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Biomedical subjects

D Belin

Publications and source records attributed to D Belin.

At least 55 records · Page 3Linked to original sources
PubMed1993

Extracellular proteolysis in the adult murine brain.

Plasminogen activators are important mediators of extracellular metabolism. In the nervous system, plasminogen activators are thought to be involved in the remodeling events required for cell migration during development and regeneration. We have now explored the expression of the plasminogen activator/plasmin system in the adult murine central nervous system. Tissue-type plasminogen activator is synthesized by neurons of most brain regions, while prominent tissue-type plasminogen activator-catalyzed proteolysis is restricted to discrete areas, in particular within the hippocampus and hypothalamus. Our observations indicate that tissue-type plasminogen activator-catalyzed proteolysis in neural tissues is not limited to ontogeny, but may also contribute to adult central nervous system physiology, for instance by influencing neuronal plasticity and synaptic reorganization. The identification of an extracellular proteolytic system active in the adult central nervous system may also help gain insights into the pathogeny of neurodegenerative disorders associated with extracellular protein deposition.

Animals↗
PubMed1992

Expression of a tumor necrosis factor alpha transgene in murine pancreatic beta cells results in severe and permanent insulitis without evolution towards diabetes.

Mice bearing a tumor necrosis factor (TNF) alpha transgene controlled by an insulin promoter developed an increasingly severe lymphocytic insulitis, apparently resulting from the induction of endothelial changes with features similar to those observed in other places of intense lymphocytic traffic. This was accompanied by dissociation of the endocrine tissue (without marked decrease in its total mass), islet fibrosis, and the development of intraislet ductules containing, by places, beta cells in their walls, suggesting a regenerative capacity. Islet disorganization and fibrosis did not result from lymphocytic infiltration, since they were also observed in SCID mice bearing the transgene. Diabetes never developed, even though a number of potentially inducing conditions were used, including the prolonged perfusion of interferon gamma and the permanent expression of a nontolerogenic viral protein on beta cells (obtained by using mice bearing two transgenes). It is concluded that (a) a slow process of TNF release in pancreatic islets induces insulitis, and may be instrumental in the insulitis resulting from local cell-mediated immune reactions, but (b) that insulitis per se is not diabetogenic, lymphocyte stimulation by cells other than beta cells being necessary to trigger extensive beta cell damage. This provides an explanation for the discrepancy between the occurrence of insulitis and that of clinical disease in autoimmune diabetes.

Animals↗
PubMed1992

Transient translational silencing by reversible mRNA deadenylation.

Tissue-type plasminogen activator (tPA) mRNA is stored, stable and untranslated, in the cytoplasm of fully grown primary mouse oocytes. Dormancy is associated with an unusually short poly(A) tail, and poly(A) tail elongation controls tPA mRNA translational activation during meiotic maturation. Here we show that the nuclear transcript of this mRNA is extensively polyadenylated and that primary oocytes contain a deadenylating activity capable of silencing the cytoplasmic message. The sequence determinants that control deadenylation and polyadenylation overlap; this AU-rich region thus serves as an adenylation control element (ACE). The translation of a reporter mRNA in primary oocytes is prevented upon inclusion of an ACE in its 3' untranslated region. Therefore, the stage-specific regulation of poly(A) tail length accounts for the regulated synthesis of tPA in oocytes, and reversible deadenylation provides a mechanism for the translational control of dormant mRNAs.

Animals↗
PubMed1992

Polarized secretion of urokinase-type plasminogen activator by epithelial cells.

Numerous epithelial cell types produce and secrete plasminogen activators (PAs) and/or PA inhibitors (PAIs). When epithelial cells were grown on polycarbonate filters and their apical and basolateral secretion products analyzed, PA activity accumulated in a highly polarized fashion; depending upon the cell line, the compartment of PA accumulation was either apical (MDCK I cells and HBL-100 cells) or basolateral (LLC-PK1, CaCo-2, and HeLa cells). By contrast, PAI-1 was recovered in roughly equal amounts in both compartments. Basolateral accumulation of urokinase-type plasminogen activator (uPA), but not its apical targeting, required an acidic compartment and the integrity of the cytoskeleton. Polarity of uPA accumulation did not result from removal of the free enzyme from the opposite compartment through its binding to the cell surface. Transfection with wild-type or mutated murine uPA demonstrated that neither the "growth factor" domain nor the kringle domain is required for the appropriate sorting of the protein. We propose that polarized secretion of PAs is one mechanism whereby cells spatially control extracellular proteolysis.

Ammonium Chloride↗
PubMed1992

Urokinase-type plasminogen activator in human aqueous humor.

In the anterior segment of the eye, fibrin clots must be rapidly resorbed to prevent further fibrosis and scarring. The aqueous humor of patients undergoing cataract surgery was analyzed for the presence of components of the fibrinolytic cascade. In 30 patients, aqueous humor and plasma were compared for their content of urokinase-type plasminogen activator (uPA), tissue-type plasminogen activator (tPA), plasminogen activators inhibitors (PAIs), plasminogen, and total proteins. With gel electrophoresis and zymographic assays of serial dilutions of plasma and aqueous humor, all these components were found to be present at lower concentrations in aqueous humor than in plasma. For total proteins, the aqueous/plasma ratio was approximately 0.003, and for plasminogen it was 0.001. Interestingly, the aqueous/plasma ratio for uPA was not as low and varied from 0.01 to 0.03. A significant proportion of the uPA in aqueous humor was present in the two-chain active form. In addition to uPA, aqueous humor contained lower levels of tPA, but no detectable levels of reactive plasminogen activators inhibitors (PAIs). The presence of a relatively high concentration of active uPA shows that the proteolytic balance of the aqueous humor in the anterior chamber of the eye is shifted toward fibrinolysis.

Aqueous Humor↗
PubMed1991

c-fos gene transcription in murine macrophages is modulated by a calcium-dependent block to elongation in intron 1.

Cultured mouse thioglycolate-elicited peritoneal macrophages exhibit a strong block to transcriptional elongation beyond the end of the c-fos gene first exon. This block is absent in freshly isolated peritoneal cells, appears slowly during culture, and does not require adherence of the cells. The extent of this block is largely responsible for the levels of c-fos mRNA in cultured macrophages, even after modulation by agents such as the tumor promoter phorbol myristate acetate and increased intracellular cyclic AMP, which also increase the activity of the c-fos promoter. When macrophages are cultured in the absence of mobilizable calcium, the block can no longer be relieved by any inducing agent. Conversely, upon calcium influxes, there is little alteration in the level of transcriptional initiation, but transcription proceeds efficiently through the entire c-fos locus. These results suggest the presence of an intragenic calcium-responsive element in the c-fos gene and illustrate its key role in the control of c-fos gene transcription.

Animals↗
PubMed1991

Sites of synthesis of urokinase and tissue-type plasminogen activators in the murine kidney.

Kidneys have long been recognized as a major source of plasminogen activators (PAs). However, neither the sites of synthesis of the enzymes nor their role in renal function have been elucidated. By the combined use of zymographies on tissue sections and in situ hybridizations, we have explored the cellular distribution of urokinase-type (u-PA) and tissue-type (t-PA) plasminogen activators and of their mRNAs in developing and adult mouse kidneys. In 17.5-d old embryos, renal tubules synthesize u-PA, while S-shaped bodies produce t-PA. In the adult kidney, u-PA is synthesized and released in urine by the epithelial cells lining the straight parts of both proximal and distal tubules. In contrast, t-PA is produced by glomerular cells and by epithelial cells lining the distal part of collecting ducts. The precise segmental distribution of PAs suggests that both enzymes may be implicated in the maintenance of tubular patency, by catalyzing extracellular proteolysis to prevent or circumvent protein precipitation.

Animals↗
PubMed1991

Differential protease expression by cutaneous squamous and basal cell carcinomas.

To assess the postulated role of plasminogen activation in tumor invasion, we have investigated the cellular sites of synthesis for urokinase-type (uPA) and tissue-type (tPA) plasminogen activators and their inhibitors (PAI-1 and PAI-2) in two human cutaneous neoplasia that differ in their metastatic potential. The combined use of zymography on tissue sections and in situ hybridization demonstrates that uPA is produced by malignant cells of squamous cell carcinomas (SCC) but not by basal cell carcinomas (BCC), whereas tPA is detected exclusively in nonmalignant dermal tissue. In addition, we show that SCC neoplastic cells simultaneously produce variable amounts of PAI-1, and that PAI-1 production correlates inversely with uPA enzymatic activity. These observations establish that invasive human malignant cells in vivo can activate plasminogen through uPA production during the early phases of tumor growth; they also demonstrate that the proteolytic activity of tumor cells can be modulated by the concomitant production of PAI-1. Because SCC have a higher invasive and metastatic potential than BCC, our findings lend further support to the involvement of plasminogen activation in malignant behavior.

Basal Cell Carcinoma↗
PubMed1990

Human and murine urokinase cDNAs linked to the murine alpha A-crystallin promoter exhibit lens and non-lens expression in transgenic mice.

cDNAs encoding either the human or the murine urokinase-type plasminogen activator (uPA) were fused downstream from the promoter-enhancer element of the murine gene encoding alpha A-crystallin, a protein found exclusively in the ocular lens. The DNAs were microinjected into fertilized mouse eggs as linear fragments free of bacterial sequences, and for each construct one line of transgenic mice was generated. In both lines transgenic uPA activity was detected in the ocular lens, in agreement with previous results reported on transgenic mice bearing genes fused to the same regulatory region. Unexpectedly however relatively high levels of this activity were found also in the retina, and furthermore, human uPA activity was found also in different parts of the brain and in the bone marrow, and to a lesser extent in the spleen, thymus and optic nerve. Transgenic uPA transcript was found in the lens, retina, brain and thymus of mice carrying the murine cDNA. Such a pattern of expression was different from that exhibited by the endogenous murine uPA gene and, excluding the lens, it appeared to be conferred by the cDNAs. The putative regulation by uPA cDNAs is suggested to be mediated through an internal enhancer-like element functioning in combination with the alpha A-crystallin promoter in a fashion independent of the specific nature of the promoter.

Animals↗
PubMed1990

Transforming growth factor-beta 1 modulates basic fibroblast growth factor-induced proteolytic and angiogenic properties of endothelial cells in vitro.

Tightly controlled proteolytic degradation of the extracellular matrix by invading microvascular endothelial cells is believed to be a necessary component of the angiogenic process. We have previously demonstrated the induction of plasminogen activators (PAs) in bovine microvascular endothelial (BME) cells by three agents that induce angiogenesis in vitro: basic FGF (bFGF), PMA, and sodium orthovanadate. Surprisingly, we find that these agents also induce plasminogen activator inhibitor-1 (PAI-1) activity and mRNA in BME cells. We also find that transforming growth factor-beta 1 (TGF-beta 1), which in vitro modulates a number of endothelial cell functions relevant to angiogenesis, also increases both PAI-1 and urokinase-type PA (u-PA) mRNA. Thus, production of both proteases and protease inhibitors is increased by angiogenic agents and TGF-beta 1. However, the kinetics and amplitude of PAI-1 and u-PA mRNA induction by these agents are strikingly different. We have used the ratio of u-PA:PAI-1 mRNA levels as an indicator of proteolytic balance. This ratio is tilted towards enhanced proteolysis in response to bFGF, towards antiproteolysis in response to TGF-beta 1, and is similar to that in untreated cultures when the two agents are added simultaneously. Using an in vitro angiogenesis assay in three-dimensional fibrin gels, we find that TGF-beta 1 inhibits the bFGF-induced formation of tube-like structures, resulting in the formation of solid endothelial cell cords within the superficial parts of the gel. These results suggest that a net positive proteolytic balance is required for capillary lumen formation. A novel perspective is provided on the relationship between extracellular matrix invasion, lumen formation, and net proteolytic balance, thereby reflecting the interplay between angiogenesis-modulating cytokines such as bFGF and TGF-beta 1.

Amino Acid Sequence↗
PubMed1990

mRNA for tissue-type plasminogen activator is present in lesional epidermis from patients with psoriasis, pemphigus, or bullous pemphigoid, but is not detected in normal epidermis.

Plasminogen activator (PA), which catalyzes the conversion of plasminogen to the proteinase plasmin, has been implicated in a variety of cutaneous disorders. Lesional epidermis from patients with psoriasis, pemphigus, bullous pemphigoid, and Hailey-Hailey disease contains elevated levels of tissue-type PA (tPA) activity compared to non-lesional epidermis or to epidermis from normal individuals. In the present study, we have used Northern blot analysis to demonstrate that mRNA for tPA is detectable in lesions from patients with psoriasis, pemphigus, and bullous pemphigoid, but is not detectable in normal epidermis. These data strongly suggest that the tPA enzymatic activity present in lesional epidermis results from enhanced synthesis of the enzyme in situ, secondary to elevated steady-state levels of tPA mRNA. Cultured keratinocytes likewise are shown to contain tPA mRNA. Previous investigators have suggested that the phenotypes of keratinocytes in culture, psoriatic epidermis, and epidermis in the process of wound reepithelialization are comparable. Our findings, combined with those of other investigators, suggest that elevated tPA expression may be another common feature of epidermis under these circumstances.

Biopsy↗
PubMed1990

Tissue plasminogen activator in psoriasis.

Elevated levels of the serine proteinase plasminogen activator are observed in psoriatic lesions. In contrast to normal epidermis, lesional psoriatic epidermis contains primarily tissue type plasminogen activator (tPA) activity and much lower levels of urokinase type plasminogen activator (uPA) activity. Tissue plasminogen activator is also detectable immunocytochemically in lesional psoriatic but not normal epidermis. Similarly, mRNA for tPA is observed in lesional epidermis only. These results suggest that lesional psoriatic epidermis synthesizes enhanced levels of tPA compared to normal. Additional data support the hypothesis that enhanced tPA may be another marker common to psoriatic epidermis, epidermis during wound repair, and keratinocytes in culture. The significance of elevated tPA in psoriatic lesions is presently unclear, but its possible relationship to epidermal proliferation and cutaneous inflammation is under study.

Humans↗
PubMed1990

Smooth muscle cells express urokinase during mitogenesis and tissue-type plasminogen activator during migration in injured rat carotid artery.

Although the level of plasminogen activator (PA) expression has been correlated with cellular proliferation and migration in vitro, this relation has not been established in tissue undergoing repair. In a rat model of arterial injury, we have measured the expression of PAs by vascular smooth muscle cells (SMCs) during entry into the growth cycle (0-24 hours) and subsequent migration from the media to the intima (starting at approximately 4 days). In normal rat carotid, low levels of urokinase-type PA (uPA) and tissue-type PA (tPA) are present; after removal of the endothelium, only uPA is detected in the media. uPA activity in extracts of carotid arteries increases and reaches a maximum between 16 and 24 hours after injury; uPA mRNA increases steadily and is maximal at 7 days. tPA activity appears at 3 days and is maximal at 7 days; tPA mRNA is present in normal vessels and reaches a maximum by 7 days. Most of the tPA in the media is associated with SMC and not with regenerating endothelium. Furthermore, tPA is present in the media before the SMCs migrate into the intima. These results demonstrate that PA expression by vascular SMCs is differentially regulated, with uPA present during mitogenesis and tPA during migration.

Animals↗
PubMed1989

Plasminogen activators in the mouse mammary gland. Decreased expression during lactation.

The enzyme content and mRNA level for both urokinase-type and tissue-type plasminogen activators have been explored during the life cycle of the adult mouse mammary gland. Both enzymes were detected, and urokinase-type plasminogen activator was the predominant form. A marked decrease in enzyme content occurred in late gestation and was maintained throughout lactation; upon weaning, the enzyme content returned to the levels found in virgin mice. These effects were entirely accounted for by changes in the respective mRNA concentrations, which were determined with respect to both total tissue RNA and poly(A+) mRNA. Thus, plasminogen activator-catalyzed proteolysis may occur at high levels throughout the life cycle of the mouse mammary gland, except during lactation.

Animals↗
PubMed1989

Characterization of the cellular binding site for the urokinase-type plasminogen activator.

Human urokinase-type plasminogen activator (uPA) binds rapidly and with high affinity to a number of human cell types; this localizes plasmin generation to the close environment of the cell surface. uPA binding to HeLa and U937 cells is mediated by a single class of sites with an affinity of 3.4 +/- 1.3 x 10(-10) M. Binding is abolished by treatment of the cells with trypsin. Chemical cross-linking of Mr 55,000 125I-uPA to the surface of HeLa and U937 cells with disuccinimidyl suberate or with formaldehyde results in the formation of a labeled complex of Mr 100,000, suggesting a Mr of 45,000 +/- 5,000 for the receptor or a subunit thereof. When cells solubilized in Triton X-114 are subjected to heat-induced phase separation, unoccupied receptor, receptor-bound 125I-uPA, and cross-linked 125I-uPA-receptor complex all partition in the detergent phase, whereas the unbound ligand remains in the aqueous phase; similar phase partitioning is observed with endogenous uPA-receptor complexes from cultured human and murine cells. Thus, uPA bound at the cell surface is tightly associated with an amphiphilic membrane protein. Interaction of uPA with this plasma membrane receptor is species-specific, since human uPA fails to bind to murine cells, and murine uPA does not bind to human cells. Finally, incubation of HeLa cells in the presence of epidermal growth factor or phorbol 12-myristate 13-acetate results, over a period of 24 h, in a progressive change in uPA binding: an approximately 10-fold increase in the number of sites is accompanied by a 10-fold decrease in their affinity. Cross-linking and phase partitioning of 125I-uPA bound to epidermal growth factor- or phorbol 12-myristate 13-acetate-treated cells indicate that, as in control conditions, it is associated with a Mr 45,000 cell surface amphiphilic polypeptide.

Cell Line↗