The RNA-Binding Protein ATXN2 is Expressed during Megakaryopoiesis and May Control Timing of Gene Expression.

Megakaryopoiesis is the process during which megakaryoblasts differentiate to polyploid megakaryocytes that can subsequently shed thousands of platelets in the circulation. Megakaryocytes accumulate mRNA during their maturation, which is required for the correct spatio-temporal production of cytoskeletal proteins, membranes and platelet-specific granules, and for the subsequent shedding of thousands of platelets per cell. Gene expression profiling identified the RNA binding protein ATAXIN2 (ATXN2) as a putative novel regulator of megakaryopoiesis. ATXN2 expression is high in CD34+/CD41+ megakaryoblasts and sharply decreases upon maturation to megakaryocytes. ATXN2 associates with DDX6 suggesting that it may mediate repression of mRNA translation during early megakaryopoiesis. Comparative transcriptome and proteome analysis on megakaryoid cells (MEG-01) with differential ATXN2 expression identified ATXN2 dependent gene expression of mRNA and protein involved in processes linked to hemostasis. Mice deficient for Atxn2 did not display differences in bleeding times, but the expression of key surface receptors on platelets, such as ITGB3 (carries the CD61 antigen) and CD31 (PECAM1), was deregulated and platelet aggregation upon specific triggers was reduced.

MEIS1 regulates early erythroid and megakaryocytic cell fate.

MEIS1 is a transcription factor expressed in hematopoietic stem and progenitor cells and in mature megakaryocytes. This biphasic expression of MEIS1 suggests that the function of MEIS1 in stem cells is distinct from its function in lineage committed cells. Mouse models show that Meis1 is required for renewal of stem cells, but the function of MEIS1 in human hematopoietic progenitor cells has not been investigated. We show that two MEIS1 splice variants are expressed in hematopoietic progenitor cells. Constitutive expression of both variants directed human hematopoietic progenitors towards a megakaryocyte-erythrocyte progenitor fate. Ectopic expression of either MEIS1 splice variant in common myeloid progenitor cells, and even in granulocyte-monocyte progenitors, resulted in increased erythroid differentiation at the expense of granulocyte and macrophage differentiation. Conversely, silencing MEIS1 expression in progenitor cells induced a block in erythroid expansion and decreased megakaryocytic colony formation capacity. Gene expression profiling revealed that both MEIS1 splice variants induce a transcriptional program enriched for erythroid and megakaryocytic genes. Our results indicate that MEIS1 expression induces lineage commitment towards a megakaryocyte-erythroid progenitor cell fate in common myeloid progenitor cells through activation of genes that define a megakaryocyte-erythroid-specific gene expression program.

Pathogen reduction treatment using riboflavin and ultraviolet light impairs platelet reactivity toward specific agonists in vitro.

BACKGROUND: Recent studies showed that Mirasol pathogen reduction treatment (PRT) leads to increased P-selectin expression and increased oxygen and glucose consumption in resting platelets (PLTs). This study investigates the effect of PRT on PLT activation. STUDY DESIGN AND METHODS: Untreated or Mirasol-treated PLTs were analyzed at different time points during storage. Microaggregation upon stimulation with phorbol myristate acetate (PMA), convulxin, and ristocetin was measured. Alpha granule contents and release upon thrombin stimulation were assessed by flow cytometry and Western blotting. PLT spreading was determined on collagen-coated glass slides. RESULTS: Mirasol PRT led to spontaneous aggregation (hyperreactivity), as measured by flow cytometry in the absence of agonist throughout storage time. PMA-induced aggregation was significantly higher in Mirasol PRT PLTs compared to controls. Aggregation in response to convulxin and ristocetin was significantly lower and directly influenced by storage time after Mirasol PRT, compared to untreated stored PLT concentrates. Despite the reported hyperreactivity of resting PLTs, PLT activation with thrombin on Day 8 after Mirasol PRT resulted in less P-selectin-positive PLTs. Furthermore, platelet factor 4 (PF4) secretion was reduced upon thrombin stimulation on Day 8 after PRT compared to controls. Significantly decreased spreading of Mirasol PRT PLTs over collagen-coated slides was observed directly after PRT and persisted throughout storage. CONCLUSION: Mirasol PRT leads to hyperreactive PLTs, probably caused by continuous basal degranulation through storage time. This results in a reduction in the degranulation capacity upon acute stimulation, which influences PLT spreading, but not overtly microaggregation. The clinical relevance needs to be investigated.

Novel role for galectin-8 protein as mediator of coagulation factor V endocytosis by megakaryocytes.

Galectin-8 (Gal8) interacts with ß-galactoside-containing glycoproteins and has recently been implicated to play a role in platelet activation. It has been suggested that Gal8 may also interact with platelet coagulation factor V (FV). This indispensable cofactor is stored in α-granules of platelets via a poorly understood endocytic mechanism that only exists in megakaryocytes (platelet precursor cells). In this study, we now assessed the putative role of Gal8 for FV biology. Surface plasmon resonance analysis and a solid phase binding assay revealed that Gal8 binds FV. The data further show that ß-galactosides block the interaction between FV and Gal8. These findings indicate that Gal8 specifically interacts with FV in a carbohydrate-dependent manner. Confocal microscopy studies and flow cytometry analysis demonstrated that megakaryocytic DAMI cells internalize FV. Flow cytometry showed that these cells express Gal8 on their cell surface. Reducing the functional presence of Gal8 on the cells either by an anti-Gal8 antibody or by siRNA technology markedly impaired the endocytic uptake of FV. Compatible with the apparent role of Gal8 for FV uptake, endocytosis of FV was also affected in the presence of ß-galactosides. Strikingly, thrombopoietin-differentiated DAMI cells, which represent a more mature megakaryocytic state, not only lose the capacity to express cell-surface bound Gal8 but also lose the ability to internalize FV. Collectively, our data reveal a novel role for the tandem repeat Gal8 in promoting FV endocytosis.

A HaemAtlas: characterizing gene expression in differentiated human blood cells.

Hematopoiesis is a carefully controlled process that is regulated by complex networks of transcription factors that are, in part, controlled by signals resulting from ligand binding to cell-surface receptors. To further understand hematopoiesis, we have compared gene expression profiles of human erythroblasts, megakaryocytes, B cells, cytotoxic and helper T cells, natural killer cells, granulocytes, and monocytes using whole genome microarrays. A bioinformatics analysis of these data was performed focusing on transcription factors, immunoglobulin superfamily members, and lineage-specific transcripts. We observed that the numbers of lineage-specific genes varies by 2 orders of magnitude, ranging from 5 for cytotoxic T cells to 878 for granulocytes. In addition, we have identified novel coexpression patterns for key transcription factors involved in hematopoiesis (eg, GATA3-GFI1 and GATA2-KLF1). This study represents the most comprehensive analysis of gene expression in hematopoietic cells to date and has identified genes that play key roles in lineage commitment and cell function. The data, which are freely accessible, will be invaluable for future studies on hematopoiesis and the role of specific genes and will also aid the understanding of the recent genome-wide association studies.

Improving the power to detect differentially expressed genes in comparative microarray experiments by including information from self-self hybridizations.

Our ability to detect differentially expressed genes in a microarray experiment can be hampered when the number of biological samples of interest is limited. In this situation, we propose the use of information from self-self hybridizations to acuminate our inference of differential expression. A unified modelling strategy is developed to allow better estimation of the error variance. This principle is similar to the use of a pooled variance estimate in the two-sample t-test. The results from real dataset examples suggest that we can detect more genes that are differentially expressed in the combined models. Our simulation study provides evidence that this method increases sensitivity compared to using the information from comparative hybridizations alone, given the same control for false discovery rate. The largest increase in sensitivity occurs when the amount of information in the comparative hybridization is limited.

Thyroid hormone receptor isoform expression in livers of critically ill patients.

OBJECTIVE: The THRA gene encodes two isoforms of the thyroid hormone receptor (TR), TRalpha1 and TRalpha2. The ratio of these splice variants could have a marked influence on T3-regulated gene expression, especially during illness. DESIGN: We studied the expression of the isoforms TRbeta1, TRalpha1, and TRalpha2 and 5'-deiodinase in postmortem liver biopsies of 58 patients who were critically ill and died in the intensive care unit (ICU). All mRNA levels were determined using real-time PCR. MAIN OUTCOME: All ratios of the biopsies were higher than those found in three normal liver biopsies due to an increased TRalpha1 level. The TRalpha1/TRalpha2 ratio increased with age and severity of illness following the equation: TRalpha1/TRalpha2 ratio = - 1.854 + (0.0323 x age) + (0.0431 x Therapeutic Intervention Scoring System score) indicating that 28% of the changed TRalpha1/TRalpha2 ratio can be predicted by these clinical variables. There was no effect of randomization to intensive insulin therapy or glucocorticoid or thyroid hormone treatment on the TRalpha1/TRalpha2 ratio or TRbeta1. Furthermore, no relation was seen between the expression levels of the 5'-deiodinase mRNA and TR isoforms or the triiodothyronine T3 levels. CONCLUSION: It appears that in critically ill patients the ratio of TRalpha1/TRalpha2 expression increases with age and severity of illness, possibly indicating a mechanism to enhance sensitivity to T3 in the oldest and sickest patients.

Comparative gene expression profiling of in vitro differentiated megakaryocytes and erythroblasts identifies novel activatory and inhibitory platelet membrane proteins.

To identify previously unknown platelet receptors we compared the transcriptomes of in vitro differentiated megakaryocytes (MKs) and erythroblasts (EBs). RNA was obtained from purified, biologically paired MK and EB cultures and compared using cDNA microarrays. Bioinformatical analysis of MK-up-regulated genes identified 151 transcripts encoding transmembrane domain-containing proteins. Although many of these were known platelet genes, a number of previously unidentified or poorly characterized transcripts were also detected. Many of these transcripts, including G6b, G6f, LRRC32, LAT2, and the G protein-coupled receptor SUCNR1, encode proteins with structural features or functions that suggest they may be involved in the modulation of platelet function. Immunoblotting on platelets confirmed the presence of the encoded proteins, and flow cytometric analysis confirmed the expression of G6b, G6f, and LRRC32 on the surface of platelets. Through comparative analysis of expression in platelets and other blood cells we demonstrated that G6b, G6f, and LRRC32 are restricted to the platelet lineage, whereas LAT2 and SUCNR1 were also detected in other blood cells. The identification of the succinate receptor SUCNR1 in platelets is of particular interest, because physiologically relevant concentrations of succinate were shown to potentiate the effect of low doses of a variety of platelet agonists.