ORP-3, a human oxysterol-binding protein gene differentially expressed in hematopoietic cells

Using differential display polymerase chain reaction, a gene was identified in CD34+-enriched populations that had with low or absent expression in CD34- populations. The full coding sequence of this transcript was obtained, and the predicted protein has a high degree of homology to oxysterol-binding protein. This gene has been designated OSBP-related protein 3 (ORP-3). Expression of ORP-3 was found to be 3- to 4-fold higher in CD34+ cells than in CD34- cells. Additionally, expression of this gene was 2-fold higher in the more primitive subfraction of hematopoietic cells defined by the CD34+38- phenotype and was down-regulated with the proliferation and differentiation of CD34+ cells. The ORP-3 predicted protein contains an oxysterol-binding domain. Well-characterized proteins expressing this domain bind oxysterols in a dose-dependent fashion. Biologic activities of oxysterols include inhibition of cholesterol biosynthesis and cell proliferation in a variety of cell types, among them hematopoietic cells. Characterization and differential expression of ORP-3 implicates a possible role in the mediation of oxysterol effects on hematopoiesis.

The zebrafish spil promoter drives myeloid-specific expression in stable transgenic fish

The spi1 (pu.1) gene has recently been identified as a useful marker of early myeloid cells in zebrafish. To enhance the versatility of this organism as a model for studying myeloid development, the promoter of this gene has been isolated and characterized. Transient transgenesis revealed that a 5.3 kilobase promoter fragment immediately upstream of the spi1 coding sequence was sufficient to drive expression of enhanced green fluorescent protein (EGFP) in injected embryos in a manner that largely recapitulated the native spi1 gene expression pattern. This fragment was successfully used to produce a germ line transgenic line of zebrafish with EGFP-expressing myeloid cells. These TG(spi1:EGFP)pA301 transgenic zebrafish represent a valuable tool for further studies of myeloid development and its perturbation.

Harnessing zebrafish for the study of white blood cell development and its perturbation

Considerable progress has been made in understanding the molecular basis of normal white blood cell development and its perturbation in disease through the use of clinical studies and traditional animal and cell line models. Despite this, however, many questions are still being answered and white blood cell disorders, including leukemia and lymphoma, remain a significant health problem. The zebrafish (Danio rerio) has emerged as a powerful alternative vertebrate model for the study of development and disease. We review the recent application of zebrafish to the study of white blood cell development and its disruption, particularly leukemogenesis. Such studies have highlighted the overall conservation of these processes throughout vertebrates, and establish zebrafish as a useful experimental model. This organism is now poised to make an important contribution to our understanding of the underlying genetic control of white blood cell development and its disruption, as well as the identification of new therapeutic agents.

Hematopoietic perturbation in zebrafish expressing a tel-jak2a fusion

Objective
Various TEL-JAK2 fusions have been identified in patients with lymphoblastic and myeloid leukemias that result in constitutive activation of the JAK2 kinase domain. Such fusions can mediate factor-independent growth of hematopoietic cell lines and induction of malignancy in mouse models.
Materials and methods
To assess whether zebrafish could be utilized as a suitable model for the study of myeloid oncogenesis, we generated a zebrafish tel-jak2a fusion oncoprotein based on that seen in a case of chronic myeloid leukemia. This was transiently expressed in zebrafish embryos under the control of the spi1 promoter, which is strongly active in myeloid precursors.
Results
Visual, histological, and molecular analysis revealed disruption of normal embryonic hematopoiesis, including perturbation of the myeloid and erythroid lineages.
Conclusion
These results indicate that the zebrafish tel-jak2a oncoprotein is functional, and suggest that this organism will be useful for the experimental study of myeloid malignancy.

Constitutive activation of zebrafish Stat5 expands hematopoietic cell populations in vivo

Objective
Constitutive activation of Stat5 has been observed in a variety of malignancies, particularly myeloid leukemias. To directly investigate the in vivo consequences of Stat5 perturbation, we expressed constitutively active forms in zebrafish.
Methods
We generated mutants of the zebrafish stat5.1 protein (N646H, H298R/N714F, and N714F) based on previously identified constitutively active mutants of murine Stat5a. The in vitro properties of these mutants were determined using phosphorylation-specific antibodies and luciferase reporter assays, and their in vivo effects were analyzed through microinjection of zebrafish embryos.
Results
Two of these stat5.1 mutants (N646H and H298R/N714F) showed increased tyrosine phosphorylation and transactivation activity compared to the wild-type protein. Expression of either mutant led to a range of hematological perturbations, which were more pronounced for the H298R/N714F mutant. Interestingly, expression of wild-type also produced generally similar phenotypes. Further analysis showed that expression of the H298R/N714F mutant led to increased numbers of early and late myeloid cells, erythrocytes, and B cells. Some nonhematopoietic developmental perturbations were also observed, but these were equally prominent with wild-type or mutant forms.
Conclusion
These data implicate Stat5 activity as a direct critical regulator of hematological cell proliferation, suggesting a causal role for constitutively-active Stat5 in the etiology of hematological malignancies.

Tyrosine residues of the granulocyte colony-stimulating factor transmit proliferation and differentiation signals in murine bone marrow cells

Granulocyte colony-stimulating factor (G-CSF) is the major regulator of granulopoiesis and acts through binding to its specific receptor (G-CSF-R) on neutrophilic granulocytes. Previous studies of signaling from the 4 G-CSF-R cytoplasmic tyrosine residues used model cell lines that may have idiosyncratic, nonphysiological responses. This study aimed to identify specific signals transmitted by the receptor tyrosine residues in primary myeloid cells. To bypass the presence of endogenous G-CSF-R, a chimeric receptor containing the extracellular domain of the epidermal growth factor receptor in place of the entire extracellular domain of the G-CSF-R was used. A series of chimeric receptors containing tyrosine mutations to phenylalanine, either individually or collectively, was constructed and expressed in primary bone marrow cells from G-CSF-deficient mice. Proliferation and differentiation responses of receptor-expressing bone marrow cells stimulated by epidermal growth factor were measured. An increased 50% effective concentration to stimulus of the receptor Ynull mutant indicated that specific signals from tyrosine residues were required for cell proliferation, particularly at low concentrations of stimulus. Impaired responses by mutant receptors implicated G-CSF-R Y764 in cell proliferation and Y729 in granulocyte differentiation signaling. In addition, different sensitivities to ligand stimulation between mutant receptors indicated that G-CSF-R Y744 and possibly Y729 have an inhibitory role in cell proliferation. STAT activation was not affected by tyrosine mutations, whereas ERK activation appeared to depend, at least in part, on Y764. These observations have suggested novel roles for the G-CSF-R tyrosine residues in primary cells that were not observed previously in studies in cell lines.

Identification of genes differentially regulated by the P210 BCR/ABL1 fusion oncogene using cDNA microarrays

Objective: The t(9;22) translocation is associated with more than 95% of cases of chronic myeloid leukemia. The resulting fusion of the BCR and ABL1 loci produces the constitutively active BCR/ABL1 tyrosine kinase. A wide range of signal transduction molecules are activated by BCR/ABL1, including MYC, PI-3 kinase, and different STAT molecules. In contrast, relatively few genes are known to be regulated by BCR/ABL1 at the level of transcription.

Materials and Methods: In an effort to better understand the transcriptional program activated by BCR/ABL1, we used cDNA microarrays to evaluate the relative expression of approximately 6450 human genes in U937 myelomonocytic cells expressing P210 BCR/ABL1 via a tetracycline-inducible promoter.

Results: We confirmed the previously reported up-regulation of the PIM1 and JUN oncogenes by BCR/ABL1. In addition, we identified 59 more genes up-regulated by BCR/ABL1. Interestingly, roughly one third of these were genes previously reported to be interferon (IFN)-responsive, including the OAS1, IFIT1, IFI16, ISGF3G, and STAT1 genes. An additional seven BCR/ABL1-regulated genes were found to be IFN-responsive in U937 cells. The expression profile also included genes encoding transcription factors, kinases, and signal transduction molecules, as well as genes regulating cell growth, differentiation, apoptosis, and cell adhesion, features previously suggested to be affected by BCR/ABL1.

Conclusion: These observations shed novel insight into the mechanism of BCR/ABL1 action and provide a range of targets for further investigation.

The role of jak2a in zebrafish hematopoiesis

Janus kinase 2 (Jak2) transduces signals from hematopoietic cytokines, and a gain-of-function mutation (Jak2617V>F) is associated with myeloproliferative diseases, particularly polycythemia vera. In this study, we examined the role of jak2a in zebrafish embryos in knock-down and overexpression studies using morpholinos (MOs) targeting the 5' untranslated region (UTR) (jak2aUTR-MO) and splice-site junction (jak2aSS-MO) of jak2a, a Jak inhibitor AG490 and a constitutive-active form of jak2a (jak2aca). At 18 and 24 hours after fertilization (hpf), jak2a is expressed predominantly in the intermediate cell mass (ICM; site of primitive hematopoiesis) of wild-type and chordin morphant embryos (characterized by expansion of ICM). Both jak2a MOs and AG490 reduced gata1+ (erythroid) cells in Tg(gata1:GFP) embryos, signal transducer and activation of transcription 5 (stat5) phosphorylation, and gene expression associated with early progenitors (scl and lmo2) and erythroid (gata1, he1 and ßhe1) and myeloid (spi1 [early] and mpo [late]) lineages. The chordin morphant is associated with increased stat5 phosphorylation, and both jak2a MOs and treatment with AG490 significantly ameliorated ICM expansion and hematopoietic gene up-regulation in these embryos. Injection of plasmid encoding jak2aca significantly increased erythropoiesis and expression of gata1, he1 and ßhe1, spi1, mpo, and l-plastin. In conclusion, zebrafish jak2a is involved in primitive hematopoiesis under normal and deregulated conditions.

The myeloproliferative disorder-associated JAK2 V617F mutant escapes negative regulation by suppressor of cytokine signaling 3

The somatic JAK2 valine-to-phenylalanine (V617F) mutation has been detected in up to 90% of patients with polycythemia and in a sizeable proportion of patients with other myeloproliferative disorders such as essential thrombocythemia and idiopathic myelofibrosis. Suppressor of cytokine signaling 3 (SOCS3) is known to be a strong negative regulator of erythropoietin (EPO) signaling through interaction with both the EPO receptor (EPOR) and JAK2. We report here that JAK2 V617F cannot be regulated and that its activation is actually potentiated in the presence of SOCS3. Instead of acting as a suppressor, SOCS3 enhanced the proliferation of cells expressing both JAK2 V617F and EPOR. Additionally, although SOCS1 and SOCS2 are degraded in the presence of JAK2 V617F, turnover of SOCS3 is inhibited by the JAK2 mutant kinase and this correlated with marked tyrosine phosphorylation of SOCS3 protein. We also observed constitutive tyrosine phosphorylation of SOCS3 in peripheral blood mononuclear cells (PBMCs) derived from patients homozygous for the JAK2 V617F mutant. These findings suggest that the JAK2 V617F has overcome normal SOCS regulation by hyperphosphorylating SOCS3, rendering it unable to inhibit the mutant kinase. Thus, JAK2 V617F may even exploit SOCS3 to potentiate its myeloproliferative capacity.

Signaling mechanisms coupled to tyrosines in the granulocyte colony-stimulating factor receptor orchestrate G-CSF-induced expansion of myeloid progenitor cells

Granulocyte colony-stimulating factor (G-CSF) is the major regulator of neutrophil production. Studies in cell lines have established that conserved tyrosines Y704, Y729, Y744, Y764 within the cytoplasmic domain of G-CSF receptor (G-CSF-R) contribute significantly to G-CSF-induced proliferation, differentiation and cell survival. However, it is unclear whether these tyrosines are equally important under more physiological conditions. Here, we investigated how individual G-CSF-R tyrosines affect G-CSF responses of primary myeloid progenitors. We generated GCSF- R deficient mice and transduced their bone marrow cells with tyrosine "null" mutant (mO), single tyrosine "add back" mutants or wild type (WT) receptors. G-CSFinduced responses were determined in primary colony assays, serial replatings and suspension cultures. We show that removal of all tyrosines had no major influence on primary colony growth. However, adding back Y764 strongly enhanced proliferativeresponses, which was reverted by inhibition of ERK activitity. Y729, which we found to be associated with the suppressor of cytokine signaling, SOCS3, had a negative effect on colony formation. After repetitive replatings, the clonogenic capacities of cells expressing mO gradually dropped compared to WT. The presence of Y729, but also Y704 and Y744, both involved in activation of STAT3, further reduced replating
efficiencies. Conversely, Y764 greatly elevated the clonogenic abilities of myeloid progenitors, resulting in a >104–fold increase of colony forming cells over mO after the fifth replating. These findings suggest that tyrosines in the cytoplasmic domain of G-CSF-R, although dispensable for G-CSF-induced colony growth, recruit signaling mechanisms that regulate the maintenance and outgrowth of myeloid progenitor cells.

Morphologic and functional characterization of granulocytes and macrophages in embryonic and adult zebrafish.

The zebrafish is a useful model organism for developmental and genetic studies. The morphology and function of zebrafish myeloid cells were characterized. Adult zebrafish contain 2 distinct granulocytes, a heterophil and a rarer eosinophil, both of which circulate and are generated in the kidney, the adult hematopoietic organ. Heterophils show strong histochemical myeloperoxidasic activity, although weaker peroxidase activity was observed under some conditions in eosinophils and erythrocytes. Embryonic zebrafish have circulating immature heterophils by 48 hours after fertilization (hpf). A zebrafish myeloperoxidase homologue (myeloid-specific peroxidase; mpx) was isolated. Phylogenetic analysis suggested it represented a gene ancestral to the mammalian myeloperoxidase gene family. It was expressed in adult granulocytes and in embryos from 18 hpf, first diffusely in the axial intermediate cell mass and then discretely in a dispersed cell population. Comparison of hemoglobinized cell distribution, mpx gene expression, and myeloperoxidase histochemistry in wild-type and mutant embryos confirmed that the latter reliably identified a population of myeloid cells. Studies in embryos after tail transection demonstrated that mpx- and peroxidase-expressing cells were mobile and localized to a site of inflammation, indicating functional capability of these embryonic granulocytes. Embryonic macrophages removed carbon particles from the circulation by phagocytosis. Collectively, these observations have demonstrated the early onset of zebrafish granulopoiesis, have proved that granulocytes circulate by 48 hpf, and have demonstrated the functional activity of embryonic granulocytes and macrophages. These observations will facilitate the application of this genetically tractable organism to the study of myelopoiesis.

Receptor activation and 2 distinct COOH- terminal motifs control G-CSF receptor distribution and internalization kinetics.

We have studied the intracellular distribution and internalization kinetics of the granulocyte colony-stimulating factor receptor (G-CSF-R) in living cells using fusion constructs of wild-type or mutant G-CSF-R and enhanced green fluorescent protein (EGFP). Under steady-state conditions the G-CSF-R localized predominantly to the Golgi apparatus, late endosomes, and lysosomes, with only low expression on the plasma membrane, resulting from spontaneous internalization. Internalization of the G-CSF-R was significantly accelerated by addition of G-CSF. This ligand-induced switch from slow to rapid internalization required the presence of G-CSF-R residue Trp650, previously shown to be essential for its signaling ability. Both spontaneous and ligand-induced internalization depended on 2 distinct amino acid stretches in the G-CSF-R COOH-terminus: 749-755, containing a dileucine internalization motif, and 756-769. Mutation of Ser749 at position –4 of the dileucine motif to Ala significantly reduced the rate of ligand-induced internalization. In contrast, mutation of Ser749 did not affect spontaneous G-CSF-R internalization, suggesting the involvement of a serine-threonine kinase specifically in ligand-accelerated internalization of the G-CSF-R. COOH-terminal truncation mutants of G-CSF-R, found in severe congenital neutropenia, lack the internalization motifs and were completely defective in both spontaneous and ligand-induced internalization. As a result, these mutants showed constitutively high cell-surface expression.

Plasmodium falciparum induces reorganization of host membrane proteins during intraerythrocytic growth

The virulence of the malaria parasite, Plasmodium falciparum, is due in large part to the way in which it modifies the membrane of its erythrocyte host. In this work we have used confocal microscopy and fluorescence recovery after photo-bleaching to examine the lateral mobility of host membrane proteins in erythrocytes infected with P falciparum at different stages of parasite growth. The erythrocyte membrane proteins band 3 and glycophorin show a marked decrease in mobility during the trophozoite stage of growth. Erythrocytes infected with a parasite strain that does not express the knob-associated histidine-rich protein show similar effects, indicating that this parasite protein does not contribute to the immobilization of the host proteins. Erythrocytes infected with ring-stage parasites exhibit intermediate mobility indicating that the parasite is able to modify its host prior to its active feeding stage.

Haematology of cultured Murray cod

Murray cod are Australia's largest freshwater fish: endangered in the wild, but supporting a boutique industry. This project studied the blood cells of Murray cod and those affected by the disfiguring chronic erosive dermatopathy. This has provided insight into the factors affecting Murray cod health.