Human Plasminogen Activator Inhibitor Type-1 Research Articles

Dual-reporter high-throughput screen for small-molecule in vivo inhibitors of plasminogen activator inhibitor type-1 yields a clinical lead candidate

Plasminogen activator inhibitor type-1 (PAI-1) is a serine protease inhibitor (serpin) implicated in numerous pathological processes, including coronary heart disease, arterial and venous thrombosis, and chronic fibrotic diseases. These associations have made PAI-1 an attractive pharmaceutical target. However, the complexity of the serpin inhibitory mechanism, the inherent metastability of serpins, and the high-affinity association of PAI-1 with vitronectin in vivo have made it difficult to identify pharmacologically effective small-molecule inhibitors. Moreover, the majority of current small-molecule PAI-1 inhibitors are poor pharmaceutical candidates. To this end and to find leads that can be efficiently applied to in vivo settings, we developed a dual-reporter high-throughput screen (HTS) that reduced the rate of nonspecific and promiscuous hits and identified leads that inhibit human PAI-1 in the high-protein environments present in vivo Using this system, we screened >152,000 pure compounds and 27,000 natural product extracts (NPEs), reducing the apparent hit rate by almost 10-fold compared with previous screening approaches. Furthermore, screening in a high-protein environment permitted the identification of compounds that retained activity in both ex vivo plasma and in vivo Following lead identification, subsequent medicinal chemistry and structure-activity relationship (SAR) studies identified a lead clinical candidate, MDI-2268, having excellent pharmacokinetics, potent activity against vitronectin-bound PAI-1 in vivo, and efficacy in a murine model of venous thrombosis. This rigorous HTS approach eliminates promiscuous candidate leads, significantly accelerates the process of identifying PAI-1 inhibitors that can be rapidly deployed in vivo, and has enabled identification of a potent lead compound.

Abstract 1922: Plasminogen activator inhibitor type-1 (PAI-1) enhances bladder cancer tumor progression.

Abstract Introduction: Bladder cancer (BCa) is among the five most common malignancies worldwide and has the highest recurrence rate of any tumor type. BCa biology is poorly understood and early detection remains one of the most urgent issues in BCa research. Previously we have identified a preliminary molecular signature of BCa in voided urine samples from BCa patients. Our results revealed aberrant overexpression of the serine protease inhibitor (serpin) plasminogen activator inhibitor type-1 (PAI-1), both at the genomic and protein level. Increasing evidence indicate that elevated levels of PAI-1 correlates to a poor prognosis in many tumor types including breast, colorectal, gastric, ovarian, and more recently bladder cancer. PAI-1 has emerged as a potential target for BCa treatment and the aim of this study is to provide a greater understanding of its role in bladder cancer progression. Methods: PAI-1 expression levels in UROtsa (benign urothelial cell), KU7 (low-grade papillary urothelial cancer cell), and UMUC14 and T24 (high-grade invasive urothelial cancer cell lines) was tested by real-time PCR (RTPCR) and Western blot analysis. Secreted PAI-1 levels were measured by ELISA. KU7 stable clones overexpressing PAI-1 was established by transfection of plasmid DNA encoding human PAI-1. T24 and UMUC14 stable clones with shRNA knockdown of PAI-1 were established by infection with retroviral particles. Cell proliferation, migration, invasion, and apoptosis assays were performed in stable clones. Results: RTPCR analysis revealed an increase of PAI-1 in T24 and UMUC14 cells (27.9 and 5.2 times, respectively) while KU7 cells exhibited significantly reduced levels as compared to the UROtsa control. Similar results were observed for PAI-1 protein concentrations in cell culture supernatants and western blot analysis. While overexpression of PAI-1 in KU7 cells promoted an increase in proliferation, migration and invasion, these were significantly reduced upon inactivation of PAI-1 in T24 and UMUC14 cells stably expressing shRNA PAI-1. Loss of PAI-1 function also led to a substantial increase in apoptotic cell death. Conclusions: These results suggest that PAI-1 overexpression in BCa promotes tumor progression and therapeutic strategies targeting PAI-1 may serve clinically beneficial outcomes. Funding provided by research grants from Florida Department of Health James and Esther King Team Science Award 10KT-01 (CJR) and National Cancer Institute RO1 CA116161 (SG). Citation Format: Evan Gomes, Steven Goodison, Makito Miyake, Shanti Ross, Ge Zhang, Charles Rosser. Plasminogen activator inhibitor type-1 (PAI-1) enhances bladder cancer tumor progression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1922. doi:10.1158/1538-7445.AM2013-1922

Posttranscriptional regulation of expression of plasminogen activator inhibitor type-1 by sphingosine 1-phosphate in HepG2 liver cells

Altered expression of plasminogen activator inhibitor type-1 (PAI-1), a major physiologic inhibitor of fibrinolysis, is implicated in the progression of atherosclerosis. Sphingosine 1-phosphate (S1P) regulates expression of diverse genes and alters expression of PAI-1 in several types of cells. However, the nature of posttranscriptional regulation of expression of PAI-1 by S1P has not yet been thoroughly elucidated. The present study was undertaken to determine whether S1P has important effects on the posttranscriptional regulation of PAI-1 expression. To evaluate this possibility, we determined promoter activity, mRNA levels, 3′-untranslated region (UTR) activity, and protein levels of PAI-1 in HepG2 cells. S1P increased PAI-1 promoter activity and the expression of PAI-1 mRNA within 4h of exposure. It decreased the expression of PAI-1 mRNA and the accumulation of PAI-1 protein into the media in 24h. Human PAI-1 mRNA exists in two subspecies (3.2 and 2.2kb). S1P decreased the baseline luciferase activity of the 1kb fragment of the 3′ terminus (+2177 to 3176nt) of the 3′-UTR of the 3.2kb PAI-1 mRNA [3′-UTR (+2177–3176)]. S1P decreased expression of PAI-1 protein, presumably by regulating PAI-1 expression at the posttranscriptional level thereby affecting mRNA stability. SERPINE1 mRNA binding protein (SERBP1) and ARE3 in the 3′-UTR were involved in the posttranscriptional regulation by S1P. Our data suggest that S1P can destabilize 3.2kb PAI-1 mRNA through specific effects on the 3′-UTR. These effects appear to involve SERBP1 leading to decreased expression of PAI-1 protein.

Posttranscriptional regulation of expression of plasminogen activator inhibitor type-1 by cAMP in HepG2 liver cells

Altered expression of plasminogen activator inhibitor type-1 (PAI-1), a physiologic fibrinolysis inhibitor, is implicated in atherosclerosis. Cyclic adenosine monophosphate (cAMP) alters PAI-1 expression in several cells. Nevertheless, posttranscriptional regulation of PAI-1 has not been elucidated. To determine whether cAMP affects PAI-1 expression at posttranscriptional level, we determined promoter activity, mRNA levels, 3'-untranslated region (UTR) activity and protein levels of PAI-1 using HepG2 cells. cAMP decreased PAI-1 promoter activity at 24 h and mRNA expression at 4 h while it increased mRNA expression and accumulation of PAI-1 protein into media at 24 h. Human PAI-1 mRNA exists in two subspecies (3.2 and 2.2 kb), and cAMP increased baseline luciferase activity of 3'-UTR of the 3.2 kb PAI-1 mRNA [3'-UTR (+1358-3176)] and 1 kb fragment of 3'-terminus of 3'-UTR of 3.2 kb mRNA [3'-UTR (+2177-3176)]. cAMP increased PAI-1 protein expression despite decrease in promoter activity, presumably by regulating PAI-1 expression at the posttranscriptional level and thereby affecting mRNA stability. The 53-nt fragment in 3'-UTR (+2591 to +2643 nt) was involved in posttranscriptional regulation by cAMP. Thus, cAMP can stabilize 3.2 kb PAI-1 mRNA mediated by specific effects on 3'-UTR, and these effects are associated with increased expression of PAI-1 protein.

Antagonistic regulation of EMT by TIF1γ and Smad4 in mammary epithelial cells

TGF-β is a potent inducer of epithelial-to-mesenchymal transition (EMT), a process involved in tumour invasion. TIF1γ participates in TGF-β signalling. To understand the role of TIF1γ in TGF-β signalling and its requirement for EMT, we analysed the TGF-β1 response of human mammary epithelial cell lines. A strong EMT increase was observed in TIF1γ-silenced cells after TGF-β1 treatment, whereas Smad4 inactivation completely blocked this process. Accordingly, the functions of several TIF1γ target genes can be linked to EMT, as shown by microarray analysis. As a negative regulator of Smad4, TIF1γ could be crucial for the regulation of TGF-β signalling. Furthermore, TIF1γ binds to and represses the plasminogen activator inhibitor 1 promoter, demonstrating a direct role of TIF1γ in TGF-β-dependent gene expression. This study shows the molecular relationship between TIF1γ and Smad4 in TGF-β signalling and EMT.

Apelin protects against angiotensin II-induced cardiovascular fibrosis and decreases plasminogen activator inhibitor type-1 production

To test the hypothesis that apelin protects against angiotensin II (Ang II)-induced cardiovascular fibrosis and vascular remodeling. Wild-type mice administered apelin or apelin along with Ang II exhibited less cardiovascular fibrosis and decreased plasminogen activator inhibitor type-1 (PAI-1) gene expression than mice receiving Ang II, N-nitro-L-arginine methyl ester (L-NAME), apelin plus L-NAME or apelin plus Ang II plus L-NAME. In-vitro analysis using a luciferase construct driven by 3.1 kb of the human PAI-1 promoter revealed that apelin blocked Ang II-mediated PAI-1 gene expression. Immunoblotting for phosphorylated myosin phosphatase subunit and myosin light chain revealed that apelin blocked Ang II activation of the Rho kinase pathway, which is associated with induction of PAI-1 gene expression by Ang II. In addition, treatment of human aortic smooth muscle cells with apelin reduced PAI-1 mRNA and protein production in the presence and absence of Ang II. Conversely, L-NAME treatment attenuated the downregulation of PAI-1 by apelin in cells. Apelin protects against cardiac fibrosis and vascular remodeling through direct regulation of PAI-1 gene expression. This protective effect is mediated through the synergistic inhibition of Ang II signaling and increased production of nitric oxide by apelin. Our data extend previous findings and provide new insight into the molecular mechanisms by which apelin elicits a cardioprotective effect.

Metals affect the structure and activity of human plasminogen activator inhibitor‐1. II. Binding affinity and conformational changes

Human plasminogen activator inhibitor type 1 (PAI-1) is a serine protease inhibitor with a metastable active conformation. The lifespan of the active form of PAI-1 is modulated via interaction with the plasma protein, vitronectin, and various metal ions. These metal ions fall into two categories: Type I metals, including calcium, magnesium, and manganese, stabilize PAI-1 in the absence of vitronectin, whereas Type II metals, including cobalt, copper, and nickel, destabilize PAI-1 in the absence of vitronectin, but stabilize PAI-1 in its presence. To provide a mechanistic basis for understanding the unusual modulation of PAI-1 structure and activity, the binding characteristics and conformational effects of these two types of metals were further evaluated. Steady-state binding measurements using surface plasmon resonance indicated that both active and latent PAI-1 exhibit a dissociation constant in the low micromolar range for binding to immobilized nickel. Stopped-flow measurements of approach-to-equilibrium changes in intrinsic protein fluorescence indicated that the Type I and Type II metals bind in different modes that induce distinct conformational effects on PAI-1. Changes in the observed rate constants with varying concentrations of metal allowed accurate determination of binding affinities for cobalt, nickel, and copper, yielding dissociation constants of ∼40, 30, and 0.09 μM, respectively. Competition experiments that tested effects on PAI-1 stability were consistent with these measurements of affinity and indicate that copper binds tightly to PAI-1.

Metals affect the structure and activity of human plasminogen activator inhibitor‐1. I. Modulation of stability and protease inhibition

Human plasminogen activator inhibitor type 1 (PAI-1) is a serine protease inhibitor with a metastable active conformation. Under physiological conditions, half of the inhibitor transitions to a latent state within 1-2 h. The interaction between PAI-1 and the plasma protein vitronectin prolongs this active lifespan by ∼50%. Previously, our group demonstrated that PAI-1 binds to resins using immobilized metal affinity chromatography (Day, U.S. Pat. 7,015,021 B2, March 21, 2006). In this study, the effect of these metals on function and stability was investigated by measuring the rate of the transition from the active to latent conformation. All metals tested showed effects on stability, with the majority falling into one of two types depending on their effects. The first type of metal, which includes magnesium, calcium and manganese, invoked a slight stabilization of the active conformation of PAI-1. A second category of metals, including cobalt, nickel and copper, showed the opposite effects and a unique vitronectin-dependent modulation of PAI-1 stability. This second group of metals significantly destabilized PAI-1, although the addition of vitronectin in conjunction with these metals resulted in a marked stabilization and slower conversion to the latent conformation. In the presence of copper and vitronectin, the half-life of active PAI-1 was extended to 3 h, compared to a half-life of only ∼30 min with copper alone. Nickel had the largest effect, reducing the half-life to ∼5 min. Together, these data demonstrate a heretofore-unknown role for metals in modulating PAI-1 stability.

Impact of statins on modulation by insulin of expression of plasminogen activator inhibitor type-1

Plasminogen activator inhibitor type-1 (PAI-1) is a physiologic inhibitor of fibrinolysis and a known determinant of cardiovascular risk. That its expression is stimulated by insulin in HepG2 human hepatoma cells has been shown earlier. Others have found that increased expression of PAI-1 is a risk factor for acute myocardial infarction. Pleiotropic (noncholesterol lowering) effects of statins seem to reduce cardiovascular risk. This study was designed to determine whether insulin stimulation of PAI-1 expression is attenuated by statins, and if so to explore mechanisms that are responsible for the attenuation. PAI-1 protein in the conditioned media was assayed by western blotting, and PAI-1 mRNA expression was measured by real-time reverse transcriptase polymerase chain reactions. Insulin (0.001-10 micromol/l) increased accumulation of PAI-1 protein in the conditioned media and PAI-1 mRNA expression in HepG2 cells. A transient transfection assay of the human PAI-1 promoter-luciferase construct demonstrated that insulin increased PAI-1 promoter activity. Increased PAI-1 mRNA was attenuated significantly by U0126 and PD98059, specific inhibitors of mitogen-activated protein kinase. In contrast, GF109203X, an inhibitor of the protein kinase C pathway, and LY294002, an inhibitor of phosphatidylinositol 3-kinase, exerted no effects. Simvastatin (10 micromol/l), known to translocate membrane-bound sterol regulatory element-binding protein to nuclei, attenuated PAI-1 mRNA expression induced by insulin. It did not affect baseline PAI-1 mRNA expression. Intraperitoneal injection of insulin (0.1 IU/kg) increased concentrations of PAI-1 antigen in plasma in mice within 3 h, correlating with hepatic PAI-1 mRNA expression. Insulin-mediated augmented expression of PAI-1 may be amenable to suppression attributable to pleiotropic effects of statins, potentially diminishing cardiovascular risk in patients with insulin resistance.

S35225 is a direct inhibitor of Plasminogen Activator Inhibitor type-1 activity in the blood

The increased risk of thrombotic events associated with disease states such as diabetes and hypertension has been correlated with elevated circulating levels of Plasminogen Activator Inhibitor type-1 (PAI-1). In the present study we evaluate the benzothiophene derivative S35225 in comparison with two recently described inhibitors of PAI-1 activity Tiplaxtinin and WAY140312 on a panel of PAI-1 activity assays in vitro and in vivo.In a direct chromogenic assay, S35225 has an IC50 value of 44±0.9 μM similar to that of Tiplaxtinin (34±7 μM) and of WAY140312 (39±1 μM). In a clot lysis assay however, S35225 has a significantly lower IC50 value than Tiplaxtinin and WAY140312 (0.6±0.3 versus 22±5 and 16±2 μM respectively). Using a tPA capture assay to quantify active PAI-1 in rat or human plasma, neither WAY140312, nor Tiplaxtinin attained 50% inhibition of PAI-1 activity at the highest concentration tested (1 mM); S35225 has an IC50 value of 194±30 μM against active rat PAI-1 and 260±41 μM against active human PAI-1. The ability of the compounds to inhibit endogenous active PAI-1 in the rat following intravenous administration was also tested using the tPA capture assay. Only S35225 reduced circulating active PAI-1 levels in vivo (maximum inhibition of 76±5% at 10 mg/kg and 53±5% at 3 mg/kg). In contrast to Tiplaxtinin and WAY140312, S35225 is a direct inhibitor of PAI-1 activity in vitro in rat and human plasmas where vitronectin is constitutively present as well as in vivo in the blood after an intravenous administration in the rat.

Large Hepatitis Delta Antigen Modulates Transforming Growth Factor-β Signaling Cascades: Implication of Hepatitis Delta Virus–Induced Liver Fibrosis

Transforming growth factor-beta (TGF-beta) has been implicated in the pathogenesis of liver disease. TGF-beta is involved in liver regeneration and in the fibrotic and cirrhotic transformation with hepatitis viral infection. Hepatitis delta virus (HDV) infection causes fulminant hepatitis and liver cirrhosis. To elucidate the molecular mechanism of HDV pathogenesis, we examined the effects of HDV-encoded-only protein, the small hepatitis delta antigen (SHDAg), and the large hepatitis delta antigen (LHDAg), on TGF-beta- and c-Jun-induced signaling cascades. The effects of either SHDAg or LHDAg on TGF-beta- and c-Jun-induced signaling cascades in Huh7 and Cos7 cells were investigated by luciferase reporter gene assay, immunoprecipitation assay, electrophoretic mobility shift assay, Western blot analysis, and confocal microscopy analysis. The LHDAg, but not the SHDAg, potentiated TGF-beta- and c-Jun-induced signal activation, and the isoprenylation of LHDAg played a major role in signaling cascades. LHDAg synergistically activated hepatitis B virus X protein-mediated TGF-beta and AP-1 signaling cascades. In addition, LHDAg enhanced the protein expression level of TGF-beta-induced plasminogen activator inhibitor-1. LHDAg may induce liver fibrosis through the regulation of TGF-beta-induced signal transductions. This regulation of TGF-beta-mediated signaling is accomplished by the isoprenylation of LHDAg, which is a novel mechanism involved in HDV pathogenesis.

Cell Type-dependent Regulation of the Hypoxia-responsive Plasminogen Activator Inhibitor-1 Gene by Upstream Stimulatory Factor-2

Transcriptional regulation of the plasminogen activator inhibitor type-1 (PAI-1) gene is an important issue since PAI-1 plays a crucial role in various pathological conditions. The transcription factor USF-2 was shown to be a negative regulator for rat PAI-1 expression, and therefore it was the aim of this study to evaluate the role of USF-2 for human PAI-1 expression. We found in human hepatoma cells (HepG2) that USF-2 induced human PAI-1 expression via two classical E-boxes and the hypoxia-responsive element (HRE) within the promoter. Gel-shift analyses showed that E-box 4 and E-box 5 bound USFs, and although the HRE contributed to the USF-dependent effects, it did not bind them. By contrast, USF-2 inhibited PAI-1 promoter activity in primary rat hepatocytes suggesting that PAI-1 expression depends on either the promoter context or USF activity which might be cell type-specific. Cotransfection of human or rat PAI-1 promoter luciferase constructs with expression vectors for wild-type USF-2 or USF-2 mutants in human HepG2 and rat H4IIE cells as well as in primary rat hepatocytes revealed that the effects of USF on PAI-1 expression depend on the cell type rather than the promoter context and that the USF-specific region domain of USF accounts for the observed cell type-specific effects.

Altered Expression of TGF-β Receptors in Hepatocellular Carcinoma – Effects of a Constitutively Active TGF-β Type I Receptor Mutant

Background/Aims: Hepatocellular carcinomas (HCC) often show resistance to the effects of transforming growth factor-β (TGF-β). This study focuses on molecular mechanisms of this resistance to explore ways to overcome it. Methods: Transcription and protein expression of TGF-β type I and type II receptors (TGF-βRI/RII) were analyzed in clinical HCCs and the human hepatoma cell lines HuH-7 and HepG2. HuH-7 cells were transiently and stably transfected with a constitutively active TGF-βRI mutant (CA TGF-βRI). Resulting growth kinetics, integrin expression, invasiveness, TGF-β-mediated activation of human plasminogen activator inhibitor type-1 (PAI-1) promoter and Smad expression were determined. Results: In clinical HCCs, there was less TGF-βRII (6/10 cases) and more TGF-βRI (8/10 cases) protein expression detectable in tumor compared to adjacent liver tissue. In HuH-7 cells, TGF-βRII expression was likewise decreased. Cells transiently transfected with CA TGF-βRI exhibited strong TGF-β-related PAI-1 promoter activation. Stably transfected cells showed an attenuated response of the PAI-1 promoter, but increased Smad7 expression. Proliferation of stable clones was decreased. There was no change in integrin expression or invasiveness. Conclusions: Decreased TGF-βRII protein expression might cause TGF-β resistance in a subset of clinical HCCs. Stable transfection with CA TGF-βRI reverses this in HuH-7 cells without increasing invasiveness.

Latency and Substrate Binding Globally Reduce Solvent Accessibility of Plasminogen Activator Inhibitor Type 1 (PAI-1): AN ADAPTATION OF PAI-1 CONFORMER CRYSTAL STRUCTURES BY HYDROGEN-DEUTERIUM EXCHANGE

Plasminogen activator inhibitor type 1 (PAI-1) plays key regulatory roles in fibrinolysis, cell migration, and tissue remodeling. A regulatory protein without known catalytic activity, PAI-1 modulates plasminogen activators through protein-protein interactions. Although global conformational alterations that occur in PAI-1 determine its regulatory activity, comprehensive assessments of concurrent dynamic, structural, and functional alterations of this critical regulatory protein have not yet been clearly defined. X-ray crystallographic studies have described four distinct PAI-1 conformational states: active, latent, reactive center loop peptide-annealed (RCL-PA), and cleaved mutant. In this study, backbone amide hydrogen-deuterium exchange detected by mass spectrometry was used to characterize dynamic and structural alterations of human PAI-1 (hPAI-1) in relation to its function. hPAI-1 conformers were defined by surface mapping the solvent-accessible sites for strategic secondary structural components of the protein. We observed a global protection from solvent for a majority of peptides in the latent conformer relative to the active conformer. Significant differences were observed in the RCL, helix A, helix D, and sheet 1C, and these regions were markedly more dynamic or solvent-exposed in the active conformation. The RCL-PA form adopts an intermediate conformational state between the active and the latent conformers. Our results demonstrate that the most dynamic regions of PAI-1 (the RCL, helices D and A, and sheet 5A) are flexible in the transition toward latency. They also show that the dynamic surface structures of the active, latent, and peptide-annealed conformers of PAI-1 are underestimated by theoretical solvent accessibility calculations derived from crystallographic data.

Tissue- and agonist-specific regulation of human and murine plasminogen activator inhibitor-1 promoters in transgenic mice

Numerous studies have described regulatory factors and sequences that control transcriptional responses in vitro. However, there is a paucity of information on the qualitative and quantitative regulation of heterologous promoters using transgenic strategies. In order to investigate the physiological regulation of human plasminogen activator inhibitor type-1 (hPAI-1) expression in vivo compared to murine PAI-1 (mPAI-1) and to test the physiological relevance of regulatory mechanisms described in vitro, we generated transgenic mice expressing enhanced green fluorescent protein (EGFP) driven by the proximal -2.9 kb of the hPAI-1 promoter. Transgenic animals were treated with Ang II, TGF-beta1 and lipopolysaccharide (LPS) to compare the relative activation of the human and murine PAI-1 promoters. Ang II increased EGFP expression most effectively in brain, kidney and spleen, while mPAI-1 expression was quantitatively enhanced most prominently in heart and spleen. TGF-beta1 failed to induce activation of the hPAI-1 promoter but potently stimulated mPAI-1 in kidney and spleen. LPS administration triggered robust expression of mPAI-1 in liver, kidney, pancreas, spleen and lung, while EGFP was induced only modestly in heart and kidney. These results indicate that the transcriptional response of the endogenous mPAI-1 promoter varies widely in terms of location and magnitude of response to specific stimuli. Moreover, the physiological regulation of PAI-1 expression likely involves a complex interaction of transcription factors and DNA sequences that are not adequately replicated by in vitro functional studies focused on the proximal -2.9 kb promoter.

A mutant, noninhibitory plasminogen activator inhibitor type 1 decreases matrix accumulation in experimental glomerulonephritis

In fibrotic renal disease, elevated TGF-β and angiotensin II lead to increased plasminogen activator inhibitor type 1 (PAI-1). PAI-1 appears to reduce glomerular mesangial matrix turnover by inhibiting plasminogen activators, thereby decreasing plasmin generation and plasmin-mediated matrix degradation. We hypothesized that therapy with a mutant human PAI-1 (PAI-1R) that binds to matrix vitronectin but does not inhibit plasminogen activators, would enhance plasmin generation, increase matrix turnover, and decrease matrix accumulation in experimental glomerulonephritis. Three experimental groups included normal, untreated disease control, and PAI-1R–treated nephritic rats. Plasmin generation by isolated day 3 glomeruli was dramatically decreased by 69%, a decrease that was reversed 43% (P < 0.02) by in vivo PAI-1R treatment. At day 6, animals treated with PAI-1R showed significant reductions in proteinuria (48%, P < 0.02), glomerular staining for periodic acid–Schiff positive material (33%, P < 0.02), collagen I (28%, P < 0.01), collagen III (34%, P < 0.01), fibronectin (48%, P < 0.01), and laminin (41%, P < 0.01), and in collagen I (P < 0.01) and fibronectin mRNA levels (P < 0.02). Treatment did not alter overexpression of TGF-β1 and PAI-1 mRNAs, although TGF-β1 protein was significantly reduced. These observations strongly support our hypothesis that PAI-1R reduces glomerulosclerosis by competing with endogenous PAI-1, restoring plasmin generation, inhibiting inflammatory cell infiltration, decreasing local TGF-β1 concentration, and reducing matrix accumulation.

A mutant, noninhibitory plasminogen activator inhibitor type 1 decreases matrix accumulation in experimental glomerulonephritis.

In fibrotic renal disease, elevated TGF-beta and angiotensin II lead to increased plasminogen activator inhibitor type 1 (PAI-1). PAI-1 appears to reduce glomerular mesangial matrix turnover by inhibiting plasminogen activators, thereby decreasing plasmin generation and plasmin-mediated matrix degradation. We hypothesized that therapy with a mutant human PAI-1 (PAI-1R) that binds to matrix vitronectin but does not inhibit plasminogen activators, would enhance plasmin generation, increase matrix turnover, and decrease matrix accumulation in experimental glomerulonephritis. Three experimental groups included normal, untreated disease control, and PAI-1R-treated nephritic rats. Plasmin generation by isolated day 3 glomeruli was dramatically decreased by 69%, a decrease that was reversed 43% (P < 0.02) by in vivo PAI-1R treatment. At day 6, animals treated with PAI-1R showed significant reductions in proteinuria (48%, P < 0.02), glomerular staining for periodic acid-Schiff positive material (33%, P < 0.02), collagen I (28%, P < 0.01), collagen III (34%, P < 0.01), fibronectin (48%, P < 0.01), and laminin (41%, P < 0.01), and in collagen I (P < 0.01) and fibronectin mRNA levels (P < 0.02). Treatment did not alter overexpression of TGF-beta1 and PAI-1 mRNAs, although TGF-beta1 protein was significantly reduced. These observations strongly support our hypothesis that PAI-1R reduces glomerulosclerosis by competing with endogenous PAI-1, restoring plasmin generation, inhibiting inflammatory cell infiltration, decreasing local TGF-beta1 concentration, and reducing matrix accumulation.

Antisense targeting of c-fos transcripts inhibits serum- and TGF-beta 1-stimulated PAI-1 gene expression and directed motility in renal epithelial cells.

Plasminogen activator inhibitor type-1 (PAI-1), the major regulator of pericellular plasmin generation, and the c-FOS transcription factor are expressed by migrating cells in response to monolayer wounding. Induced c-fos and PAI-1 transcripts were evident within 30 min and 2 h, respectively, of scrape injury to confluent, growth-arrested, cultures of NRK epithelial cells. Since c-FOS/AP-1 DNA-binding activity modulates both basal and inducible modes of PAI-1 gene control, and AP-1 motif binding factors were present in quiescent as well as stimulated NRK cells, a model of directionally regulated cell movement (migration into scrape-denuded "wounds") was used to assess the consequences of c-fos transcript targeting on PAI-1 expression and cell motility. This in vitro model of epithelial injury closely approximated in vivo wound repair with regard to the spatial and temporal emergence of cohorts of cells involved in migration, proliferation, and PAI-1 expression. Stable cell lines (NRKsof) were generated by transfection of parental NRK cells with a c-fos antisense expression vector. Serum-inducible c-fos transcripts and PAI-1 protein levels were significantly attenuated in NRKsof transfectants relative to parental controls or cells transfected with a neo(R) vector without the sof insert. NRKsof cells had a markedly impaired ability to repair scrape-generated monolayer wounds under basal, serum-stimulated, or TGF-beta 1-supplemented culture conditions. Since injury closure and PAI-1 induction were attenuated in c-fos antisense cells, it was important to clarify the role of specific AP-1 sites in serum-mediated PAI-1 transcription. PAI-1 "promoter"-driven CAT reporter expression was assessed within the real time of serum-stimulated PAI-1 induction. A segment of the PAI-1 promoter corresponding to nucleotides -533 to -764 upstream of the transcription start site functioned as a prominent serum-responsive region (SSR). The 9-fold increase in CAT mRNA levels attained with the -533 to -764 bp PAI-1 SRR ligated to a minimal PAI-1 promoter (i.e., 162 bp of 5' flanking sequence containing the basal transcription complex) closely approximated the serum-induced transcriptional activity of a fully responsive (1,230 bp) PAI-1 promoter construct as well as the endogenous PAI-1 gene. AP-1-like, CTF/NF-1-like, and AP-2 sites were identified in the SRR. The SRR AP-1 motif was homologous to the sequence TGACACA that mapped between nucleotides -740 and -703 in the human PAI-1 gene, a region essential for growth factor-inducible PAI-1 transcription. While the functionality of this AP-1 site in wound-regulated PAI-1 synthesis remains to be determined, antisense c-fos transcripts effectively attenuated PAI-1 induction and basal as well as growth factor-stimulated cell locomotion, indicating that expression of both the PAI-1 and c-fos genes is necessary for wound-initiated NRK cell migration.

Identification and Localization of a Fatty Acid Response Region in the Human Plasminogen Activator Inhibitor-1 Gene

The increased expression of plasminogen activator inhibitor type-1 (PAI-1) is associated with increased concentrations of fatty acids in blood and may accelerate atherogenesis in diabetes. The present study was designed to define mechanisms by which nonesterified (free) fatty acids (FFAs) augment the expression of PAI-1. FFAs increased PAI-1 protein and mRNA expression by HepG2 cells. To identify potential regulatory elements, we constructed chimeric genes by fusing 1313, 853, 610, or 328 bp of human PAI-1 5'-flanking DNA to a luciferase reporter (PAI-LUC). A 2-fold increase in luciferase activity was seen when cells were transfected with PAI-LUC 1313, 863, or 610 and exposed to FFAs. No response to FFAs was seen with PAI-LUC 328 and after deletion of a 72-bp (-599 to -528) fragment from PAI-LUC 1313. This 72-bp fragment conferred FFA responsiveness to a different (simian virus 40) promoter. Two footprinted regions were demonstrated by DNase I analysis. Gel mobility shift assays indicated specific binding of extracted proteins to an FFA response element: 5'-TG(G/C)(1-2)CTG-3'. This sequence is repeated 4 times and is similar to an Sp1-binding site. Sp1 consensus oligonucleotides inhibited binding of extracted proteins to the regulatory elements. Accordingly, FFA-induced increased expression of PAI-1 in HepG2 cells is mediated by the binding of a transcription factor or factors to the repeated fatty acid response element, 5'-TG(G/C)(1-2)CTG-3', that is highly homologous to an Sp1-binding site.

Sp1-like activity mediates angiotensin-II-induced plasminogen-activator inhibitor type-1 (PAI-1) gene expression in mesangial cells

Angiotensin II (Ang II) up-regulates plasminogen-activator inhibitor type-1 (PAI-1) expression in mesangial cells to enhance extracellular matrix formation. The proximal promoter region (bp -87 to -45) of the human PAI-1 gene contains several potent binding sites for transcription factors [two phorbol-ester-response-element (TRE)-like sequences; D-box (-82 to -76) and P-box (-61 to 54), and one Sp1 binding site-like sequence, Sp1-box 1 (-72 to -67)]. We studied this region to determine the transcription factor(s) that mediates Ang-II-induced transcriptional activation of the PAI-1 gene. Various double-stranded decoy oligodeoxynucleotides (ODNs) corresponding to various sequences in the proximal promoter region were transfected to mesangial cells to examine the effects on Ang-II-induced PAI-1 mRNA expression. Transfection with the full-length decoy (bp -87 to -45, D-P-ODN) markedly attenuated Ang-II-induced PAI-1 mRNA expression by up to 70%. Transfection with D-ODN (-87 to -71) and P-ODN (-66 to -45), which correspond to each of the two TRE-like sequences, did not attenuate the expression. Gel-shift assays using nuclear extracts prepared from Ang-II-treated mesangial cells and D-P-ODN showed three specific complexes. The major complex was supershifted by anti-Sp1 antibody. The methylation-interference experiment demonstrated that human recombinant Sp1 bound to the so-called GT box (TGGGTGGGGCT, -78 to -69), which contains the Sp1-box 1. The complex that migrated with anti-Sp1 antibody was enhanced in the cells treated with Ang II. Further, D-Sp1-ODN (-85 to -63) containing the GT box attenuated up-regulation of PAI-1 mRNA expression induced by Ang II to a level (68±9% inhibition) comparable to D-P-ODN, whereas ODN with four mutations in the GT box had no effect. Our findings suggest that binding of Sp1 or an Sp1-like transcription factor to the GT box in the PAI-1 promoter up-regulates PAI-1 gene transcription in mesangial cells stimulated with Ang II. This transcription-factor binding site may be targeted to control Ang-II-dependent extracellular matrix formation by mesangial cells.