Primary Megakaryocytes Research Articles

Lactoferrin Inhibits Platelet Production from Human Megakaryocytes in Vitro

The mechanism of megakaryopoiesis, proplatelet formation (PPF) and platelet (PLT) production is not fully elucidated. Lactoferrin (LF) has been reported to have many biological functions including cell proliferation and differentiation, and the LF receptor is present on megakaryocytic cells. In the present study, we examined the effect of human LF (hLF) on PLT production from primary megakaryocytes (MKs). At first, we developed a PLT production system derived from human CD34+ cells by thrombopoietin (TPO) stimulation. Because the number of proplatelets, PLTs and CD41+ MKs was remarkably increased after day 5, we employed the TPO-induced CD34+ cells on day 5. Then, the effect of hLF on PLT production from human primary MKs was examined. In the range of 3-30 micrg/ml, hLF significantly inhibited PLT production up to about 60%. However, it did not significantly change the intensity of CD41 expression in MKs and the ploidy of MKs. In addition, it did not inhibit MK progenitors. These results suggest that LF directly inhibits PLT production from matured MKs, but does not inhibit megakaryopoiesis, including proliferation/maturation processes.

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.

Interferon-α2b-Induced Thrombocytopenia Is Caused by Inhibition of Cytoplasmic Maturation and Platelet Production, but Not Proliferation and Endomitosis in Human Megakaryocytes.

Human interferon (IFN)-α is a standard treatment for the patients with chronic hepatitis C. It is well known that chronic hepatitis C can transform into liver cirrhosis and hepatocellular carcinoma, and the risk of progression to liver cirrhosis is around 40% in 5 years. Thus, it is important for chronic hepatitis C patients to receive IFN-α treatment to prevent its malignant transformation. Thrombocytopenia is one of the major adverse effects of IFN-α and often leads to a dose reduction or discontinuation of IFN-α therapy. However, there is little information how IFN-α inhibits human megakaryopoiesis. In this study, we demonstrated that IFN-α does not inhibit colony formation of megakaryocytes (CFU-MK) from human CD34-positive hematopoietic stem cells. IFN-α also does not inhibit endomitosis (DNA duplication without cytokinesis), but inhibits cytoplasmic maturation of megakaryocytes and platelet production in vitro. The inhibitory effects of IFN-α on the development of demarcation membrane in human megakaryocytes were visualized by staining primary megakaryocytes with di-8-ANEPPS dye under a confocal laser microscope. Loss of platelet production by IFN-α was analyzed by measuring proplatelet formation (PPF) and counting the human platelets produced in the supernatant of human primary megakaryocytes by flow cytometry. The expression of transcription factors regulating late stage megakaryopoiesis such as GATA-1, p45 NF-E2 and MafG was suppressed by IFN-α. To confirm these in vitro observations in vivo system, we transplanted human CD34-positive hematopoietic cells into the immunodeficient NOD/Shi-scid/IL-2Rγnull (NOG) mice (hu-NOG) after 2.4 Gy irradiation. Recombinant human(rh) IFN-α significantly reduced the number of human platelets but did not decrease the number of human megakaryocytes in hu-NOG mice. rhIFN-α did not change murine platelet counts in hu-NOG mice, as IFN-α has species specificity. The DNA ploidy of human megakaryocytes in the bone marrow of hu-NOG mice was not altered after treatement with rhIFN-α. We also confirmed the life time of human platelets was not shortend by rhIFN-α in hu-NOG mice, indicating IFN-α does not promote the clearance of human platelets. We have also confirmed that a novel thrombopoietin mimetics, NIP-004, prevented rhIFN-α-induced thrombotytopenia and increased the number of polyploid human megakaryocytes in hu-NOG mice, suggesting NIP-004 might be useful for the patients with hepatitis C to avoid IFN-α-induced thrombocytopenia. In conclusion, these results clarified that IFN-α induces thrombocytopenia by inhibiting the cytoplasmic maturation of megakaryocytes but not proliferation and endomitosis in human megakaryocytes.

TPO-Induced miR-150 Down-Regulates c-Myb in Primary Megakaryocytes and a Megakaryocytic Cell Line.

Mice harboring c-Myb hypomorphic mutations display enhanced thrombopoiesis because of increased numbers of megakaryocytic progenitors (CFU-MK) and mature megakaryocytes (MK). Thrombopoietin (Tpo), the primary regulator of megakaryopoiesis, induces these same effects, which lead us to hypothesize that Tpo might act, at least in part, through modulation of c-Myb expression. We found using quantitative (Q)-PCR that c-Myb mRNA levels were 13-fold reduced during Tpo-induced MK maturation. Micro RNAs (miRs) are ∼22 nucleotide species that down-regulate gene expression by binding to the 3′ untranslated region (UTR) of specific mRNAs, enhancing mRNA degradation, or by reducing mRNA translation efficiency. We noted that the 3′UTR of c-Myb contains a number of miR target sites, including four that bind miR150; using a specific Q-PCR assay we also found that Tpo increased mir-150 expression to 160% of baseline at 24 hr and 250% at 48 hr in UT7/TPO cells (n=2 experiments). To test if miR150 affects c-Myb expression, we introduced the 3′UTR of c-Myb into a luciferase reporter gene (pCMV-luc-3′UTRcMyb), in which CMV promoter-driven luciferase activity would reflect the stability of the 3′UTR of c-Myb, and allow us to test the effects of miR150 on c-Myb expression in transduced cells; Q-PCR and western blotting were used to simultaneously assess endogenous c-Myb mRNA and protein levels in the cells treated with miR-150 and anti-miR-150, and their respective controls (Ambion, ABI). Co-transfection of UT7/TPO cells with pCMV-luc-3′UTRcMyb and miR-150 significantly down-regulated luciferase activity to 40% of baseline 24 hr following transfection (p = 0.035; n=2 experiments) compared to a miR negative control. Luciferase activity in cells transfected with a control luc plasmid lacking the 3′UTR of c-Myb was not modulated by introduction of miR-150. Q-PCR analysis revealed that endogenous c-Myb mRNA was significantly down-regulated to 60% of baseline upon transfection of miR-150 compared to the negative control (p = 0.043), while the essential megakaryocytic transcription factor, AML1/RUNX1, remained unaltered. Western blotting of these cell lysates revealed that c-Myb protein expression was down-regulated to 30% of baseline (n=3 experiments) following transduction with miR150 but not with the miR negative control. Converse experiments utilizing anti-miRs, which inhibit expression of endogenous miRs, revealed that anti-miR150 significantly upregulated luciferase activity to 180% of baseline compared to an anti-miR-negative control (p=0.003; n=2 experiments). These findings establish that miR-150 down-modulates c-Myb mRNA, and to a greater extent protein levels, suggesting effects on both mRNA stability and protein translation efficiency. And since Tpo affects miR-150 expression, our results also suggest that in addition to direct effects on the survival and growth of MK progenitor cells, mediated by the JAK/STAT, PI3K/Akt and MAPK pathways, Tpo down-modulates c-Myb expression during megakaryopoiesis through the induction of miR150. We are currently ascertaining the in vivo role of miR-150 in Tpo-induced megakaryopoiesis, but these studies already establish that hematopoietic growth factors such as Tpo can influence transcription factor expression through modulation of microRNA species.

Tescalcin is an essential factor in megakaryocytic differentiation associated with Ets family gene expression

We show here that the process of megakaryocytic differentiation requires the presence of the recently discovered protein tescalcin. Tescalcin is dramatically upregulated during the differentiation and maturation of primary megakaryocytes or upon PMA-induced differentiation of K562 cells. This upregulation requires sustained signaling through the ERK pathway. Overexpression of tescalcin in K562 cells initiates events of spontaneous megakaryocytic differentiation, such as expression of specific cell surface antigens, inhibition of cell proliferation, and polyploidization. Conversely, knockdown of this protein in primary CD34+ hematopoietic progenitors and cell lines by RNA interference suppresses megakaryocytic differentiation. In cells lacking tescalcin, the expression of Fli-1, Ets-1, and Ets-2 transcription factors, but not GATA-1 or MafB, is blocked. Thus, tescalcin is essential for the coupling of ERK cascade activation with the expression of Ets family genes in megakaryocytic differentiation.

Chemotherapy-Induced Thrombocytopenia Derives from the Selective Death of Megakaryocyte Progenitors and Can Be Rescued by Stem Cell Factor

Thrombocytopenia is a common side effect of chemotherapy, responsible for increased risk of bleeding and delay of treatment schedules in cancer patients. It is currently unknown how chemotherapeutic agents affect platelet production and whether the platelet precursors megakaryocytes represent a direct target of cytotoxic drugs. We investigated the effects of chemotherapeutic agents on primary megakaryocytes by using a culture system that recapitulates in vitro human megakaryopoiesis and found that cytotoxic drugs predominantly destroyed megakaryocytic progenitors at early stages of differentiation. Immature megakaryocytes could be protected from chemotherapeutic agents by the cytokine stem cell factor (SCF), which binds the c-kit receptor expressed on hematopoietic stem and progenitor cells. In chemotherapy-treated megakaryocytes, SCF activated Akt, neutralized the mitochondrial apoptotic machinery, and inhibited caspase activity. Interfering with Akt activation abrogated the antiapoptotic effects of SCF, whereas exogenous expression of constitutively active Akt inhibited drug-induced apoptosis of primary megakaryocytes, indicating the Akt pathway as primarily responsible for SCF-mediated protection of megakaryocyte progenitors. These results indicate apoptosis of megakaryocyte progenitors as a major cause of chemotherapy-induced thrombocytopenia and suggest that SCF may be used to prevent platelet loss in cancer patients with c-kit-negative tumors.

Peroxisome Proliferator-Activated Receptor gamma (PPARγ) Regulates CD36 Gene Expression in Megakaryocytic Cell Lines.

CD36 is a receptor for thrombospondin and is present on megakaryocytes and platelets. It may be critical for normal megakaryocyte-formation and platelet function. The human CD36 gene locus extends 38.3 kb along chromosome 7 at position 7q11.2. Six distinct promoters (1A-1F) have been described that flank corresponding alternatively spliced first exons. To date the activity of these six promoters has not been tested in the megakaryocytic lineage and understanding the regulated expression of the CD36 gene may provide additional insights into the biology of this receptor. The 1B promoter, which is active in monocytes and adipocytes possesses a PPAR γ Direct Repeat (DR-1) binding site at position -260bp that is inducible in those same lineages with PPAR γ ligands. Recent studies have shown that primary megakaryocytes, platelets and the megakaryocyte line MEG-01 cells express PPARγ. To elucidate the molecular basis for CD36 gene expression in megakaryocytes and to query whether PPARγ plays a role in CD36 expression in this lineage we treated two megakaryocytic cell lines HEL and CHRF with increasing doses of two PPARγ ligands, 15DPGJ2, an arachadonic acid (AA) metabolite and Troglitazone, a thiazolidinedione drug administered to diabetics. RT-PCR studies using an exon 1A/exon 3 primer set or an exon 1B/exon 3 primer set established that CD36 mRNA from both promoters was expressed in these two lineages. Further these studies showed that HEL cells exhibited ligand dependent transactivation of the 1B but not the 1A promoter to both 15DPGJ2 and Troglitazone. CHRF cells did not demonstrate a drug dependent induction of either promoter. However, flow cytometry studies using a FITC conjugated anti-human CD36 antibody revealed that both HEL and CHRF cells had significant increased surface expression of the CD36 receptor. CHRF cells in particular had a 23 fold increase in surface CD36 over vehicle treated controls. Western Blot Analysis demonstrated that this increase in surface receptor quantity was secondary to drug-induced increases in the steady state protein levels. The relative increase in protein level was consistent with the mRNA levels determined by our RT-PCR results. We conclude that the 1B promoter is PPARγ responsive in HEL cells and that an alternate promoter perhaps 1C, 1D, 1E, 1F or a novel promoter is PPAR gamma responsive in CHRF cells. These data may have clinical implications for diabetics currently undergoing thiazolidinedione drug treatment with troglitazone as CD36 increases on the platelet surface lead to increased AA uptake and circulating platelet hyperactivation. These represent the first studies showing the nuclear receptor PPARγ's involvement in regulating gene expression in megakaryocytes. [Display omitted] [Display omitted]

Characterisation Erythroid-Specific Cis-Elements Regulating the Key Transcription Factor GATA1.

The critical myeloid transcription factor GATA1 can specify erythroid cells at expense of granulocytes/macrophages (GM) from the common myeloid progenitor (CMP). In red cells sustained GATA1 expression is required for terminal maturation. Conversely, GATA1 expression has to be extinguished to allow GM differentiation. Therefore, one component in dissecting myeloid specification will be to define how GATA1 expression is regulated. The level of GATA1 is mainly controlled transcriptionally. Here, we show that murine(m) GATA1 mRNA rises 6-fold as CMPs differentiate to MEPs (megakaryocyte-erythroid progenitor) and another 4-fold as MEPs differentiate to Ter119+ erythroid cells. As a step towards understanding the molecular basis of erythroid-specific GATA1 expression, we have been characterising GATA1 cis-elements. Previously, we and others showed that an upstream enhancer (mHS−3.5) is required to direct erythroid-specific GATA1 expression in cooperation with sequences near the mGata1 gene (IE promoter and intron element mHS+3.5) in transgenic mice. Though a mGata1 transgene regulated by mHS−3.5-IE-mHS+3.5 grossly rescues erythropoiesis in GATA1 knock out mice, germ line deletion of mHS−3.5 leaves red cell GATA1 expression unaffected. This suggests other cis-elements in the mGata1 locus can substitute for mHS−3.5 in red cells. Recently, we identified an erythroid-specific DNase I hypersensitive site (DHS), mHS-25, with enhancer activity in erythroid cell lines and where the chromatin associated with it is hyperacetylated at histone H3/H4. We now demonstrate by fine DHS mapping that mHS-25 is more complex, being composed of two adjacent DHSs ~500 bp apart (mHS-25 and mHS-26). These DHSs are present only in primary red cells and not other primary cells. Chromatin immunoprecipitation (ChiP) shows that H3 and H4 associated with both sites is hyperacetylated only in Ter119+ cells. We tested mHS25/6 function in mice transgenic for a mGata1-LacZ reporter construct regulated by mHS-25/6-IE-mHS+3.5. β-galactosidase expression was quantitated in myeloid lineages by FACS analysis using lineage-specific antibodies and the β-galactosidase substrate FDG. 6 out of 7 F0 transgenic embryos expressed β-galactosidase in 5–23% of fetal liver Ter119+ cells. FDG staining was not detected in CD61+Mac1- megakaryocytes or Mac-1+ macrophages. <0.2% of Ter119+ cells stained with FDG when mHS-25/6 was not present. Erythroid-specific reporter gene expression was confirmed in bone marrow samples in 2 independent lines of mice transgenic for this reporter construct. ChiP analysis demonstrated that GATA1, SCL, E2A, the SCL-interacting protein Ldb1 and LMO2 bind in vivo to mHS-25/6 only in Ter119+ cells (and not primary megakaryocytes or neutrophils). Previously, we and others have shown that these transcription factors exist in a multi-protein complex to activate gene expression. Taken together, these findings suggest mHS-25/6 is an erythroid-specific GATA1 enhancer in primary murine cells. Finally, to begin to understand the relative roles of mHS−3.5 and mHS-25/6 during erythroid differentiation, we show by DHS mapping and ChiP analysis that in the multi-potential myeloid cell line FDCP-mix, only mHS−3.5 is present (but not mHS-25/6) and that it binds GATA1/SCL/E2A/Ldb1/LMO2 in vivo. Later in Ter119+ cells both mHS−3.5 and mHS-25/6 are seen with GATA1/SCL/E2A/Ldb1/LMO2 detected at these sites. This suggests a hierarchical utilisation of these two mGata1 cis-elements during erythroid differentiation.

Endomitotic MKs Undergo Cortical Contraction during Anaphase but Do Not Complete Furrow Ingression.

Megakaryocytes (MKs) develop high degrees of polyploidy in the course of normal maturation through a process called endomitosis. Previous studies have shown that endomitotic MKs abort anaphase after sister chromatid separation, and studies using fixed cells have shown that a midzone is formed in anaphase but that a well-developed cleavage furrow is not. However, studies in fixed cells may miss transient dynamic events that are critical for evaluating potential mechanisms of failed cytokinesis in endomitosis. Therefore we have used live cell imaging as a tool to further define the ability of primary endomitotic MKs to furrow during anaphase. We imaged mouse marrow MKs expressing tubulin fused to YFP, acquiring brightfield and fluorescent images every 2 minutes. Endomitotic cells were identified by the formation of a multipolar spindle, and anaphase was defined as the point at which separation of spindle poles was visible. The extent of cell shape change following anaphase was determined visually and also by measuring the circularity of the cell, with a perfectly round cell having a circularity value of 1. We found that 56% of endomitotic MKs underwent some shape change (n=25), visible as localized indentation, protrusion or elongation of the cell cortex, with an average reduction in circularity of 0.043 occurring a mean of 14.6 minutes after the initiation of anaphase. After an average of 7.8 minutes the cells generally reverted to a round shape without completing furrow ingression. In contrast, a typical mitotic control cell showed a reduction in circularity of 0.443 occurring 8 minutes after the start of anaphase and resulting in the formation of two cells (see Figure 1, representative cells). To determine if the shape change seen in endomitosis after anaphase is dependent on RhoA, we imaged MKs in the presence of the RhoA inhibitor C3 toxin. Although 2 μg/ml C3 toxin blocked cytokinesis in diploid control cells, it did not measurably alter the frequency (44%, n=16) or degree of shape change (0.039) in endomitotic MKs following anaphase, although treatment did appear to delay shape change (onset occurring a mean of 24.8 min after anaphase) and prolong the period of cortical contractility. Therefore although endomitotic MKs exhibit a phase of increased cortical contractility following anaphase, it is not solely dependent on the activity of RhoA and it does not result in sustained furrow ingression. Live cell imaging represents a powerful new tool for dissecting the mechanisms of endomitosis. [Display omitted]

P2Y12-Independent Transient Activation and P2Y12-Dependent Prolonged Activation of Platelet Integrin αIIbβ3.

Affinity of platelet integrin αIIbβ3 for ligands is dynamically regulated and the activation of αIIbβ3 has been analyzed by binding assays using fibrinogen or ligand mimetic antibodies such as PAC1. Ligand binding assays are suitable for the condition when the number of receptors would reach a plateau phase before the end of incubation period. However, these assays would not give us useful information when the receptors are exposed only transiently followed by rapid inactivation within a few minutes. We found transient aggregation of platelets from a P2Y12-deficient patient (OSP-1) when platelets were stimulated with PAR1-activating peptide (PAR1-AP), PAR4-AP or U46619. Therefore, we hypothesized that transient αIIbβ3 activation occurs under P2Y12-deficient conditions. To estimate time-dependent changes of activated αIIbβ3, we analyzed PAC1 binding rates by measuring PAC1 binding every 30 sec (or 1 min) after the addition of PAC1 with different post-stimulation lag periods because the initial velocity of ligand binding is known to correlate with the number of activated receptors (Frojmovic et al., Biophys J, 59, 1991). When control platelets were stimulated with PAR1-AP (50μM), PAC1-binding rates did not change up to 5 min. In P2Y12-deficient platelets, the binding rate in the initial 30 sec was equivalent to control platelets and estimated values of activated αIIbβ3 in the first time fraction was, at least, about 50 % of PMA-treated platelets regardless of P2Y12-signals. In sharp contrast, only a subtle increase of PAC1 binding was seen in the sixth time fraction (2.5–3min), indicating that majority of activated αIIbβ3 was rapidly inactivated within 3 min in P2Y12-deficient platelets. We also found clear, but transient PAC1 binding induced by PAR1 stimulation in megakaryocytic cell line, CMK. When FITC-PAC1 binding was analyzed in CMK expressing high levels of glycoprotein Ib, substantial PAC1 binding was observed 0.5–10 min after PAR1 stimulation. Similar transient PAC1 binding profiles were also observed in primary megakaryocytes derived from cultured umbilical cord blood mononuclear cells. Initial velocities of PAC1 binding with different lag periods confirmed the highest levels of αIIbβ3 activation in the first time fraction (0 to 1 min) both in CMK and in megakaryocytes. Next, we found that P2Y12-dependent signals are defective in CMK because intracellular cyclic AMP levels did not change by ADP in PGE1-treated CMK. Overexpression of P2Y12 by nucleofection, however, resulted in ADP-dependent increase of PAC1 binding by PAR1 stimulation and the extent of ADP-dependent effects correlated with the expression levels of P2Y12 both in CMK and in megakaryocytes.It has been believed that agonist-induced αIIbβ3 activation is absent under P2Y12-defective conditions or in megakaryocytic cell line because bound ligand is not observed in “usual” ligand binding assays. However, analyzing PAC1 binding rates in our experimental systems unravels P2Y12-independent transient αIIbβ3 activation. Furthermore, we demonstrated that the duration of αIIbβ3 activation is prolonged by P2Y12-signals in platelets, CMK and megakaryocytes. Here, this new strategy would let us overcome serious problems existing in “usual” ligand binding assays, providing precise information about dynamic behaviors of integrin αIIbβ3.

Molecular and electrophysiological characterization of transient receptor potential ion channels in the primary murine megakaryocyte.

The molecular identity of platelet Ca(2+) entry pathways is controversial. Furthermore, the extent to which Ca(2+)-permeable ion channels are functional in these tiny, anucleate cells is difficult to assess by direct electrophysiological measurements. Recent work has highlighted how the primary megakaryocyte represents a bona fide surrogate for studies of platelet signalling, including patch clamp recordings of ionic conductances. We have now screened for all known members of the transient receptor potential (TRP) family of non-selective cation channels in murine megakaryocytes following individual selection of these rare marrow cells using glass micropipettes. RT-PCR detected messages for TRPC6 and TRPC1, which have been reported in platelets and megakaryocytic cell lines, and TRPM1, TRPM2 and TRPM7, which to date have not been demonstrated in cells of megakaryocytic/platelet lineage. Electrophysiological recordings demonstrated the presence of functional TRPM7, a constitutively active cation channel sensitive to intracellular Mg(2+), and TRPM2, an ADP-ribose-dependent cation channel activated by oxidative stress. In addition, the electrophysiological and pharmacological properties of the non-selective cation channels stimulated by the physiological agonist ADP are consistent with a major role for TRPC6 in this G-protein-coupled receptor-dependent Ca(2+) influx pathway. This study defines for the first time the principal TRP channels within the primary megakaryocyte, which represent candidates for Ca(2+) influx pathways activated by a diverse range of stimuli in the platelet and megakaryocyte.

Characterization of a Megakaryocyte-specific Enhancer of the Key Hemopoietic Transcription Factor GATA1

Specification and differentiation of the megakaryocyte and erythroid lineages from a common bipotential progenitor provides a well studied model to dissect binary cell fate decisions. To understand how the distinct megakaryocyte- and erythroid-specific gene programs arise, we have examined the transcriptional regulation of the megakaryocyte erythroid transcription factor GATA1. Hemopoietic-specific mouse (m)GATA1 expression requires the mGata1 enhancer mHS-3.5. Within mHS-3.5, the 3' 179 bp of mHS-3.5 are required for megakaryocyte but not red cell expression. Here, we show mHS-3.5 binds key hemopoietic transcription factors in vivo and is required to maintain histone acetylation at the mGata1 locus in primary megakaryocytes. Analysis of GATA1-LacZ reporter gene expression in transgenic mice shows that a 25-bp element within the 3'-179 bp in mHS-3.5 is critical for megakaryocyte expression. In vitro three DNA binding activities A, B, and C bind to the core of the 25-bp element, and these binding sites are conserved through evolution. Activity A is the zinc finger transcription factor ZBP89 that also binds to other cis elements in the mGata1 locus. Activity B is of particular interest as it is present in primary megakaryocytes but not red cells. Furthermore, mutation analysis in transgenic mice reveals activity B is required for megakaryocyte-specific enhancer function. Bioinformatic analysis shows sequence corresponding to the binding site for activity B is a previously unrecognized motif, present in the cis elements of the Fli1 gene, another important megakaryocyte-specific transcription factor. In summary, we have identified a motif and a DNA binding activity likely to be important in directing a megakaryocyte gene expression program that is distinct from that in red cells.

Thrombopoietin regulates IEX-1 gene expression through ERK-induced AML1 phosphorylation

AbstractThe extracellular signal-regulated kinases (ERKs) are required for thrombopoietin (TPO) functions on hematopoietic cells, but the ERKs targets involved remain unknown. Here we show that the regulation of the immediate early gene X-1 (IEX-1), identified as an ERK substrate in response to TPO, was mediated by an ERK-dependent phosphorylation of AML1. The addition of TPO to UT7-Mpl cells and primary megakaryocytes induced gene expression of IEX-1. Neither erythropoietin (EPO) nor granulocyte macrophage-colony stimulating factor (GM-CSF) was able to activate IEX-1 gene expression in UT7-Mpl cells. The induced expression was mediated by a transcriptional activation of the IEX-1 promoter and required an AML1-binding site located at –1068. The direct involvement of AML1 in the regulation of IEX-1 gene expression was shown by both the use of AML1 mutants and by shRNA experiments targeting endogenous AML1. Finally, the ability of TPO to induce the IEX-1 gene expression was inhibited by U0126, a specific inhibitor of the ERKs activator MEK and AML1 transcriptional activity was shown to be modulated by TPO through ERK-dependent phosphorylation. Taken together, these data suggest that AML1 plays a role in modulating the IEX-1 expression and that the ERK-dependent AML1 phosphorylation regulates the TPO-mediated activation of IEX-1.

Mammalian Target of Rapamycin Is Required for Thrombopoietin-Induced Proliferation of Megakaryocyte Progenitors

Thrombopoietin (TPO) is a potent regulator of megakaryopoiesis and stimulates megakaryocyte (MK) progenitor expansion and MK differentiation. In this study, we show that TPO induces activation of the mammalian target of rapamycin (mTOR) signaling pathway, which plays a central role in translational regulation and is required for proliferation of MO7e cells and primary human MK progenitors. Treatment of MO7e cells, human CD34+, and primary MK cells with the mTOR inhibitor rapamycin inhibits TPO-induced cell cycling by reducing cells in S phase and blocking cells in G0/G1. Rapamycin markedly inhibits the clonogenic growth of MK progenitors with high proliferative capacity but does not reduce the formation of small MK colonies. Addition of rapamycin to MK suspension cultures reduces the number of MK cells, but inhibition of mTOR does not significantly affect expression of glycoproteins IIb/IIIa (CD41) and glycoprotein Ib (CD42), nuclear polyploidization levels, cell size, or cell survival. The downstream effectors of mTOR, p70 S6 kinase (S6K) and 4E-binding protein 1 (4E-BP1), are phosphorylated by TPO in a rapamycin- and LY294002-sensitive manner. Part of the effect of the phosphatidyl inositol 3-kinase pathway in regulating megakaryopoiesis may be mediated by the mTOR/S6K/4E-BP1 pathway. In conclusion, these data demonstrate that the mTOR pathway is activated by TPO and plays a critical role in regulating proliferation of MK progenitors, without affecting differentiation or cell survival.

Thrombopoietin (TPO) induces c- myc expression through a PI3K- and MAPK-dependent pathway that is not mediated by Akt, PKC ζ 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) ζ 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, PKC ζ or mTOR.

PI3K/Akt/FOXO3a Pathway Contributes to Thrombopoietin-Induced Proliferation of Primary Megakaryocytes In Vitro and In Vivo Via Modulation of p27Kip1.

Thrombopoietin (TPO), the primary regulator of megakaryocyte (MK) and platelet formation, modulates the activity of multiple signal transduction molecules, including those in the Jak/STAT, p42/p44 MAPK, and phosphatidylinositol 3-kinase (PI3K)/Akt pathways. In the previous study, we reported that PI3K and Akt are necessary for TPO-induced cell cycle progression of primary MK progenitors. The absence of PI3K activity results in a block of transition from G1 to S phase in these cells (Geddis AE et al. JBC 2001 276:34473–34479). However, the molecular events secondary to the activation of PI3K/Akt responsible for MK proliferation remain unclear. In this study we show that FOXO3a and its downstream target p27Kip1 play an important role in TPO-induced proliferation of MK progenitors. TPO induces phosphorylation of Akt and FOXO3a in both UT-7/TPO, a megakaryocytic cell line, and primary murine MKs in a PI3K dependent fashion. Cell cycle progression of UT-7/TPO cells is blocked in G1 phase by inhibition of PI3K. We found that TPO down-modulates p27Kip1 expression at both the mRNA and protein levels in UT-7/TPO cells and primary MKs in a PI3K dependent fashion. UT-7/TPO stably expressing constitutively active Akt or a dominant-negative form of FOXO3a failed to induce p27Kip1 expression after TPO withdrawal. Induced expression of an active form of FOXO3a resulted in increased p27Kip1 expression in this cell line. In an attempt to assess whether FOXO3a has an effect of MK proliferation in vivo, we compared the number of MKs in Foxo3a-deficient mice and in wild type controls. Although peripheral blood cell counts of erythrocytes, neutrophils, monocytes and platelets were normal in the Foxo3a-deficient mice, total nucleated marrow cell count of Foxo3a-deficient mice were 60% increased compared with wild type controls. In addition, the increase of MKs was more profound than that of total nucleated marrow cells; CD41+ MKs from Foxo3a-deficient mice increased 2.1-fold, and mature MKs with 8N and greater ploidy increased 2.5-fold, compared with wild type controls. Taken together with the previous observation that p27Kip1-deficient mice also display increased numbers of MK progenitors, our findings strongly suggest that the effect of TPO on MK proliferation is mediated by PI3K/Akt-induced FOXO3a inactivation and subsequent p27Kip1 down-regulation in vitro and in vivo.

Characterization of a Megakaryocyte-Specific Enhancer of the Key Hemopoietic Transcription Factor GATA1.

Specification and differentiation of the megakaryocyte and erythroid lineages from a common bipotential progenitor provides a well-studied model to dissect binary cell fate decisions. To understand how the distinct megakaryocyte- and erythroid-specific gene programs arise, we have examined the transcriptional regulation of the transcription factor GATA1, that is required for normal maturation of these two lineages. Megakaryocyte- and erythroid-specific mouse (m)GATA1 expression requires the mGata1 enhancer mHS-3.5. Within mHS-3.5, we previously showed that the 3′ 179 base pairs (bp) of mHS-3.5 are required for megakaryocyte but not red cell expression. Here, we show that mHS-3.5 binds key hemopoietic transcription factors in vivo (GATA1, SCL/TAL-1) and is required to maintain histone acetylation in the mGata1 locus in primary megakaryocytes. When deletional constructs containing mHS-3.5 were used to direct GATA1-LacZ reporter gene expression in transgenic mice, a 25 bp element within the 3′ 179bp in mHS-3.5, was critical for megakaryocyte expression. In vitro three uncharacterized DNA-binding activities A, B and C bind to the core of the 25 bp element, and these binding sites are conserved through evolution. Of these, only activity B is present in primary megakaryocytes but not red cells. Furthermore, mutation analysis in transgenic mice reveals that activity B is required for megakaryocyte-specific enhancer function. Bioinformatic analysis shows that sequence corresponding to the binding site for activity B is a previously unrecognised motif present in the cis-elements of other megakaryocyte-specific genes. In summary, we have identified a motif and a DNA-binding activity that are likely to be important in directing a megakaryocyte gene expression program distinct from that in red cells.

Reciprocal Interaction between Megakaryocytes and Myeloma Cells Results in Increased Survival and Proliferation of Myeloma Cells.

Megakaryocytes are often expanded in bone marrow (BM) areas infiltrated with myeloma (MM) cells while thrombocytopenia is uncommon in untreated MM patients. The aim of this study was to investigate the reciprocal interaction between primary megakaryocytes and MM cells. Cultures of megakaryocytes and their precursors were prepared by incubating mobilized peripheral blood in IMDM media supplemented with BSA, thrombopoeitin (TPO), IL-6 and IL-3. Following removal of all non-adherent cells, the remaining adherent megakaryocytes (>90%) highly expressed c-MPL, CD41a and factor VIII, and had various degree of ploidy. We initially demonstrated that purified MM cell-conditioned media from 7 patients increased migration of megakaryocyte precursors across a 8 μM pore size membrane 3.6±0.9 fold (p=0.001), an effect that was inhibited by anti-CXCR4 neutralizing antibody (p<0.003). To study whether TPO is produced in myelomatous BM, mesenchymal stem cells (MSCs) were cultured on the backside of 1 μM pore-size transwell inserts' membranes while MM cells were incubated in the upper chamber of the inserts. Histologic examination demonstrated that the MSC's cytoplasmic villi pass through the membrane pores, allowing contact with tumor cells. Using qRT-PCR, we demonstrated upregulation of TPO expression by 5±3 and 3±1 folds in co-cultured MSCs and MM cells, respectively. Co-culture of megakaryocyte precursors with CD138-selected MM cells stimulated their differentiation into mature megakaryocytes as determined by flow cytometry DNA content. This suggests that megakaryopoiesis and platelet maintenance are locally and systemically regulated in MM. To study the effect of megakaryocytes on tumor cells, purified MM cells from 14 patients were co-cultured with megakaryocytes in media containing FBS and TPO at an estimated ratio of 1000:1 for 5–7 days. Myeloma cells did not firmly adhere to megakaryocytes and were easily removed by gentle pipetting. Survival and proliferation of MM cells were determined by trypan blue exclusion, annexin V/PI flow cytometry, MTT assay and BrdU labeling index. MM cells in co-cultures with megakaryocytes had more viable cells (225,000±22,000 vs. 482,000±46,000, p<0.0001), higher MTT OD reading (0.4±0.1 vs. 1.1±0.2, p<0.03), fewer annexin V positive cells (79%±6 vs. 33%±5, p<0.0008) and higher labeling index (2.1±0.5 vs. 6.7±1.3, p<0.01) compared to MM cells cultured alone. Culture of MM cells in transwell inserts and megakaryocytes on the bottom of wells (non-contact conditions), partially supported survival and proliferation of MM cells, indicating that both soluble factors and physical contact are involved in MM-megakaryocyte interactions. Co-culture of MM cells from 5 patients with either megakaryocytes or osteoclasts resulted in similar stimulatory effects on MM cells. Since bortezomib is increasingly used for MM treatment and known to induce thrombocytopenia in patients with MM we evaluated its effect on isolated megakaryocytes using MTT assay. Bortezomib at concentrations of 0.2–1 nM effectively inhibited growth of megakaryocytes and MM cell lines, CAG and ARP-1, but not MSCs, suggesting an additional mechanism for its anti-MM response. We conclude that megakaryocytes are an important microenvironmental component directly supporting MM growth. Therapeutic interventions that interfere with MM-megakaryocyte interaction may help control MM.

Phosphatidyl Inositol (4,5)P2 Marks Megakaryocyte Internal Membranes and Is Associated with Megakaryocyte Maturation and Platelet Release.

To produce blood platelets, the megakaryocyte (MK) cytoplasm elaborates proplatelets, accompanied by expansion of membrane surface area and dramatic cytoskeletal rearrangements. Invaginated demarcation membranes (DMS) are thought to be the source for the proplatelet and platelet membranes, however, they have THUS far BEEN INSUFFICIENTLY characterized. We first used a mouse model where the cDNA encoding enhanced yellow fluorescence protein (EYFP) with a C-terminally introduced myristoyl acceptor site has been introduced into the GPIIb locus. Heterozygous knock-in mice reveal yellow fluroescent MKs with an internal staining pattern that resembles the reticiulated pattern of the DMS as found in micrographs. Proplatelet-forming MKs reveal contiguous membrane connection between the internally stained membranes and the outlines of the proplatelet shaft resulting in production of fluorescent platelets. We next sought to characterize the internal membranes biochemically and retrovirally infected MKs to express the green fluorescence protein (EGFP) tagged with the pleckstrin homology domain of phospholipase Cδ1 (PLCδ1) which binds with high specificity to phosphatidylinositol(4,5)P2 (PIP2). Young MKs stain the cell periphery as described for most other cell types. Mature MKs, however, stain the internal membranes, whereas the plasma membrane becomes PIP2-negative as shown by co-staining with CD41. Proplatelet membranes emanate from these internal PIP2-positive membranes, proving that the DMS is indeed the membrane reservoir during platelet biogenesis. Appearance of PI-4,5-P2 in the DMS occurs in proximity to PI-5-P-4-kinaseα (PI4Kα), a protein highly expressed in MKs and platelets, as shown by overexpressing EGFP-tagged kinase in primary MKs. In addition, shRNA-mediated loss of PIP4Kα or depletion of its presumptive substrate block DMS development and expansion of MK size. Thus, PI-4,5-P2 is a marker and essential component of internal membranes and is most likely introduced about the non-canonical pathway using PI5P as the substrate. PI-4,5-P2 promotes actin polymerization by activating small GTPases from the Rac/Rho superfamily as well as Wiskott-Aldrich Syndrome (WASp) family proteins. Indeed, PIP2 is associated with filamentous actin when MKs are co-stained with phalloidin. Expression of a dominant-negative N-WASp C-terminal fragment (CA-domain) that inactivats all WASp/WAVE family members leads to Arp3 binding without assembling the complete Arp2/3 complex, thus inhibiting actin filament nucleation. F-Actin staining in the infected MKs reveals a pattern similar to that of MKs treated with pharmacologic dosage of actin polymerization-antagonists like cytochalasin D, which disrupts actin filaments and inhibits proplatelet formation when administered early in MK culture. Dominant-negative WASp impairs proplatelet elaboration similarly, acting at a step past expansion of the cell volume. These observations implicate a signaling pathway wherein PI-4,5-P2 facilitates DMS development and suggests a pathway that links a DMS lipid marker with local assembly of actin fibers as a requirement for platelet biogenesis.

GATA1-Mediated Megakaryocyte Differentiation and Growth Control Can Be Uncoupled and Mapped to Different Domains in GATA1.

The zinc finger transcription factor GATA1 plays a critical non-redundant role in megakaryopoiesis in mice and human patients with both germline and acquired GATA1 mutations. Germline deletion of the mouse regulatory element, HSI (ΔneoΔHS mice), specifically reduces megakaryocyte GATA1 expression to 5% of wild-type levels. ΔneoΔHS mice are viable but have defective megakaryocyte maturation and growth control. Mutant megakaryocytes fail to shed proplatelets in vitro and the platelet count is ~5 fold lower in mutant mice. Moreover, mutant megakaryocyte precursors hyperproliferate in vivo and in vitro.Here, we show there is a specific ~10-fold increase in abnormal CD41+ FcγRlowc-kit+CD9+ megakaryocyte-erythroid progenitor cells in fetal liver. These progenitors form abnormally large colonies in vitro and are unable to form proplatelets. In contrast, earlier myeloid progenitors (the common myeloid progenitor CMP) were present at expected frequencies and differentiated to all non-megakaryocyte myeloid lineages normally. Consistent with this GATA1 expression was selectively reduced in the abnormal progenitors and immature megakaryocyte precursors.We then used these GATA1-deficient primary megakaryocyte progenitors as a cellular substrate to define the amino acid domains within GATA1 required to facilitate proplatelet release and retard immature megakaryocyte growth. Previous data suggested that the bi-functional GATA1 zinc fingers could bind both DNA and interact with protein partners. The C-terminal zinc finger is essential for binding to all GATA sites whereas the N-terminal finger helps binding to a subset of GATA sites and mediates interaction with the critical GATA1 cofactor, FOG-1. Finally, the N-terminal 84 amino acids of GATA1 are important for megakaryopoiesis. Neonates and children with Down Syndrome (DS) can develop megakaryocyte leukaemia acquire mutations in GATA1 exon 2 in fetal life that result in the production of a short form of GATA1 protein (GATA1s) lacking the N-terminal 84 amino acids. In this study, we have systematically dissected GATA1 domains required for platelet release and control of megakaryocyte growth by using retroviral-mediated gene transfer to ectopically express modified GATA1 molecules in primary GATA1-deficient fetal megakaryocyte CD41+FcγRlowc-kit+CD9+progenitors. In addition to the GATA1-FOG-1 interaction and C-terminal zinc finger, two domains within the first N-terminal 110 amino acids are required for platelet release. To restrict megakaryocyte growth, two domains are required; the C-terminal zinc finger and residues in the N-terminus between amino acids 54–110. Importantly, interaction with FOG-l is not required to restrain megakaryocyte growth. Furthermore, we showed that chicken GATA1 was able to rescue both, platelet release and growth control. As only a few residues are conserved in the N-terminus between mouse and chicken GATA1, this potentially pinpoints critical functional residues in the N-terminus. In summary, distinct GATA1 domains regulate terminal megakaryocyte gene expression leading to platelet release and restrain megakaryocyte growth. Analysis of distinct mutations in the N-terminus and a modified version of GATA1 that is crippled in its interaction with FOG-1 shows platelet production and megakaryocyte growth can be uncoupled. Finally, these results have implications for how GATA1s may function in megakaryocytic leukemia in DS.