Jean-Dominique Vassalli

The plasminogen activator/plasmin system.

proteases and inhibitors has been shown to be under the control of hormones and growth factors. Plasminogen activators (PAs) and their inhibitors (PAIs) are thought to be key participants in the balance ofproteolytic and antiproteolytic activities that regulates matrix turnover. This article summarizes the evidence that supports this contention, discusses the role ofPAspecific cell surface binding sites, and also draws attention to a number of instances in which the presence ofPAs cannot be reconciled with an exclusive function in ECM degradation.

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.

Amiloride selectively inhibits the urokinase‐type plasminogen activator

The diuretic drug amiloride, an inhibitor of Na+ uptake, competitively inhibits the catalytic activity of the urokinase‐type plasminogen activator (u‐PA), with a K i of 7 × 10−6 M. Generation of plasmin, cleavage of peptide substrates, and interaction of u‐PA with a specific macromolecular proteinase inhibitor are all prevented in the presence of the drug. In contrast, amiloride does not affect the activity of either tissue‐type plasminogen activator, plasmin, plasma kallikrein or thrombin. The inhibition of u‐PA by amiloride may be related to the previously reported inhibition of u‐PA‐type enzymes by Na+. Amiloride or related compounds could prove useful in selectively controlling u‐PA‐catalyzed extracellular proteolysis.

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.

Facultative polypeptide translocation allows a single mRNA to encode the secreted and cytosolic forms of plasminogen activators inhibitor 2

Two forms of plasminogen activators inhibitor 2 (PAI‐2) are synthesized by human and murine monocytes/macrophages: one accumulates in the cytosol, while the other is translocated into the endoplasmic reticulum, glycosylated and secreted. We show here that a single mRNA encodes both forms of PAI‐2. Firstly, a single PIA‐2 mRNA was detected by Northern blot hybridization and by RNase protection. Secondly, transfection of a PAI‐2 cDNA led to the synthesis of both forms of PAI‐2. Finally, in vitro translation of an mRNA transcript of the PAI‐2 cDNA in the presence of microsomal membranes generated two topologically distinct forms of PAI‐2. The cytosolic and secreted forms of PAI‐2 do not result from the use of two translation start sites, since their synthesis initiates at the same AUG, in a sequence context that is conserved between the human and murine genes. Thus, the accumulation of one polypeptide into two topologically distinct cellular compartments can be achieved by facultative translocation.

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.

Urokinase-catalyzed plasminogen activation at the monocyte/macrophage cell surface: a localized and regulated proteolytic system

In the adult organism, monocytes and macrophages are among the few cell types that can migrate within and between body compartments. To do so, they must have the capacity to clear for themselves a path through the macromolecular barriers of basement membranes and other extracellular matrices. This requires the controlled and localized degradation of matrix proteins by extracellular proteases. Mononuclear phagocytes can produce a number of such enzymes, including collagenolytic, elastinolytic, and gelatinolytic hydrolases (Takemura and Werb 1984). Because they can, directly or indirectly, catalyze the degradation of most components of extracellular matrices, plasminogen activators (PAs) are thought to play a key role in the proteolytic events that accompany the migration of a wide variety of cell types, during ontogeny as well as in pathologic circumstances. Monocytes and macrophages can produce PAs, and the regulation of their PA-dependent proteolytic activity has been a focus of attention in recent years. The findings of a number of investigators converge to suggest that the expression of PA activity is a tightly controlled phenotypic property of human and murine mononuclear phagocytes, and that multiple mechanisms act concurrently to achieve the exquisitely focused and regulated generation of plasmin precisely where and when it is needed to allow cell migration in the context of inflammatory reactions.

Protease-nexin I as an androgen-dependent secretory product of the murine seminal vesicle.

A search for inhibitors of urokinase‐type plasminogen activator (uPA) in the male and female murine genital tracts revealed high levels of a uPA ligand in the seminal vesicle. This ligand is functionally, biochemically and immunologically indistinguishable from protease‐nexin I (PN‐I), a serpin ligand of thrombin and uPA previously detected only in mesenchymal cells and astrocytes. A survey of murine tissues indicates that PN‐I mRNA is most abundant in seminal vesicles, where it represents 0.2–0.4% of the mRNAs. PN‐I is synthesized in the epithelium of the seminal vesicle, as determined by in situ hybridization, and is secreted in the lumen of the gland. PN‐I levels are much lower in immature animals, and strongly decreased upon castration. Testosterone treatment of castrated males rapidly restores PN‐I mRNA levels, indicating that PN‐I gene expression is under androgen control.

Translational Control: Awakening dormant mRNAs

The translational control of many maternal mRNAs in oocytes and early embryos relies on changes in poly(A) tail length; the factors controlling poly(A) tail length are being identified in a range of species.

PLASMINOGEN ACTIVATOR INHIBITOR-1 IS INDUCED IN MICROVASCULAR ENDOTHELIAL CELLS BY A CHONDROCYTE-DERIVED TRANSFORMING GROWTH FACTOR-BETA

We have previously demonstrated that a chondrocyte-derived TGF-beta inhibits spontaneous endothelial sprout formation in an in vitro model of angiogenesis (Pepper et al., J.Cell.Physiol. 146:170-179, 1991). We suggested that the inhibition might be mediated by an antiproteolytic effect. In this paper, we describe the induction of PAI-1 and PAI-1 mRNA in microvascular endothelial cells by chondrocyte conditioned medium. This effect can be significantly reduced by the addition of anti-TGF-beta antibodies to the conditioned medium, and can be mimicked by the addition of exogenous TGF-beta 1. Taken together with our previous observations, these results demonstrate that the inhibition of endothelial sprout formation occurs concomitantly with an increase in the production of PAI-1, a physiological plasminogen activator inhibitor. This suggests that TGF-beta-induced antiproteolysis is responsible for the inhibition of sprout formation.