Anne Filipe

P45 NF-E2 REGULATES EXPRESSION OF THROMBOXANE SYNTHASE IN MEGAKARYOCYTES

Transcription factor p45 NF‐E2 is highly expressed in the erythroid and megakaryocytic lineages. Although p45 recognizes regulatory regions of several erythroid genes, mice deficient for this protein display only mild dyserythropoiesis but have abnormal megakaryocytes and lack circulating platelets. A number of megakaryocytic marker genes have been extensively studied, but none of them is regulated by NF‐E2. To find target genes for p45 NF‐E2 in megakaryopoiesis, we used an in vivo immunoselection assay: genomic fragments bound to p45 NF‐E2 in the chromatin of a megakaryocytic cell line were immunoprecipitated with an anti‐p45 antiserum and cloned. One of these fragments belongs to the second intron of the thromboxane synthase gene (TXS). We demonstrate that the TXS gene, which is mainly expressed in megakaryocytes, is indeed directly regulated by p45 NF‐E2. First, its promoter contains a functional NF‐E2 binding site; second, the intronic NF‐E2 binding site is located within a chromatin‐dependent enhancer element; third, p45‐null murine megakaryocytes do not express detectable TXS mRNA, although TXS expression can be detected in other cells. These data, and the structure of the TXS promoter and enhancer, suggest that TXS belongs to a distinct subgroup of genes involved in platelet formation and function.

Regulation of embryonic/fetal globin genes by nuclear hormone receptors: a novel perspective on hemoglobin switching

The CCAAT box is one of the conserved motifs found in globin promoters. It binds the CP1 protein. We noticed that the CCAAT‐box region of embryonic/fetal, but not adult, globin promoters also contains one or two direct repeats of a short motif analogous to DR‐1 binding sites for non‐steroid nuclear hormone receptors. We show that a complex previously named NF‐E3 binds to these repeats. In transgenic mice, destruction of the CCAAT motif within the human ϵ‐globin promoter leads to substantial reduction in ϵ expression in embryonic erythroid cells, indicating that CP1 activates ϵ expression; in contrast, destruction of the DR‐1 elements yields striking ϵ expression in definitive erythropoiesis, indicating that the NF‐E3 complex acts as a developmental repressor of the ϵ gene. We also show that NF‐E3 is immunologically related to COUP‐TF orphan nuclear receptors. One of these, COUP‐TF II, is expressed in embryonic/fetal erythroid cell lines, murine yolk sac, intra‐embryonic splanchnopleura and fetal liver. In addition, the structure and abundance of NF‐E3/COUP‐TF complexes vary during fetal liver development. These results elucidate the structure as well as the role of NF‐E3 in globin gene expression and provide evidence that nuclear hormone receptors are involved in the control of globin gene switching.

Structure-function analysis of the extracellular domains of the Duffy antigen/receptor for chemokines: characterization of antibody and chemokine binding sites.

Summary. The Duffy antigen/receptor for chemokines (DARC), a seven‐transmembrane glycoprotein carrying the Duffy (Fy) blood group, acts as a widely expressed promiscuous chemokine receptor. In a structure–function study, we analysed the binding of chemokines and anti‐Fy monoclonal antibodies (mAbs) to K562 cells expressing 39 mutant forms of DARC with alanine substitutions spread out on the four extracellular domains (ECDs). Using synthetic peptides, we defined previously the Fy6 epitope (22‐FEDVW‐26), and we characterized the Fya epitope as the linear sequence 41‐YGANLE‐46. In agreement with these results, mutations of F22‐E23, V25 and Y41, G42, N44, L45 on ECD1 abolished the binding of anti‐Fy6 and anti‐Fya mAbs to K562 cells respectively, Anti‐Fy3 binding was abolished by D58–D59 (ECD1), R124 (ECD2), D263 and D283 (ECD4) substitutions. Mutations of C51 (ECD1), C129 (ECD2), C195 (ECD3) and C276 (ECD4 severely reduced anti‐Fy3 and CXC‐chemokine ligand 8 (CXCL‐8) binding. CXCL‐8 binding was also abrogated by mutations of F22–E23, P50 (ECD1) and D263, R267, D283 (ECD4). These results defined the Fya epitope and suggested that (1) two disulphide bridges are involved in the creation of an active chemokine binding pocket; (2) a limited number of amino acids in ECDs 1–4 participate in CXCL‐8 binding; and (3) Fy3 is a conformation‐dependent epitope involving all ECDs. We also showed that N‐glycosylation of DARC occurred on N16SS and did not influence antibody and chemokine binding.

Lutheran/basal cell adhesion molecule accelerates progression of crescentic glomerulonephritis in mice

Migration of circulating leukocytes from the vasculature into the surrounding tissue is an important component of the inflammatory response. Among the cell surface molecules identified as contributing to leukocyte extravasation is VCAM-1, expressed on activated vascular endothelium, which participates in all stages of leukocyte–endothelial interaction by binding to leukocyte surface expressed integrin VLA-4. However, not all VLA-4-mediated events can be linked to VCAM-1. A novel interaction between VLA-4 and endothelial Lutheran (Lu) blood group antigens and basal cell adhesion molecule (BCAM) proteins has been recently shown, suggesting that Lu/BCAM may have a role in leukocyte recruitments in inflamed tissues. Here, we assessed the participation of Lu/BCAM in the immunopathogenesis of crescentic glomerulonephritis. High expression of Lu/BCAM in glomeruli of mice with rapidly progressive glomerulonephritis suggests a potential role for the local expression of Lu/BCAM in nephritogenic recruitment of leukocytes. Genetic deficiency of Lu/BCAM attenuated glomerular accumulation of T cells and macrophages, crescent formation, and proteinuria, correlating with reduced fibrin and platelet deposition in glomeruli. Furthermore, we found a pro-adhesive interaction between human monocyte α4β1 integrin and Lu/BCAM proteins. Thus, Lu/BCAM may have a critical role in facilitating the accumulation of monocytes and macrophages, thereby exacerbating renal injury.

Red cell and endothelial Lu/BCAM beyond sickle cell disease☆

Recent studies shed new lights on the biological function of blood group antigens, such as the adhesion properties of the Lutheran (Lu) blood group antigens carried by the Lu/BCAM glycoproteins. The Lu/BCAM adhesion glycoproteins were first identified as laminin-10/11 erythroid receptors involved in RBC adhesion to endothelium in sickle cell anemia. Lu/BCAM mediated cell adhesion to laminin is stimulated by epinephrine, a physiological stress mediator, and is dependent of phosphorylation by protein kinase A. More recently, we demonstrated that constitutive phosphorylation of Lu/BCAM is also involved in abnormal RBC adhesion to endothelium in patients with polycythemia vera (PV), a frequent myeloproliferative disorders associated with the V617F mutation of the tyrosine kinase JAK2 leading to continuous stimulation of erythropoiesis. This observation suggests that Lu/BCAM could participate to the high incidence of vascular thrombosis that also characterizes PV disease. In mice, which do not express Lu/BCAM in erytroid tissues, invalidation of the Lu/BCAM gene provided evidence that Lu/BCAM gps, as laminin-alpha5 receptors, are involved in vivo in the maintenance of normal basement membrane organization in different non erythroid tissues since up to 90% of the mutant kidney glomeruli exhibited a reduced number of visible capillary lumens and irregular thickening of the glomerular basement membrane, while intestine exhibited smooth muscle coat thickening and disorganization. All these results further illustrate that minor blood group antigens might have important role under physiological and physiopathological conditions in erythroid and non erythroid tissues as well.