Protease inhibitors prevent plasminogen-mediated,but not pemphigus vulgaris-induced,acantholysis in human epidermis

Pemphigus is an autoimmune blistering disease of the skin and mucous membranes. It is caused by autoantibodies directed against desmosomes, which are the principal adhesion structures between epidermal keratinocytes. Binding of autoantibodies leads to the destruction of desmosomes resulting in the loss of cell-cell adhesion (acantholysis) and epidermal blisters. The plasminogen activator system has been implicated as a proteolytic effector in pemphigus. We have tested inhibitors of the plasminogen activator system with regard to their potential to prevent pemphigus-induced cutaneous pathology. In a human split skin culture system, IgG preparations of sera from pemphigus vulgaris patients caused histopathologic changes (acantholysis) similar to those observed in the original pemphigus disease. All inhibitors that were tested (active site inhibitors directed against uPA, tPA, and/or plasmin; antibodies neutralizing the enzymatic activity of uPA or tPA; substances interfering with the binding of uPA to its specific cell surface receptor uPAR) failed to prevent pemphigus vulgaris IgG-mediated acantholysis. Plasminogen-mediated acantholysis, however, was effectively antagonized by the synthetic active site serine protease inhibitor WX-UK1 or by p-aminomethylbenzoic acid. Our data argue against applying anti-plasminogen activator/anti-plasmin strategies in the management of pemphigus.     

Plasminogen activators augment endothelial cell organization in vitro by two distinct pathways

Endothelial cell differentiation into capillary structures is a complex process that requires the concerted effects of several extracellular matrix proteases, including plasminogen activators. Here, the role of tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) was evaluated in an in vitro model of endothelial morphogenesis involving organization of human umbilical vein endothelial cells into tubular structures when they are cultured on the basement membrane preparation, Matrigel. Both uPA and tPA were detected in HUVEC cultures on Matrigel, and inhibitors of plasminogen activators or of serine proteases decreased the extent of the tube network formed by the cells. The decrease resulting from serine protease inhibitors was additive to that from matrix metalloproteinase inhibitors which have previously been shown to decrease tube formation in this model, suggesting that the two classes of proteases modulate tube formation by distinct mechanisms. Plasminogen activator inhibitor (PAl)-1 decreased tube formation by 50% when added up to 4.5 h after the initiation of an 18 h assay and caused 25% inhibition when added 9.5 h after culture initiation, indicating that the effects of plasminogen activators are not limited to an early event in the differentiation process. Steady-state expression of mRNA for uPA increased during the first several hours of culture on Matrigel, further supporting a role for PA activity throughout the process of tube formation. These findings suggested that PAs may affect multiple events during tube-forming activity. A fucosylated peptide comprising the amino-terminal domain of uPA that binds to the uPA receptor (uPAR) but lacking proteolytic activity enhanced tube formation. In contrast, a defucosylated form of the same peptide had no effect. Since fucosylation of this fragment has been shown to be essential in other models of cell stimulation by uPA-uPAR interaction, these data support the hypothesis that uPA enhances endothelial morphogenesis both through proteolytic activity and via uPAR occupancy. Plasminogen activators could facilitate angiogenesis in vivo. © 1995 Wiley-Liss Inc.     

Phorbol ester activation of a proteolytic cascade capable of activating latent transforming growth factor-betaL a process initiated by the exocytosis of cathepsin B

12-O-Tetradecanoylphorbol-13-acetate (TPA) suppresses the proliferation of the human breast epithelial cell line MCF10A-Neo by initiating proteolytic processes that activate latent transforming growth factor (TGF)-beta in the serum used to supplement culture medium. Within 1 h of treatment, cultures accumulated an extracellular activity capable of cleaving a substrate for urokinase-type plasminogen activator (uPA) and tissue plasminogen activator (tPA). This activity was inhibited by plasminogen activator inhibitor-1 or antibodies to uPA but not tPA. Pro-uPA activation was preceded by dramatic changes in lysosome trafficking and the extracellular appearance of cathepsin B and beta-hexosaminidase but not cathepsins D or L. Co-treatment of cultures with the cathepsin B inhibitors CA-074 or Z-FA-FMK suppressed the cytostatic effects of TPA and activation of pro-uPA. In the absence of TPA, exogenously added cathepsin B activated pro-uPA and suppressed MCF10A-Neo proliferation. The cytostatic effects of both TPA and cathepsin B were suppressed in cells cultured in medium depleted of plasminogen/plasmin or supplemented with neutralizing TGF-beta antibody. Pretreatment with cycloheximide did not suppress the exocytosis of cathepsin B or the activation of pro-uPA. Hence, TPA activates signaling processes that trigger the exocytosis of a subpopulation of lysosomes/endosomes containing cathepsin B. Subsequently, extracellular cathepsin B initiates a proteolytic cascade involving uPA, plasminogen, and plasmin that activates serum-derived latent TGF-beta.     

Structure-function studies of the SERPIN plasminogen activator inhibitor type 1. Analysis of chimeric strained loop mutants.

Three chimeric mutants of plasminogen activator inhibitor 1 (PAI-1) have been constructed where the strained loop of wild type PAI-1 (wtPAI-1) has been replaced with a 19-amino acid region from either plasminogen activator inhibitor 2 (PAI-2), antithrombin III, or with an artificial serine protease inhibitor superfamily consensus strained loop. The inhibitors were expressed in Escherichia coli, and the purified proteins had specific activities toward urokinase-type plasminogen activator (uPA) or the single- and two-chain forms of tissue type plasminogen activator (tPA) that were similar to wtPAI-1. Experiments suggest that the strained loop of PAI-1 is not responsible for the transition between the latent and the active conformations or for binding to vitronectin. Second-order rate constants for the interactions with uPA and single- or two-chain tPA were similar to those of wtPAI-1. Values range from a low of 1.8 x 10(5) M-1 s-1 for the interaction of the PAI-2 chimera with single-chain tPA to a high value of 1.6 x 10(7) M-1 s-1 for the consensus mutant with two-chain tPA. This former value is 200 times higher than the reported rate constant for the interaction between PAI-2 and single-chain tPA, suggesting that structures outside of the strained loop are responsible for the major differences in specificity between PAI-1 and PAI-2.     

A high-affinity receptor for urokinase plasminogen activator on human keratinocytes: characterization and potential modulation during migration.

Low passage cultures of normal human keratinocytes produce several components of the plasminogen activator/plasmin proteolytic cascade, including urokinase plasminogen activator (uPA), tissue plasminogen activator (tPA), and two specific inhibitors. Studies here presented demonstrate that these cells also contain a high-affinity (Kd = 3 x 10(-10) M) plasma membrane-binding site for uPA. High molecular weight uPA, either as the single-chain precursor or two-chain activated form, bound to the receptor; however, low molecular weight (33 kD) uPA, tPA, or epidermal growth factor did not compete for binding, demonstrating specificity. Acid treatment, which removed endogenous uPA from the receptor, was required to detect maximal binding (45,000 sites per cell). To investigate the possibility that the uPA receptor on keratinocytes may be involved in epithelial migration during wound repair, cultures were wounded and allowed to migrate into the wounded site. Binding sites for uPA were localized by autoradiographic analysis of 125I-uPA binding as well as by immunocytochemical studies using anti-uPA IgG. With both techniques uPA binding sites were detected selectively on the plasma membrane of cells at the leading edge of the migrating epithelial sheet. This localization pattern suggests that uPA receptor expression on keratinocytes may be coupled to cell migration during cutaneous wounding.     

Proteases with plasminogen activator activity in hamster oviduct

At present the physiological role of most oviductal proteins remains unknown. In this work, we present evidence that the oviductal secretion as well as the crude oviductal tissue-extract show proteolytic-like esterase and amidase activity. The proteolytic activity of the oviductal enzymes was higher in the oviducts of superovulated hamster females than in those of normal ones, indicating that gonadotrophic hormones would stimulate the synthesis and secretion of these enzymes. Some of their properties were analyzed in the 15,600-g supernatant of both oviductal tissue extracts (OE) and oviductal fluid (OF). The enzymatic activity toward the synthetic substrates p-tosyl-l-arginine methyl ester-HCl (TAME) and alpha-N-benzoyl-dl-arginine-p-nitroanilide HCl (BAPNA) was activated by calcium ions, reached a maximum at pH 7.5, and was inhibited by soybean trypsin inhibitor (SBTI), N-alpha-p-tosyl-l-lysine chloromethyl ketone HCl (TLCK), phenyl methyl sulfonyl fluoride (PMSF), and benzamidine. The OE glycoprotein fraction recognized by WGA-Sepharose affinity columns (37% total proteins) showed proteolytic activity with properties similar to the OE and OF enzymes. The protease activity could be ascribed to a plasminogen activator (PA) detected in the Triton X-100 treated tissue crude membrane fraction (Triton-CMF) and in the oviductal secretion of the superovulated females. In the Triton-CMF fraction, 100% of the proteolytic activity was plasminogen-dependent. The use of amiloride, a selective urokinase-type plasminogen activator (uPA) inhibitor, shows that 90% of this activity was due to a tissue-type plasminogen activator (tPA) and 10% to uPA whereas in the uterus 100% of the activity was tPA. Only a small percentage of the OF proteolytic activity was plasminogen-dependent, probably due to the presence of PA inhibitors in this medium.     

Interaction of heparin with plasminogen activators and plasminogen: effects on the activation of plasminogen

The amidolytic plasmin activity of a mixture of tissue plasminogen activator (tPA) and plasminogen is enhanced by heparin at therapeutic concentrations. Heparin also increases the activity in mixtures of urokinase-type plasminogen activator (uPA) and plasminogen but has no effect on streptokinase or plasmin. Direct analyses of plasminogen activation by polyacrylamide gel electrophoresis demonstrate that heparin increases the activation of plasminogen by both tPA and uPA. Binding studies show that heparin binds to various components of the fibrinolytic system, with tight binding demonstrable with tPA, uPA, and Lys-plasminogen. The stimulation of tPA activity by fibrin, however, is diminished by heparin. The ability of heparin to promote plasmin generation is destroyed by incubation of the heparin with heparinase, whereas incubation with chondroitinase ABC or AC has no effect. Also, stimulation of plasmin formation is not observed with dextran sulfate or chondroitin sulfate A, B, or C. Analyses of heparin fractions after separation on columns of antithrombin III-Sepharose suggest that both the high-affinity and the low-affinity fractions, which have dramatically different anticoagulant activity, have similar activity toward the fibrinolytic components.     

Overexpression of plasminogen activators in the nucleus accumbens enhances cocaine-, amphetamine- and morphine-induced reward and behavioral sensitization

Urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) are extracellular proteases that play a role in synaptic plasticity and remodeling. Psychostimulants induce both tPA and uPA in acute and chronic drug delivery, but cocaine induces preferentially uPA, whereas morphine and amphetamine induce preferentially tPA. Specific doxycline-regulatable lentiviruses expressing these extracellular proteases have been prepared and stereotaxically injected into the nucleus accumbens. We show that tPA-overexpressing animals show greater locomotor activity and behavioral sensitization upon morphine and amphetamine treatments. These effects could be fully suppressed by doxycycline or when tPA had been silenced using small interfering RNAs (siRNAs)-expressing lentiviruses. Furthermore, animals infected with lentiviruses expressing uPA show enhanced conditional place preference for cocaine compared with tPA-overexpressing animals. In contrast, tPA-overexpressing animals when administered amphetamine or morphine showed greater place preference compared with uPA-overexpressing animals. The effects are suppressed when tPA has been silenced using specific siRNAs-expressing vectors. Tissue-type plasminogen activator and uPA possibly induce distinct behaviors, which may be interpreted according to their differential pattern of activation and downstream targets. Taken together, these data add further evidence for a significant function of extracellular proteases tPA and uPA in addiction and suggest a differential role of plasminogen activators in this context.     

Structural requirements for the growth factor activity of the amino-terminal domain of urokinase.

High molecular weight urokinase-type plasminogen activator (uPA) in which proteolytic activity was inactivated (diisopropyl fluorophosphate (DFP)-uPA), its amino-terminal fragment (ATF, amino acids (aa) 1-143), and fucosylated and defucosylated growth factor domains (GFD, aa 4-43) were tested for growth-promoting effects and binding in human SaOS-2 osteosarcoma cells and U-937 lymphoma cells. DFP-uPA, ATF, and both the fucosylated and defucosylated GFD were capable of competing with 125I-ATF for binding to both SaOS-2 and U-937 cells. DFP-uPA, ATF, and fucosylated GFD were also mitogenic in SaOS-2 cells and increased cell numbers. However, defucosylated GFD was nonmitogenic in SaOS-2 cells and did not stimulate cell proliferation, even though it bound to these cells in a manner equivalent to the fucosylated GFD. A nonglycosylated high molecular weight uPA expressed and purified from Escherichia coli inhibited 125I-ATF binding to SaOS-2 cells but was also nonmitogenic. No mitogenic activity was observed in U-937 cells treated with the uPA forms capable of eliciting a mitogenic response in SaOS-2 cells. Proteolytically prepared kringle domain (aa 47-135) and low molecular weight uPA (aa 144-411) did not compete for 125I-ATF binding and did not elicit any mitogenic response in either of the cell lines tested. In addition, tissue plasminogen activator (tPA), which has been shown to be homologous to uPA in its growth factor domain and is also fucosylated, did not inhibit 125I-ATF binding nor elicit any mitogenic response. These results demonstrate that the GFD, implicated in binding to the uPA receptor, is also responsible for growth factor like activity in SaOS-2 cells and that the fucosylation at Thr18 within this domain may serve as a molecular trigger in eliciting this response.     

Activators and inhibitors of the plasminogen system in Alzheimer's disease

Accumulation and deposition of Aβ is one of the main neuropathological hallmarks of Alzheimer's disease (AD) and impaired Aβ degradation may be one mechanism of accumulation. Plasmin is the key protease of the plasminogen system and can cleave Aβ. Plasmin is activated from plasminogen by tissue plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). The activators are regulated by inhibitors which include plasminogen activator inhibitor-1 (PAI-1) and neuroserpin. Plasmin is also regulated by inhibitors including α2-antiplasmin and α2-macroglobulin. Here, we investigate the mRNA levels of the activators and inhibitors of the plasminogen system and the protein levels of tPA, neuroserpin and α2-antiplasmin in post-mortem AD and control brain tissue. Distribution of the activators and inhibitors in human brain sections was assessed by immunoperoxidase staining. mRNA measurements were made in 20 AD and 20 control brains by real-time PCR. In an expanded cohort of 38 AD and 38 control brains tPA, neuroserpin and α2-antiplasmin protein levels were measured by ELISA. The activators and inhibitors were present mainly in neurons and α2-antiplasmin was also associated with Aβ plaques in AD brain tissue. tPA, uPA, PAI-1 and α2-antiplasmin mRNA were all significantly increased in AD compared to controls, as were tPA and α2-antiplasmin protein, whereas neuroserpin mRNA and protein were significantly reduced. α2-macroglobulin mRNA was not significantly altered in AD. The increases in tPA, uPA, PAI-1 and α2-antiplasmin may counteract each other so that plasmin activity is not significantly altered in AD, but increased tPA may also affect synaptic plasticity, excitotoxic neuronal death and apoptosis.     

Insulin-like growth factor-binding protein-5 activates plasminogen by interaction with tissue plasminogen activator, independently of its ability to bind to plasminogen activator inhibitor-1, insulin-like growth factor-I, or heparin

Transgenic mice expressing IGFBP-5 in the mammary gland exhibit increased cell death and plasmin generation. Because IGFBP-5 has been reported to bind to plasminogen activator inhibitor-1 (PAI-1), we determined the effects of this interaction in HC11 cells. PAI-1 prevented plasmin generation from plasminogen and inhibited cleavage of focal adhesions, expression of caspase 3, and cell death. IGFBP-5 could in turn prevent the effects of PAI-1. IGFBP-5 mutants with reduced affinity for IGF-I (N-term) or deficient in heparin binding (HEP- and C-term E and F) were also effective. This was surprising because IGFBP-5 reportedly interacts with PAI-1 via its heparin-binding domain. Biosensor analysis confirmed that, although wild-type IGFBP-5 and N-term both bound to PAI-1, the C-term E had greatly decreased interaction with PAI-1. This suggests that IGFBP-5 does not antagonize the actions of PAI-1 by a direct molecular interaction. In a cell-free system, using tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA) to activate plasminogen, PAI-1 inhibited plasmin generation induced by both activators, whereas IGFBP-5 prevented the effects of PAI-1 on tPA but not uPA. Furthermore, we noted that IGFBP-5 activated plasminogen to a greater extent than could be explained solely by inhibition of PAI-1, suggesting that IGFBP-5 could directly activate tPA. Indeed, IGFBP-5 and the C-term E and F were all able to enhance the activity of tPA but not uPA. These data demonstrate that IGFBP-5 can enhance the activity of tPA and that this can result in cell death induced by cleavage of focal adhesions. Thus IGFBP-5 can induce cell death by both sequestering IGF-I and enhancing plasmin generation.     

Measuring plasminogen activator inhibitor activity in plasma by two enzymatic assays

We compared two methods that measure plasminogen activator inhibitor (PAI) activity in plasma based on the ability of PAI to inhibit tissue plasminogen activator (tPA) or urokinase (uPA) in order to determine which method most accurately measures plasma PAI activity after stressors, like hemorrhage. Plasma PAI activity was significantly elevated after hemorrhage in both assays. Using standard curves derived from rhPAI-1, we found that the tPA-PAI assay was more sensitive than the uPA-PAI assay. However, we measured a 10-fold difference in PAI activity as measured between assays, suggesting that some endogenous plasma constituents (tPA, uPA, plasminogen or plasmin) may interfere with the accurate determination of PAI activity. Increasing the amount of plasma in each assay led to a progressive increase in PAI activity. However, removing either tPA or plasminogen from the tPA-PAI assay unmasked the presence of some endogenous tPA and plasminogen. Furthermore, increasing plasma volume in either assay increases measured plasma tPA, but not uPA. Finally, plasma tPA is elevated after hemorrhage, whereas plasma uPA is not. These results suggest that endogenous tPA and plasminogen may interfere with the measurement of plasma PAI activity in the tPA-PAI assay after hemorrhage or other stresses. The uPA-PAI assay does not have this confounding problem because endogenous uPA does not interfere with the assay, nor does it rise during hemorrhage.     

Regulation of serine protease inhibitor-E2 and plasminogen activator expression and secretion by follicle stimulating hormone and growth factors in non-luteinizing bovine granulosa cells in vitro.

During ovarian follicle growth, there is expansion of the basal lamina and changes in the follicular extracellular matrix (ECM) that are mediated in part by proteolytic enzyme cascades regulated by tissue-type plasminogen activator (tPA) and urokinase plasminogen activator (uPA). One PA inhibitor, serine protease inhibitor-E2 (SERPINE2) is expressed in granulosa but not theca cells, and expression changes with follicle development. In this study, we hypothesized that PA and SERPINE2 expression/secretion by granulosa cells are regulated by FSH and growth factors. SERPINE2 mRNA and protein levels, tPA gene expression and uPA secretion were stimulated by FSH. Insulin-like growth factor-I stimulated SERPINE2 secretion and uPA activity, and decreased secreted tPA activity and gene expression. Bone morphogenetic protein-7 increased SERPINE2 secretion and expression and tPA secretion. In contrast, fibroblast growth factor-2 inhibited tPA secretion and SERPINE2 secretion and expression. Epidermal growth factor inhibited SERPINE2 secretion and expression, but increased secreted tPA activity. Estradiol and SERPINE2 secretion were highly positively correlated, but estradiol did not alter SERPINE2 expression. These data demonstrate that SERPINE2 expression and protein secretion are regulated by FSH and growth factors in non-luteinizing bovine granulosa cells. As estradiol is a known marker of follicle health, and SERPINE2 is an anti-apoptotic factor, we propose that SERPINE2 is involved in the regulation of atresia in bovine follicles.     

Tissue-specific and time-coordinated hormone regulation of plasminogen-activator-inhibitor type I and tissue-type plasminogen activator in the rat ovary during gonadotropin-induced ovulation

The plasminogen-activator system provides proteolytic activity in many biological processes. The regulation of plasminogen activation may occur at many levels including the synthesis and secretion of plasminogen activators (PA) and the specific inhibition of PA activity by inhibitors. PA-inhibitor type-1 (PAI-1) is an efficient inhibitor of tissue-type PA (tPA) and urokinase-type PA (uPA) that may therefore be instrumental for the control of plasminogen activation. To investigate if coordinated regulation of PA and PA inhibitors take place in vivo in response to physiological signals, we have examined the regulation of PAI-1 and tPA in the ovary during gonadotropin-induced ovulation. We found that PAI-1, as well as tPA activity and mRNA levels, were coordinately regulated by gonadotropins in a time-dependent and cell-specific manner, such that a surge of PA-activity was obtained just prior to ovulation. Both theca-interstitial and granulosa cells synthesized PAI-1, but their maximal PAI-1 expression occurred at different times during the periovulatory period, ensuring inhibition of proteolytic activity in ovarian extra cellular compartments both before and after ovulation. The coordinated regulation of tPA and PAI-1 in the ovary may fine-tune the peak of PA activity which may be important for the regulation of the ovulatory process.     

Synthesis and preliminary evaluation of amiloride analogs as inhibitors of the urokinase-type plasminogen activator (uPA)

A known side-activity of the oral potassium-sparing diuretic drug amiloride is inhibition of the enzyme urokinase-type plasminogen activator (uPA, K(i)=7 μM), a promising anticancer target. Several studies have demonstrated significant antitumor/metastasis properties for amiloride in animal cancer models and it would appear that these arise, at least in part, through inhibition of uPA. Selective optimization of amiloride's structure for more potent inhibition of uPA and loss of diuretic effects would thus appear as an attractive strategy towards novel anticancer agents. The following report is a preliminary structure-activity exploration of amiloride analogs as inhibitors of uPA. A key finding was that the well-studied 5-substituted analogs ethylisopropyl amiloride (EIPA) and hexamethylene amiloride (HMA) are approximately twofold more potent than amiloride as uPA inhibitors.     

Generation of cell surface-bound plasmin by cell-associated urokinase-type or secreted tissue-type plasminogen activator: a key event in melanoma cell invasiveness in vitro.

Recently, we have shown that plasminogen activators (PAs) of both types, urokinase-type (uPA) as well as tissue-type (tPA), are involved in the in vitro invasiveness of human melanoma cells. The present study is focused on the generation and importance of cell surface-bound plasmin in this process. The human melanoma cell lines MelJuso and MeWo expressed plasminogen binding sites on the cell surface. Plasminogen binding was saturable and not species-specific, since human and bovine plasminogen bound to the cells with comparable efficiency. The activation of the proenzyme plasminogen bound on MelJuso cells, which expressed surface-associated uPA activity, occurred almost synchronously with binding to the cell surface. Removal of cell-associated uPA considerably reduced plasmin generation on these cells. In contrast, plasminogen activation on MeWo cells, which secreted tPA into the culture supernatant and which were devoid of surface-associated PA activity, was by far less effective. The efficiency of the activation process could be increased by addition of exogenous tPA. With both cell lines, plasmin generation on the cell surface was suppressed by inhibitory monoclonal antibodies specific for the respective PA type. Selective inhibition of cell surface-associated plasmin by preincubating the cells with an inhibitory monoclonal antibody or with aprotinin, as well as removal of plasmin from the cell surface, led to a significant decrease in cellular invasiveness of both cell lines into various biological substrates such as fibrin gel, the basement membrane extract Matrigel, or intact extracellular matrix. Both cell lines were able to penetrate an intact cell layer of the human keratinocyte line HaCaT, a process, which also proved to be dependent on cell-associated plasmin. In conclusion, these data provide evidence that plasminogen activation associated with the surface of human melanoma cells is catalyzed much more efficiently by cell-associated uPA (MelJuso) than by secreted tPA (MeWo). Cell-associated plasmin, which is protected from inactivation by serum inhibitors, represents the essential component of the proteolytic cascade of plasminogen activation during in vitro invasiveness of human melanoma cells.     

Effect of compressive force on the expression of MMPs, PAs, and their inhibitors in osteoblastic Saos-2 cells

Bone matrix turnover is regulated by matrix metalloproteinases (MMPs), tissue inhibitors of matrix metalloproteinases (TIMPs), and the plasminogen activation system, including tissue-type plasminogen activator (tPA), urokinase-type plasminogen activator (uPA), and plasminogen activator inhibitor type-1 (PAI-1). We previously demonstrated that 1.0g/cm(2) of compressive force was an optimal condition for inducing bone formation by osteoblastic Saos-2 cells. Here, we examined the effect of mechanical stress on the expression of MMPs, TIMPs, tPA, uPA, and PAI-1 in Saos-2 cells. The cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and with or without continuously compressive force (0.5-3.0g/cm(2)) for up to 24h. The levels of MMPs, TIMPs, uPA, tPA, and PAI-1 gene expression were estimated by determining the mRNA levels using real-time PCR, and the protein levels were determined using ELISA. The expression levels of MMP-1, MMP-2, MMP-14, and TIMP-1 markedly exceeded the control levels at 1.0g/cm(2) of compressive force, whereas the expression levels of MMP-3, MMP-13, TIMP-2, TIMP-3, TIMP-4, tPA, uPA, and PAI-1 markedly exceeded the control levels at 3.0g/cm(2). These results suggest that mechanical stress stimulates bone matrix turnover by increasing these proteinases and inhibitors, and that the mechanism for the proteolytic degradation of bone matrix proteins differs with the strength of the mechanical stress.     

Regulation of the plasminogen activator system in the ovary

Extracellular matrix (ECM) not only provides a structural support for the organism, but also actively conducts cell-to-cell signal transduction and regulates cell proliferation, migration, development and metabolism. The targeted ECM degradation generated by plasminogen activator (PA) and regulated by plasminogen activator inhibitor (PAI) is, therefore, an event that affects a wide variety of physiological and pathological processes. The ovary is the best model to study the regulation and function of extracellular proteolysis mediated by multicomponents like the PA system. Studies carried out over the past 10 years in a number of laboratories have elucidated some of the biochemical events related to the function and regulation of the PA system in the ovary: hormone-induced proteolytic activity provided by tissue-type PA(tPA) and modulated by PAI-1 in the preovulatory follicles is responsible for a controlled and directed proteolysis leading to rupture of selected follicles during ovulation, whereas the coordinated expression of urokinase-type PA (uPA) and PAI-1 in the early growing follicle may be important in ECM degradation during cell proliferation and migration; the PA system may also play a role in the control of corpus luteum (CL) development through an autocrine or paracrine mechanism. Increase in tPA and PAI-1 expression in CL at a later stage is well correlated with a sharp decrease in CL progesterone production, while the increase in uPA mRNA levels and activity in the early stage of CL development is correlated with an increase in progesterone secretion.     

Effects of extracellular DNA on plasminogen activation and fibrinolysis

The increased levels of extracellular DNA found in a number of disorders involving dysregulation of the fibrinolytic system may affect interactions between fibrinolytic enzymes and inhibitors. Double-stranded (ds) DNA and oligonucleotides bind tissue-(tPA) and urokinase (uPA)-type plasminogen activators, plasmin, and plasminogen with submicromolar affinity. The binding of enzymes to DNA was detected by EMSA, steady-state, and stopped-flow fluorimetry. The interaction of dsDNA/oligonucleotides with tPA and uPA includes a fast bimolecular step, followed by two monomolecular steps, likely indicating slow conformational changes in the enzyme. DNA (0.1-5.0 μg/ml), but not RNA, potentiates the activation of Glu- and Lys-plasminogen by tPA and uPA by 480- and 70-fold and 10.7- and 17-fold, respectively, via a template mechanism similar to that known for fibrin. However, unlike fibrin, dsDNA/oligonucleotides moderately affect the reaction between plasmin and α(2)-antiplasmin and accelerate the inactivation of tPA and two chain uPA by plasminogen activator inhibitor-1 (PAI-1), which is potentiated by vitronectin. dsDNA (0.1-1.0 μg/ml) does not affect the rate of fibrinolysis by plasmin but increases by 4-5-fold the rate of fibrinolysis by Glu-plasminogen/plasminogen activator. The presence of α(2)-antiplasmin abolishes the potentiation of fibrinolysis by dsDNA. At higher concentrations (1.0-20 μg/ml), dsDNA competes for plasmin with fibrin and decreases the rate of fibrinolysis. dsDNA/oligonucleotides incorporated into a fibrin film also inhibit fibrinolysis. Thus, extracellular DNA at physiological concentrations may potentiate fibrinolysis by stimulating fibrin-independent plasminogen activation. Conversely, DNA could inhibit fibrinolysis by increasing the susceptibility of fibrinolytic enzymes to serpins.