bluuebunny/crossref_metadata_2025 huggingface dataset:
Akt2 Supports aIIbb3-Dependent Reorganization of the Actin Cytoskeleton in Platelets.
Authors: August Shelley , Woulfe Donna S.
Published: April 2020

Akt is a serine-threonine kinase with well-described roles in growth and metabolism. Previous studies from our laboratory have shown that Akt also plays important roles in platelet function in vitro and in thrombus formation in vivo. Two isoforms are present in mouse platelets, Akt1 and Akt2, with Akt2 being the dominant isoform. Our previous studies have shown that platelets from Akt2−/− mice have marked defects in aggregation, a- and dense granule secretion, and fibrinogen binding. Each of these platelet functions depends in part on the function of the major platelet integrin, aIIbb3. However, whether Akt might regulate the function of aIIbb3 is still unknown. To determine whether Akt regulates aIIbb3-dependent signaling, first the rate of thrombin-initiated clot retraction was compared in platelet-rich plasma (PRP) from wild-type versus Akt2−/− mice. Akt2−/− platelets have a delay in the aIIbb3-dependent retraction of the fibrin clot compared to their wildtype counterparts. Akt2−/− platelets also have a delay in aIIbb3-dependent spreading on immobilized fibrinogen. However, adhesion to immobilized fibrinogen was normal in these platelets, suggesting that the spreading defect is due to a defect in outside-in signaling by the integrin, rather than in fibrinogen binding. Furthermore, unstimulated platelets expressing a constitutively active form of Akt spread more rapidly on fibrinogen-coated slides and generate more filopodial extensions than wildtype platelets, suggesting that Akt activation may be sufficient to induce outside-in signaling and /or cytoskeletal remodeling. Interestingly, integrin activation was not sufficient to induce Akt phoshorylation, suggesting that integrin activity is downstream rather than upstream of Akt activation. Taken together, these results suggest that integrin aIIbb3 is not necessary for Akt activation; however, Akt promotes outside-in signaling and cytoskeletal remodeling by aIIbb3.
Mechanism of Activation and Role of Extracellular Signal Regulated Kinase in ADP-Induced Thromboxane A2 Generation in Platelets.
Authors: Garcia Analia , Shankar Haripriya , Kunapuli Satya P.
Published: April 2020

We have previously shown that ADP-induced thromboxane A2 generation in platelets requires co-ordinated signaling events from the Gq-coupled P2Y1 receptor and the Gi-coupled P2Y12 receptor in addition to outside-in signaling. It is also known that ADP-induced thromboxane A2 generation is completely abolished in the presence of extracellular calcium, but the mechanism of this negative regulation is not known. In this study we sought to identify the important signaling molecules in ADP-induced thromboxane A2 generation in platelets and characterize the regulation of these molecules by extracellular calcium. Erk2 activation occurred when outside-in signaling was blocked, indicating that it is a downstream event from the P2Y receptors. However, blockade of either P2Y1 or the P2Y12 receptors with corresponding antagonists completely abolished Erk phosphorylation, indicating that both P2Y receptors are required for ADP-induced Erk activation. However, blockade of Erk upstream kinase MEK had not effect on ADP-induced aggregation in aspirin-treated platelets, but dramatically inhibited aggregation as well as secretion in non-aspirinated platelets, suggesting that Erk might be important for thromboxane A2 generation. Finally, PP1 and PP2, inhibitors of Src family kinases, but not PP3, an inactive analog, abolished ADP-induced Erk phosphorylation and thromboxane A2 generation. Interestingly, ADP-induced Erk phosphorylation was completely inhibited in the presence of extracellular calcium, indicating that Erk is a key signaling molecule regulated by extracellular calcium in the negative regulation of thromboxane A2 generation. We conclude that Erk2 is activated downstream of P2Y receptors through a complex mechanism involving Src kinases and plays an important role in ADP-induced thromboxane A2 generation. We also conclude that extracellular calcium blocks ADP-induced thromboxane A2 generation through the inhibition of Erk activation.
Human Plasma Gelsolin Inhibits Cellular Responses to Platelet Activating Factor (PAF).
Authors: Magnuson-Osborn Teresia A. , Dahlgren Claes , Hartwig John H. , Stossel Thomas P.
Published: April 2020

Gelsolin is a highly conserved intracellular actin-binding protein with an extracellular isoform named plasma gelsolin (pGSN). Relatively high (250 mg /L) blood concentrations of pGSN decrease in response to trauma, major surgery, sepsis, burns, ionizing radiation, and hyperoxia. Depletion of pGSN to a critical (~20%) level precedes and predicts complications of primary injuries such as lung permeability changes, ARDS, assisted ventilation and death. Administration of recombinant pGSN ameliorates such complications and reduces mortality in animal models. A proposed mechanism for pGSN’s protective effects is that it inhibits inflammatory mediators generated during primary injuries, since pGSN binds bioactive mediators, including lysophospatidic acid (LPA) and endotoxin in vitro. Because of its structural similarity we hypothesized that plasma gelsolin binds also to the potent lipid mediator platelet activating factor (PAF) and report here on the inhibition of PAF-induced cellular activation. Recombinant pGSN inhibited PAF-induced P-selectin up-regulation by human platelets as measured by flow cytometry. A ten- to 40-fold molar excess (0.5–20 μM) of pGSN over PAF inhibits P-selectin expression by 40 to 80%. The concentrations of plasma gelsolin used approximate the ~2–3 μM concentrations in plasma, and the molar excess of pGSN over PAF is probably greater in biological systems, where PAF has nanomolar affinity for its receptor. pGSN also inhibited PAF-induced superoxide anion (O2-) production (measured by chemiluminescence) of human neutrophils (PMN) in a concentration-dependent manner. The inhibition was up to 80% at a concentration of 10 μM (tenfold molar excess over PAF). A phospholipid-binding peptide derived from pGSN (QRLFQVKGRR) also inhibited PAF-mediated O2- generation by PMN. The inhibition was 65% at a 1:1 molar ratio (1 μM). In conclusion pGSN interferes with PAF-induced cellular activation in vitro, suggesting a mechanism for the protective role of plasma gelsolin that has been observed in vivo.
Effect of DMT1 Mutations at Birth and in the First Years of Life.
Authors: Achille Iolascon , Maria d’Apolito , Veronica Servedio , Luigia De Falco , Antonio Piga , Clara Camaschella
Published: April 2020

Divalent metal transporter 1 (DMT1) is involved in dietary iron uptake on the luminal side of duodenal enterocytes and transfers iron from the endosome to the cytosol in the marrow erythroblasts. Spontaneous (mk mice and Belgrade rats) or acquired (DMT1 -/- mice) inactivation of DMT1 in rodents produces a severe microcytic anemia at birth, caused by inefficient intestinal iron absorption and defective iron utilization in erythroid cells. The first reported patient with DMT1 mutations had microcytic anemia and iron overload in adult life. We here report the hematological phenotype of a newborn with a severe mycrocytic anemia (Hb 4 g/dL, MCV 71 fL) at birth and during the first months of life. Serum iron, transferrin saturation and serum ferritin were 160 microg/L, 100% and 846 ng/ml respectively at 3 months of age. Hepatic iron overload wad documented at the age of 5 years by both non invasive SQUID and liver biopsy. Sequence analysis of genomic DNA of the family revealed that the child was compound heterozygote for two novel DMT1 mutations, inherited by the asymptomatic parents. The first change deleted 3 bp (c.310 - 3_5del CTT) in intron 4 resulting in a splicing abnormality and the skipping of exon 5. The second was C>T 1246 substitution that causes arginine > cysteine replacement at position 416 (p. R416C) in the protein. This missense affects an highly conserved residue in one of the putative transmembrane domains. A striking reduction of the protein in peripheral blood cells of the proband was demonstrated by western blot using an anti-DMT1 antibody. The child required blood transfusions at birth and in the first two months of life. Thereafter, treatment with subcutaneous erythropoietin mantained hemoglobin levels between 7.5–9.5 g/dL, allowing transfusion-independence. The haematological phenotype of this patient highlights the essential role of DMT1 in erythropoiesis. The early and significant hepatic iron accumulation indicates that, as in animal models, DMT1 is dispensable for liver iron uptake. Finally DMT1 inactivation in the gut is likely bypassed by other pathways of iron absorption.
Urinary Hepcidin in Thalassemic Syndromes.
Authors: Nemeth Elizabeta , Origa Raffaella , Ganz Tomas , Galanello Renzo
Published: April 2020

Hepcidin, a 25 amino-acid peptide hormone synthesized in the liver, is the key regulator of iron homeostasis. Hepcidin inhibits intestinal iron absorption, recycling of iron in the macrophages and mobilization of iron from hepatic stores. Hepcidin expression is induced by iron loading and inflammation and is suppressed by anemia and hypoxia, but the relative influences of these modifiers are not well understood. Thalassemia syndromes represent a clinical setting where hepcidin is regulated by opposing influences of ineffective erythropoiesis and elevated iron load. We evaluated urinary hepcidin levels in 10 thalassemia intermedia (TI) patients who had no or very few transfusions (less than 5, and all completed more than 15 years ago), and 11 thalassemia major (TM) patients who were regularly transfused and iron chelated. All patients had beta-zero thalassemia (beta 39C→G non-sense mutation). When compared to the unrelated controls, urinary hepcidin was decreased in TI and increased in TM [median (interquartile range) in ng hepcidin/mg creatinine: controls 44 (27–66); TI 6 (5–9); TM 218 (116–470); all comparisons p<0.001 by One Way ANOVA with Dunn’s]. However, assessment of the hepcidin-to-ferritin ratio, an index of the appropriateness of hepcidin expression relative to the degree of iron loading, showed that the ratio was low in both thalassemia syndromes when compared to controls. The result suggests that even in TM patients, hepcidin is inappropriately low relative to the patients’ iron load. Importantly, in TM when measured over 1 week, hepcidin levels decreased in correlation with the patients’ rapidly decreasing Hb levels. In considering all the thalassemia patients together, urinary hepcidin levels correlated positively with serum ferritin and hemoglobin, and negatively with sTfR and serum erythropoetin. Multivariate analysis showed the strongest correlation with sTfR (r2=0.83).

The results indicate that in TI, the strong erythropoietic drive is the main regulator of hepcidin. The resulting hepcidin deficiency may be the cause of the increased iron absorption in TI. In TM, transfusions partially relieve the erythropoetic drive and increase the iron loading of macrophages thus raising hepcidin levels above those seen in TI. In the future, therapeutic use of hepcidin could restore normal iron homeostasis in some thalassemics, especially those not requiring transfusions.
Erythrocyte Adenylate Kinase Deficiency: First In-Depth Biochemical Characterization of the Y164C Mutant Enzyme Causing Hemolytic Anemia.
Authors: Abrusci Patrizia , Chiarelli Laurent R. , Fermo Elisa , Galizzi Alessandro , Zanella Alberto , Valentini Giovanna
Published: April 2020

Adenylate kinases (AK, EC 2.7.4.3) are monomeric enzymes that catalyze the reversible transfer of the γ-phosphate group from a phosphate donor (normally ATP) to a phosphate acceptor (normally AMP) releasing two molecules of nucleoside diphosphates (ADP). Therefore, the AK enzymes play a crucial role in the synthesis of nucleotides that are required for a variety of cellular metabolic processes. At present, six isoforms of AK with different tissue localization and substrate specificity have been characterized in man. AK1 is a cytosolic enzyme, mainly expressed in skeletal muscle, brain and erythrocytes. Adenylate kinase deficiency is a rare red blood cell enzymopathy causing hemolysis; the genetic disorder is transmitted as an autosomal recessive trait. To date 7 different mutations have been reported. To analyze the mutations at the protein level, we have undertaken the expression, purification and enzymological characterization of the human AK1 in the wild-type and Y164C mutant form. The cDNA encoding the AK1 was obtained from a blood sample of a healthy donor, with normal AK1 activity. Maximal expression (about 100–120 mg of AK1 per liter of E.coli culture) was obtained after 6 hours of induction with 0.5 mM IPTG at 37°C. The recombinant enzyme was purified to homogeneity following a simple procedure. The specific activity was greater than 2000 U/mg. AK1 exhibited a relatively high stability (T50 after 10 min of incubation, 53°C). Moreover, Mg-ATP protected enzyme activity from heat inactivation (T50 about 20 degrees higher). The catalytic efficiency values towards ATP and AMP were essentially identical and similar to that of the authentic enzyme. The Y164C mutation has been found in an Italian patient with congenital hemolytic anemia and undetectable erythrocyte AK1 activity. By site directed-mutagenesis we have produced the Y164C mutant protein and we have performed the first in-depth functional analysis of AK1 causing hemolytic anemia. The mutant enzyme essentially exhibited Km values unchanged, but drastic reductions of the catalytic efficiency towards both substrates (2 orders of magnitude). The Y164C mutant displayed little alterations in the thermostability properties. However, Mg-ATP failed to protect the enzyme activity from heat inactivation. Our data strongly suggest that the absence of AK1 activity observed in the patient homozygous for the Y164C mutation is due to inability of the enzyme to undergo conformational changes essential for the catalytic cycle.
The Allantois and Chorion, Isolated before Chorio-Allantoic Fusion, Have Hematopoietic Potential.
Authors: Zeigler Brandon M. , Daisuke Sugiyama , Guo Y. , Karen Downs M. , Nancy Speck A.
Published: April 2020

The chorio-allantoic placenta provides for the exchange of nutrients, gas, and waste between the fetus and mother. The murine placenta is also enriched for long term repopulating hematopoietic stem cells, and is therefore a stem cell niche (Gekas et al. Dev. Cell8, 365, 2005 and Ottersbach et al. Dev. Cell8, 377, 2005). However, it is not known if the placenta is colonized by hematopoietic stem cells that originate from other sites in the conceptus, or whether the placenta itself is also a site of hematopoietic cell emergence. The placenta is formed through the fusion of the allantois, a mesodermal extension from the primitive streak, with the chorionic plate, which is comprised of extraembryonic ectoderm and mesoderm. Here we show that the allantois and chorion, isolated before chorio-allantoic fusion and prior to the establishment of circulation, contain hematopoietic potential. Both allantois and chorion explants produced CD45 positive cells, some of which also expressed Mac-1 and Gr-1, when cultured on OP9 stromal cells in the presence of cytokines that promote myeloid cell differentiation. Cultures of allantois and chorion explants in the presence of lymphoid promoting cytokines induced B220 and CD19 positive B cells. Spatial and temporal analysis of Runx1, a marker for definitive hematopoiesis, showed Runx1 is not expressed in either the allantois or the chorion prior to fusion, but is expressed at the base of the allantois and at the chorio-allantoic junction following fusion. Both allantoic explant cultures, and cultures of whole embryos from which allantoises were removed in order to prohibit chorio-allantoic fusion showed that Runx1 expression in neither tissue is dependent upon fusion. We conclude that the allantois and chorion have intrinsic hematopoietic potential prior to the establishment of circulation. Our results furthermore suggest that the placenta is both a niche and a source of hematopoietic cells.
BMP4 Favors Hematopoietic Differentiation from Murine Embryonic Stem Cells and Acts by Activation of the cdx-hox Pathway.
Authors: Lengerke Claudia , Wang Yuan , Yates Frank , Maouche-Chretien Leila , Daley George Q.
Published: April 2020

Cdx4 and cdx1, members of the caudal family of homeodomain-containing transcriptional regulators, are important for specifying the hematopoietic fate of mesoderm in the zebrafish. We have shown that the cdx4 gene plays a role in enhancing hematopoietic fate during in vitro differentiation of murine ESCs (Davidson et al., Nature 2003). Cdx4 induces hox genes, and genetic modification of mESCs with a combination of cdx4 and hoxb4 promotes long-term engraftment of ESC-derived HSCs in lethally irradiated primary and secondary mice (Wang et al, submitted). While cdx1 is known to be a direct target of signaling by the embryonic morphogens fgf, wnt3a, and retinoids, morphogens acting upstream of cdx4 have not yet been defined. Our goal is to determine optimal morphogen conditions for hematopoietic commitment from murine embryonic stem cells by evaluating activation of the cdx-hox pathway. We have developed quantitative RT-PCR assays for the cdx genes (cdx4, cdx1 and cdx2) and multiple hox genes as well as markers specific to hematopoietic stem cells and lineages. We have used these assays, together with a reporter line engineered to express GFP from the brachury locus (Fehling et al., Development 2003), to characterize the conditions for mesodermal induction and hematopoietic fate specification following addition of morphogens to differentiating cultures of ES cells under serum-free conditions. Among all morphogens tested (BMP4, activin, nodal, wnt3a, wnt5a, sonic hedgehog, indian hedgehog, retinoic acid), only BMP4 has been found to strongly induce CDX4 gene expression within the developing embryoid bodies, while addition of the BMP4 inhibitor noggin to serum suppressed CDX4 expression. Addition of BMP4 significantly increases the number of emerging CD41+ and CD45+ cells, the precursors of definitive hematopoietic stem cells. We are currently analyzing the functional changes following BMP4 exposure, and correlating hematopoietic maturation with changes in the Hox gene expression pattern. Analysis of the cdx-hox gene pathway provides a means of otpimizing induction of hematopoietic fate by application of embryonic morphogens.
Methyl Binding Domain Protein 2 Mediates gamma-Globin Silencing in Adult beta-YAC Transgenic Mice.
Authors: Rupon Jeremy W. , Wang ShouZhen , Gaensler Karin , Lloyd Joyce , Ginder Gordon D.
Published: April 2020

The genes of the vertebrate beta-globin locus undergo a switch in expression during development whereby embryonic/fetal genes of the cluster are sequentially silenced and the adult genes are activated during erythroid development. DNA methylation has been shown to be associated with developmentally silenced globin genes, and compounds that inhibit cytosine methylation have been shown to activate transcription from developmentally silenced globin genes in several species, including humans. Previously, we have shown that the methyl domain binding protein 2 (MBD2) is involved in maintaining embryonic rho-globin gene silencing in adult avian erythroid cells. We describe here a role for MBD2 in the DNA methylation mediated silencing and maintenance of silencing of the human fetal gamma-globin gene in a transgenic mouse model. We confirmed the previously published report by Pace et al that the gamma-globin gene is reactivated, upon treatment with the DNA methyltransferase inhibitor, 5-azacytidine, of mice containing the entire beta-globin locus as a yeast artificial chromosome (BetaYAC) transgene. In order to elucidate the mechanism through which DNA methylation represses the gamma-globin gene in adult erythroid cells, betaYAC/MBD2−/− mice were generated by breeding BetaYAC mice with MBD2−/− mice. Anemic adult betaYAC/MBD2−/− mice continue to express the gamma-globin gene at a level commensurate with animals treated with 5-azacytidine, which is10–20 fold over those treated with 1-acetyl-2-phenylhydrazine alone, as measured by both quantitative PCR and by RNase protection assays. In addition, the level of gamma-globin gene expression is consistently several fold higher in MBD2−/− compared to wild type BetaYAC mice in 14.5 and 16.5 dpc fetal liver erythroblasts. Furthermore, transcriptional activation of the gamma-globin gene in adult erythroblasts is associated with a modest decrease in DNA methylation around the gamma-globin promoters and a ~4-fold enrichment of histone H3 trimethylated at lysine 4 (TriK4), as measured by chromatin immunoprecipitation assay using quantitative PCR. Finally, treatment of MBD2 null mice with 5-azacytidine induces only a small, non-additive induction of gamma-globin expression indicating that DNA methylation acts primarily through MBD2 to maintain gamma-globin suppression in adult erythroid cells. These results suggest that MBD2 is a potential target for therapeutic induction of gamma-globin gene expression in the settings of sickle cell anemia and beta-thalassemia.
Large-Scale Liquid Culture Production of Erythroid Cells from Human Embryonic Stem Cells.
Authors: Olivier Emmanuel N. , Qiu Caihong , Bouhassira Eric E.
Published: April 2020

Early human erythropoiesis is difficult to study because the material is hard to access experimentally. Hence, relatively little is known about the gene expression profiles or the mechanism of globin gene expression in these early cells. We report here a system to produce large quantities in liquid culture of virtually pure erythroid cells starting from H1 human embryonic stem cells (hESCs). The system is adapted from methods to produce enucleated red blood cells from cord blood and consist of five steps. During the first step, hESCs are differentiated by co-culture on immortalized human fetal hepatocytes (FH-B-hTERT) for two weeks to produce hematopoietic cells. CD34 positive cells are then magnetically sorted and placed in step 2 for seven days in serum free medium in the presence of SCF, Epo, hydro-cortisone, flt-3 ligand, BMP-4 and IL3. In step 3, the cells are incubated for seven days in the same medium and cytokine cocktail but with IGF-1 and without flt-3-ligand. In step 4, the cells are incubated with Epo for 3 days, and in step 5 the cells are incubated without cytokine on a feeder layer of MS-5 cells. In a typical experiment, 2 millions hESCs (two 10cm2 wells) yield 50,000 sorted CD34 positive cells. Culture of these cells for about three weeks yields about 5 millions erythroid cells. This corresponds to a 5 to 10,000-fold amplification of the sorted hematopoietic cells since we estimate that only a few percent of the cells recovered with the CD34 magnetic beads are hematopoietic. Flow cytometry analysis revealed that at the beginning of the second step the CD34+ cells are CD45−, CD71low and CD235a−. After 7 days in liquid culture CD34 expression is less than 10%, CD45 and CD71 expressions are more than 95% and CD235a is less than 20%. Eight days later the cells are 95% CD34− CD45− CD71high and CD235a+. Finally at the end of the culture the cells become CD34−, CD45−, CD71− and CD235a+. Morphological analysis by Wright-Giemsa staining revealed that the differentiation process in the liquid culture is relatively synchronous and that at the end of the culture the majority of the cells are orthochromatic erythroblasts. In contrast to cord blood derived cells placed in similar differentiation conditions, very few enucleated red blood cells could be obtained from hESCs. Hemoglobin can first be detected spectrophotometrically after day 10 of liquid culture and reach a concentration of 20 pmol/106 cells at the end of the culture. Globin chain analysis by PCR and HPLC reveals that ξ, α, ε, and γ globin chains are synthesized by these cells but not β-globin could be detected. A detailed analysis of globin expression in early human erythroid cells will be presented in an accompanying abstract. This experimental system will be useful to study early erythropoiesis, to test gene therapy vectors, and to create genetically modified red blood cells.
High Throughput Screen To Identify Small Molecules That Differentially Regulate Expression of the Globin Genes.
Authors: Ebert Benjamin L. , Mak Raymond , Pretz Jennifer L. , Peck David , Haggerty Stephen , Tolliday Nicola , Golub Todd R.
Published: April 2020

Several lines of evidence indicate that the pharmacological activation of fetal hemoglobin is an effective therapy for sickle cell anemia and beta thalassemia, but novel treatments for these diseases are needed. We developed and validated a high throughput assay to detect differential regulation of the globin genes and utilized this assay in a small molecule screen to identify novel compounds that increase the relative expression of gamma globin. In our assay, transcripts for the alpha, beta, delta, epsilon, gamma, theta, and zeta globin genes are amplified by multiplexed ligation-mediated PCR. Labeled amplicons are captured on different fluorescent microspheres using molecular barcodes, and the relative abundance of labeled amplicons is detected by high speed flow cytometry. To recapitulate the activity of compounds in the bone marrow of patients as accurately as possible, the screen was performed using primary human erythroid progenitor cells cultured in vitro. The assay was adapted to 384-well format with robotic liquid handling. In validation studies, the assay detected the expected increases in globin gene expression during erythroid differentiation, increased gamma globin expression in umbilical cord blood progenitor cells, and increased gamma globin expression in cells treated with known inducers of fetal hemoglobin including hydroxyurea and sodium butyrate. We screened a library of 1040 known bioactive compounds, 75% of which are FDA approved drugs, and a library of 600 compounds produced by diversity oriented synthesis that have been shown to inhibit histone deacetylase (HDAC) activity. In the screen, we rediscovered previously identified globin gene regulators, further validating our globin assay. For example, corticosteroids, known activators of fetal hemoglobin, increased the relative expression of gamma globin. Thyroid hormone specifically increased expression of delta globin, consistent with clinical observations that hemoglobin A2 levels are increased in hyperthyroidism and decreased in hypothyroidism. We identified ten novel compounds from the diversity oriented synthesis library that powerfully induce expression of the gamma globin gene relative to beta globin. Moreover, HDAC inhibition reversed the ontogeny of globin gene expression, coordinately increasing expression of fetal and embryonic relative to the adult globin genes. Relative to beta globin gene expression, gamma and epsilon globin were induced while delta globin was unaffected by HDAC inhibitors; relative to alpha globin expression, zeta globin was increased and theta globin was unaffected. The identification of compounds that differentially regulate globin gene expression may provide lead compounds for the development of novel therapies for sickle cell disease and beta thalassemia and may help elucidate the molecular events underlying switching of the globin genes during normal development.
Genomic Organization, Chromatin Structure, and Transcriptional Regulation of the Murine Alpha Hemoglobin Stabilizing Protein (AHSP) Gene.
Authors: Dore Louis C. , Vakoc Christopher R. , Blobel Gerd A. , Hardison Ross C. , Bodine David M. , Gallagher Patrick G. , Weiss Mitchell J.
Published: April 2020

Alpha Hemoglobin Stabilizing Protein (AHSP, Eraf) is an abundant erythroid protein that binds and stabilizes alpha globin and alpha hemoglobin (Hb). In mice, loss of AHSP causes hemolytic anemia, with elevated levels of reactive oxygen species and Hb precipitation in erythrocytes. Loss of AHSP exacerbates beta thalassemia phenotypes in mice, presumably by enhancing the toxicity of excessive free alpha Hb. Based on these findings, AHSP is a candidate modifier gene for beta thalassemia in humans. No mutations in the AHSP coding region have been identified in patients to date. However, several groups reported an inverse correlation between beta thalassemia severity and erythroid AHSP expression levels, raising the possibility that AHSP is a quantitative trait modifier of beta thalassemia. To address this possibility, it is important to define the mechanisms that control expression of the AHSP gene. Transcripts of murine Ahsp are inducible by GATA-1. The goals of the current studies are to investigate the mechanisms of this induction and to define the DNA domain that regulates the locus. Using phylogenetic comparisons, we identified a hotspot for mammalian chromosomal rearrangement just downstream of the Ahsp gene. This hotspot is located at the end of a syntenic block of approximately 350 kb that is conserved in mammals and likely marks the 3′ end of the gene regulatory domain. We focused our initial functional studies on a 7 kb genomic region bounded at the 5′ (centromeric) end of Ahsp by the nearest adjacent gene, an EST expressed in multiple tissues, and at the 3′ (telomeric) end by the rearrangement hotspot. In transient transfection assays, the Ahsp promoter region conferred erythroid-specific expression to a linked reporter gene. In heterologous cells, GATA-1 transactivated the Ahsp promoter in a dose-dependent fashion. To examine GATA-1 binding and its subsequent effects on the Ahsp gene in vivo, we used G1E-ER4 cells, a GATA-1 null erythroblast line that undergoes terminal erythroid maturation after activation of an estradiol-inducible form of GATA-1. We made several findings with regards to the role of GATA-1 in Ahsp gene regulation. First, GATA-1 and its cofactor, Friend of GATA-1 (FOG-1), bind directly to the Ahsp locus at regions that contain conserved GATA consensus motifs and are predicted to be important erythroid regulatory elements by our bioinformatic studies. Second, GATA-1 induces epigenetic changes in chromatin structure that are associated with gene activation, including formation of a DNase I hypersensitive site, hyperacetylation of histones H3 and H4, and methylation of histone H3 lysine-4. Together, these findings begin to establish the DNA region and mechanisms that control Ahsp transcription, allowing for further studies to map the cis elements responsible for population variations in gene expression.
Expression of Fetal Hemoglobin among Adult Human Erythroblasts Is Associated with an Altered Expression Pattern for the Transcription Factor c-Myb.
Authors: Rudrabhatla Parvathi , Miller Jeffery L.
Published: April 2020

The c-Myb transcription factor plays a central role in regulating growth and differentiation during definitive erythropoiesis. In addition, c-Myb expression has been previously linked to GATA-1 expression and hereditary persistence of fetal hemoglobin. Here we examined a potential role for c-Myb in signaled expression of fetal hemoglobin (HbF) among primary adult human erythroblasts. Initial experiments were performed to determine the expression pattern of c-Myb under conditions of low fetal hemoglobin production (CD34 cells cultured over 14 days in media supplemented with erythropoietin (EPO) alone). Quantitative PCR performed on cells sampled on days 2, 5, 8, 11, and 14 demonstrated highly regulated c-Myb expression. A dramatic increase (>30 fold) in c-Myb mRNA was measured between days 5 and 8 as the committed progenitor cells differentiated to the proerythroblast stage. That increase was followed by a 3-fold reduction in c-Myb during terminal differentiation. Western blotting of nuclear extracts obtained on days 7 and 14 in EPO cultures corroborated with the quantitative PCR analyses. Electrophoretic mobility shift assays performed using day 7 and day 14 nuclear extracts showed a shifted major band with a c-Myb consensus probe. That major band was competed by unlabeled c-Myb probe on both days. Moreover, day 14 extracts repeatedly revealed two additional bands that were not competed suggesting the possibility of other complexes binding to the Myb motif or changes in c-Myb conformation during terminal differentiation. Chromatin immunoprecipitation assays also revealed preliminary evidence for Myb binding directly to beta globin locus. When compared to low HbF culture conditions (EPO; HbF <1%), cells grown in high HbF conditions (erythropoietin + stem cell factor + TGF-beta (EST); HbF>35%) demonstrated a distinct pattern of c-Myb expression. Relative to the low HbF conditions, high HbF expressing cells had reduced c-Myb levels on day 7 followed by increased levels on day 14. These results suggest that c-Myb possesses highly regulated patterns of expression and DNA binding during erythroid maturation. In addition, the association of changes in c-Myb expression patterns with HbF production suggests that this growth-related transcription factor may help regulate fetal hemoglobin synthesis in patients with beta-hemoglobinopathies.
Antagonistic Roles of ERK1/2 and p38 MAP Kinases in Hemoglobin Synthesis.
Authors: Blank Volker , Lehalle Damien , Mardini Louay , Boudia Mansouria Merad , Derjuga Anna , Moore Amy
Published: April 2020

Murine erythroleukemia (MEL) cells provide a valuable model to study the molecular events leading to erythroid differentiation. Maturing erythroid cells synthesize large quantities of hemoglobin, a process requiring the coordinated synthesis of heme and globin. Here, we investigated the role of the ERK and p38 mitogen-activated protein kinase (MAPK) signaling pathways in the differentiation of MEL cells. We determined the effect of the MEK1/2 inhibitor U0126 that blocks the ERK1/2 pathway, and the p38 inhibitor SB202190 on the differentiation potential of MEL cells induced by hexamethylene bisacetamide (HMBA). We found that treatment of HMBA induced MEL cells with the ERK1/2 pathway inhibitor U0126 results in higher hemoglobin levels. Using a fluorometric assay, we determined that intracellular heme levels also increased. Immunoblot studies showed an increase in globin protein levels. In contrast, treatment of MEL cells with the p38 inhibitor SB202190 has the opposite effect, leading to decreased amounts of heme and hemoglobin. In addition, inhibition of the p38 pathways results in lower transferrin receptor levels. Our results suggest that the ERK1/2 and p38 pathways play antagonistic roles in HMBA induced erythroid differentiation in MEL cells. This data also provides a novel link between MAPK signaling and the regulation of heme biosynthesis and iron uptake in erythroid cells.
Surveyor™ Nuclease: A New Rapid and Effective Strategy for Detection of Single Nucleotide Changes in Alpha Globin Genes.
Authors: Chandy Mammen , Sindhuvi Eunice E. , Shaji R.V. , Mathews Vikram , George Biju , Vishwabandya Auro , Srivastava Alok
Published: April 2020

Alpha thalassemia is the most common globin gene disorder in the world and the disease is caused by mutations in either the alpha2 or alpha1 genes. Deletions of one (-α) or both (--) of these genes are the most common cause of alpha-thalassemia, while point mutations and small insertions/deletions are present but at a much lower frequency (5%). The number of point mutations described in these genes has been steadily increasing, with greater than 40 identified to date. Identifying these point mutations acquires importance as patients with non-deletional hemoglobin (Hb) H disease are more severely affected. Comprehensive evaluation of α globin genes is also essential while determining the molecular basis of the thalassemia intermedia phenotype. Mutations in the alpha globin gene can be detected either by direct DNA sequencing or by screening methods. Although small (about 1kb, 3 exons and 2 introns), the alpha-globin genes are duplicate and highly G-C rich, which makes them difficult to denature and therefore reducing sequencing efficiency and causing frequent artifacts. We have developed a simple and robust method based on mismatch-specific DNA endonuclease, SurveyorTM Nuclease. Genomic DNA control samples (n=21) harboring 12 different nucleotide changes characterized by direct DNA sequencing in either the alpha2 or alpha1 samples were used to validate this method. These samples were heterozygous or homozygous for each point mutation, or compound heterozygous for a deletion and the point mutation. Controls were amplified by gene specific primers designed by Primer3 software followed by digestion with SurveyorTM Nuclease. This endonuclease is a member of the CEL nuclease family of mismatch-specific nucleases derived from celery. It recognizes all insertions, deletions and base substitutions, and cleaves the mismatch of any heteroduplex DNA. After digestion, cleavage products are analyzed by agarose gel electrophoresis. The size of the digestion products indicates the location of the mutation, which is then confirmed and characterized by sequencing. All the 12 different nucleotide changes [α1 CD 14 TàC, α2 CD 104 TàG, α2 IVS II-55 TàG, α1 IVS II 141 TàC, α1 IVS I 117 GàT, α2 IVS I 1 GàA, Poly A (-AA), α1 CD 119 CàT, α2 IVS I 116, α2 CD 22 CàT, α2 CD 29 TàC, α2 +15 CàG] in the alpha globin genes were detected by this assay indicating a high sensitivity and specificity of this method. Mutation screening by SurveyorTM Nuclease is rapid (~ 5 hours), cost effective ($8/sample) and sensitive (100%). This assay in combination with the multiplex-PCR for screening the common alpha globin gene deletions will be the most comprehensive and economical approach for genetic diagnosis of alpha thalassaemia. Mismatch specific nucleases may have increasing application as a mutation detection strategy for human genetic diseases.
CREB Transgenic Mice Develop Myeloproliferative Disease/Myelodysplastic Syndrome after a Prolonged Latency.
Authors: Shankar Deepa B. , Kinjo Kentaro , Chang Jenny , Sakamoto Kathleen M.
Published: April 2020

The cyclic AMP Response Element Binding Protein, CREB, is a basic leucine zipper transcription factor that induces genes that regulate cell proliferation and survival. CREB is a downstream target of GM-CSF signaling pathways in myeloid cells, resulting in transactivation of critical target genes. We previously demonstrated that both CREB protein and mRNA levels are increased in the bone marrow cells of patients with AML at diagnosis. In contrast, CREB is expressed at low levels in normal bone marrow cells. To examine the expression of CREB in specific bone marrow and leukemia stem cell populations, we performed quantitative real-time PCR. AML blast cells were sorted and were shown to have increased CREB mRNA expression in CD34+CD33+ and CD34-CD33+ population compared to normal bone marrow progenitor cells. To understand the role of CREB in normal hematopoiesis and leukemogenesis, we created transgenic mice in which the myeloid specific hMRP8 promoter controls CREB expression. Within months, these mice developed increased monocytes and neutrophils in the peripheral blood. The increased cells in the peripheral blood and bone marrow were Gr1+Mac1+. We did not observe increases in other cell lineages using FACS analysis and the markers, B220, CD3, and Ter119. Bone marrow cells from CREB transgenic mice displayed increased colony size, greater numbers of colonies, and immortalization in colony replating assays with methylcellulose containing IL-3, IL-6, and Stem Cell Factor, compared to age-matched littermate controls. Bone marrow from CREB transgenic mice grew in the absence of cytokines, demonstrating factor-independent growth. Increased numbers of CFU-M was observed with CREB transgenic mouse bone marrow in colony assays with methylcellulose containing M-CSF. Although the mice did not develop acute leukemia, 7 out of 8 CREB transgenic mice compared to 0 out of 13 control mice developed enlarged spleens and myeloproliferative disease (MPD) after 12 months of age. Histology of the spleens showed destruction of the normal architecture with aberrant myeloid cells, suggestive of a myeloproliferative disease/myelodysplastic syndrome. Both the indolent course and phenotype of our mice were similar to Chronic Myelomonocytic Leukemia (CMML) observed in humans. Interestingly, myeloid cells from patients with CML in chronic phase also express higher levels of CREB. Studies are underway to characterize CREB expression in peripheral blood or bone marrow cells from patients with MPD/MDS, including CML, CMML, and transient myeloproliferative disease of Down Syndrome. We are also analyzing the spleens from CREB transgenic mice that develop MPD/MDS. Our results demonstrate that overexpression of CREB is sufficient to induce myeloid cell transformation to a preleukemic state in vivo. Therefore, the CREB transgenic mouse provides a useful model to test novel therapies to treat MPD/MDS.
Allogeneic Stem Cell Transplantation (SCT) with Reduced-Intensity Conditioning (RIC) in Children with Malignancies: A Retrospective Study from the Societe Francaise de Greffe de Moelle et de Therapie Cellulaire (SFGM-TC).
Authors: Sirvent Anne , Baruchel Andre , Paillard Catherine , Esperou Helene , Bordigoni Pierre , Bay Jacques-Olivier , Michel Gerard , Tabrizi Reza , Vannier Jean-Pierre , Galambrun Claire , Buzyn Agnes , Dalle Jean-Hugues
Published: April 2020

RIC regimens followed by allogeneic SCT have demonstrated to induce complete donor chimerism and long-term survival with decreased transplant-related mortality in adults with poor-risk hematological malignancies. Pediatric experience of RIC regimens is mostly reported in children with non malignant diseases in whom late long-term complications should be reduced. There are only limited data in children with malignancies, ineligible for conventional SCT. We conducted a retrospective survey of 28 children who underwent a RIC regimen for a malignant hematological disease (18 AL, 2 RAEBt, 1 CML, 3 NHL, 2 HD) or a solid tumor (1 Ewing sarcoma, 1 rhabdomyosarcoma) from 1998 to 2004 in 11 participating hospitals. All children had contraindications against myeloablative regimen. Median age was 14 years. Fourteen patients (pts) had an advanced disease (> CR2, refractory or progressive).Ten pts had already undergone at least one transplantation. Most RIC were fludarabine-based (n= 26). Nineteen children received additional anti-thymocyte globulin and 4 were transplanted with a T-depleted graft. Source of hematopoietic stem cell was marrow (n= 17), peripheral blood (n= 9) or cord blood (n= 2). Donor was matched related (n= 12) or unrelated (n= 8), mismatched related (n= 2) or unrelated (n= 6). Median follow-up after SCT is 142 days (range: 22–2345). Engraftment was observed in all 25 valuable pts. Day-90 chimerism analysis was performed in 18 pts and showed complete donor cells in 12 and autologous reconstitution in 3 pts. Grade II–IV aGVHD and cGVHD developed in 8/25 and 3/16 valuable pts, respectively. The day-100 transplant-related mortality rate was 18%. Disease progression or relapse occurred in 13 pts. Nineteen pts died: 11 from initial disease, 2 from infection, 2 from GVHD, 4 from other toxic complication. Four pts underwent a second transplantation for relapse or autologous reconstitution (3 myeloablative, 1RIC SCT). Five pts are alive in CR 145, 336, 782, 993, 2345 days after a first transplantation, 2 pts 272 and 1062 days after a second transplantation. Only 1 child among the 14 with an advanced disease is alive versus 6 among those with a lower-risk disease. In conclusion, allogeneic SCT with RIC is feasible in heavily pre-treated children. In order to reduce the high incidence of relapse, further prospective studies are needed to determine best pts selection criteria (initial diagnosis, treatment prior to transplant, chemosensitivity of the disease) and the best transplantation strategy (timing, type of the RIC, post-transplant additional treatment).
Lymphoid Chimerism (LC) Kinetic after Reduced Intensity Conditioning (RIC) Allogeneic Stem Cell Transplantation (allo-SCT).
Authors: Mohty Mohamad , Avinens Odile , Faucher Catherine , Furst Sabine , Blaise Didier , Eliaou Jean-Francois
Published: April 2020

In contrast to standard myeloablative allo-SCT, the kinetic of lineage-specific chimerism of donor origin is an important issue after RIC-allo-SCT, because of its major impact on transplant-related events and outcome. Predictive factors for full CD3+ T cell LC are not yet well defined in this setting. Here, we investigated the impact of different factors on the establishment of full LC after RIC-allo-SCT. 102 patients from a single center receiving allo-SCT from an HLA-identical sibling were analyzed. Patients and graft characteristics are: age 49 y. (range, 18–67), diagnoses: 41 myeloid malignancies (40%), 43 lymphoid malignancies (42%) and 18 metastatic solid tumors (18%). 83 patients (81%) were considered as high risk. 65 patients (64%) received a fludarabine, busulfan and ATG-based RIC, while 36 pts (36%) received a low dose irradiation-based RIC. All patients received a PBSC graft, with 55 (54%) receiving CSA alone for GVHD prophylaxis and 47 (46%) receiving CSA and MMF. Chimerism of immunomagnetically CD3+ T cells was assessed by VNTR at day 30, 60, 90 and >90 after allo-SCT. Full LC was defined as >95% of CD3+ sorted T cells of donor origin. At day 30, only 30% (95%CI, 21–39%) of patients achieved a full donor LC, and this increased to 50% (95%CI, 40–60%) by day 60. At day 90, a majority of 77% of the patients (95%CI, 69–85%) had a full donor LC. In this series, none of the patients’, graft, RIC regimen or disease characteristics could be predictive of establishment of a full LC at day 30 after allo-SCT. When looking for predictive factors for full donor LC at day 90, univariate analysis including patients and disease characteristics, RIC type and GVHD prophylaxis regimen, PBSC graft composition (CD34+, CD4+, CD8+, CD19+, NK cells), and early transplant-related events such as acute GVHD and time to engraftment, showed that a female donor, diagnosis (lymphoid malignancy or solid tumor vs. myeloid malignancy), CD34+ stem cell dose, and CD4+ T cell dose infused with the graft, were significantly associated with the establishment of full LC by day 90. In the multivariate analysis, a diagnosis other than a myeloid malignancy, was the strongest parameter significantly predictive of establishment of full LC at day 90 after RIC-allo-SCT (P=0.007; OR=3.82; 95%CI, 1.4–10.1). Overall, these results suggest that graft composition may represent an attractive tool towards harnessing chimerism of donor origin after RIC allo-SCT. Most importantly, disease-related features (type, previous courses of chemotherapy, previous autologous transplantation,≡) are the strongest predictors of the kinetic of donor LC that may impact transplant-related events after RIC allo-SCT.
αvβ3 Integrin Activation and Matrix Binding Mediate Cellular Responses to Mechanical Stretch.
Authors: Katsumi Akira , Matsushita Tadashi , Naoe Tomoki , Schwartz Martin A.
Published: April 2020

Mechanical tension is a critical determinant of vascular cell growth, differentiation, apoptosis, migration and development. Integrins have been implicated in sensing force but little is known about how forces are transduced to biochemical signals. We now show that mechanical strain stimulates conformational activation of integrin αvβ3 in NIH3T3 cells by using conformation specific antibody WOW-1. LM609, an antibody against αvβ3 integrin that is insensitive to integrin conformation, showed no change in binding after stretch, excluding changes in surface expression. Stretch also induced an increase of LIBS-6 binding, evidence that conformational activation leads to new binding of integrins to ECM. PI3K and Akt are both rapidly activated by strain, and PI3K inhibition decreases integrin activation. These data strongly suggest that PI3K mediates αvβ3 integrin activation. The blocking anti-fibronectin antibody 16G3 strongly inhibited the stretch-induced activation of JNK under conditions where existing adhesions did not appear to be disrupted, indicating that new integrin-ECM binding is required for JNK activation. These data define a pathway by which early activation of PI3K, through induction of integrin activation and ECM binding, stimulates a cytoplasmic signaling pathway implicated in cellular responses.