Plasminogen activator regulation in osteoblasts: parathyroid hormone inhibition of type-1 plasminogen activator inhibitor and its mRNA.

In order to determine the mechanism by which parathyroid hormone (PTH) stimulates plasminogen activator (PA) activity in rat osteoblasts, we investigated the effect of human PTH(1-34) [hPTH(1-34)] on the synthesis of mRNAs for tissue-type PA (tPA), urokinase-type PA (uPA), and PA inhibitor-1 (PAI-1), and on release of PA activity and PAI-1 protein in both normal rat calvarial osteoblasts and UMR 106-01 osteogenic sarcoma cells. hPTH(1-34) (0.25-25 nM) decreased PAI-1 mRNA and protein, and increased PA activity in both cell types in a dose-dependent manner with ED50 of about 1 nM for both responses. Forskolin and isobutylmethylxanthine also stimulated PA activity and decreased PAI-1 protein and mRNA in both cell types. hPTH(1-34) did not show any consistent effect on tPA and uPA mRNA in calvarial osteoblasts, but a modest (two-fold) increase of both mRNAs was observed in UMR 106-01 cells treated with 25 nM hPTH(1-34). However, when protein synthesis was inhibited with 100 microM cycloheximide, the increase of tPA and uPA mRNA by hPTH(1-34) was enhanced in UMR 106-01 cells and became evident in calvarial osteoblasts. Fibrin autography also revealed that hPTH(1-34) increases tPA and uPA activity, especially after cycloheximide treatment in UMR 106-01 cells. These results strongly suggest that PTH increases PA activity predominantly by decreasing PAI-1 protein production through a cyclic adenosine monophosphate (cAMP)-dependent mechanism in rat osteoblasts. The reduction of PAI-1 protein by PTH results in enhanced action of both tPA and uPA, and would contribute to the specific roles of these PAs in bone.     

Prostaglandin E2 regulates production of plasminogen activator isoenzymes,urokinase receptor,and plasminogen activator inhibitor-1 in primary cultures of rat calvarial osteoblasts

The bone resorbing agent, prostaglandin E₂ (PGE₂), was found to alter several components of the plasminogen activator (PA)/plasmin pathway in primary cultures of rat neonatal osteoblast-like cells. The mRNA and activities of both urokinase-type PA (uPA) and tissue-type PA (tPA) were enhanced by PGE₂ treatment. The presence of mRNA for the uPA receptor (uPAR) has been demonstrated in these cells and steady-state levels shown to be greatly enhanced, the response being rapid and sustained for at least 24 hours. mRNA for plasminogen activator inhibitor 1 (PAI-1) was modulated in a biphasic manner, with inhibition of the constitutive level apparent at 4 hours of treatment and stimulation apparent at 12 hours and longer, while PAI-1 protein, measured by an ELISA assay for rat PAI-1, was diminished over this period. Neither PAI-2 mRNA nor mRNA for the broad spectrum protease inhibitor, protease nexin-1 (PN-1), was found to be modulated by PGE₂. Therefore, PGE₂ is likely to stimulate cell surface proteolytic activity, since uPA mRNA and cell-associated activity were elevated, as was mRNA for the cellular receptor for uPA. Although it was not possible to measure uPAR number and affinity it seems likely that elevated uPAR mRNA would translate into increased uPARs which would localize the increased uPA activity to the pericellular region. tPA mRNA and activity were also increased transiently with the activity inhibited with prolonged incubations, apparently by PAI-1. Elevation of tPA mRNA and activity may result in elevated activity within the extracellular matrix as tPA has been reported to associate with several matrix proteins. Thus the early effect of PGE₂ would be to promote proteolysis, both pericellularly and in the extracellular matrix. The inhibition of PAI-1 mRNA and protein, which would contribute to the elevation of activity, is due to PGE₂, but the later stimulatory effect on PAI-1 mRNA may be due to feedback regulation by transforming growth factor beta (TGFβ), secreted by osteoblasts and activated by elevated levels of PA. © 1995 Wiley-Liss Inc.     

Plasminogen-dependent activation of latent transforming growth factor beta (TGFβ) by growing cultures of osteoblast-like cells

Osteoblasts secrete transforming growth factor beta (TGFβ) as a biologically inert, latent complex that must be dissociated before the growth factor can exert its effects. We have examined the production and proteolytic activation of latent TGFβ (LTGFβ) by clonal UMR 106-01 rat osteosarcoma cells and neonatal mouse calvarial (MC) osteoblast-like cells in vitro. Synthetic bPTH-(1–34) increased the activity of tissue-type (tPA) and urokinase-type (uPA) plasminogen activators (PA) in cell lysates (CL) of UMR 106-01 cells. The concentration of active TGFβ in serum-free CM from cultures treated with bPTH-(1–34) and plasminogen was significantly greater than in CM from untreated controls and cultures treated with either bPTH-(1–34) or plasminogen alone. This effect occurred at concentrations of PTH-(1–34) that increased PA activity and was prevented by aprotinin, an inhibitor of plasmin activity. Treatment with bPTH-(1–34) had no effect on the concentration of TGFβ in acid-activated samples of CM. Functional consequences of proteolytically activated TGFβ was examined in primary cultures of neonatal MC osteoblast-like cells. Human platelet TGFβ1 caused a dose-dependent increase in the migration of these cells in an in vitro wound healing assay. Cell migration was also stimulated in cultures treated with bPTH-(1–34) and plasminogen together. This effect was blocked by an anti-TGFβ1 antibody. The results of these studies demonstrate that (1) LTGFβ secreted by osteoblasts in vitro is activated under conditions where the plasmin activity in the cultures is increased, and (2) the TGFβ generated by plasmin-mediated proteolysis is biologically active. We suggest that the local concentration of TGFβ in bone may be controlled by the osteoblast-associated plasminogen activator/plasmin system. Furthermore, since several calciotropic factors influence osteoblast PA activity, this system may have an important role in mediating their anabolic and/or catabolic effects. © 1993 Wiley-Liss, Inc.     

The THP-1 cell line is a urokinase-secreting mononuclear phagocyte with a novel defect in the production of plasminogen activator inhibitor-2

Mononuclear phagocytes regulate the generation of plasmin by secreting urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor-2 (PAI-2). We investigated the production of plasminogen activator (PA) and PA inhibitor by the human monocytic leukemia cell line, THP-1. Similar to U937 monoblast-like cells and peripheral blood monocytes (PBM), THP-1 cells produce a PA that is specifically neutralized by anti-uPA antibody and comigrates with human high molecular mass uPA (54 kDa) on casein-plasminogen zymogaphy. PA activity could be dissociated from intact THP-1 cells by brief treatment with a weak acid-glycine buffer, indicating that the uPA is secreted and bound to receptors on the plasma membrane. Regulation of uPA proceeds normally in THP-1 cells, with cell-associated PA activity increasing from 77 +/- 20 to 163 +/- 26 and 325 +/- 30 mPU/10(6) cells in response to PMA and LPS, respectively; parallel increases in steady state levels of uPA mRNA were observed. In contrast to normal expression of uPA activity, functional PAI-2 could not be demonstrated in either the conditioned media or cell lysates of THP-1 under basal or stimulated conditions. Both U937 and PBM secrete low levels of PA inhibitor activity that increase substantially in response to stimulation with PMA and LPS. Immunoreactive PAI-2, measured by ELISA, was undetectable in THP-1 lysates or conditioned medium, but was consistently present in U937 and PBM, paralleling the presence of PA inhibitor activity. THP-1 cells express low levels of an abnormally sized mRNA for PAI-2 and demonstrate a regulatory defect whereby steady state levels of PAI-2 mRNA are markedly reduced upon stimulation with PMA or LPS. By contrast, U937 and PBM respond to identical stimulation with increases in PAI-2 mRNA. We conclude that THP-1 cells express a structurally abnormal species of PAI-2 mRNA, with complete loss of inhibitory activity as well as altered function of PMA- and LPS-responsive regulatory elements.     

Different mechanisms contribute to simultaneous inhibition of urokinase and tissue-type plasminogen activators by glucocorticoids in human ovarian carcinoma cells

Previous studies from our laboratory have demonstrated that OVCA 433 human ovarian carcinoma cells are glucocorticoid responsive by several criteria and contain high affinity, saturable, steroid-specific glucocorticoid receptors. These cells secrete both mammalian plasminogen activators (PAs), urokinase (uPA) and tissue-type PA (tPA). Treatment of OVCA 433 cells with 1 x 10(-7) M dexamethasone (Dex) for 4 days led to 77% and 83% reductions in the extracellular activities of uPA and tPA, respectively, released into serum-free conditioned medium during a 1-h period. Dex treatment led to a 71% decrease in the rate of extracellular uPA antigen accumulation, as determined by enzyme-linked immunosorbent assay, as well as a 73% reduction in steady state uPA mRNA levels. In contrast, Dex treatment led to only a 42% decrease in the rate of extracellular tPA antigen accumulation and a 48% decrease in tPA mRNA levels; such decreases were insufficient to account for the 83% reduction in tPA activity. Thus, while Dex-induced decreases in uPA antigen and mRNA levels accounted for all but 6% of the decrease in uPA activity, a large discrepancy existed between the magnitudes of decreased tPA activity and decreased tPA antigen and mRNA levels. OVCA 433 cells produce both PAI-1 and PAI-2, two specific PA inhibitors. Treatment of cells with 1 x 10(-7) M Dex for 4 days led to a 3.3-fold increase in the rate of extracellular PAI-1 accumulation, with little or no effect on PAI-2 accumulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Reciprocal regulation of tissue-type and urokinase-type plasminogen activators in the differentiation of murine preadipocyte line 3T3-L1 and the hormonal regulation of fibrinolytic factors in the mature adipocytes

Adipose tissue expresses a variety of genes including tumor necrosis factor alpha and type-1 plasminogen activator inhibitor (PAI-1); and these factors, produced by adipocytes, may be associated with the risk of coronary events in obesity. In this study, we characterized the production of fibrinolytic factors including tissue-type plasminogen activator (tPA), urokinase-type PA (uPA), and PAI-1 in the differentiation of preadipocytes, and examined the hormonal regulation of these fibrinolytic factors in mature adipocytes. Mouse 3T3-L1 preadipocytes were employed as a model of adipocytes. Adipocyte differentiation was induced by insulin, dexamethasone, and 3-isobutyl-1-methyl xanthine (IBMX). alpha-Glycerophosphate dehydrogenase (GPDH) activity and glucose transporter 4 (GLUT4) mRNA, indices for adipocyte maturation, were induced on Day 4, and gradually increased. GPDH activity reached its maximum level on Day 14. The level of tPA, a major PA in preadipocytes, dramatically decreased with differentiation. On the other hand, that of uPA reciprocally increased. PAI-1 production was also dramatically induced concomitant with differentiation. In mature adipocytes, uPA production was dominant (25 microg/ml/24 h vs. 0.8 microg/ml/24 h for tPA). Total PA activity in the mature adipocytes was reduced by insulin or dexamethasone, but not by glucagon. Insulin, IBMX, and dexamethasone significantly decreased both uPA and tPA production, and increased PAI-1 production. Glucagon had no effect on the production of these fibrinolytic factors. Our results reveal that uPA is one of the markers for the differentiation of 3T3-L1 cells and that insulin, IBMX, and dexamethasone are potent regulators of the fibrinolytic activity in differentiated 3T3-L1 cells, reciprocally affecting PA and PAI-1 levels in them.     

Osteoblasts display receptors for and responses to leukemia-inhibitory factor

Specific binding of leukemia-inhibitory factor (LIF) to osteoblasts, but not multinucleated osteoclasts, was demonstrated by receptor autoradiography by using cells isolated from newborn rat long bones. The clonal rat osteogenic sarcoma cells, UMR 106-06, which have several phenotypic properties of osteoblasts, expressed 300 LIF receptors per cell, with an apparent KD of 60 pM. Treatment of calvarial osteoblasts or UMR 106-01 cells with LIF resulted in a dose-dependent inhibition of plasminogen activator (PA) activity. Both calvarial osteoblasts and osteogenic sarcoma cells were shown by Western blotting and reverse fibrin autography to produce plasminogen activator inhibitor-1 (PAI-1), the production of which was increased by LIF treatment. Northern blot analysis revealed that LIF treatment resulted in a rapid (peak 1 hour), dose-dependent increase in mRNA for PAI-1. LIF treatment of the preosteoblast cell line, UMR 201, enhanced the alkaline phosphatase response of these cells to retinoic acid. Each of the osteoblast-like cell types (calvarial osteoblasts, UMR 106-06, and UMR 201) was shown to produce LIF by bioassay and, by using the polymerase chain reaction (PCR), was shown to express low levels of mRNA for LIF. These data establish that cells of the osteoblast lineage are targets for LIF action. The reported anabolic effects of this cytokine on bone formation in vivo could be related to inhibition of protease activity. LIF may be an important paracrine modulator in bone, or perhaps an autocrine one, based on the evidence for its production by osteoblasts and osteoblast-like cells.     

Urokinase-type and tissue-type plasminogen activators are essential for in vitro invasion of human melanoma cells

This study evaluates the contribution of two types of plasminogen activators (PAs; tissue-type PA (tPA) versus urokinase-type PA (uPA) toward the invasiveness of human melanoma cells in a novel in vitro assay. We identified two human melanoma cell lines, MelJuso and MeWo, expressing uPA or tPA as shown at mRNA, protein, and enzyme activity level. MelJuso cells produced uPA as well as plasminogen activator inhibitor-1 (PAI-1). The latter was, however, not sufficient to neutralize the cell-associated or secreted uPA activity. MeWo cells secreted tPA, but the enzyme was not found to be cell-associated. PAI-1 production by these cells was not detectable. Plasminogen activation and fibrinolytic capacity of both cell lines were reduced by anticatalytic monoclonal antibodies specific for the respective type of PA or by aprotinin. In a novel in vitro invasion assay, antibodies to PA as well as aprotinin decreased the invasiveness of both cell lines into a fibrin gel, Matrigel, or intact extracellular matrix. Our results confirm the importance of uPA-catalyzed plasminogen activation in tumor cell invasiveness. Furthermore, we provide evidence that tPA, beyond its key role in thrombolysis, can also be involved in in vitro invasion of human melanoma cells.     

Extracellular matrix degradation by cultured mesangial cells: mediators and modulators

Decreased degradation of the glomerular extracellular matrix (ECM) is thought to contribute to the accumulation of glomerular ECM that occurs in diabetic nephropathy and other chronic renal diseases. Several lines of evidence indicate a key role for the plasminogen activator/plasminogen/plasmin system in glomerular ECM degradation. However, which of the two plasminogen activators (PAs) present in renal tissue, tissue plasminogen activator (tPA) or urokinase-type plasminogen activator (uPA), is responsible for plasmin generation and those factors that modulate the activity of this system remain unclear. This study utilized mesangial cells isolated from mice with gene deletions for tPA, uPA, and plasminogen activator inhibitor 1 (PAI-1) to further delineate the role of the PA/plasminogen/plasmin system in ECM accumulation. ECM degradation by uPA-null mesangial cells was not significantly different from controls (92% +/- 1%, n = 12). In contrast, ECM degradation by tPA-null mesangial cells was markedly reduced (-78 +/- 1%, n = 12, P < 0.05) compared with controls, whereas tPA/uPA double-null mesangial cells degraded virtually no ECM. Previous studies from this laboratory have established that transforming growth factor-beta1 (TGFbeta1) inhibits ECM degradation by cultured mesangial cells by increasing the production of PAI-1, the major physiological PA inhibitor. In keeping with this observation, TGFbeta1 (1 ng/ml) had no effect on ECM degradation by PAI-1-null MC. High glucose levels (30 mM) in the presence or absence of insulin (0.1 mM) caused a moderate increase in ECM degradation by normal human mesangial cells. In contrast, glycated albumin, whose concentration is known to increase in diabetes, produced a dose-dependent (0.2-0.5 mg/ml) inhibition of ECM degradation by normal human mesangial cells. Taken together, these results document the importance of tPA versus uPA in renal plasmin production and indicate that in contrast to elevated glucose, glycated albumin may contribute to ECM accumulation in diabetic nephropathy.     

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.     

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.     

Cyclic AMP-dependent and -independent effects on tissue-type plasminogen activator activity in osteogenic sarcoma cells; evidence from phosphodiesterase inhibition and parathyroid hormone antagonists

The plasminogen activator (PA) in clonal osteogenic sarcoma cells of rat origin (UMR 106-01 and UMR 106-06) and in osteoblast-rich rat calvarial cells has been characterized using specific antibodies to be tissue-type PA (tPA). An Mr value of 75,000 by SDS-polyacrylamide gel electrophoresis and fibrin autoradiography supports this characterization. There was also evidence for an Mr 105,000 component, which could be due to a proteinase-inhibitor complex. The mechanism of regulation of this tPA activity has been studied in the clonal osteogenic sarcoma cells. Parathyroid hormone (PTH) and prostaglandin E2, which increase cyclic AMP production in the sarcoma cells, also increased tPA activity. The sensitivity and magnitude of the tPA response to PTH and prostaglandin E2 were increased by simultaneous treatment with isobutylmethylxanthine (IBMX) at drug concentrations which had little effect themselves on tPA activity. In UMR 106-06 cells, which unlike UMR 106-01 cells show a cyclic AMP response to calcitonin, tPA activity was also increased in response to calcitonin, and the effect was enhanced by IBMX. 1,25-Dihydroxyvitamin D-3 also increased tPA activity in the cells, but this response was not modified by IBMX. Synthetic peptide antagonists of PTH-responsive adenylate cyclase, [34Tyr]-hPTH (3-34) amide and [34Tyr]-hPTH (5-34) amide, inhibited the PTH-induced increase in tPA activity over the same concentration range at which they inhibited cyclic AMP production, but the antagonist peptides had no effect on the tPA responses to prostaglandin E2, calcitonin or 1,25-dihydroxyvitamin D-3. These data indicate that cyclic AMP mediates the actions of PTH, prostaglandin E2 and calcitonin in increasing tPA activity in the clonal osteogenic sarcoma cells. 1,25-Dihydroxyvitamin D-3, on the other hand, increases tPA activity through a mechanism independent of cyclic AMP.     

Detection of cathepsin B, plasminogen activators and plasminogen activator inhibitor in human non-small lung cancer cell lines

Human non-small lung cancer cell lines HS-24 (established from a primary squamous cell carcinoma) and SB-3 (established from a metastasis of a primary adenocarcinoma of the lung into the adrenal gland) were analysed for the proteinases tissue-type plasminogen activator (tPA), urokinase-type plasminogen activator inhibitor (PAI-1). The proteinases were characterized by activity measurements, inhibition studies, enzyme-linked immunosorbent assay (ELISA), and Western blot analysis. Cell-associated proteinases were determined in cell lysates, secreted proteinases in cell conditioned culture media. Both cell lines were found to secrete uPA and PAI-1, whereas tPA could be detected only in HS-24 conditioned media. No cathepsin B activity could be detected in media of both cell lines. However, activation experiments and western blot analysis showed, that at least HS-24 secrete an inactive precursor. Cell lysates of HS-24 and SB-3 show PA activity, but on a low level. Cathepsin B activity was also found to be low in HS-24 lysates. However, SB-3 lysates show high cathepsin B activity. Further characterization of the proteinases by their sensitivity against several inhibitors suggests that they are similar to the corresponding proteinases of normal, nonmalignant cells.     

Sesamol regulates plasminogen activator gene expression in cultured endothelial cells: a potential effect on the fibrinolytic system

Sesamol is a component in the nutritional makeup of sesame that was identified as an antioxidant. In recent years, the importance of the plasminogen activator (PA) and its adjustment factor, plasminogen activator inhibitor-1 (PAI-1), in the prevention of atherosclerosis has gradually received recognition. The objective of this in vitro study was to demonstrate the effects of sesamol on PA and PAI-1. We also compared the effects of sesamol with two well-known antioxidants, vitamins C and E, by using human umbilical vein endothelial cells as an experimental model and by treating them with the above-mentioned three nutrients with doses up to 100 micromol/L. After 24 h, cells and cultural medium were collected for analysis. The concentrations of tissue PA (tPA), urokinase PA (uPA) and PAI-1 were measured by an enzymatic immunity method. Northern blot method was used to analyze the expression of mRNA of these three types of proteins. The results showed that sesamol increased the production of uPA and tPA significantly and also up-regulated the mRNA expressions of these proteins. On the other hand, vitamins C and E could induce tPA but not uPA. As for PAI-1, none of the nutrients induced any evident response. These findings suggest that the overall vascular fibrinolytic capacity may be enhanced by using sesamol to regulate PA gene expression.     

Elimination of hydrocortisone from the medium enables tissue plasminogen activator gene expression by normal and immortalized nonmalignant human epithelial cells.

Human cervical epithelial cells transfected and immortalized with human papillomavirus type 16 DNA (HCE16/3) can be, like many other epithelial cells, normally grown in medium supplemented with epidermal growth factor, cholera toxin, hydrocortisone, insulin, transferrin, thyroid hormone and serum. We found that hydrocortisone diminished tissue plasminogen activator (tPA) production to an undetectable level. The removal of hydrocortisone increased urokinase plasminogen activator (uPA) activity within 24-48 h and tPA activity within 48-72 h, and converted the cells to a more elongated and fibroblastic phenotype. Upregulation of uPA mRNA was seen as early as at 3 h and of tPA mRNA within 48-72 h. Higher molecular weight forms (97-110 kDa) of plasminogen activators were seen in zymograms, apparently complexed with PAI-1, starting at 6 h both in the presence and absence of hydrocortisone. Immunoprecipitation with a PAI-1 monoclonal antibody confirmed that both uPA and tPA were complexed. We also studied normal diploid human bronchial epithelial cells (NHBE) and NHBE cells transformed with an adeno-12/SV40 hybrid virus (BEAS-2B). In both types of nonmalignant epithelial cells, the removal of hydrocortisone increased uPA activity. The omission of hydrocortisone increased tPA levels significantly in BEAS-2B cell cultures, and in NHBE cell cultures tPA became detectable at 72 h. No PA complexes were seen in these two cell types. We conclude that normal and immortalized nonmalignant epithelial cells produce tPA, but only if hydrocortisone is omitted in the growth medium.     

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.     

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.     

促性腺激素诱导猕猴排卵周期中卵巢纤溶酶...

Changes of plasminogen activator (PA) and its inhibitor (PAI-1) activity and antigen have been investigated during PMSG/hCG induced ovulation in rhesus monkeys. It has been demonstrated that the ovarian tissue type PA (tPA) activity, which reaches maximum prior to ovulation and declines thereafter, is closely related to follicular rupture; significant increases in urokinase type PA (uPA) only occurs in granulosa cells after ovulation. Since the secretory activity of ovarian PAI-1 reaches its peak level 12-24 h earlier than tPA the rapid decrease in PAI-1 activity in the approach of ovulation is correlated with the elevation of tPA activity. It is, therefore, suggested that a counterbalance of tPA and PAI-1 activity within the ovary may play an important role in the ovulation mechanism, whereas uPA may be involved in the regulation of corpus luteum formation.