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J Hagège

Publications and source records attributed to J Hagège.

18 recordsLinked to original sources
PubMed1998

Soluble latent membrane-type 1 matrix metalloprotease secreted by human mesangial cells is activated by urokinase.

BACKGROUND: Matrix metalloprotease 2 (MMP2) is secreted in a latent inactive form (pro-MMP2) that is activated on the cell surface by a membrane-type 1 MMP (MT1-MMP) in the presence of the tissue inhibitor of MMP (TIMP2). In spite of evidence for the synthesis of MT1-MMP shown by immunoblotting, immunocytochemistry and RT-PCR, and of TIMP2, MMP2 was found exclusively in a latent form in human mesangial cells (HMC) serum-free culture medium. METHODS AND RESULTS: On purified membranes of HMC, MT1-MMP was found in a 63 kD latent form and as a faint band of 55 kD. The 55 kD band was also present in the ultracentrifuged conditioned medium and likely represented MT1-MMP cleaved from its transmembrane domain, since Northern blot analysis showed only one transcription product. The addition of urokinase plasminogen activator (uPA, 100 nM) to HMC membranes induced the activation of pro-MMP2 via the activation of latent membrane-associated MT1-MMP as reflected by the cleavage of the 63 and 55 kD forms. In addition, when the conditioned medium was successively incubated with uPA and alpha 2-macroglobulin and analyzed by immunoblotting, MT1-MMP decreased, indicating that the soluble MT1-MMP was in a latent form and was activated by uPA. CONCLUSION: Our results provide the first evidence, to our knowledge, of the existence of a soluble latent form of MT1-MMP secreted by primary human cells in culture, confirming that MT1-MMP is an ectoenzyme, and show that uPA can regulate MT1-MMP activity in a soluble phase.

Cell Membrane↗
PubMed1998

PAI-1 secretion and matrix deposition in human peritoneal mesothelial cell cultures: transcriptional regulation by TGF-beta 1.

BACKGROUND: Plasminogen activator inhibitor-1 (PAI-1), the main inhibitor of plasminogen activators in plasma and in peritoneum, impairs plasmin formation that is essential for the repair processes of the mesothelium damaged by peritoneal dialysis fluids and peritonitis. The fibrogenetic cytokine transforming growth factor-beta (TGF-beta) displays variable effects on extracellular matrix remodeling enzymes and their inhibitors depending on tissues and cell lines. We previously found an unexpected stimulating effect of TGF-beta 1 on matrix metalloproteinase-9 in peritoneal mesothelial cells. In this study, we analyzed the effects of TGF-beta 1 on PAI-1 production and deposition in extracellular matrix. METHODS: We used primary cultured mesothelial cells and a recently established human peritoneal mesothelial cell line (HMrSV5). Cell-associated and secreted plasminogen activators and their inhibitors were detected and characterized by substrate gel zymography. PAI-1 was identified by reverse zymography and by Western blotting, and total PAI-1 was measured by ELISA. Secreted and cell-associated PA activity was measured by its ability to activate plasminogen into plasmin, that is, by the release of paranitroaniline from the plasmin synthetic substrate S-2251. PAI-1 mRNA accumulation was assessed by Northern blot. In vitro nuclear run-on assays were carried out to determine whether TGF-beta 1 had transcriptional effects on PAI-1 expression. Finally, the subcellular distribution of PAI-1 was analyzed by immunofluorescence and by immunogold silver staining. RESULTS: TGF-beta 1 increased PAI-1 antigen in the conditioned media of HMrSV5 cells, in a time- and concentration-dependent manner. This induced a dramatic decrease of free tPA in the cell medium and of membrane-bound uPA, and a parallel increase of high molecular weight PA-PAI complexes. Consequently, secreted and cell-associated plasminogen activator activities were considerably reduced. In primary cultured peritoneal mesothelial cells, TGF-beta 1 also induced PAI-1 secretion and the shift of tPA toward high molecular weight complexes. TGF-beta 1 increased PAI-1 mRNA in a time- and concentration-dependent manner. This effect was at least in part transcriptional since an approximately threefold increase in the rate of PAI-1 gene transcription was observed in nuclei sampled after a four-hour cell exposure to 5 ng/ml TGF-beta 1. Finally, TGF-beta 1 substantially increased the amount of intracellular and matrix-associated PAI-1. CONCLUSIONS: These results suggest that excessive TGF-beta 1 stimulated PAI-1 could prevent appropriate peritoneal healing by impairing the degradation of fibrin and of unorganized matrix components, and by interfering with cell migration.

Blotting, Northern↗
PubMed1995

Thrombin, phorbol ester, and cAMP regulate thrombin receptor protein and mRNA expression by different pathways.

Human mesangial cells have been used to study the regulation of thrombin receptor protein and mRNA expression during cross-talk between different signal transduction pathways. Persistent activation of thrombin receptor by thrombin led to homologous down-regulation of thrombin receptor protein. However, thrombin receptor mRNA expression was not affected, suggesting that increased receptor degradation is responsible for homologous down-regulation. Chronic activation of protein kinase C by phorbol 12-myristate 13-acetate (PMA) and of adenylylcyclase by prostaglandin E1 (PGE1) resulted in heterologous down-regulation of thrombin receptor protein. In contrast to thrombin, PMA and PGE1 reduced in parallel thrombin receptor mRNA levels to 51% and 24% of control, respectively, indicating that heterologous down-regulation of thrombin receptor protein is, at least in part, due to inhibition of receptor mRNA expression. The mechanisms of heterologous down-regulation of thrombin receptor protein have been studied in detail and compared to homologous down-regulation. PMA-induced down-regulation was completely blocked by GF 109 203 X, an inhibitor of protein kinase C. However, the loss of thrombin receptor induced by thrombin was not prevented by GF 109 203 X, indicating that homologous regulation is not dependent on protein kinase C activation. The heterologous effect of PGE1 was mimicked by 8-bromo-cAMP, isobutylmethylxanthine, and forskolin, suggesting that an increase in intracellular cAMP level is involved in heterologous regulation. Interestingly, heterologous down-regulation induced by PGE1 seems not to require previous internalization of thrombin receptor. These data indicate that thrombin receptor protein and mRNA expression can be regulated in homologous and heterologous ways by different mechanisms.

Cells, Cultured↗
PubMed1995

Thrombin and phorbol ester induce internalization of thrombin receptor of human mesangial cells through different pathways.

Thrombin is a potent activator of human mesangial cells probably by activation of its functional receptor. Northern blot analysis demonstrates the presence of mRNA encoding the functional thrombin receptor in mesangial cells, and surface expression of thrombin receptor antigen has been confirmed by immunocytochemistry. Using 125I-labeled ATAP2, a monoclonal antibody against the functional thrombin receptor, we found that thrombin and thrombin receptor agonist peptide (TRAP) induce homologous internalization of thrombin receptor in a dose-dependent manner. Redistribution of thrombin receptor from the cell surface to vesicular structures in the cytoplasm has been followed by immunocytochemistry. Additionally, a dose-dependent loss of cell surface thrombin receptor is induced by phorbol 12-myristate 13-acetate (PMA), suggesting that thrombin receptor undergoes heterologous internalization in response to PMA. The time course of thrombin-induced receptor internalization is different from that observed with TRAP and PMA. Protein kinase C inhibitors, staurosporine and GF 109 203 X, do not affect thrombin receptor internalization induced by thrombin and TRAP but block receptor internalization stimulated by PMA. These data suggest that heterologous thrombin receptor internalization induced by PMA is mediated by protein kinase C. However, activation of protein kinase C is not responsible for homologous thrombin receptor internalization caused by thrombin and TRAP.

Alkaloids↗
PubMed1995

Characterization of pra, a gene for replication control in pSAM2, the integrating element of Streptomyces ambofaciens.

pSAM2 is a genetic element found integrated in Streptomyces ambofaciens (B2) and additionally in a replicating form in two mutants B3 and B4. The presence of the pSAM2 replicating form in these mutants was the result of mutations located on pSAM2 in the pra locus, named pra3 and pra4, respectively. The pra gene is not directly involved in replication, but its inactivation led to the disappearance of the pSAM2 free form; therefore, it was considered as a replication regulator. The pra3 and pra4 mutations were located in the pra promoter and were shown to be point substitutions that increase the promoter strength. The replication regulator role of pra was demonstrated by the fact that its constitutive expression in cells harbouring pSAM2B2, which is normally only integrated, led to the appearance of the pSAM2 replicating form. Northern analysis showed that the pra gene transcript can be detected only for the replicating mutants B3 and B4 and that the three adjacent genes korSA, pra and traSA were transcribed separately. As replication of pSAM2 is not needed for its maintenance but is an indispensable stage of its transfer, the pra gene, described formally as an activator of pSAM2 replication, is patently involved in pSAM2 transfer.

Amino Acid Sequence↗
PubMed1994

Identification of a gene encoding the replication initiator protein of the Streptomyces integrating element, pSAM2.

pSAM2 is an 11-kilobase integrating element from Streptomyces ambofaciens which was previously shown to generate single-stranded DNA during replication, indicating that it probably replicates by a rolling-circle replication (RCR) mechanism. Two separate regions are involved in its replication, one of which was shown to contain the plus origin of replication (ds origin). We report here the study of the second region. Its nucleotide sequence was determined and analysed for open reading frames (ORFs). Three putative ORFs were identified: orf183 (183 amino acids (aa)), orf50 (50 aa), and repSA (459 aa). orf183 is not necessary for replication. The function of orf50 is unknown. repSA is essential for pSAM2 replication; it could encode a protein, RepSA, presenting similarities to the replication initiator proteins (Rep) of elements that replicate by an RCR mechanism. A derivative consisting of repSA, the region containing ds origin, a Streptomyces antibiotic resistance marker, and pBR322, could replicate in Streptomyces, further demonstrating that this ORF encodes the major replication protein of pSAM2. repSA might be co-transcribed with the genes involved in integration and excision of pSAM2.

Amino Acid Sequence↗
PubMed1994

Receptor binding and degradation of urokinase-type plasminogen activator by human mesangial cells.

The binding of [125I] labeled urokinase-type plasminogen activator (u-PA) was studied on human mesangial cells (MC) in culture. The binding of active [125I]u-PA at 37 degrees C reached a plateau after 30 minutes of incubation and remained stable for at least four hours. When the supernatant was analyzed with trichloracetic acid (TCA), TCA soluble radioactive material could be detected after a lag phase of 30 minutes, and then increased linearly for four hours. Analysis by electrophoresis on SDS PAGE and autoradiography of the cell associated radioactivity and of the intracellular content showed that active u-PA and u-PA complexed to plasminogen activator inhibitor type-1 (PAI-1) were bound to the cell surface, but only u-PA/PAI-1 complexes were internalized and degraded. Therefore, the Kd and the number of binding sites were determined by competitive inhibition curves at 4 degrees C using diisopropyl-fluorophosphate (DFP) u-PA. Scatchard plots showed a Kd = 400 +/- 30 pM, and Bmax = 240,000 +/- 25,000 sites/cell. Excess of the amino terminal fragment of u-PA (ATF) completely blocked the specific binding of [125I]u-PA, confirming that the binding of u-PA was independent of the presence of the active site and/or of the formation of complexes with PAI-1. 3H thymidine incorporation by mesangial cells after stimulation with 100 nM active u-PA showed that u-PA had a moderate but significant mitogenic effect, in contrast to inactive u-PA and ATF. However, this mitogenic effect was not accompanied by a proliferative effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1994

Heparin selectively inhibits synthesis of tissue type plasminogen activator and matrix deposition of plasminogen activator inhibitor 1 by human mesangial cells.

BACKGROUND: Mesangial changes in a variety of pathologic conditions involve mesangial cell proliferation and mesangial matrix remodelling. Heparin has been shown to prevent these processes in vivo. In vitro, heparin interferes with cell growth, proto-oncogene expression, synthesis of specific proteins, and extracellular matrix composition. In some cell types, it seems to interact with intracellular protein kinase C-dependent pathways. The effect of heparin on the mesangial plasminogen activating system (tissue type plasminogen activator, t-PA, and plasminogen activator inhibitor type 1, PAI-1), which is thought to be involved in matrix remodelling, has not been previously reported. EXPERIMENTAL DESIGN: Cultured human mesangial cells were stimulated by 10% fetal calf serum (FCS) or 16 nM phorbol myristate acetate (PMA) in the presence or absence of anticoagulant or nonanticoagulant heparins. Cell proliferation, synthesis of t-PA and PAI-1, cell morphology, and PAI-1 matrix deposition were studied using cell counting, [3H]thymidine incorporation, specific t-PA and PAI-1 enzyme-linked immunosorbent assay, Northern blot analysis, light microscopy, immunofluorescence and immunogold silver staining with combined bright-field and epipolarization microscopy. RESULTS: Heparin partially inhibited FCS-stimulated cell growth but not PMA-induced thymidine incorporation. FCS and PMA stimulated t-PA (p < 0.05 and p < 0.01, respectively) and PAI-1 synthesis (p < 0.05 and p < 0.01 respectively). Heparin selectively and partially inhibited FCS-stimulated t-PA, but not PAI-1 synthesis. It has no effect on PMA-stimulated t-PA or PAI-1 synthesis but prevented cell shape-changes induced by PMA, suggesting that heparin inhibits some but not all protein kinase C (PKC)-dependent effects and that heparin block in t-PA synthesis is distal to PKC activation. Heparin decreased PAI-1 matrix accumulation. Similar distal to PKC activation. Heparin decreased PAI-1 matrix accumulation. Similar results were observed with anticoagulant and nonanticoagulant heparin fragments. CONCLUSIONS: In human mesangial cells, anticoagulant and nonanticoagulant heparin exert an antiproliferative effect and may prevent mesangial matrix changes by decreasing FCS-stimulated t-PA synthesis and PAI-1 deposition in the matrix. Heparin is able to inhibit PKC-dependent cell shape changes but not PKC-dependent t-PA or PAI-1 synthesis. It also inhibits PKC-independent cell proliferation and t-PA synthesis. These results suggest multiple intracellular sites of action for heparin, unrelated or distal to PKC activation.

Blotting, Northern↗
PubMed1993

Mode and origin of replication of pSAM2, a conjugative integrating element of Streptomyces ambofaciens.

pSAM2 is an 11 kb integrating element from Streptomyces ambofaciens that is capable of replication. It generates single-stranded DNA during replication, and is therefore the first Streptomyces integrating element to be described that may belong to the family of elements, called the ssDNA elements, that replicate by a rolling-circle mechanism. The direction of replication has been identified. The plus origin (ori) of replication and minus origin (M-O) have been located. Streptomyces lividans harbouring replicating pSAM2 also contain numerous small covalently closed circular DNA molecules (scm) derived from pSAM2. These scm contain ori and extend on both sides of the putative nick site. Sequences at the junction points of these scm are heterogeneous but short direct repeats were always found in the vicinity of these junctions.

Amino Acid Sequence↗
PubMed1993

Transfer functions of the conjugative integrating element pSAM2 from Streptomyces ambofaciens: characterization of a kil-kor system associated with transfer.

pSAM2 is an 11-kb integrating element from Streptomyces ambofaciens. During matings, pSAM2 can be transferred at high frequency, forming pocks, which are zones of growth inhibition of the recipient strain. The nucleotide sequences of the regions involved in pSAM2 transfer, pock formation, and maintenance have been determined. Seven putative open reading frames with the codon usage typical of Streptomyces genes have been identified: traSA (306 amino acids [aa]), orf84 (84 aa), spdA (224 aa), spdB (58 aa), spdC (51 aa), spdD (104 aa), and korSA (259 aa). traSA is essential for pSAM2 intermycelial transfer and pock formation. It could encode a protein with similarities to the major transfer protein, Tra, of pIJ101. TraSA protein contains a possible nucleotide-binding sequence and a transmembrane segment. spdA, spdB, spdC, and spdD influence pock size and transfer efficiency and may be required for intramycelial transfer. A kil-kor system similar to that of pIJ101 is associated with pSAM2 transfer: the korSA (kil-override) gene product could control the expression of the traSA gene, which has lethal effects when unregulated (Kil phenotype). The KorSA protein resembles KorA of pIJ101 and repressor proteins belonging to the GntR family. Thus, the integrating element pSAM2 possesses for transfer general features of nonintegrating Streptomyces plasmids: different genes are involved in the different steps of the intermycelial and intramycelial transfer, and a kil-kor system is associated with transfer. However, some differences in the functional properties, organization, and sizes of the transfer genes compared with those of other Streptomyces plasmids have been found.

Amino Acid Sequence↗
PubMed1992

Cell-specific regulation of plasminogen activator inhibitor 1 and tissue type plasminogen activator release by human kidney mesangial cells.

Human mesangial cells in culture synthesize and secrete plasminogen activator inhibitor 1 (PAI-1) and tissue-type plasminogen activator (t-PA). Phorbol myristate acetate (PMA), a known activator of protein kinase C, induces a three to four-fold increase in t-PA and PAI-1 release over a period of 24 h, whereas cell-associated t-PA and PAI-1 levels remain relatively stable. A similar effect is obtained with oleylacetyl glycerol, a more physiologic protein kinase C activator. The effect of PMA is suppressed in the presence of H7, an inhibitor of cellular protein kinases, and by cycloheximide and actinomycin D, indicating a requirement for de novo protein and RNA synthesis, respectively. Northern blot analysis of PMA-treated cells reveals a rapid and transient increase in PAI-1 mRNA reaching a maximum after 4-8 h, whereas increase in t-PA mRNA levels requires 24 h. Activation of protein kinase A by addition of 8-bromocyclic AMP (8-bromo cAMP) has no significant effect on PAI-1 release but inhibits the PMA-mediated increases in PAI-1 antigen and mRNA. Addition of 8-bromo cAMP alone does not affect t-PA release. When added to PMA-stimulated cells, 8-bromo cAMP inhibits t-PA release in a dose-dependent manner, but causes a superinduction of t-PA mRNA. 8-bromo cAMP also induces a decrease in PMA-stimulated intracellular t-PA release. Similar inhibition is observed after stimulation of endogenous adenylate cyclase with prostaglandin E1 or isoproterenol. This indicates that protein kinase A activation may inhibit PMA-stimulated t-PA release via a post-transcriptional effect, e.g. inhibition of protein synthesis or activation of protein degradation. In conclusion, hormones or mediators which activate protein kinase C can stimulate t-PA and PAI-1 synthesis in human mesangial cells. Protein kinase A activation has no effect on the basal release of PAI-1 and t-PA by human mesangial cells, and, in contrast to endothelial cells, it inhibits both PMA-stimulated PAI-1 and t-PA releases. This cell-specific regulation of t-PA and PAI-1 seems to be mediated by differential transcriptional and post transcriptional mechanisms.

Blotting, Northern↗
PubMed1992

Plasminogen activator inhibitor-1 deposition in the extracellular matrix of cultured human mesangial cells.

Human mesangial cells secrete tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor 1 (PAI-1), the latter being secreted in large excess in vitro. We demonstrate that PAI-1 is a major component of the extracellular matrix of cultured human mesangial cells, where its deposition is dependent on cell density. By immunogold silver staining, epipolarization microscopy and dispersive X-ray spectrometry, we have shown that matrix-associated PAI-1 is synthesized by spreading human mesangial cells, as indicated by the time-dependent accumulation of PAI-1 and the inhibitory effect of cycloheximide. Furthermore, by in situ hybridization, PAI-1 mRNA was detected in cultured mesangial cells. t-PA is present inside the cells, or at the cell surface, but is never associated with the extracellular matrix. Exogenous t-PA can remove matrix-associated PAI-1 without affecting cell adhesion. A similar effect was obtained by addition of urokinase-type plasminogen activator (u-PA) but not with fibrinolysis unrelated enzymes. In conclusion, PAI-1 is synthesized by human cultured mesangial cells and is deposited in the extracellular matrix by nonconfluent cells, whereas less PAI-1 is seen between confluent cells. This can explain the absence of detectable PAI-1 in normal human kidney biopsies. t-PA released by mesangial cells can bind and detach matrix PAI-1.

Cells, Cultured↗
PubMed1987

Extraneuronal production of dopamine by kidney slices in normo and hypertensive rats.

Synthesis of dopamine (DA) was studied in cortical slices of Sprague-Dawley (SD) rat kidneys incubated with L-DOPA (10(-6) M). In morphological studies, formaldehyde-induced histofluorescent deposits were detected at the apical pole of the convoluted parts of the proximal tubules. Elsewhere along the nephron, no positive reaction appeared. DOPA decarboxylase inhibitors suppressed the advent of the deposits, but prior denervation did not. Biochemical studies showed: (a) slices produced 2.2 nmol mg-1 protein, glomeruli 0.2 and cytosol 43 nmol. In absence of L-DOPA or in presence of DOPA decarboxylase inhibitors, DA production was suppressed. Prior denervation did not influence DA production; (b) DA synthesis increased with sodium concentration of the milieu (range 95 to 152 mmol). Adult spontaneously hypertensive rats (SHR) produced less DA than SD or normotensive Wistar-Kyoto, at any sodium concentration from 95 to 142 mmol. This discrepancy could be related to the pathogenesis of hypertension.

Animals↗