PAI-1 in tissue fibrosis

Fibrosis is defined as a fibroproliferative or abnormal fibroblast activation-related disease. Deregulation of wound healing leads to hyperactivation of fibroblasts and excessive accumulation of extracellular matrix (ECM) proteins in the wound area, the pathological manifestation of fibrosis. The accumulation of excessive levels of collagen in the ECM depends on two factors: an increased rate of collagen synthesis and or decreased rate of collagen degradation by cellular proteolytic activities. The urokinase/tissue type plasminogen activator (uPA/tPA) and plasmin play significant roles in the cellular proteolytic degradation of ECM proteins and the maintenance of tissue homeostasis. The activities of uPA/tPA/plasmin and plasmin-dependent MMPs rely mostly on the activity of a potent inhibitor of uPA/tPA, plasminogen activator inhibitor-1 (PAI-1). Under normal physiologic conditions, PAI-1 controls the activities of uPA/tPA/plasmin/MMP proteolytic activities and thus maintains the tissue homeostasis. During wound healing, elevated levels of PAI-1 inhibit uPA/tPA/plasmin and plasmin-dependent MMP activities, and, thus, help expedite wound healing. In contrast to this scenario, under pathologic conditions, excessive PAI-1 contributes to excessive accumulation of collagen and other ECM protein in the wound area, and thus preserves scarring. While the level of PAI-1 is significantly elevated in fibrotic tissues, lack of PAI-1 protects different organs from fibrosis in response to injury-related profibrotic signals. Thus, PAI-1 is implicated in the pathology of fibrosis in different organs including the heart, lung, kidney, liver, and skin. Paradoxically, PAI-1 deficiency promotes spontaneous cardiac-selective fibrosis. In this review, we discuss the significance of PAI-1 in the pathogenesis of fibrosis in multiple organs.     

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.     

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.     

The plasminogen activator inhibitor PAI-1 controls in vivo tumor vascularization by interaction with proteases, not vitronectin. Implications for antiangiogenic strategies

The plasminogen (Plg)/plasminogen activator (PA) system plays a key role in cancer progression, presumably via mediating extracellular matrix degradation and tumor cell migration. Consequently, urokinase-type PA (uPA)/plasmin antagonists are currently being developed for suppression of tumor growth and angiogenesis. Paradoxically, however, high levels of PA inhibitor 1 (PAI-1) are predictive of a poor prognosis for survival of patients with cancer. We demonstrated previously that PAI-1 promoted tumor angiogenesis, but by an unresolved mechanism. We anticipated that PAI-1 facilitated endothelial cell migration via its known interaction with vitronectin (VN) and integrins. However, using adenoviral gene transfer of PAI-1 mutants, we observed that PAI-1 promoted tumor angiogenesis, not by interacting with VN, but rather by inhibiting proteolytic activity, suggesting that excessive plasmin proteolysis prevents assembly of tumor vessels. Single deficiency of uPA, tissue-type PA (tPA), uPA receptor, or VN, as well as combined deficiencies of uPA and tPA did not impair tumor angiogenesis, whereas lack of Plg reduced it. Overall, these data indicate that plasmin proteolysis, even though essential, must be tightly controlled during tumor angiogenesis, probably to allow vessel stabilization and maturation. These data provide insights into the clinical paradox whereby PAI-1 promotes tumor progression and warrant against the uncontrolled use of uPA/plasmin antagonists as tumor angiogenesis inhibitors.     

Plasminogen Activator Inhibitor-1 Expression by Brain Microvessel Endothelial Cells Is Inhibited by Elevated Glucose

Abstract: Patients with diabetes are predisposed to microvascular disease. In the retina and brain, this is characterized by neovascularization and new capillary formation. Because of the potential importance of plasmin generation in these processes, we evaluated the effect of elevated glucose concentrations on expression of plasminogen activator inhibitor-1 (PAI-1), tissue plasminogen activator (tPA), and urokinase (uPA) in cultured bovine brain endothelial cells (BBEC) versus cultured bovine aortic endothelial cells (BAEC). We observed that BBEC PAI-1 mRNA levels were decreased fivefold in cells cultured in media containing 20 m M glucose compared with BBEC cultured in media with 5.5 m M glucose, whereas expression of PAI-1 mRNA in BAEC, bovine mesenteric endothelial cells, and human umbilical vein endothelial cells was not modulated under these conditions. Expression of PAI-1 protein was also inhibited by growth of BBEC in elevated glucose, but the effect was less marked than at the mRNA level. Elevated glucose did not decrease expression of PAI-1 protein by BAEC. Withdrawal of acidic fibroblast growth factor enhanced expression of PAI-1 mRNA and protein in BBEC. Expression of tPA mRNA was not affected by the glucose concentration of the medium, and uPA mRNA was not detected in our BBEC cultures. A decrease in the local tissue activity of PAI-1 by elevated glucose concentrations, with no effect on tPA or uPA expression, would lead to an increase in the plasmin activity and thereby predispose neural tissues, such as the cerebrum and retina, of diabetic patients to neovascularization.     

A truncated plasminogen activator inhibitor-1 protein induces and inhibits angiostatin (kringles 1-3), a plasminogen cleavage product

Plasminogen activator inhibitor-1 (PAI-1) is a serpin protease inhibitor that binds plasminogen activators (uPA and tPA) at a reactive center loop located at the carboxyl-terminal amino acid residues 320-351. The loop is stretched across the top of the active PAI-1 protein maintaining the molecule in a rigid conformation. In the latent PAI-1 conformation, the reactive center loop is inserted into one of the beta sheets, thus making the reactive center loop unavailable for interaction with the plasminogen activators. We truncated porcine PAI-1 at the amino and carboxyl termini to eliminate the reactive center loop, part of a heparin binding site, and a vitronectin binding site. The region we maintained corresponds to amino acids 80-265 of mature human PAI-1 containing binding sites for vitronectin, heparin (partial), uPA, tPA, fibrin, thrombin, and the helix F region. The interaction of "inactive" PAI-1, rPAI-1(23), with plasminogen and uPA induces the formation of a proteolytic protein with angiostatin properties. Increasing amounts of rPAI-1(23) inhibit the proteolytic angiostatin fragment. Endothelial cells exposed to exogenous rPAI-1(23) exhibit reduced proliferation, reduced tube formation, and 47% apoptotic cells within 48 h. Transfected endothelial cells secreting rPAI-1(23) have a 30% reduction in proliferation, vastly reduced tube formation, and a 50% reduction in cell migration in the presence of VEGF. These two studies show that rPAI-1(23) interactions with uPA and plasminogen can inhibit plasmin by two mechanisms. In one mechanism, rPAI-1(23) cleaves plasmin to form a proteolytic angiostatin-like protein. In a second mechanism, rPAI-1(23) can bind uPA and/or plasminogen to reduce the number of uPA and plasminogen interactions, hence reducing the amount of plasmin that is produced.     

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.     

Purified alpha 2-macroglobulin receptor/LDL receptor-related protein binds urokinase.plasminogen activator inhibitor type-1 complex. Evidence that the alpha 2-macroglobulin receptor mediates cellular degradation of urokinase receptor-bound complexes.

Complexes between 125I-labeled urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type-1 (PAI-1) bound to purified alpha 2-macroglobulin (alpha 2M) receptor (alpha 2MR)/low density lipoprotein receptor-related protein (LRP). No binding was observed when using uPA. The magnitude of uPA.PAI-1 binding was comparable with that of the alpha 2MR-associated protein (alpha 2MRAP). Binding of uPA.PAI-1 was blocked by natural and recombinant alpha 2MRAP, and about 80% inhibited by complexes between tissue-type plasminogen activator (tPA) and PAI-1, and by a monoclonal anti-PAI-1 antibody. In human monocytes, uPA.PAI-1, like uPA and its amino-terminal fragment, bound to the urokinase receptor (uPAR). Degradation of uPAR-bound 125I-uPA.PAI-1 was 3-4-fold enhanced as compared with uncomplexed uPAR-bound uPA. The inhibitor-enhanced uPA degradation was blocked by r alpha 2MRAP and inhibited by polyclonal anti-alpha 2MR/LRP antibodies. This is taken as evidence for mediation of internalization and degradation of uPAR-bound uPA.PAI-1 by alpha 2MR/LRP.     

Transforming growth factor beta1 stimulates urokinase plasminogen activator system on prostate cancer cells

The effect of TGFbeta1 on the proliferation and plasminogen activator system (PA) of two prostate carcinoma cell lines, PC3 and DU145, was investigated. PA, particularly urokinase plasminogen activator (uPA), has been implicated in extracellular proteolysis, local invasiveness, metastatic spread and angiogenesis. High levels of uPA and plasminogen activator inhibitor-1 (PAI-1) correlate with poor prognosis in several cancers. TGFbeta1 had no significant effect on the proliferation of either cell line. TGFbeta1 increased the production of uPA in PC3 and DU145 cells. Despite the very low PAI-1 protein levels in both cell lines, TGFbeta1 treatment resulted in a remarkable increase in PAI-1 secretion. PAI-2 protein was also increased by 59% in the PC3 cells. A divergent effect of TGFbeta1 on the uPA enzyme activity was observed (28% decrease in PC3 and 131% increase in DU145 cells). Overall, TGFbeta1 treatment did not affect the invasion of reconstituted basement membrane of PC3 cells. In addition to the uPA:PAI-1 ratio, the presence of PAI-2 may be an important factor in the determination of metastatic sites for prostate cancer cells. In conclusion, the potential contribution of TGFbeta1 to tumor invasion may be considered as positive, based on both loss of growth inhibition and stimulation of components of the invasive system of prostate carcinoma.     

Binding of type 1 plasminogen activator inhibitor to the extracellular matrix of cultured bovine endothelial cells

The binding of type 1 plasminogen activator inhibitor (PAI-1) to the extracellular matrix (ECM) of cultured bovine aortic endothelial cells was investigated using purified 125I-labeled or L-[35S]methionine-labeled PAI-1 as probes. Little specific binding of latent PAI-1 to ECM previously depleted of endogenous PAI-1 could be demonstrated. In contrast, the guanidine-activated form of PAI-1 bound to ECM in a dose- and time-dependent manner, and binding was saturable. The dissociation constant (Kd) for this interaction was estimated to be 60 nM by Scatchard analysis, and approximately 6 pmol of activated PAI-1 was bound per cm2 of ECM. Binding was relatively specific since unlabeled, activated PAI-1 competed with 35S-labeled PAI-1 for binding to ECM, but latent PAI-1 did not. Moreover, PAI-2, protein C inhibitor (i.e. PAI-3), protease nexin-1, and alpha 2-antiplasmin were not able to compete. Tissue-type plasminogen activator (tPA) also inhibited binding, but diisopropyl fluorophosphate-inactivated tPA did not. Pretreatment of ECM with tPA, urokinase-type PA, or thrombin had no effect on its ability to subsequently bind PAI-1, whereas trypsin, plasmin, and elastase pretreatment greatly reduced its ability to bind PAI-1. Guanidine-activated, radiolabeled PAI-1 resembled active endogenous PAI-1 since it was unstable in solution but stable when bound to ECM. In addition, it formed complexes with tPA that had a relatively low affinity for ECM. These data suggest that ECM of bovine aortic endothelial cells contains a protease-sensitive structure that binds active PAI-1 tightly and relatively selectively and that this association stabilizes PAI-1 against the spontaneous loss of activity that occurs in solution.     

PAI-1 deficiency reduces liver fibrosis after bile duct ligation in mice through activation of tPA

Plasminogen activator inhibitor-1 (PAI-1) increases injury in several liver, lung and kidney disease models. The objective of this investigation was to assess the effect of PAI-1 deficiency on cholestatic liver fibrosis and determine PAI-1 influenced fibrogenic mechanisms. We found that PAI-1(-/-) mice had less fibrosis than wild type (WT) mice after bile duct ligation. This change correlated with increased tissue-type plasminogen activator (tPA) activity, and increased matrix metalloproteinase-9 (MMP-9), but not MMP-2 activity. Furthermore, there was increased activation of the tPA substrate hepatocyte growth factor (HGF), a known anti-fibrogenic protein. In contrast, there was no difference in hepatic urokinase plasminogen activator (uPA) or plasmin activities between PAI-1(-/-) and WT mice. There was also no difference in the level of transforming growth factor beta 1 (TGF-beta1), stellate cell activation or collagen production between WT and PAI-1(-/-) animals. In conclusion, PAI-1 deficiency reduces hepatic fibrosis after bile duct obstruction mainly through the activation of tPA and HGF.     

Synthesis of Urokinase-Type Plasminogen Activator and of Type- 1 Plasminogen Activator Inhibitor in Neuronal Cultures of Human Fetal Brain: Stimulation by Phorbol Ester

Human neuronal brain cultures established from 12- and 14-week-old fetuses synthesize and secrete urokinase-type plasminogen activator (uPA) and limited amounts of tissue-type plasminogen activator (tPA). These cells also produce and secrete the endothelial cell-type PA inhibitor (PAI-1), which forms sodium dodecyl sulfate-stable tPA/PAI-1 complexes in the culture medium. Immunocytochemistry shows a predominant localization of uPA, tPA, and PAI-1 in neuronal cells, with only a very weak positivity detectable in the few glial cells present in these cultures. The protein kinase C (PKC) activator 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulates the synthesis of both uPA and PAI-1, resulting in a final increase in the plasmin-generating capacity of neuronal cell cultures. No significant effect is observed, however, when cells are treated with the TPA analogue 4 alpha-phorbol 12,13-didecanoate, which is inactive as a PKC inducer, or with the neurotrophic polypeptide basic fibroblast growth factor. These data represent the first characterization of the plasmin-generating system in human fetal brain neurons and suggest a role for PKC in the modulation of uPA and PAI-1 synthesis.     

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.     

Plasminogen activator inhibitor type-1-deficient mice have an enhanced IFN-gamma response to lipopolysaccharide and staphylococcal enterotoxin B

Plasminogen activator inhibitor type-1 (PAI-1) is a major inhibitor of fibrinolysis by virtue of its capacity to inhibit urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA). Systemic inflammation is invariably associated with elevated circulating levels of PAI-1, and during human sepsis plasma PAI-1 concentrations predict an unfavorable outcome. Knowledge about the functional role of PAI-1 in a systemic inflammatory response syndrome is highly limited. In this study, we determined the role of endogenous PAI-1 in cytokine release induced by administration of LPS or staphylococcal enterotoxin B (SEB). Both LPS and SEB elicited secretion of PAI-1 into the circulation of normal wild-type (Wt) mice. Relative to Wt mice, PAI-1 gene-deficient (PAI-1(-/-)) mice demonstrated strongly elevated plasma IFN-gamma concentrations after injection of either LPS or SEB. In addition, PAI-1(-/-) splenocytes released more IFN-gamma after incubation with LPS or SEB than Wt splenocytes. Both PAI-1(-/-) CD4+ and CD8+ T cells produced more IFN-gamma upon stimulation with SEB. LPS-induced IFN-gamma release in mice deficient for uPA, the uPA receptor, or tPA was not different from IFN-gamma release in LPS-treated Wt mice. These results identify a novel function of PAI-1 during systemic inflammation, where endogenous PAI-1 serves to inhibit IFN-gamma release by a mechanism that does not depend on its interaction with uPA/uPA receptor or tPA.     

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.     

Collagen modulates gene activation of plasminogen activator system molecules

Skin extracellular matrix (ECM) molecules regulate a variety of cellular activities, including cell movement, which are central to wound healing and metastasis. Regulated cell movement is modulated by proteases and their associated molecules, including the serine proteases urinary-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) and their inhibitors (PAIs). As a result of wounding and loss of basement membrane structure, epidermal keratinocytes can become exposed to collagen. To test the hypothesis that during wounding, exposed collagen, the most abundant ECM molecule in the skin, regulates keratinocyte PA and PAI gene expression, we utilized an in vitro model in which activated keratinocytes were cultured in dishes coated with collagen or other ECM substrates. tPA, uPA, and PAI-1 mRNA and enzymatic activity were detected when activated keratinocytes attached to fibronectin, vitronectin, collagen IV, and RGD peptide. In contrast, adhesion to collagen I and collagen III completely suppressed expression of PAI-1 mRNA and protein and further increased tPA expression and activity. Similarly, keratinocyte adhesion to laminin-1 suppressed PAI-1 mRNA and protein expression and increased tPA activity. The suppressive effect of collagen I on PAI-1 gene induction was dependent on the maintenance of its native fibrillar structure. Thus, it would appear that collagen- and laminin-regulated gene expression of molecules associated with plasminogen activation provides an additional dimension in the regulation of cell movement and matrix remodeling in skin wound healing.     

Regulation of tissue-type plasminogen activator and plasminogen activator inhibitor type-1 in cultured rat Sertoli and Leydig cells

New data are provided to show that (i) rat Sertoli cells produce two types of plasminogen activators, tissue type (tPA) and urokinase type (uPA), and a plasminogen activator inhibitor type-1 (PAI-1); (ii) both tPA (but not uPA) and PAI-1 secretion in the culture are modified by FSH, forskolin, dbcAMP, GnRH, PMA and growth factors (EGF and FGF), but not by hCG and androstenedione (△4); (iii) in vitro secretion of tPA and PA-PAI-1 complexes of Sertoli cells are greatly enhanced by presence of Leydig cells which produce negligible tPA but measurable PAI-1 activity;(iv) combination culture of Sertoli and Leydig cells remarkably increases FSH-induced PAI-1 activity and decreases hCG- and forskolin-induced inhibitor activity as compared with that of two cell types cultured alone. These data suggest that rat Sertoli cells, similar to ovarian granulosa cells, are capable of secreting both tPA and uPA, as well as PAI-1. The interaction of Sertoli cells and Leydig cells is essential for the cells to response to     

The effect of IL-1alpha on the expression of matrix metalloproteinases, plasminogen activators, and their inhibitors in osteoblastic ROS 17/2.8 cells

Interleukin-1 (IL-1) plays key roles in altering bone matrix turnover. This turnover is regulated by matrix metalloproteinases (MMPs), tissue inhibitor 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). In this study, we examined the effect of IL-1alpha on the expression of the MMPs, TIMPs, tPA, uPA, and PAI-1 genes in osteoblasts derived from the rat osteosarcoma cell line ROS 17/2.8. The cells were cultured in alpha-minimum essential medium containing 10% fetal bovine serum with 0 or 100 U/ml of IL-1alpha for up to 14 days. The levels of MMPs, TIMPs, uPA, tPA, and PAI-1 expression were estimated by determining the mRNA levels using real-time RT-PCR and by determining protein levels using ELISA. In IL-1alpha cultures, the expression levels of MMP-1, -2, -3, -13, and -14 exceeded that of the control through day 14 of culture, and the expression of MMPs increased markedly from the proliferative to the later stages of culture. The TIMP-1, -2, and -3 expression levels increased from the initial to the proliferative stages of culture. The expression of tPA increased greatly during the proliferative stage of culture, and uPA expression increased throughout the culture period, increasing markedly from the proliferative to the later stages of culture. In contrast, PAI-1 expression decreased in the presence of IL-1alpha through day 14. These results suggest that IL-1alpha stimulate bone matrix turnover by increasing MMPs, tPA, and uPA production and decreasing PAI-1 production by osteoblasts, and incline the turnover to the resolution.     

Tissue plasminogen activator and its receptor in the human amnion, chorion, and decidua at preterm and term

The plasminogen activator system consists of two proteins: tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA), which act upon their specific receptors to generate plasmin from plasminogen located on the cell surface. Plasmin then acts directly and indirectly to degrade the components of the extracellular matrix (ECM). This process is likely to be important in the normal turnover of the ECM of fetal membranes and in its premature weakening in preterm premature rupture of the fetal membranes. Quantitative Northern analysis and in situ hybridization have shown that the decidua expresses mRNA for tPA. However, the immunolocalized tPA protein was most strongly associated with the amnion and chorion, as was its receptor annexin II, suggesting that the amnion and chorion are the targets for decidual tPA. At term, decidual tPA expression was unaffected by labor, and the tPA receptor was elevated both before and after labor. At preterm, the converse was found: decidual tPA expression was significantly (p < 0. 05) up-regulated by labor, but the tPA receptor was not. The results suggest that the generation of plasmin at term would be controlled by an increased concentration of the tPA receptor in the amnion and chorion, whereas at preterm a pathological increase in plasmin would be generated by an overexpression of tPA, initiated by labor.     

Expression of plasminogen activator and plasminogen activator inhibitor mRNA in human fibroblasts grown on different substrates

mRNA levels for urokinase type plasminogen activator (uPA), tissue type plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1) and plasminogen activator inhibitor-2 (PAI-2) were examined in human diploid (neonatal foreskin) fibroblasts grown in 200-ml microcarrier suspension culture. Four different substrates were used. These included gelatin-coated polystyrene plastic, DEAE-dextran, glass-coated polystyrene plastic and uncoated polystyrene plastic. Our previous studies have shown that culture fluids from diploid fibroblasts grown on DEAE-dextran contained higher levels of plasminogen-dependent fibrinolytic activity than culture fluids from the same cells grown on other substrates. The increased plasminogen activator activity was due largely to elevated amounts of tPA (In Vitro Cell. Develop. Biol. 22: 575–582, 1986). The present study shows that there is a corresponding elevation of tPA mRNA in diploid fibroblasts cultured on DEAE-dextran relative to the other substrates. There does not appear to be any difference in uPA mRNA or in mRNA for PAI-1 or PAI-2 produced by the same cells on the four substrates. These data suggest that the influence of the substrate on plasminogen activator production is mediated at the genetic level.