Ets-1 p27: a novel Ets-1 isoform with dominant-negative effects on the transcriptional properties and the subcellular localization of Ets-1 p51.

The transcription factor Ets-1 is implicated in various physiological processes and invasive pathologies. We identified a novel variant of ets-1, ets-1Delta(III-VI), resulting from the alternative splicing of exons III to VI. This variant encodes a 27 kDa isoform, named Ets-1 p27. Ets-1 p27 lacks the threonine-38 residue, the Pointed domain and the transactivation domain, all of which are required for the transactivation of Ets-1 target genes. Both inhibitory domains surrounding the DNA-binding domain are conserved, suggesting that Ets-1 p27, like the full-length Ets-1 p51 isoform, is autoinhibited for DNA binding. We showed that Ets-1 p27 binds DNA in the same way as Ets-1 p51 does and that it acts both at a transcriptional and a subcellular localization level, thereby constituting a dual-acting dominant negative of Ets-1 p51. Ets-1 p27 blocks Ets-1 p51-mediated transactivation of target genes and induces the translocation of Ets-1 p51 from the nucleus to the cytoplasm. Furthermore, Ets-1 p27 overexpression represses the tumor properties of MDA-MB-231 mammary carcinoma cells in correlation with the known implication of Ets-1 in various cellular mechanisms. Thus the dual-acting dominant-negative function of Ets-1 p27 gives to the Ets-1 p27/Ets-1 p51 ratio a determining effect on cell fate.

Stromelysin-1 expression is activated in vivo by Ets-1 through palindromic head-to-head Ets binding sites present in the promoter.

Regulation of the gene expression of Stromelysin-1 (matrix metalloproteinase-3), a member of the matrix metalloproteinase family, is critical for tissue homeostasis. The Stromelysin-1 promoter is known to be transactivated by Ets proteins through palindromic head-to-head Ets binding sites (EBS), an unusual configuration among metalloproteinase promoters. Patterns of increased co-expression of Stromelysin-1 and Ets-1 genes have been observed in pathological processes such as rheumatoid arthritis, glomerulonephritis and tumor invasion. In this context, we show in a synovial fibroblastic model cell line (HIG-82), which is able to co-express Stromelysin-1 and Ets-1, that the EBS palindrome is essential for the expression of Stromelysin-1. More precisely, using electrophoretic mobility shift assays, DNA affinity purification and chromatin immunoprecipitation, we demonstrate that endogenous Ets-1, but not Ets-2, is present on this palindrome. The use of a dominant-negative form of Ets-1 and the decrease of Ets-1 amount either by fumagillin, an antiangiogenic compound, or by short interfering RNA show that the activation rate of the promoter and the expression of Stromelysin-1 correlate with the level of endogenous Ets-1. Thus, it is the first demonstration, using this cellular model, that endogenously expressed Ets-1 is actually a main activator of the Stromelysin-1 promoter through its effective binding to the EBS palindrome.

Endocan is a novel chondroitin sulfate/dermatan sulfate proteoglycan that promotes hepatocyte growth factor/scatter factor mitogenic activity.

Proteoglycans that modulate the activities of growth factors, chemokines, and coagulation factors regulate in turn the vascular endothelium with respect to processes such as inflammation, hemostasis, and angiogenesis. Endothelial cell-specific molecule-1 is mainly expressed by endothelial cells and regulated by pro-inflammatory cytokines (Lassalle, P., Molet, S., Janin, A., Heyden, J. V., Tavernier, J., Fiers, W., Devos, R., and Tonnel, A. B. (1996) J. Biol. Chem. 271, 20458-20464). We demonstrate that this molecule is secreted as a soluble dermatan sulfate (DS) proteoglycan. This proteoglycan represents the major form either secreted by cell lines or circulating in the human bloodstream. Because this proteoglycan is specifically secreted by endothelial cells, we propose to name it endocan. The glycosaminoglycan component of endocan consists of a single DS chain covalently attached to serine 137. Endocan dose-dependently increased the hepatocyte growth factor/scatter factor (HGF/SF)-mediated proliferation of human embryonic kidney cells, whereas the nonglycanated form of endocan did not. Moreover, DS chains purified from endocan mimicked the endocan-mediated increase of cell proliferation in the presence of HGF/SF. Overall, our results demonstrate that endocan is a novel soluble dermatan sulfate proteoglycan produced by endothelial cells. Endocan regulates HGF/SF-mediated mitogenic activity and may support the function of HGF/SF not only in embryogenesis and tissue repair after injury but also in tumor progression.

Human endothelial-cell specific molecule-1 binds directly to the integrin CD11a/CD18 (LFA-1) and blocks binding to intercellular adhesion molecule-1.

ICAMs are ligands for LFA-1, a major integrin of mononuclear cells involved in the immune and inflammatory processes. We previously showed that endothelial cell specific molecule-1 (ESM-1) is a proteoglycan secreted by endothelial cells under the control of inflammatory cytokines. Here, we demonstrate that ESM-1 binds directly to LFA-1 onto the cell surface of human blood lymphocytes, monocytes, and Jurkat cells. The binding of ESM-1 was equally dependent on Ca(2+), Mg(2+), or Mn(2+) divalent ions, which are specific, saturable, and sensitive to temperature. An anti-CD11a mAb or PMA induced a transient increase in binding, peaking 5 min after activation. Direct binding of ESM-1 to LFA-1 integrin was demonstrated by specific coimmunoprecipitation by CD11a and CD18 mAbs. A cell-free system using a Biacore biosensor confirmed that ESM-1 and LFA-1 dynamically interacted in real time with high affinity (K(d) = 18.7 nM). ESM-1 consistently inhibited the specific binding of soluble ICAM-1 to Jurkat cells in a dose-dependent manner. These results suggest that ESM-1 and ICAM-1 interact with LFA-1 on binding sites very close to but distinct from the I domain of CD11a. Through this mechanism, ESM-1 could be implicated in the regulation of the LFA-1/ICAM-1 pathway and may therefore influence both the recruitment of circulating lymphocytes to inflammatory sites and LFA-1-dependent leukocyte adhesion and activation.

Characterization of the heparin-binding site of the mycobacterial heparin-binding hemagglutinin adhesin.

The mycobacterial adhesin heparin-binding hemagglutinin (HBHA) contains several lysine-rich repeats at its carboxyl-terminal end. Using truncated recombinant HBHA forms and hybrid proteins containing HBHA repeats grafted onto the Escherichia coli maltose-binding protein (MBP), we found that these repeats are responsible for heparin binding. Immunofluorescence microscopy studies revealed that their deletion abrogates binding of HBHA to human pneumocytes. Conversely, when fused to MBP, the HBHA repeats confer pneumocyte adherence properties to the hybrid protein. Treatment of pneumocytes with glycosaminoglycan-degrading enzymes showed that HBHA binding depends on the presence of heparan sulfate chains on the cell surface. The epitope of a monoclonal antibody that inhibits mycobacterial adherence to epithelial cells was mapped within the lysine-rich repeats, confirming their involvement in mycobacterial adherence to epithelial cells. Surface plasmon resonance analyses showed that recombinant HBHA binds to immobilized heparin with fast association kinetics (k(a) = 5.62 (+/- 0.10) x 10(5) m(-1) s(-1)), whereas the dissociation kinetics were slower (k(d) = 0.015 (+/- 0.002) s(-1)), yielding a K(D) value of 26 nm. Similar analyses with grafted MBP indicated similar kinetic constants, indicating that the carboxyl-terminal repeats contain the entire heparin-binding site of HBHA. The molecular characterization of the interactions of HBHA with epithelial glycosaminoglycans should help to better understand mycobacterial adherence within the lungs and may ultimately lead to new approaches for therapy or immunoprophylaxis.

Involvement of poly (ADP-ribose)-polymerase in the Pax-6 gene regulation in neuroretina.

The quail Pax-6 gene is expressed from two promoters named P0 and P1. P0 promoter is under the control of a neuroretina-specific enhancer (EP). This enhancer activates the P0 promoter specifically in neuroretina cells and in a developmental stage-dependent manner. The EP enhancer binds efficiently, as revealed by southwestern experiments, to a 110 kDa protein present in neuroretina cells but not in Quail Embryos Cells and Retinal Pigmented Epithelium which do not express the P0-initiated mRNAs. To study the role of p110 in Pax-6 regulation, we have purified the p110 from neuroretina cells extracts. Based on the peptide sequence of the purified protein, we have identified the p110 as the poly(ADP-ribose) polymerase (PARP). Using bandshift experiments and footprinting studies, we present evidence that PARP is a component of protein complexes bound to the EP enhancer that increases the on rate of the protein complex formation to DNA. Using PARP inhibitors (3AB and 6.5 Hphe), we show that these products are able to inhibit EP enhancer activity in neuroretina cells. Finally, we demonstrate that these inhibitors are able to decrease the expression of the P0-initiated mRNA in the MC29-infected RPE cells which, in contrast to the RPE cells, accumulated the PARP in response to v-myc expression. Our results suggest that PARP is involved in the Pax-6 regulation.

Recombinant human IL-16 inhibits HIV-1 replication and protects against activation-induced cell death (AICD).

The chemoattractant cytokine IL- 16 has been reported to suppress lymphocyte activation and to inhibit HIV-1 replication in acutely infected T cells. We have cloned and expressed human IL-16 in Escherichia coli and investigated whether the recombinant protein could regulate the level of lymphocyte apoptosis from HIV-1-infected subjects. After purification and refolding, only 2-10% of the recombinant cytokine was present in a biologically active homotetrameric form. This could explain the need for high concentrations of the bacterially derived IL- 16 to induce significant inhibition of HIV-1 replication. Addition of IL-16 to unstimulated peripheral blood mononuclear cell (PBMC) cultures from HIV-1-infected subjects did not modify the observed level of spontaneous lymphocyte apoptosis. In contrast, IL-16 added to PBMC cultures stimulated with anti-CD3, anti-CD95 or dexamethasone reduced significantly the percentage of lymphocytes undergoing AICD. This effect was found to correlate with the ability of the cytokine to decrease CD95 expression on activated CD4+ T cells. Comparative studies on PBMC from healthy individuals indicated that the regulation of apoptosis levels by IL-16 is a complex phenomenon and could depend on the nature of the activator used and/or the immune status of lymphocytes tested. The outcome of CD4 cross-linking on T cells by various ligands is discussed in the context of the observed beneficial activities of IL- 16 and its potential role in the treatment of HIV disease.

Back-mutation of the V-Ets to the C-Ets carboxy-terminal amino acids in the P135gag-myb-ets results in chicken neuroretina cells transformation and loss of basic fibroblast growth factor responsiveness.

The v-Myb, v-Ets containing E26 retrovirus (called in this work E26ABC) induces the proliferation of chicken neuroretina (CNR) cells in minimal medium, strongly stimulated by basic Fibroblast Growth Factor (bFGF) which confers on them the ability to form colonies in soft agar. V-Ets differs from its cellular counterpart c-Ets-1 by two point mutations and by the replacement of the 13 last C-terminal amino acids by 16 unrelated residues as a consequence of DNA segment inversion in the viral sequence. It has been documented that this different C-terminal sequence influences DNA binding activity and specificity. Replacement in E26ABC virus of the sequence encoding the 16 v-Ets last C-terminal amino acids by the sequence encoding the 13 c-Ets-1 derived C-terminus (virus E26ABO), results in the production of a P135gag-myb-ets with modified biological properties on CNR cells. E26ABO infected CNR cells proliferate in minimal medium more efficiently than E26ABC, are unresponsive to bFGF and able to grow in soft agar. In contrast, CNR cells infected by viruses encoding Myb and Ets proteins either in the E26ABO or in the E26ABC configuration are bFGF responsive. Since Myb alone is sufficient to induce bFGF responsiveness on CNR cells, these results suggest that the c-Ets-1 C-terminus interferes with the Myb activity of the E26ABO P135gag-myb-ets protein in CNR cells.

2-Amino diphenylsulfides as new inhibitors of trypanothione reductase.

Trypanothione reductase (TR) is the primary enzyme responsible for the reduction of trypanothione, the analog of glutathione found in trypanosomatidae. We have discovered a series of diphenylsulfides which are potent inhibitors of TR and have no activity on mammalian glutathione reductase. These compounds are also active in vitro on various stages of the parasite. Although structurally related to phenothiazines, which are known to be TR inhibitors, these compounds are devoided of any neuroleptic activity, making them attractive leads to develop specific and non toxic anti-chagasic drugs.

Compared recognition of di- and trisulfide substrates by glutathione and trypanothione reductases.

Trypanothione trisulfide was synthesized according to two strategies. It was found to be recognized and reduced by trypanothione reductase as the natural disulfide substrate. At the difference with the mechanism observed for the reduction of glutathione trisulfide by glutathione reductase, the intermediate trypanothione persulfide was rapidly reduced. The enzymatic reduction of another trisulfide derived from an alternative substrate of trypanothione reductase was also studied. The structure of the trisulfide bridge of the substrate (intra- or intermolecular) appeared to be a determining factor in the enzymatic reduction pattern. Moreover, in the case of the alternative substrate of trypanothione reductase, differences of kinetics appeared for the first time between a di- and a trisulfide species. All kinetic parameters are given.

Purification and characterization of a trypanothione-glutathione thioltransferase from Trypanosoma cruzi.

Although trypanothione [T(S)2] is the major thiol component in trypanosomatidae, significant amounts of glutathione are present in Trypanosoma cruzi. This could be explained by the existence of enzymes using glutathione or both glutathione and T(S)2 as cofactors. To assess these hypotheses, a cytosolic fraction of T. cruzi epimastigotes was subjected to affinity chromatography columns using as ligands either S-hexylglutathione or a non-reducible analogue of trypanothione disulphide. A similar protein of 52 kDa was eluted in both cases. Its partial amino acid sequence indicated that it was identical with the protein encoded by the TcAc2 cDNA previously described [Schoneck, Plumas-Marty, Taibi et al. (1994) Biol. Cell 80, 1-10]. This protein showed no significant glutathione transferase activity but surprisingly catalysed the thiol-disulphide exchange between dihydrotrypanothione and glutathione disulphide. The kinetic parameters were in the same range as those determined for trypanothione reductase toward its natural substrate. This trypanothione-glutathione thioltransferase provides a new target for a specific chemotherapy against Chagas' disease and may constitute a link between the glutathione-based metabolism of the host and the trypanothione-based metabolism of the parasite.

Reduction of a trisulfide derivative of glutathione by glutathione reductase.

Glutathione trisulfide was synthesized from glutathione disulfide and its reduction by glutathione reductase was studied. A two-step reaction was observed. In a first step, the rate of reduction was similar to that observed with glutathione disulfide. In addition to glutathione, a persulfide intermediate was detected by an electrochemical method and was carboxymethylated by iodoacetate to be identified by Plasma Desorption Mass Spectrometry. During the second step the reduction of this intermediate led to the formation of hydrogen sulfide and a second equivalent of glutathione.

RP 59500: a proposed mechanism for its bactericidal activity.

RP 59500 is a combination of RP 57669 and RP 54476, which are semisynthetic water soluble derivatives of pristinamycin IA (PIA) and pristinamycin IIA (PIIA), respectively. Like their precursors, these molecules are bacteristatic in their own right. In association, they exert bactericidal activity against a variety of Gram-positive bacteria. Experiments involving the binding of these antibiotics to the target bacterial ribosome showed that both the binding sites and the mechanism of action of the components of RP 59500 are identical to those of the parent molecules. By affinity-labelling with a structural analogue of RP 57669, it was demonstrated that L24, a protein of the 70S ribosomal subunit, was specifically labelled. Experiments using radioactive N-ethylmaleimide to label proteins possessing a thiol residue, indicated that proteins L24, L10 and L11 are not only close to each other in the ribosomal structure, but are also adjacent (if not actually part of) the channel through which newly synthesized proteins are extruded. We propose that the mechanism of action of these compounds is to close or narrow the extrusion channel of these proteins, which could lead to their accumulation on the ribosome. We cannot exclude, of course, the possibility that this accumulation disturbs peptidyl-tRNA hydrolase (PHT) activity, thereby depleting free tRNAs within the cell and inhibiting protein synthesis.

Potentiation by salicylate and salicyl alcohol of cadmium toxicity and accumulation in Escherichia coli.

The toxicity of Cd2+ in Escherichia coli K-12 was potentiated by salicylate and several related compounds. The efficiency of plating on Luria broth plates was reduced by more than 10(5)-fold when 10 mM salicylate and 200 microM CdCl2 were present simultaneously but was unaffected when either compound was present by itself. Synergistic effects were found at pH 7.4 with certain other weak acids (acetyl salicylate [aspirin], benzoate, and cinnamate) and with a nonacidic salicylate analog, salicyl alcohol, but not with acetate or p-hydroxy benzoate. Thus, the synergism with Cd2+ is determined by the structure of the compounds and not merely by their acidity. The kinetics of 109Cd2+ uptake by cells grown and assayed in broth indicated the presence of two uptake systems with Kms of 1 and 52 microM Cd2+ and Vmaxs of 0.059 and 1.5 mumol of Cd2+ per min per g of cells, respectively. The kinetics of uptake for cells grown and assayed with 20 mM salicyl alcohol showed 2.5-fold increases in the Vmaxs of both systems but no change in the Kms. Salicylate-grown cells also exhibited increased rates of 109Cd2+ uptake by both systems. Thus, enhanced uptake of Cd2+ may be responsible for the potentiation of Cd2+ toxicity by salicylate and salicyl alcohol.

Potentiation of susceptibility to aminoglycosides by salicylate in Escherichia coli.

Susceptibility of Escherichia coli to kanamycin and seven other aminoglycosides has been found to be strongly potentiated by salicylate. At pH 7.5, in the presence of 15 mM salicylate and 0.5 micrograms of kanamycin per ml, the efficiency of plating of the bacteria was 2 x 10(-5), whereas there was no significant killing in the presence of kanamycin or salicylate alone. With 0.75 micrograms of kanamycin per ml, the addition of 2.5 mM salicylate was sufficient to reduce the efficiency of plating by more than 10(4)-fold. Synergistic effects were found also at pHs 6.5 and 8.5. To determine whether the action of salicylate resulted from its behavior as a weak acid or its salicyl structure, similar experiments were carried out with acetate and salicyl alcohol. Acetate, a membrane-permeating weak acid, showed a synergistic effect on kanamycin susceptibility at pH 6.5 that was comparable to the effect seen with salicylate at pH 6.5. However, acetate had no synergistic effect with kanamycin at pH 7.5 or 8.5. This is consistent with the ability of acetate to increase the membrane potential of cells and the dependence of susceptibility to kanamycin and other aminoglycosides on the membrane potential. Salicyl alcohol, which has a hydroxyl group in the place of the carboxyl group that is present in salicylate, was an effective synergist with kanamycin. It was equally effective at pHs 6.5 and 7.5 and somewhat more effective at pH 8.5. These results support the hypothesis that two effects are involved in the synergy between aminoglycosides and salicylate: a weak acid effect, possibly to increase the membrane potential, and an uncharacterized effect related to the salicyl structure.

Studies on pristinamycin synergism in Staphylococcus aureus.

Binding experiments were performed with both components of the pristinamycin complex (pristinamycin IA (PIA) and pristinamycin IIA (PIIA] using ribosomes from sensitive and resistant Staphylococcus aureus. Fluorescence polarization was used to measure PIA binding. The results obtained show a direct correlation between inhibition, synergy and the enhancement of the affinity of PIA for its receptor in the presence of PIIA. The uptake of PIA by intact cells seems to be directly correlated with affinity between PIA and ribosomes, a phenomenon which is probably shared with the macrolide antibiotics.

Irreversible binding of pristinamycin IIA (streptogramin A) to ribosomes explains its "lasting damage" effect.

In vitro and in vivo studies are presented to test the hypothesis that the synergistic action of the pristinamycins is not due to a catalytic effect of pristinamycin IIA (PIIA) on the bacterial ribosome. We demonstrate that there is a proportionality between the quantity of PIIA bound on the ribosome and pristinamycin IA (PIA) retained by it. Moreover in vitro and in vivo experiments correlated to biological effects (growth and protein synthesis) demonstrate that pristinamycin IIA is tightly bound on 70S ribosome, which satisfactory explains the so called "lasting damage effect".

Autoradiography of tissue distribution of the IIA constituent of the pristinamycins.

The tissue distribution of a radioactive analogue of the IIA constituent of the pristinamycins was studied in female mice by autoradiography. Examination of slides and photographs discloses the presence of the antibiotic on the skin and in the bone marrow only a short time after injection. Elimination of the antibiotic is quick and is mainly through the digestive tract.