Josef Brock

Galectin-1, a natural ligand for the receptor-type protein tyrosine phosphatase CD45.

Galectin-1 binds preferentially to N-acetyllactosamine residues on oligosaccharides associated with several cell surface glycoconjugates. In the present work, placental galectin-1 has been identified to be a natural ligand for the receptor-type protein tyrosine phosphatase CD45. The binding of galectin-1 to CD45 was detected by affinity chromatography of NP 40 solubilized Jurkat T cell membranes on galectin-1 agarose followed by immunoblotting of the galectin-1 agarose bound fraction applying monoclonal antibodies to CD45 isoforms. The PTPase activity of the galectin-1 agarose binding membrane fraction could be inhibited by sodium orthovanadate. Preincubation of Jurkat T cell membrane preparations with galectin-1 decreased the membrane-associated PTPase activity in a concentration-dependent manner. Incubation of Jurkat cells with galectin-1 suppressed the immunoprecipitated PTPase activity of CD45. Galectin-1 stimulates the cell surface expression of phosphatidylserine an early indicator of apoptosis. In CD45+ Jurkat T cells, galectin-1 induces higher levels of phosphatidylserine when compared with CD45- Jurkat cells. These observations indicate that galectin-1-mediated ligation of CD45 is involved in the induction of apoptosis in Jurkat T cells.

Activation of the transcription factor NF-κB by the erythropoietin receptor: Structural requirements and biological significance

Abstract The transcription factor nuclear factor kappa B (NF-κB) has been implicated in the regulation of genes mainly involved in inflammation and immune response. We analysed the role of NF-κB in signalling pathways induced by the hematopoietic growth factor erythropoietin (EPO). Our data, obtained by electrophoretic mobility shift assays (EMSA) and reporter gene assays, show that the intracellular domain of the EPO receptor (EPOR) transmits signals leading to the activation of NF-κB. Studies employing an inhibitor specific for the EPOR-associated tyrosine kinase JAK2 suggest that JAK2-dependent pathways are not involved. The induction of an NF-κB-triggered reporter gene construct was inhibited by cotransfection of dominant negative forms of the src kinase Lyn, but not by dominant negative JAK2. Using epidermal growth factor (EGF)/EPOR hybrids containing mutant forms of the EPOR intracellular domain, we were able to further define the critical structures for the induction of NF-κB. The data show that although the activity of JAK2 seems to be dispensable, its association to the receptor, as well as the phosphorylation of membrane proximal tyrosine residues, are essential. Furthermore, the functional analysis of different receptor forms revealed a correlation of the abilities to induce NF-κB activity and to generate antiapoptotic signals.

Rapid activation of the MAP kinase pathway in hematopoietic cells by erythropoietin, granulocyte-macrophage colony-stimulating factor and interleukin-3.

MAP kinases are a family of serine/threonine specific protein kinases becoming activated in response to different proliferative stimuli by phosphorylation at both threonine and tyrosine residues. We report the involvement of MAP kinases in the signal transduction of the hematopoietic growth factors erythropoietin (EPO), granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) in the factor dependent human erythroleukemic cell line TF-1, suggesting a crucial role of these enzymes in the regulation of proliferation of hematopoietic cells. Both time course and degree of MAP kinase activation were similar for all three cytokines. A slightly lower stimulation effect of EPO corresponds to the observation that EPO stimulated cells proliferate at a lower rate.

SHP1 protein tyrosine phosphatase negatively modulates erythroid differentiation and suppression of apoptosis in J2E erythroleukemic cells.

Abstract The SH2 domain-containing tyrosine phosphatase SHP1 is known to play a crucial role in the regulation of hematopoiesis. It has been shown previously that SHP1 associates with the activated erythropoietin receptor (EPOR) and negatively regulates mitogenic signaling. To further elucidate the role of SHP1 in erythropoietin (EPO)-induced cellular responses we employed J2E erythroleukemic cells as a model for erythroid maturation and cytokine-triggered suppression of apoptosis. Our data indicate that overexpressed SHP1 inhibits both EPO-induced differentiation as well as prevention of apoptosis. The specific signaling pathways responsible are not unraveled so far. Therefore, we analyzed the involvement of SHP1 in two established EPO-stimulated pathways, the JAK/STAT and the MAP kinase cascades, by transient coexpression of reporter constructs containing binding sites for transcription factors targeted by these pathways and a SHP1 cDNA. Both pathways are inhibited by SHP1 as indicated by the lower induction of reporter gene activity. In conclusion, SHP1 regulates the transcriptional activity stimulated by the EPO-induced JAK/STAT and MAPK pathways and is involved in the signaling machinery responsible for erythroid differentiation and suppression of apoptosis.

Requirement for JAK2 inErythropoietin-Induced Signalling Pathways

Erythropoietin (EPO) exerts its activities by the induction of multiple signalling pathways through interaction with the erythropoietin receptor (EPOR). Previous studies have suggested that the Ras/MAP kinase as well as the JAK/STAT signalling cascades play significant roles in the induction of EPO-responsive genes. Here we show that, in HCD-57 erythroleukemic cells, both pathways are activated by EPO in a dose-dependent manner with similar sensitivities and kinetics. The activation of signalling molecules is closely related to the proliferative status of the cells. Using an antisense strategy, we were able to show that the downregulation of the JAK2 protein level in HCD-57 cells results in a distinct reduction of the ability to induce not only STAT5 DNA-binding, but also MAP kinase activity. Our results thus provide evidence for a significant contribution of the cytosolic tyrosine kinase JAK2 to the EPO-induced activation of the Ras/MAP kinase cascade.

INDUCTION OF LONG-TERM SURVIVAL OF RAT SKIN ALLOGRAFTS BY A NOVEL, HIGHLY EFFICIENT ANTI-CD4 MONOCLONAL ANTIBODY

The new monoclonal antirat CD4 antibody RIB 5/2, which detects another epitope than those covered by W3/25 and MRC OX35, was tested for its immunosup-pressive potency following skin allografting by using strain combinations with different genetic barriers in the MHC and genetic low- or high-responder background. High-dose and long-term therapy of the grafted rats led to a significant delay of the acute rejection (P<0.01) in the strain combination Wistar Furth—to—BDX as well as in LEW1W-to-LEW1A. No significant prolongation of the mean allograft survival time was obtained for the high-responder rats (LEW1A-to-LEW1W). Cytofluorometric analysis revealed that RIB 5/2 exerts the immunosuppressive activity predominantly by modulation of the CD4 glycoprotein. Furthermore, the dependence of the humoral immune response against the mouse-globulins upon the administered protein quantity could be demonstrated.

Galectin-induced activation of the transcription factors NFAT and AP-1 in human Jurkat T-lymphocytes.

Galectin-mediated ligation of glycoepitopes on T-cell activation markers induces an increase in the cytosolic calcium concentration ([Ca(2+)](i)) originating from a transient Ca(2+) release of internal stores as well as a sustained influx across the plasma membrane. In transiently transfected Jurkat T-lymphocytes, galectins [galectin-1 (gal-1), recombinant human galectin-1 (rec gal-1), nematode 32-kDa galectin (LEC-1), nematode 16-kDa galectin (LEC-6)] differentially stimulate the expression of the luciferase reporter gene constructs pNFAT-TA-Luc and pAP1(PMA)-TA-Luc, which are activated by the nuclear factor of activated T-cells (NFAT) or the transcription factor, activator protein 1 (AP-1), respectively. The galectin-stimulated expression of the reporter constructs is completely inhibited by lactose (30 mM) and asialofetuin (30 microM) carrying Galbeta1-4GlcNAc sequences. Preincubation of pNFAT-TA-Luc-transfected cells with cyclosporine A (0.1 microM) and FK506 (0.01 microM) abrogated the gal-1-induced expression of the reporter luciferase (Luc). Electrophoretic mobility shift assays (EMSAs) provided evidence for gal-1-stimulated increase in the binding of nuclear extracts to a synthetic oligonucleotide with an AP-1 consensus sequence.

Activation of STAT5 during EPO-directed suppression of apoptosis

The ligand-dependent activation of the JAK/STAT (Januskinase/Signal Transducer and Activator of Transcription) pathway has been implicated in the explanation of cytokine-specific regulation of gene expression. Previous studies have reported conflicting results on the role of the transcription factor STAT5 in erythropoietin (EPO)-induced cellular responses. In this study we focused on the functional importance of STAT5 docking sites in the intracellular EPO receptor (EPOR) domain for the mediation of antiapoptotic activities. We demonstrate that EPO-dependent survival of erythroleukemic cell lines is accompanied by sustained STAT5 DNA-binding activity. The role of single tyrosine residues was dissected by the analysis of myeloid FDCP-1 cells stably expressing mutant EPOR proteins. The data show that receptors having a high potential to mediate antiapoptotic signals also effectively activate STAT5, whereas receptors lacking STAT5 docking sites are diminished in both activities. We conclude that the transcription factor STAT5 is functionally implicated in the EPO-dependent survival of cells.

Inhibition of proliferation but not erythroid differentiation of J2E cells by rapamycin

During erythropoiesis, replication and maturation are tightly coupled processes. Here, we show that the immunosuppressant rapamycin inhibited basal- as well as erythropoietin-stimulated proliferation of the erythroid cell line J2E. In addition, it enhanced the antiproliferative effect of sodium butyrate. Although rapamycin suppressed erythroid cell division, it did not affect terminal differentiation induced by erythropoietin or sodium butyrate. The proliferative status of J2E cells correlated well with the activity of the ribosomal S6 kinase p70S6k, an enzyme effectively blocked by rapamycin. It was concluded from this study that erythroid maturation proceeded normally despite the rapamycin-induced inhibition of mitosis and of p70S6k activity. These data provide further evidence that separate signalling pathways for proliferation and differentiation exist in erythroid cells.

Immunohistochemical and glycohistochemical localization of the β-galactoside-binding S-type lectin in human placenta

The localization of the beta-galactoside binding lectin was studied immunohistochemically on acetone-fixed cryostat sections of full-term placental tissue using a biotinylated monoclonal antibody and glycohistochemically applying biotinylated asialofetuin and lactosylated bovine serum albumin. On blots of placental tissue lysates the lectin is recognized by the biotinylated lactosylated bovine serum albumin. The glycoconjugate recognition of the lectin on blots was inhibited in the presence of 0.1 M lactose showing the specificity of the interactions. The anti-lectin monoclonal antibody stained syncytiotrophoblast and trophoblastic cells. Both reagents applied for glycohistochemistry stained syncytiotrophoblast and trophoblastic cells of placental villi and the trophoblastic layer of extraplacental membranes. A strong uniform cytoplasmic staining was characteristic for syncytiotrophoblast and to a lower extent for cytotrophoblastic cells. The localization of the lectin is discussed with respect to a possible immunosuppressive function.