Erythroblasts are a source of angiogenic factors.

In adult bone marrow, mature erythroblasts are produced within structures called erythroblastic islands and then cross the endothelial barrier to reach circulation. Erythroblastic islands are composed of a central macrophage surrounded by maturing erythroblasts. In this study, it is shown that erythroid cells, but not the other mature hematopoietic cells, coexpress 2 angiogenic factors, vascular endothelial growth factor A (VEGF-A) and placenta growth factor (PlGF). Secretion of both VEGF-A and PlGF increases during in vitro erythroid differentiation. Erythroblast-conditioned medium can induce both migration of monocytes and endothelial cells and the permeability of endothelial cells. These effects are inhibited by anti-PlGF and/or anti-VEGF antibodies. Finally, it is shown that VEGF-A and PlGF proteins are expressed by bone marrow erythroblasts in vivo. Angiogenic factors secreted by erythroblasts may promote interactions either with macrophages in erythroblastic islands or with endothelial cells that would facilitate the passage of erythroid cells through the endothelial barrier. (Blood. 2001;97:1968-1974)

Lmo2 and GATA-3 associated expression in intraembryonic hemogenic sites.

It is now widely accepted that hemopoietic cells born intraembryonically are the best candidates for the seeding of definitive hemopoietic organs. To further understand the mechanisms involved in the generation of definitive hemopoietic stem cells, we analysed the expression of the hemopoietic-related transcription factors Lmo2 and GATA-3 during the early steps of mouse development (7-12 dpc), with a particular emphasis on intraembryonic hemogenic sites. We show here that both Lmo2 and GATA-3 are present in the intraembryonic regions known to give rise to hemopoietic precursors in vitro and in vivo, suggesting that they act together at key points of hemopoietic development. (1) Lmo2 and GATA-3 are expressed in the caudal mesoderm during the phase of intraembryonic precursors determination. (2) A highly transient concomitant expression is observed in the caudal intraembryonic definitive endoderm, suggesting that these factors are involved in the specification of intraembryonic hemopoietic precursors. (3) Lmo2 and GATA-3 are expressed within the hemopoietic clusters located in the aortic floor during fetal liver colonisation. Furthermore, a strong GATA-3 signal allowed us to uncover previously unreported mesodermal aggregates beneath the aorta. A combined in situ and immunocytological analysis strongly suggests that ventral mesodermal GATA-3 patches are involved in the process of intraembryonic stem cell generation.

Neuropilin-1 is expressed on bone marrow stromal cells: a novel interaction with hematopoietic cells?

In adult bone marrow, hematopoietic stem cells are found in close association with distinctive stromal cell elements. This association is necessary for maintenance of hematopoiesis, but the precise mechanisms underlying the cross-talk between stromal cells and hematopoietic stem cells are poorly understood. In this study, we used a bone marrow stromal cell line (MS-5) that is able to support human long-term hematopoiesis. This hematopoietic-promoting activity cannot be related to expression of known cytokines and is abolished by addition of hydrocortisone. Using a gene trap strategy that selects genes encoding transmembrane or secreted proteins expressed by MS-5 cells, we obtained several insertions that produced fusion proteins. In one clone, fusion protein activity was downregulated in the presence of hydrocortisone, and we show that insertion of the trap vector has occurred into the neuropilin-1 gene. Neuropilin-1 is expressed in MS-5 cells, in other hematopoietic-supporting cell lines, and in primary stromal cells but not in primitive hematopoietic cells. We show that neuropilin-1 acts as a functional cell-surface receptor in MS-5 cells. Two neuropilin-1 ligands, semaphorin III and VEGF 165, can bind to these cells, and the addition of VEGF 165 to MS-5 cells increases expression of 2 cytokines known to regulate early hematopoiesis, Tpo and Flt3-L. Finally, we show that stromal cells and immature hematopoietic cells express different neuropilin-1 ligands. We propose that neuropilin-1 may act as a novel receptor on stromal cells by mediating interactions between stroma and primitive hematopoietic cells.

Inhibitory interactions controlling organizer activity in fish.

An experimental system allowing the observation of 2 active organizing centers during zebrafish development is described. It was achieved by injection into a single marginal cell at the 16-cell stage of TARAM-A-D mRNA. TARAM-A-D was previously described as the mutated constitutive form of a type I receptor for transforming growth factor beta. In 80% of the injected embryos, 2 distinct organizers were observed at the onset of gastrulation. At the end of gastrulation, these embryos showed duplicated axial structures. Nevertheless, only 25% of the injected embryos displayed a recognizable axis duplication after 1 day of development. This paradox is taken as an evidence for suppressive effects exerted in a reciprocal manner when more than 1 organizing center is present.

An activated form of type I serine/threonine kinase receptor TARAM-A reveals a specific signalling pathway involved in fish head organiser formation.

The role of Transforming Growth Factor beta (TGF-beta)-related molecules in axis formation and mesoderm patterning in vertebrates has been extensively documented, but the identity and mechanisms of action of the endogenous molecules remained uncertain. In this study, we isolate a novel serine/threonine kinase type I receptor, TARAM-A, expressed during early zebrafish embryogenesis first ubiquitously and then restricted to dorsal mesoderm during gastrulation. A constitutive form of the receptor is able to induce the most anterior dorsal mesoderm rapidly and to confer an anterior organizing activity. By contrast, the wild-type form is only able to induce a local expansion of the dorsal mesoderm. Thus an activated form of TARAM-A is sufficient to induce dorsoanterior structures and TARAM-A may be activated by dorsally localized signals. Our data suggest the existence in fish of a specific TGF-beta-related pathway for anterior dorsal mesoderm induction, possibly mediated by TARAM-A and activated at the late blastula stage by localized dorsal determinant.

Analysis of the thrombopoietin receptor (MPL) promoter implicates GATA and Ets proteins in the coregulation of megakaryocyte-specific genes.

The MPL gene codes for the thrombopoietin receptor, whose ligand specifically controls megakaryocytic differentiation. To understand the molecular basis for the megakaryocyte-specific expression of MPL, we analyzed the promoter of this gene. A 200 bp fragment is sufficient for high-level specific expression. This fragment can bind several trans-acting factors in vitro, including GATA-1 and members of the Ets family. GATA-1 binds with low affinity to a unique GATA motif at -70 in the MPL promoter, and destruction of this site yields only a modest decrease in expression level in HEL cells. Ets proteins also bind with low affinity to two sites. One is located at position -15 and its destruction reduces expression to 50%; the other is located immediately downstream of the GATA motif and plays a crucial role in expression of the promoter in HEL cells, as its inactivation reduces expression to 15%. Furthermore, GATA-1 and two Ets proteins, Ets-1 and Fli-1, can trans-activate the MPL promoter in heterologous cells. The effects of GATA-1 and these two Ets proteins are additive. Together with our previous results on the glycoprotein IIb (GpIIb) promoter, this study indicates a molecular basis for the coregulation of early markers of megakaryocyte differentiation.

Structure and transcription of the human c-mpl gene (MPL).

The human c-mpl proto-oncogene encodes a member of the cytokine receptor superfamily, expressed mainly in CD 34-positive hematopoietic progenitors and in the megakaryocytic lineage. To investigate the elements required for this tissue-specific expression, we cloned the human c-mpl gene (MPL) as well as the 5' end of the mouse gene. The human c-mpl gene contains 12 exons distributed over 17 kb of DNA. Each of the two "cytokine receptor domains" of Mpl is encoded by a set of four exons, the transmembrane domain by a single exon and the cytoplasmic domain by two exons. We also describe how three types of mRNA, encoding different proteins, are generated. The major species contains all 12 exons; mRNAs encoding a protein with a smaller cytoplasmic domain are produced by termination of the transcript within intron 10, and mRNAs encoding a putative soluble form of the c-Mpl protein lack exons 9 and 10. The promoter regions of the human and mouse genes were characterized. These promoters are GC-rich and contain putative binding sites for proteins of the Ets and GATA families. Finally, we show that a 700-bp fragment of the human c-mpl promoter is active in the HEL and K562 cell lines, which express erythroid and megakaryocytic markers, but is inactive in the nonhematopoietic HeLa cell line and the Jurkat T lymphoid cell line.

The Fli-1 proto-oncogene, involved in erythroleukemia and Ewing's sarcoma, encodes a transcriptional activator with DNA-binding specificities distinct from other Ets family members.

The late stages of the erythroleukemias induced by either the replication-defective Friend spleen focus-forming virus (SFFV) or the Friend murine leukemia virus (F-MuLV) are associated with the insertional activation of one of two members (Spi-1 or Fli-1) of the Ets protooncogene family of transcriptional factors. Fli-1 is not rearranged or activated in the erythroleukemias induced by SFFV, and similarly Spi-1 is not rearranged or activated in the leukemic cell clones induced by F-MuLV. This strict specificity of integration sites suggests that Fli-1 and Spi-1 may be functionally distinct and transactivate different downstream genes during the progression of multistage Friend erythroleukemia. In this study, we show that the Fli-1 protein, like other Ets proteins, has DNA-binding activity and can act as a sequence-specific transcriptional activator. We also show that the Fli-1 and Spi-1 proteins are functionally distinct in that they recognize and transactivate through distinct DNA binding sites. Furthermore, we have identified an octanucleotide core sequence that is required in vitro for optimal binding of Fli-1 to the Drosophila E74 target and the promoter sequence of the human GPIIB gene.

Amino acid residues involved in the catalytic site of human erythrocyte bisphosphoglycerate mutase. Functional consequences of substitutions of His10, His187 and Arg89.

Human bisphosphoglycerate mutase (GriP2 mutase) is a trifunctional enzyme which synthesizes and degrades GriP2 in red cells. Among the amino acid residues involved in its active site there are two conserved histidine residues, His10 which is phosphorylated during the catalytic process and His187 for which only speculative data have been made about the potential role during the reactions. Another amino acid residue, Arg89, had not been described as part of this active site but we have recently shown that a natural mutant Arg89-->Cys was highly thermolabile and showed severe perturbations of its enzymatic properties. To understand better the exact role of these residues, replacements of His10 by Gly (H10G) or Asp (H10D), His187 by Asn (H187N), Tyr (H187Y) or Asp (H187D) and Arg89 by Cys (R89C), Ser (R89S), Gly (R89G) or Lys (R89K) were performed by site-directed mutagenesis. The results obtained in this report show that replacement of the His10 residue completely abolished the enzymatic activities. Concerning the His187 residue, our results afford arguments that it plays an essential role in the three catalytic activities. Indeed all these activities are abolished in the two H187Y and H187D variants, whereas they are detectable though strongly diminished, for the H187N variant. In addition mutations at His187 could be distinguishable from those at His10 since the former resulted in a thermolabile enzyme, whereas no significant change in heat stability was observed for the latter. It is noteworthy that the H187N variant is protected against thermal instability by glycerate 2,3-bisphosphate (GriP2). Concerning the Arg89 mutants, R89C, R89S and R89G, the three variants showed characteristics identical to those found in the natural R89C mutant, i.e. loss of 99% of synthase activity, consistent decrease of mutase and 2-phosphoglycolate-stimulated phosphatase activities whereas the unstimulated phosphatase activity was normal. Moreover these mutants were unstable at 55 degrees C but GriP2 was able to protect them against thermal instability. In contrast, the R89K mutant was stable at 55 degrees C. Its synthase and unstimulated phosphatase activities were normal but its mutase and 2-phosphoglycolate-stimulated phosphatase activities were decreased. In addition, Km values for monophosphoglycerates were increased (3.2-fold) in the synthase but normal in mutase activities, whereas Km values for GriP2 were normal in mutase and phosphatase activities.(ABSTRACT TRUNCATED AT 400 WORDS)

GATA and Ets cis-acting sequences mediate megakaryocyte-specific expression.

The human glycoprotein IIB (GPIIB) gene is expressed only in megakaryocytes, and its promoter displays cell type specificity. We show that this specificity involved two cis-acting sequences. The first one, located at -55, contains a GATA binding site. Point mutations that abolish protein binding on this site decrease the activity of the GPIIB promoter but do not affect its tissue specificity. The second one, located at -40, contains an Ets consensus sequence, and we show that Ets-1 or Ets-2 protein can interact with this -40 GPIIB sequence. Point mutations that impair Ets binding decrease the activity of the GPIIB promoter to the same extent as do mutations that abolish GATA binding. A GPIIB 40-bp DNA fragment containing the GATA and Ets binding sites can confer activity to a heterologous promoter in megakaryocytic cells. This activity is independent of the GPIIB DNA fragment orientation, and mutations on each binding site result in decreased activity. Using cotransfection assays, we show that c-Ets-1 and human GATA1 can transactive the GPIIB promoter in HeLa cells and can act additively. Northern (RNA) blot analysis indicates that the ets-1 mRNA level is increased during megakaryocyte-induced differentiation of erythrocytic/megakaryocytic cell lines. Gel retardation assays show that the same GATA-Ets association is found in the human GPIIB enhancer and the rat platelet factor 4 promoter, the other two characterized regulatory regions of megakaryocyte-specific genes. These results indicate that GATA and Ets cis-acting sequences are an important determinant of megakaryocytic specific gene expression.

Erythroid-specific activity of the glycophorin B promoter requires GATA-1 mediated displacement of a repressor.

We have performed a detailed analysis of the cis-acting sequences involved in the erythroid-specific expression of the human glycophorin B (GPB) promoter and found that this promoter could be divided into two regions. The proximal region, -1 to -60, contains a GATA binding sequence around -37 and an SP1 binding sequence around -50. This region is active in erythroid and non-erythroid cells. The distal region, -60 to -95, contains two overlapping protein binding sites around -75, one for hGATA-1 and one for ubiquitous proteins. This distal region completely represses the activity of the proximal promoter in non-erythroid cells and defines the -95 GPB construct as a GPB promoter that displays erythroid specificity. Using site directed mutagenesis, we show that the -37 GATA and the -50 SP1 binding sites are necessary for efficient activity of the -95 GPB construct. Mutations that impair the -75 GATA-1 binding result in extinction of the -95 GPB construct activity if the -75 ubiquitous binding site is not altered, or in loss of erythroid specificity if the -75 ubiquitous binding site is also mutated. Using a cotransfection assay, we found that hGATA-1 can efficiently activate transcription of the -95 GPB construct in non-erythroid cells. This transactivation is abolished by mutations that impair either the -37 GATA-1 or the -50 SP1 binding. Mutations that impair the -75 GATA-1 binding and still allow the -75 ubiquitous binding also abolish the transactivation of the -95 GPB construct, indicating that hGATA-1 can remove repression of the GPB promoter by displacement of the ubiquitous proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Compound heterozygosity in a complete erythrocyte bisphosphoglycerate mutase deficiency.

Erythrocyte bisphosphoglycerate mutase (BPGM) deficiency is a rare disease associated with a decrease in 2,3-diphosphoglycerate concentration. A complete BPGM deficiency was described in 1978 by Rosa et al (J Clin Invest 62:907, 1978) and was shown to be associated with 30% to 50% of an inactive enzyme detectable by specific antibodies and resulting from an 89 Arg-->Cys substitution. The propositus' three sisters exhibited the same phenotype, while his two children had an intermediate phenotype. Samples from the family were examined using polymerase chain reaction and allele-specific oligonucleotide hybridization and sequencing techniques. Amplification of erythrocyte total RNA from the propositus' sister around the 89 mutation indicated the presence of two forms of messenger RNAs, a major form with the 89 Arg-->Cys mutation and a minor form with a normal sequence. Sequence studies of the propositus' DNA samples indicated heterozygosity at locus 89 and another heterozygosity with the deletion of nucleotide C 205 or C 206. Therefore, the total BPGM deficiency results from a genetic compound with one allele coding for an inactive enzyme (mutation BPGM Créteil I) and the other bearing a frameshift mutation (mutation BPGM Créteil II). Examination of the propositus' two children indicated that they both inherited the BPGM Créteil I mutation.

Spleen focus-forming virus long terminal repeat insertional activation of the murine erythropoietin receptor gene in the T3Cl-2 friend leukemia cell line.

We have characterized the structure of the erythropoietin receptor gene promoter in normal murine erythroid tissues and in Friend-induced tumor cells. Using primer extension analysis, we identified two distinct transcriptional start sites, which were located 2 base pairs apart in anemic spleens, fetal liver, Friend-induced tumoral spleens, and mouse erythro-leukemia cells. In contrast, transcription was initiated 37 base pairs upstream of the normal cap sites in T3Cl-2, a Friend virus-induced murine erythroleukemia cell line. Also, the erythropoietin receptor mRNA in T3Cl-2 was overexpressed when compared with other erythroleukemia cell lines. We found that abnormal transcription occurring in T3Cl-2 cells resulted from an erythropoietin receptor gene alteration. Indeed, one erythropoietin receptor allele was rearranged by insertion of a spleen focus-forming virus long terminal repeat within the noncoding region of the first exon, 45 bases upstream of the ATG initiation codon and in the same 5'----3' orientation. The transcription of the rearranged allele was shown to be directed from the long terminal repeat promoter, leading to a long terminal repeat-erythropoietin receptor fusion transcript, whereas the normal erythropoietin receptor allele was weakly transcribed. Such altered receptor gene activation may provide a positive pressure in the development of tumorigenic erythroleukemia.

Natural and artificial mutants of the human 2,3-bisphosphoglycerate as a tool for the evaluation of structure-function relationships.

2,3-bisphosphoglycerate mutase is a multifunctional enzyme which catalyses in red blood cells the synthesis and the degradation of 2,3-bisphosphoglycerate, the allosteric effector of hemoglobin. In order to study the structure-function relationships in BPGM, an expression vector was constructed which yielded an active protein, but with a modified electrophoretic mobility, due to a non-blocked N-terminal residue. Using site directed mutagenesis, mutants were produced with shortened chains. Results indicated the importance of residues 252-256 for the function. A natural deficient mutant with the substitution 89 Arg----Cys was described. Artificial mutant with the same substitution reproduced the same defect, as well as mutants Arg----Gly and Arg----Ser, indicating the key role of Arg 89 in the enzymatic mechanism.