factor and is involved in leukemogenesis of T-cell acute lymphoblastic leukemia. Our results raised the possibility that NKX2-5 plays a role in malignant transformation partly through expression of RCAN1 in myeloid leukemia cells. Finally, we searched for RCAN1-interacting proteins in leukemia cells. By screening a cDNA library from K562 cells using yeast two-hybrid assay, we found that RCAN1 interacts with HINT1, HINT2 and RINT1. HINT1 has been reported to be a tumor suppressor against hepatocellular carcinoma, and HINT2 and RINT1 have also been found to function as tumor suppressors. It would be interesting to clarify whether RCAN1 affects the activity of these molecules, and it is important to reveal the involvement of these molecules in leukemogenesis. ConclusionRCAN1 is extraordinarily expressed in a wide range of hematological malignant cells, and NKX2-5 might be important for its ectopic expression. It is possible that RCAN1 is biologically significant in leukemogenesis through affecting certain tumor suppressors. DisclosuresNo relevant conflicts of interest to declare.

Characterization and transplantation of induced megakaryocytes from hematopoietic stem cells for rapid platelet recovery by a two-step serum-free procedure

A complete process for mass generation of megakaryocytes from hematopoietic stem cells under serum-free conditions has great clinical potential for rapid platelet reconstruction in thrombocytopenia patients. We have previously reported on the generation of an optimized serum-free medium (serum-free hematopoietic stem cell medium) for ex vivo expansion of CD34(+) cells. Here, we further generated large amounts of functional megakaryocytes from serum-free expanded CD34(+) cells under a complete and optimal serum-free condition for complying with clinical regulations. Serum substitutes and cytokines were screened and optimized for their concentration for megakaryocyte generation by systemically methods. Serum-free induced megakaryocytes were characterized by surface antigens, gene expression, ex vivo megakaryocyte activation ability, and ability of megakaryocyte and platelet recovery in nonobese diabetic/severe combined immunodeficient mice. The optimal serum-free megakaryocyte induction medium was Iscove's modified Dulbecco's medium containing serum substitutes (i.e., human serum albumin, human insulin, and human transferrin) and a cytokine cocktail (i.e., thrombopoietin, stem cell factor, Fms-like tyrosine kinase 3 ligand, interleukin-3, interleukin-6, interleukin-9, and granulocyte-macrophage colony-stimulating factor). After induction, induced megakaryocytes expressed CD41a and CD61 surface antigens, nuclear factor erythroid-derived 2 and GATA-1 transcription factors and megakaryocyte activation ability. Importantly, transplantation of induced megakaryocytes could accelerate megakaryocyte and platelet recovery in irradiated nonobese diabetic/severe combined immunodeficient mice. In conclusion, we have developed a serum-free megakaryocyte induction medium, and the combination of serum-free megakaryocyte and serum-free hematopoietic stem cell media can generate a large amount of functional megakaryocytes efficiently. Our method represents a promising source of megakaryocytes and platelets for future cell therapy.

Regulation of Down Syndrome Critical Region 1 expression by Nuclear Factor of Activated T cells in megakaryocytes

As precursors of platelets, megakaryocytes must fulfil the complex tasks of protein synthesis and platelet assembly. Megakaryocytic dysfunction can lead to neoplastic disorders, such as acute megakaryoblastic leukaemia, an entity with a 500-fold increased incidence in children with Down syndrome (DS). Down Syndrome Critical Region 1 (DSCR1), a member of the calcipressin family of calcineurin inhibitors, is overexpressed in DS, and destabilization of the calcineurin/Nuclear Factor of Activated T cells (NFAT) pathway by overexpression of DSCR1 has been implicated in some of the pathophysiological features of the disease. The roles of NFAT and DSCR1 in megakaryocyte signalling and gene expression, however, are unknown. In this study, we show that calcineurin and NFAT are components of a calcium-induced signalling cascade in megakaryocytes. NFAT activation in megakaryocytes was induced by fibrillar collagen type I and was completely sensitive to the calcineurin inhibitor cyclosporin A. We established DSCR1 as a calcium-induced NFAT target gene in these cells and show that overexpression of DSCR1 in megakaryocytes strongly inhibits NFAT activation as well as NFAT-dependent expression of the Fas ligand gene (FASLG). These results suggest that DSCR1 acts as an endogenous feedback inhibitor of NFAT signalling in megakaryocytes, and may have implications for megakaryocytic gene expression in DS.

Reciprocal Regulation of DSCR1 (Down Syndrome Critical Region 1) Expression and NFAT (Nuclear Factor of Activated T Cells) Activation in Megakaryocytes.

NFAT (Nuclear Factor of Activated T cells) is a family of calcium-induced, calcineurin-dependent transcription factors, well characterized as central regulators of inducible gene expression in T lymphocytes but now known to function also in several other cell types in various adaptation and differentiation processes. Activation of NFAT by the phosphatase calcineurin is counteracted by several inhibitory kinases and can be completely blocked by the immunosuppressant Cyclosporin A. The Down syndrome critical region 1 (DSCR1; also termed CSP1, MCIP1 or RCAN1) gene belongs to the calcipressin family of endogenous calcineurin inhibitors and is expressed in several isoforms, one of which (isoform C, coded by exons 4–7) has been described to be a transcriptional target for NFAT in striated muscle, endothelial, and neural cells. The DSCR1 gene is located within the Down syndrome critical region of human chromosome 21 and is, together with 200–300 other genes, overexpressed about 1.5-fold in patients with Down syndrome (DS). Previously, dysregulation of NFAT signaling by overexpression of DSCR1 has been implicated in causing various of the pathophysiological features observed in DS patients. Children with DS also suffer from an about 500-fold increased incidence of acute megakaryocytic leukemia; the respective roles of NFAT or DSCR1 in megakaryocytes of either normal individuals or those with DS, however, has not yet been established. Here we show that DSCR1 is upregulated during megakaryocytic differentiation in a lineage-specific manner, and in mature megakaryocytes is further strongly induced by calcineurin stimulation. DSCR1 expression in megakaryocytes is regulated by NFAT, since overexpression of NFATc2 enhances, while overexpression of the specific inhibitor of NFAT activation, VIVIT, suppresses expression of the gene. We further demonstrate that DSCR1 does not only represent an NFAT target in megakaryocytes, but itself acts an inhibitor of NFAT signaling in these cells. Overexpression of DSCR1 in CMK cells as well as in primary megakaryocytes by retroviral transduction profoundly suppressed ionomycin-induced dephosphorylation and nuclear translocation of NFATc2, as well as transactivation of an NFAT-dependent promoter construct. Finally, overexpression of DSCR1 in megakaryocytes markedly downregulated both the constitutive and induced expression of Fas Ligand, a pro-apoptotic gene recently established as a NFAT target in megakaryocytes. Together, these results suggest that DSCR1 acts as an NFAT-induced NFAT inhibitor in megakaryocytes and, when overexpressed, interferes with the expression of NFAT-dependent megakaryocytic genes.

The Calcineurin/NFAT Signaling Pathway Is a Target for the Collagen Type I-Induced Calcium Response in Megakaryocytes and Mediates Expression of Megakaryocytic Genes.

Megakaryocytes, as precursor cells of platelets, comprise a cell population crucial for the maintenance of adult hematopoiesis. Since platelets are anuclear cellular fragments, the platelet transcriptome as well as the proteome are largely determined by the megakaryocyte. Calcium signaling in response to agonists such as collagen or thrombin has a central function in platelet activation and therefore likely represents an important signaling pathway in megakaryocytes as well. Indeed, megakaryocytes show considerable elevation of intracellular calcium levels in response to selected platelet agonists, but the downstream effector molecules of calcium signaling in these cells or the transcriptional responses induced are unknown. We here establish calcineurin and the NFAT (Nuclear Factor of Activated T cells) family of transcription factors as components of a calcium-induced signaling cascade in megakaryocytes. In resting megakaryocytes, NFAT is cytoplasmic and inactive, but can be activated by fibrillar collagen type 1, a physiological agonist of platelets and megakaryocytes known to induce a sustained increase in intracellular calcium levels in these cells. In contrast, treatment with SDF-1a, thrombopoietin, or VEGF remained without noticeable effect, presumably because of the short duration of the calcium transients induced by these agents. Collagen-induced NFAT activation in megakaryocytes requires dephosphorylation by calcineurin and is completely sensitive to the calcineurin inhibitor cyclosporin A. Activation of NFAT by collagen was paralleled by the induction of the expression of Down Syndrome Critical Region I (DSCR1) and Fas Ligand (FasL), two genes recently identified as NFAT targets in megakaryocytes. Collagen-induced expression of DSCR1 and FasL occurred in a calcineurin- and NFAT-dependent manner, as it was blocked by both cyclosporin A as well as the specific peptide inhibitor of NFAT, VIVIT. These experiments show that the calcineurin pathway is a target for selected physiological ligands capable of inducing sustained calcium mobilization in megakaryocytes and regulates megakaryocyte gene expression in a cyclosporin A- and NFAT-dependent manner.

Blockade of Chemotherapy-Induced Thrombocytopenia by Bax Inhibiting Peptides (BIPs) in Mouse Model.

Thrombocytopenia is a major side effect of cancer chemotherapy producing bleeding problems as well as cancer treatment delays. To preclude it, the platelets progenitors megakaryocytes need to be protected from drug-induced apoptosis. According to previous reports, anti-apoptotic molecules such as Bcl-2, Bcl-xL, and caspase inhibitors were found to interfere platelet formation by megakaryocytes, thus these apoptosis inhibitors are not appropriate therapeutic candidates for the prevention of drug-induced thrombocytopenia. Recently we developed a new type of cell permeable apoptosis inhibitor, Bax Inhibiting Peptides (BIPs), which are derived from the Bax binding domain of Ku70. Bax is a key mediator of apoptosis, and Ku70 is a multifunctional protein playing roles in cell survival and DNA repair. In the present study, we determined whether BIPs prevent chemotherapy-induced thrombocytopenia both in vitro and in vivo. Importantly, there are several cancer cell types that acquired mutations in genes of Bax and/or Bax activating factors. Therefore, we hypothesize that protection of non-tumorigenic essential cells (such as megakaryocytes) by BIPs is beneficial for patients suffering from Bax-negative cancer. For in vitro study, Dami cells (human pro-megakaryocyte cell line) and mouse primary cultured megakaryocytes were used. BIPs significantly inhibited apoptosis induction by anti-cancer drugs such as etoposide, cisplatin, doxorubicin, or paclitaxel. Importantly, BIPs did not interfere the activity of megakaryocytes to produce platelet-like particle in cell culture, suggesting that BIPs protect megakaryocytes from drug-induced apoptosis without suppressing their platelets production. For in vivo study, C57BL/6J mice and Bax-/- mice (backcrossed with C57BL/6J) were used. Etoposide (150 mg/kg, i.p.) induced a significant decrease of platelet count in whole blood within 2 days after the drug administration in wild type C57BL/6J mice. Pretreatment of mice with BIPs (166 mg/kg, i.p., 30 min before etoposide injection) blocked etoposide-induced platelet count decrease in these wild type mice. The same dose of etoposide did not induce significant platelet count decrease in Bax-/- mice. These results suggest that chemotherapy-induced thrombocytopenia, at least in part, is due to the stimulation of Bax-mediated cell death probably both in megakaryocytes and platelets. In our preliminary experiments, three times injection of caspase inhibitor (z-VAD-fmk, 166 mg/kg, i.p. every 24 hrs) caused prolongation of bleeding time from the wound (tail cut). Thus, as previously reported by other groups using in vitro system, caspase inhibitor may have its own side effect to inhibit platelet formation or/and activation in vivo. On the other hand, BIPs (166 mg/kg, 3 times every 24 hrs, i.p.) did not extend bleeding time from the tail. Taken together, BIP may become a useful supplemental therapeutic agent to block chemotherapy-induced thrombocytopenia.

Thrombocytopoiesis Is Not Regulated by Thrombopoietin (TPO) but Proplatelet Formation (PPF) Stimulating Factor.

TPO is believed to be the physiological humoral hematopoietic factor in megakaryopoiesis and thrombopoiesis. Some investigators reported the inverted relationship between platelet mass and serum TPO levels, resulting in the conclusion that the platelet mass may directly play a role of regulating the circulating TPO levels. In the previous ASH meeting, we reported that serum TPO levels were not elevated in Immune thrombocytopenic purpura (ITP) patients. In this study, we measured platelet count, the serum TPO level, % reticulated platelet (%RP) and megakaryocytes mass in patients with refractory anemia in myelodysplastic syndrome (MDS-RCMD). The mean plasma TPO level in 20 healthy persons was 57.3±15.2 pg/ ml, the percentage of RPs was 1.51±0.71%.Plasma TPO levels were widely distributed (799.9±883.0 pg/ ml, n=35) in MDS-RCMD, while they were less than 131.0 pg/ml in ITP (48.5±37.0 pg/ ml, n=37). There were no significant relationship between platelet counts and plasma TPO levels in MDS-RCMD or ITP. These results indicated that plasma TPO level was not regulated by the platelet mass at least in thrombocytopenic patient with MDS-RCMD or ITP. We evaluated the thrombopoiesis in thrombocytopenic patients with MDS-RCMD or ITP. %RPs were 1.9±0.2 % in MDS-RCMD (n=34), indicating that increased thrombopoiesis was not observed in MDS-RCMD, in which serum TPO levels were increased. While %RPs were 6.6±4.9 % in ITP (n=45), indicating that thrombopoiesis was observed in ITP, in which serum TPO levels were not increased. These results indicated that thrombopoiesis was not regulated by plasma TPO levels but some thrombopoiesis stimulating factor(s). Therefore, we evaluated the thrombopoiesis stimulating activity in plasma of patients with MDS-RCMD or ITP as the indicator of PPF stimulating activity of murine megakaryocytes. PPF stimulating activity was measured as the ratio of PPF bearing megakaryocytes/ viable megakaryocytes after 48 hrs serum-free culture of murine megakaryocytes.As the plasma have some inhibitory effects of PPF activiy, the HDL fraction, isolated from plasma, was used as the methods described previously in Ishida Y et al. (Thrombosis and Haemostasis , 2001; 85:349–55).The higher PPF stimulating activity was observed in patients with ITP (n=15) or MDS-RCMD (n=15) than that with healthy donors (n=10) {healthy donors 26.1±5.4 % versus ITP 38.0± 6.0% (p<0.01), MDS-RCMD 41.9±5.3%(p<0.01)}. There was a significant correlation between the PPF activity and platelet counts (R=0.68; p<0.01) in these thrombopenic patients. These data strongly suggest that thrombopoiesis may not be regulated by TPO but some PPF stimulating factor(s).

Septic peripheral embolization from Haemophilus parainfluenzae endocarditis

A 40-year-old, previously healthy lady with good dentition, presented with pyrexia of unknown origin for 6 weeks. Physical examination was unremarkable. Initial cultures were negative. Rheumatoid factor was elevated but she did not have any joint symptoms. Because of severe thrombocytopenia which developed during hospitalization, bone marrow examination was done, which showed normal megakaryocytic activity and reactive haemophagocytosis. …

Proliferative Activity of Megakaryocytes in Myelo- and Lymphoproliferative Diseases.

Aim of the study: Evaluation of significance of investigation of Ki67 and AgNOR staining as an indices of proliferative activity of megakaryocytes.Material and methods: Bone marrow smears from 151 patients suffering from hematological malignancies: polycythemia vera (PV) − 14 cases, myelofibrosis (MF) − 14, essential thrombocythemia (ET) − 15, chronic myeloid leukemia (CML) − 11, myelodysplastic syndromes (MDS) − 18; acute myeloid leukemia (AML) − 30, acute lymphoblastic leukemia (ALL) − 10, low grade non-Hodgkin's lymphomas (NHL) with bone marrow infiltration − 20, multiple myeloma (MM) − 19. Cytological slides were stored in the room temperature more than 24 hours and then stained using immunocytochemical method for Ki67 and cytochemical method for AgNOR. Slides were analyzed using illuminating microscope with digital image analysis (software LUCIA). Mean particles area, mean nuclear area and maturity of nucleus in AgNOR staining as well as intensity of Ki67 expression were evaluated, according to arbitrary scale (strong, medium, light, no reaction).Results: Look at the table 1 and table 2.Conclusions:There is a good correlation between expression of Ki-67 and AgNOR's in megakaryocytes as well as between nuclear maturity and indices of Ki-67 and AgNOR in myeloproliferative disorders. Differences in AgNOR's and Ki 67 staining area between myeloproliferative diseases and another hematological malignancies are also significant.Table 1:Values of indices of the Ki67 and AgNOR in megakaryocytesDiseasesMean nucleus surface μm2Mean AgNOR particle area in nucleus of megakaryocytes μm2Mean Ki67 particle area in nucleus of megakaryocytes μm2Intensity of Ki67 expression in megakaryocytesGrade of megakaryocytes maturityPV667,290,680,19MediumIII and IVET812,320,820,29Strong / MediumIII and IVMF767,920,550,26Strong / MediumIII / IVCML734,300,570,15Medium / LightIII / IVMDS645,670,060,03LightIII / IVAML679,040,04No reactionNo reactionIVALL712,230,03No reactionNo reactionIVNHL798,560,04No reactionNo reactionIVMM629,490,04No reactionNo reactionIVTable 2:Intensity of Ki67 expression in megakaryocytes in hematological disordersDiseasesStrong – % of cellsMedium – % of cellsLight – % of cellsNo reaction – % of cellsPV3643147ET5633110MF5132161CML1643347MDS9126811AML0000ALL0000NHL0000MM0000

Megakaryocyte apoptosis: sorting out the signals

British Journal of Pharmacology (2005) 145, 271–273. doi:10.1038/sj.bjp.0706202