Megakaryocyte Gene Expression Research Articles

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.

GATA1-mediated megakaryocyte differentiation and growth control can be uncoupled and mapped to different domains in GATA1.

The DNA-binding hemopoietic zinc finger transcription factor GATA1 promotes terminal megakaryocyte differentiation and restrains abnormal immature megakaryocyte expansion. How GATA1 coordinates these fundamental processes is unclear. Previous studies of synthetic and naturally occurring mutant GATA1 molecules demonstrate that DNA-binding and interaction with the essential GATA1 cofactor FOG-1 (via the N-terminal finger) are required for gene expression in terminally differentiating megakaryocytes and for platelet production. Moreover, acquired mutations deleting the N-terminal 84 amino acids are specifically detected in megakaryocytic leukemia in human Down syndrome patients. In this study, we have systematically dissected GATA1 domains required for platelet release and control of megakaryocyte growth by ectopically expressing modified GATA1 molecules in primary GATA1-deficient fetal megakaryocyte progenitors. In addition to DNA binding, distinct N-terminal regions, including residues in the first 84 amino acids, promote platelet release and restrict megakaryocyte growth. In contrast, abrogation of GATA1-FOG-1 interaction leads to loss of differentiation, but growth of blocked immature megakaryocytes is controlled. Thus, distinct GATA1 domains regulate terminal megakaryocyte gene expression leading to platelet release and restrain megakaryocyte growth, and these processes can be uncoupled.

Differential requirements for the activation domain and FOG-interaction surface of GATA-1 in megakaryocyte gene expression and development

AbstractGATA1 is mutated in patients with 2 different disorders. First, individuals with a GATA1 mutation that blocks the interaction between GATA-1 and its cofactor Friend of GATA-1 (FOG-1) suffer from dyserythropoietic anemia and thrombocytopenia. Second, children with Down syndrome who develop acute megakaryoblastic leukemia harbor mutations in GATA1 that lead to the exclusive expression of a shorter isoform named GATA-1s. To determine the effect of these patient-specific mutations on GATA-1 function, we first compared the gene expression profile between wild-type and GATA-1–deficient megakaryocytes. Next, we introduced either GATA-1s or a FOG-binding mutant (V205G) into GATA-1–deficient megakaryocytes and assessed the effect on differentiation and gene expression. Whereas GATA-1–deficient megakaryocytes failed to undergo terminal differentiation and proliferated excessively in vitro, GATA-1s–expressing cells displayed proplatelet formation and other features of terminal maturation, but continued to proliferate aberrantly. In contrast, megakaryocytes that expressed V205G GATA-1 exhibited reduced proliferation, but failed to undergo maturation. Examination of the expression of megakaryocyte-specific genes in the various rescued cells correlated with the observed phenotypic differences. These studies show that GATA-1 is required for both normal regulation of proliferation and terminal maturation of megakaryocytes, and further, that these functions can be uncoupled by mutations in GATA1.

Ets-dependent Regulation of Target Gene Expression during Megakaryopoiesis

Megakaryopoiesis is the process by which hematopoietic stem cells in the bone marrow differentiate into mature megakaryocytes. The expression of megakaryocytic genes during megakaryopoiesis is controlled by specific transcription factors. Fli-1 and GATA-1 transcription factors are required for development of megakaryocytes and promoter analysis has defined in vitro functional binding sites for these factors in several megakaryocytic genes, including GPIIb, GPIX, and C-MPL. Herein, we utilize chromatin immunoprecipitation to examine the presence of Ets-1, Fli-1, and GATA-1 on these promoters in vivo. Fli-1 and Ets-1 occupy the promoters of GPIIb, GPIX, and C-MPL genes in both Meg-01 and CMK11-5 cells. Whereas GPIIb is expressed in both Meg-01 and CMK11-5 cells, GPIX and C-MPL are only expressed in the more differentiated CMK11-5 cells. Thus, in vivo occupancy by an Ets factor is not sufficient to promote transcription of some megakaryocytic genes. GATA-1 and Fli-1 are both expressed in CMK11-5 cells and co-occupy the GPIX and C-MPL promoters. Transcription of all three megakaryocytic genes is correlated with the presence of acetylated histone H3 and phosphorylated RNA polymerase II on their promoters. We also show that exogenous expression of GATA-1 in Meg-01 cells leads to the expression of endogenous c-mpl and gpIX mRNA. Whereas GPIIb, GPIX, and C-MPL are direct target genes for Fli-1, both Fli-1 and GATA-1 are required for formation of an active transcriptional complex on the C-MPL and GPIX promoters in vivo. In contrast, GPIIb expression appears to be independent of GATA-1 in Meg-01 cells.

Identification of Signaling-Induced Transcription Pathway Determinants that Influence Erythro-Megakaryocytic Lineage Commitment.

Erythroid and megakaryocytic cells are derived from a common precursor and share many of the same transcriptional regulators. We and others have demonstrated a critical role for sustained activation of extracellular-related (ERK) kinase in PKC-induced megakaryocytic differentiation of K562 cells. However, the transcriptional mechanisms underlying this process are largely unknown. Our prior studies have focused on late stage megakaryocyte specific genes such as glycoprotein IIb/CD41. In order to understand the early transcriptional events regulating this process, we have begun to examine the mechanisms regulating the expression of CD9, a tetraspanin molecule that is currently the earliest identified marker of megakaryocyte differentiation. The PKC agonist ingenol 3,20 dibenzoate (IDB) rapidly induced CD9 expression in both K562 cells as well as in primary human CD34+ progenitor cells, much earlier than the kinetics for CD41 induction. CD9 induction by PKC was dependent on ERK activation. Recently, it has been shown that immediate early gene expression, including the transcription factor Egr-1, is highly sensitive to the duration and amplitude of ERK activity. Egr-1 has been previously implicated in the regulation of hematopoietic lineage commitment. PKC/ERK activation rapidly and strongly induced Egr-1 expression, which remained elevated for up to 96 hrs after IDB treatment of K562 cells. Egr-1 induction was closely followed by CD9 induction, suggesting a causal relationship. This was supported by chromatin immunoprecipitation (ChIP) analysis, which showed strong recruitment of Egr-1 to the CD9 promoter during PKC/ERK-induced differentiation. KLF5, a known downstream target of Egr-1, was identified as an additional target of PKC/ERK signaling during megakaryocytic differentiation. KLF5, a novel GC binding factor, was found to be expressed in primary megakaryocytes and not in erythroid cells. KLF5 expression was induced by IDB treatment, and ChIP analysis showed that Egr-1 was recruited to the KLF5 promoter during PKC/ERK-induced differentiation. KLF5 has been shown previously to regulate known megakaryocyte-expressed genes PDGF-A and TGF-β expression in vascular smooth muscle, but a role in megakaryocyte gene expression has not been previously suggested. To better understand the regulation of Egr-1 in erythromegakaryocytic differentiation, Nab2, a natural co-repressor of Egr-1 was also evaluated. Control K562 cells strongly expressed c-myc and Nab2. ChIP studies show that c-myc binds to the Nab2 promoter in control K562 cells and both c-myc and Nab2 expression strongly declined during the first 24 hours after PKC stimulation. Enforced expression of Nab2 not only blocked CD9 induction by IDB, but also induced marked hemoglobinization of K562 cells. Taken together, these findings suggest a novel and critical role for the balance in Egr-1/Nab2 activity as a regulatory mechanism in erythroid versus megakaryocytic differentiation pathways.

Upstream stimulatory factors stimulate transcription through E-box motifs in the PF4 gene in megakaryocytes

AbstractPlatelet factor 4 (PF4) is expressed during megakaryocytic differentiation. We previously demonstrated that the homeodomain proteins (myeloid ecotropic integra tion site 1 [MEIS1], Pbx-regulating protein 1 [PREP1], and pre-B-cell leukemia transcription factors [PBXs]) bind to the novel regulatory element tandem repeat of MEIS1 binding element [TME] and transactivate the rat PF4 promoter. In the present study, we investigated and identified other TME binding proteins in megakaryocytic HEL cells using mass spectrometry. Among identified proteins, we focused on upstream stimulatory factor (USF1) and USF2 and investigated their effects on the PF4 promoter. USF1 and 2 bound to the E-box motif in the TME and strongly transactivated the PF4 promoter. Furthermore, physiologic bindings of USF1 and 2 to the TME in rat megakaryocytes were demonstrated by the chromatin immunoprecipitation (ChIP) assay. Interestingly, the E-box motif in the TME was conserved in TME-like sequences of both the human and mouse PF4 promoters. USF1 and 2 also bound to the human TME-like sequence and transactivated the human PF4 promoter. Expressions of USF1 and 2 were detected by reverse-transcriptase–polymerase chain reaction (RT-PCR) in the human megakaryocytes derived from CD34+ cells. Thus, these studies demonstrate that the novel TME binding transcription factors, USF1 and 2, transactivate rat and human PF4 promoters and may play an important role in megakaryocytic gene expression.

Manipulation of gene expression in megakaryocytes.

The process of megakaryocyte generation in the bone marrow and subsequent differentiation leading to platelet production is little understood. Known as megakaryo-poiesis, it involves a number of unique biological features, including an increase in the nuclear DNA content (endoreplication) and partitioning of cytoplasm and membranes into platelets. Abnormalities of thrombosis and hemostasis can occur as a result of alterations in the number of platelets generated and maintained in the circulation or through aberrations in the functional behavior of the platelet itself. Although some of these abnormalities are most likely indirect in their etiology (for example, immune thrombocytopenias), many are directly linked to an inherited or acquired genetic effect operating at some point during megakaryopoiesis. Inherited mutations affecting megakaryocytes and platelets are relatively rare, although there is increasing interest in the association between genetic polymorphisms in platelet proteins that have no profound phenotype but may be linked to an increased thrombotic risk. Specific genetic changes leading to deregulation of proliferation and/or differentiation at some point during megakaryopoiesis are also implicated in several acquired conditions, including leukemias, preleukemic states, and dysplasias.

Homeodomain proteins MEIS1 and PBXs regulate the lineage-specific transcription of the platelet factor 4 gene

AbstractPlatelet factor 4 (PF4) is expressed during megakaryocytic differentiation. We previously reported that GATA-1 and ETS-1 regulate the rat PF4 promoter and transactivate the PF4 gene. For the present study, we investigated the regulatory elements and their transcription factors responsible for the lineage-specific expression of the PF4 gene. The promoter activities of deletion constructs were evaluated, and a novel regulatory element termed TME (tandem repeat of MEIS1 binding element) (–219 to –182) was defined. Binding proteins to TME were strongly detected in HEL nuclear extracts by electrophoresis mobility shift assay (EMSA), and they were purified by DNA affinity chromatography. By performing Western blottings and supershift assays, the binding proteins were identified as homeodomain proteins, MEIS1, PBX1B, and PBX2. These factors are expressed in megakaryocytes differentiated from CD34+ cells in human cord blood. MEIS1 and PBXs bind to the TME as MEIS1/PBX complexes and activate the PF4 promoter. In nonmegakaryocytic HepG2 cells, GATA-1 and ETS-1 activate the PF4 promoter approximately 10-fold. Surprisingly, we found that additional expression of both MEIS1 and PBX2 multiplied this major activation another 2-fold. This activation was not observed when MEIS1 binding sites in the TME were disrupted. Furthermore, inhibition of the binding of endogenous MEIS1/PBX complexes to the TME decreased the promoter activity by almost one half, in megakaryocytic HEL cells. Thus, these studies demonstrate that the homeodomain proteins, MEIS1, PBX1B, and PBX2, play an important role in megakaryocytic gene expression.

Gene expression profile of megakaryocytes from human cord blood CD34(+) cells ex vivo expanded by thrombopoietin.

Previously, we investigated the process of megakaryocytopoiesis during ex vivo expansion of human cord blood (CB) CD34(+) cells using thrombopoietin (TPO) and found that megakaryocytopoiesis was closely associated with apoptosis. To understand megakaryocytopoiesis at the molecular level, we performed a microserial analysis of gene expression (microSAGE) in megakaryocytes (MKs) and nonmegakaryocytes (non-MKs) derived from human CB CD34(+) cells by ex vivo expansion using TPO, and a total of 38909 tags, representing 8976 unique genes, were identified. In MKs, many of the known genes, including coagulation factor VII, P-selectin (CD62P), pim-1, azurocidin, defensin, and CD48 were highly expressed; meanwhile, those genes encoding some small G proteins of the Ras family (Rab 7 and Rab 11A) and glutathione S transferase family (1, 4, A2, omega, and pi) showed lower expression levels in MKs. These gene expression profiles will be useful to understand megakaryocytopoiesis at the molecular level, including apoptosis and related signal transduction pathways.

Transcriptional regulation of granulocyte and monocyte development.

Granulocytes and monocytes develop from a common myeloid progenitor. Early granulopoiesis requires the C/EBPalpha, PU.1, RAR, CBF, and c-Myb transcription factors, and terminal neutrophil differentiation is dependent upon C/EBPepsilon, PU.1, Sp1, CDP, and HoxA10. Monopoiesis can be induced by Maf-B, c-Jun, or Egr-1 and is dependent upon PU.1, Sp1, and ICSBP. Signals eminating from cytokine receptors modulate factor activities but do not determine cell fates. Orchestration of the myeloid developmental program is achieved via cooperative gene regulation, via synergistic and inhibitory protein-protein interactions, via promoter auto-regulation and cross-regulation, via regulation of factor levels, and via induction of cell cycle arrest: For example, c-Myb and C/EBPalpha cooperate to activate the mim-1 and NE promoters, PU.1, C/EBPalpha, and CBF, regulate the NE, MPO, and M-CSF Receptor genes. PU.1:GATA-1 interaction and C/EBP suppression of FOG transcription inhibits erythroid and megakaryocyte gene expression. c-Jun:PU.1, ICSBP:PU.1, and perhaps Maf:Jun complexes induce monocytic genes. PU.1 and C/EBPalpha activate their own promoters, C/EBPalpha rapidly induces PU.1 and C/EBPepsilon RNA expression, and RARalpha activates the C/EBPepsilon promoter. Higher levels of PU.1 are required for monopoiesis than for B-lymphopoiesis, and higher C/EBP levels may favor granulopoiesis over monopoiesis. CBF and c-Myb stimulate proliferation whereas C/EBPalpha induces a G1/S arrest; cell cycle arrest is required for terminal myelopoiesis, perhaps due to expression of p53 or hypo-phosphorylated Rb.

Absence of mutations in the HoxA10, HoxA11 and HoxD11 nucleotide coding sequences in thrombocytopenia with absent radius syndrome.

Recent studies have suggested the HoxA10, HoxA11 and HoxD11 homeobox genes as candidate loci for the thrombocytopenia with absent radius (TAR) syndrome. For example, targeted disruptions of these Hox genes result in abnormal development of the mouse radius, while overexpression of HoxA10 stimulates mouse megakaryocyte (MK) development in vitro. To examine the expression of Hox genes in human MK cells, we utilized reverse transcription polymerase chain reaction with degenerate oligonucleotides to study megakaryocytic cell lines (MEG-01, DAMI), and primary human MK purified from adult and cord blood. Using this approach, 13 out of 40 clones isolated from cell lines, 10 out of 21 from cord MK, and 11 out of 21 from adult MK were identified as HoxA10, while HoxA11 and HoxD11 sequences were not detected. The normal genomic sequences for the human HoxA10, -A11, and -D11 genes were then determined and sequenced in 10 unrelated individuals with TAR syndrome. In all patients the derived amino acid sequence for the three Hox genes was identical to normal controls. Southern blotting did not reveal genomic rearrangements or deletions at these loci, and in two patients intact HoxA10 transcripts were detected by amplification in myeloid cells. Although these studies cannot completely exclude the possibility that the TAR syndrome results from non-coding mutations that affect the level of Hox gene expression in megakaryocytes, mutations in the coding sequence of the Hox genes known to affect radial development are not a common cause of TAR syndrome.

Mpl ligand increases P2Y1 receptor gene expression in megakaryocytes with no concomitant change in platelet response to ADP.

The P2Y(1) receptor is responsible for the initiation of platelet aggregation in response to ADP and plays a key role in thrombosis. Although this receptor is expressed early in the platelet lineage, the regulation of its expression during megakaryocyte differentiation is unknown. In the mouse megakaryocytic cell line Y10/L8057, we detected P2Y(1) mRNA of three sizes (2.5, 4.4, and 7.4 kb). These cells have previously been shown to respond to Mpl ligand, the pivotal regulator of megakaryocytopoiesis, by increasing their expression of differentiation markers. Mpl ligand enhanced levels of P2Y(1) mRNAs in Y10/L8057 cells and this effect was selective: the same cytokine did not increase levels of A2a adenosine receptor mRNA. Although Mpl ligand did not affect the short half-lives of the P2Y(1) mRNAs, it enhanced transcription of the P2Y(1) gene. It also increased cell size and the number of cell surface P2Y(1) receptors, but not P2Y(1) receptor density. Injection of Mpl ligand into mice up-regulated P2Y(1) receptor mRNAs in megakaryocytes, as shown by in situ hybridization. However, platelets isolated from these mice did not exhibit a higher P2Y(1) receptor density or increased reactivity to ADP. This correlates with the finding that Mpl ligand increases GPIIb mRNA in megakaryocytes but not the density of the protein per platelet. Thus, the enhancement of P2Y(1) receptor expression induced by Mpl ligand in megakaryocytes may be an integral feature of their differentiation, whereas clinical use of this compound might not be associated with platelet hyper-reactivity to ADP.

Stimulation of NF-E2 DNA Binding by CREB-binding Protein (CBP)-mediated Acetylation

The hematopoietic transcription factor NF-E2 is an important regulator of erythroid and megakaryocytic gene expression. The transcription cofactor cAMP-response element-binding protein (CREB)-binding protein (CBP) has previously been implicated in mediating NF-E2 function. In this report, we examined the role of CBP, a coactivator with intrinsic acetyltransferase activity, in the regulation of NF-E2. We found that both the hematopoietic-specific subunit of NF-E2, p45, and the widely expressed small subunit, MafG, interact with CBP in vitro and in vivo. CBP acetylates MafG, but not p45, predominantly in the basic region of MafG. Immunoprecipitation experiments with anti-acetyl lysine antibodies demonstrate that MafG is acetylated in vivo in erythroid cells. Transfection experiments further show that CBP stimulates MafG acetylation in intact cells in an E1A-sensitive manner. Acetylation of MafG augments DNA binding activity of NF-E2, and mutations at the major acetylation sites markedly reduce DNA binding and transcriptional activation by NF-E2. Together, these results suggest that recruitment of CBP by NF-E2 to specific erythroid/megakaryocytic promoters might regulate transcription by at least two mechanisms involving both modification of chromatin structure and modulation of transcription factor activity.

Unexpected and coordinated expression of Spi-1, Fli-1, and megakaryocytic genes in four Epo-dependent cell lines established from transgenic mice displaying erythroid-specific expression of a thermosensitive SV40 T antigen

Most erythroleukemic cell lines established in vitro coexpress erythrocytic and megakaryocytic markers that often are associated with expression of Spi-1 and/or Fli-1 transcription factors known as transactivators of megakaryocyte-specific promoters. In the present study, we examined the possibility of establishing new cell lines keeping strictly erythroid-specific properties in vitro through the targeted and conditional immortalization of erythrocytic progenitors. For that purpose, we established several lines of transgenic mice displaying erythroid-specific expression of a thermosensitive SV40 T antigen. As expected, these transgenic mice developed splenomegaly due to the massive amplification of Ter 119 positive erythroid nucleated cells expressing T antigen. Despite this drastic effect in vivo, the in vitro immortalization of erythropoietin-dependent erythroid progenitors unexpectedly occurred at low frequency, and all four cell lines established expressed both erythrocytic (globins) and megakaryocytic markers (glycoprotein IIb, platelet factor 4) as well as Spi-1 and Fli-1 transcripts at permissive temperature. Switching the cells to the nonpermissive temperature led to a marked increase in globin gene expression and concomitant decrease in expression of Spi-1, Fli-1, and megakaryocytic genes in an erythropoietin-dependent manner. Interestingly, enhanced expression of Spi-1 and Fli-1 genes already was detected in the Ter 119 positive cell population of transgenic mice spleen in vivo. However, like normal Ter 119 erythroid cells, these Ter 119 positive cells from transgenic mice still expressed high levels of β-globin and very low or undetectable glycoprotein IIb and platelet factor 4 megakaryocytic transcripts. Taken together, these data indicate that the unexpected expression of megakaryocytic genes is a specific property of immortalized cells that cannot be explained only by enhanced expression of Spi-1 and/or Fli-1 genes.

In Vivo Expression of Murine Platelet Glycoprotein Ibα

AbstractWe have performed a systematic in vivo evaluation of gene expression for the glycoprotein (GP) Ibα subunit of the murine platelet adhesion receptor, GP Ib-IX-V. This study is warranted by in vitro observations of human GP Ibα expression in cells of nonhematopoietic lineage and reports of regulation of the GP Ibα gene by cytokines. However, an in vivo role for a GP Ib-IX-V receptor has not been established beyond that described for normal megakaryocyte/platelet physiology and hemostasis. Our Northern analysis of mouse organs showed high levels of GP Ibα mRNA in bone marrow with a similar expression pattern recapitulated in mice containing a luciferase transgene under the control of the murine GP Ibα promoter. Consistently high levels of luciferase activity were observed in the two hematopoietic organs of mice, bone marrow (1,400 relative light units/μg of protein [RLUs]) and spleen (500 RLUs). Reproducible, but low-levels of luciferase activity were observed in heart, aorta, and lung (30 to 60 RLUs). Among circulating blood cells, the luciferase activity was exclusively localized in platelets. No increase in GP Ibα mRNA or luciferase activity was observed after treatment of mice with lipopolysaccharides (LPS) or tumor necrosis factor-α (TNF-α). We conclude the murine GP Ibα promoter supports a high level of gene expression in megakaryocytes and can express heterologous proteins allowing an in vivo manipulation of platelet-specific proteins in the unique environment of a blood platelet.

In Vivo Expression of Murine Platelet Glycoprotein Ibα

We have performed a systematic in vivo evaluation of gene expression for the glycoprotein (GP) Ibα subunit of the murine platelet adhesion receptor, GP Ib-IX-V. This study is warranted by in vitro observations of human GP Ibα expression in cells of nonhematopoietic lineage and reports of regulation of the GP Ibα gene by cytokines. However, an in vivo role for a GP Ib-IX-V receptor has not been established beyond that described for normal megakaryocyte/platelet physiology and hemostasis. Our Northern analysis of mouse organs showed high levels of GP Ibα mRNA in bone marrow with a similar expression pattern recapitulated in mice containing a luciferase transgene under the control of the murine GP Ibα promoter. Consistently high levels of luciferase activity were observed in the two hematopoietic organs of mice, bone marrow (1,400 relative light units/μg of protein [RLUs]) and spleen (500 RLUs). Reproducible, but low-levels of luciferase activity were observed in heart, aorta, and lung (30 to 60 RLUs). Among circulating blood cells, the luciferase activity was exclusively localized in platelets. No increase in GP Ibα mRNA or luciferase activity was observed after treatment of mice with lipopolysaccharides (LPS) or tumor necrosis factor-α (TNF-α). We conclude the murine GP Ibα promoter supports a high level of gene expression in megakaryocytes and can express heterologous proteins allowing an in vivo manipulation of platelet-specific proteins in the unique environment of a blood platelet.

Constitutive and inducible expression of megakaryocyte-specific genes in Friend erythroleukaemia cells.

Friend murine erythroleukaemia cells (MELC) were analysed by semiquantitative RT-PCR for the constitutive and inducible expression of megakaryocyte-specific genes. Uninduced MELC expressed detectable levels of mRNAs for acethylcholinesterase (AChE), platelet factor-4 (PF4), glycoprotein IIb (GPIIb) and von Willebrand factor (VWF), whereas the erythroid alpha- and beta-globin genes were not transcribed appreciably. However, MELC exposed to 5 mM hexamethylene bisacetamide (HMBA) or 1.5% dimethyl sulphoxide (DMSO) seemed to be channelled towards a mixed erythroid/megakaryocytic phenotype characterized by unaltered levels of VWF mRNA, increased levels of AChE, GPIIb and PF4 mRNA. and simultaneous induction of the globin genes. Megakaryocyte-related genes were expressed. in the absence of globin gene transcription, by MELC treated with either phorbol-12-myristate acetate (PMA; 100 ng/ml) or colcemid (40 nM), an antimicrotubule agent capable of promoting polyploidization in this model. Moreover, PMA and colcemid induced also de novo expression of the thrombopoietin receptor c-mpl. PMA and colcemid did not affect high basal c-myb mRNA levels which, in turn, were down-regulated upon HMBA or DMSO induction. Additionally, both uninduced and induced MELC exhibited significant levels of Epo-R and IL-3R mRNAs, whereas no expression of granulocyte/macrophage-related genes was detected. Megakaryocyte gene expression of MELC was also compared to that of other haemopoietic cell populations from normal mice and mice infected with the anaemic strain of the Friend virus. According to our results, MELC should be seen as an unique erythro-megakaryocytic model of differentiation, potentially useful for studying molecular events governing lineage commitment as well as some steps of megakaryocytopoiesis.

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

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

Alzheimer's presenilin 1 gene expression in platelets and megakaryocytes: Identification of a novel splice variant

The presenilin 1 (PSI) gene located on chromosome 14 has been linked with the majority of early-onset FAD. The normal biological role of PS1 as well as the mechanism by which mutations in PS1 cause FAD remains unknown. PS1 expression in platelets and the Dami megakaryocytic cell line was examined by Western blot analysis and RT-PCR. Using an anti-N-terminus PS1 antibody we detected PS1 immunoreactive bands of 44, 32 and 27 kDa in both cell types. After RT-PCR we observed that platelets and megakaryocytes carry at least four different PS1 transcripts. One of them is a novel PS1 splice variant that lacks the coding sequence for exon 10 resulting in a shorter 409 amino acid protein.

Identification of Essential GATA and Ets Binding Motifs within the Promoter of the Platelet Glycoprotein Ibα Gene

Platelet glycoprotein (GP) Ib-IX-V is a multisubunit adhesion receptor that supports platelet attachment to thrombogenic surfaces at sites of vascular injury. The congenital absence of the receptor results in a bleeding disorder associated with "giant" platelets, a condition linking the expression of the complex to platelet morphogenesis. To understand better the expression of the GP Ib-IX-V complex, studies were undertaken to define the essential genetic elements supporting the expression of the alpha-subunit of the complex (GP Ib alpha). GP Ib alpha promoter activity was evaluated by transfection of human erythroleukemia cells with reporter plasmids coding for the enzyme, luciferase. Studies were initiated with a fragment extending 2,738 nucleotides 5' to the transcription start site and lead to the identification of 253 nucleotides retaining full promoter activity in human erythroleukemia cells. In cells of nonhematopoietic lineage, human endothelial and HeLa cells, the GP Ib alpha promoter activity was no greater than background levels obtained with promoterless constructs. Gel shift assays and site-directed mutagenesis studies defined essential GATA and Ets binding motifs 93 and 150 nucleotides upstream of the transcription start site, a finding which further substantiates these elements as important determinants of megakaryocytic gene expression. The results define essential cis-acting elements responsible for the expression of GP Ib alpha and provide insights into molecular events coinciding with the release of normal platelets into the bloodstream.