Plasminogen Activator Inhibitor-1 Transcription Research Articles

Small leucine zipper protein negatively regulates liver fibrosis by suppressing the expression of plasminogen activator inhibitor-1

Liver fibrosis, which is caused by viral infection, toxic exposure, and autoimmune diseases, is a chronic liver disease. Plasminogen activator inhibitor-1 (PAI-1) is a serine protease inhibitor of tissue-type plasminogen activator (tPA) and urokinase plasminogen activator, which convert plasminogen into plasmin. Therefore, PAI-1 suppresses fibrinolysis by blocking plasmin synthesis and is involved in liver fibrosis via extracellular matrix deposition. Small leucine zipper protein (sLZIP) acts as a transcription factor and plays critical roles in many cellular processes. However, the role of sLZIP in liver fibrosis remains unclear. In this study, we investigated the role of sLZIP in regulating PAI-1 transcription and liver fibrosis. sLZIP knockdown enhanced the expression of PAI-1 at the mRNA and protein levels. sLZIP knockdown also increased PAI-1 secretion and suppressed blood clot lysis by blocking tPA activity. Moreover, conditioned medium derived from sLZIP knockdown cells downregulated the expression of matrix metalloprotease (MMP)-2 and MMP-9 in the presence of tPA in hepatic stellate cells (HSCs). Liver-specific sLZIP knockout mice showed deteriorated liver fibrosis compared to control mice in a bile duct ligation-induced fibrosis model. These findings demonstrate that sLZIP functions as a negative regulator of liver fibrosis by suppressing PAI-1 transcription and HSC activation.

Chuanxiong Rhizoma extracts prevent cholestatic liver injury by targeting H3K9ac-mediated and cholangiocyte-derived secretory protein PAI-1 and FN.

Chuanxiong Rhizoma (CX, the dried rhizome of Ligusticum wallichii Franch.), a well-known traditional Chinese medicine, is clinically used for treating cardiovascular, cerebrovascular and hepatobiliary diseases. Cholestatic liver damage is one of the chronic liver diseases with limited effective therapeutic strategies. Currently, little is known about the mechanism links between CX-induced anti-cholestatic action and intercellular communication between cholangiocytes and hepatic stellate cells (HSCs). The study aimed to evaluate the hepatoprotective activity of different CX extracts including the aqueous, alkaloid, phenolic acid and phthalide extracts of CX (CXAE, CXAL, CXPA and CXPHL) and investigate the intercellular communication-related mechanisms by which the most effective extracts work on cholestatic liver injury. The active compounds of different CX extracts were identified by UPLC-MS/MS. A cholestatic liver injury mouse model induced by bile duct ligation (BDL), and transforming growth factor-β (TGF-β)-treated human intrahepatic biliary epithelial cholangiocytes (HIBECs) and HSC cell line (LX-2 cells) were used for in vivo and in vitro studies. Histological and other biological techniques were also applied. The results indicated that CXAE, CXAL and CXPHL significantly reduced ductular reaction (DR) and improved liver fibrosis in the BDL mice. Meanwhile, both CXAE and CXPHL suppressed DR in injured HIBECs and reduced collagen contraction force and the expression of fibrosis biomarkers in LX-2 cells treated with TGF-β. CXPHL suppressed the transcription and transfer of plasminogen activator inhibitor-1 (PAI-1) and fibronectin (FN) from the 'DR-like' cholangiocytes to activated HSCs. Mechanistically, the inhibition of PAI-1 and FN by CXPHL was attributed to the untight combination of the acetyltransferase KAT2A and SMAD3, followdd by the suppression of histone 3 lysine 9 acetylation (H3K9ac)-mediated transcription in cholangiocytes. In conclusion, CXPHL exerts stronger anti-cholestatic activity in vivo and in vitro than other CX extracts, and its protective effect on the intracellular communication between cholangiocytes and HSCs is achieved by reducing KAT2A/H3K9ac-mediated transcription and release of PAI-1 and FN.

PAI-1: A Major Player in the Vascular Dysfunction in Obstructive Sleep Apnea?

Obstructive sleep apnea is a chronic and prevalent condition that is associated with endothelial dysfunction, atherosclerosis, and imposes excess overall cardiovascular risk and mortality. Despite its high prevalence and the susceptibility of CVD patients to OSA-mediated stressors, OSA is still under-recognized and untreated in cardiovascular practice. Moreover, conventional OSA treatments have yielded either controversial or disappointing results in terms of protection against CVD, prompting the need for the identification of additional mechanisms and associated adjuvant therapies. Plasminogen activator inhibitor-1 (PAI-1), the primary inhibitor of tissue-type plasminogen activator (tPA) and urinary-type plasminogen activator (uPA), is a key regulator of fibrinolysis and cell migration. Indeed, elevated PAI-1 expression is associated with major cardiovascular adverse events that have been attributed to its antifibrinolytic activity. However, extensive evidence indicates that PAI-1 can induce endothelial dysfunction and atherosclerosis through complex interactions within the vasculature in an antifibrinolytic-independent matter. Elevated PAI-1 levels have been reported in OSA patients. However, the impact of PAI-1 on OSA-induced CVD has not been addressed to date. Here, we provide a comprehensive review on the mechanisms by which OSA and its most detrimental perturbation, intermittent hypoxia (IH), can enhance the transcription of PAI-1. We also propose causal pathways by which PAI-1 can promote atherosclerosis in OSA, thereby identifying PAI-1 as a potential therapeutic target in OSA-induced CVD.

Inhibition of plasminogen activator inhibitor-1 attenuates against intestinal fibrosis in mice.

Background/AimsIntestinal fibrosis is a major complication of Crohn’s disease (CD). The profibrotic protein transforming growth factor-β (TGF-β) has been considered to be critical for the induction of the fibrotic program. TGF-β has the ability to induce not only the expression of extracellular matrix (ECM) including collagen, but also the production of plasminogen activator inhibitor-1 (PAI-1) that prevents enzymatic degradation of the ECM during the onset of fibrotic diseases. However, the significance of PAI-1 in the developing intestinal fibrosis has not been fully understood. In the present study, we examined the actual expression of PAI-1 in fibrotic legion of intestinal inflammation and its correlation with the abnormal ECM deposition.MethodsChronic intestinal inflammation was induced in BALB/c mice using 8 repeated intrarectal injections of 2,4,6-trinitrobenzene sulfonic acid (TNBS). TM5275, a PAI-1 inhibitor, was orally administered as a carboxymethyl cellulose suspension each day for 2 weeks after the sixth TNBS injection.ResultsUsing a publicly available dataset (accession number, GSE75214) and TNBS-treated mice, we observed increases in PAI-1 transcripts at active fibrotic lesions in both patients with CD and mice with chronic intestinal inflammation. Oral administration of TM5275 immediately after the onset of intestinal fibrosis upregulated MMP-9 (matrix metalloproteinase 9) and decreased collagen accumulation, resulting in attenuation of the fibrogenesis in TNBS-treated mice.ConclusionsPAI-1-mediated fibrinolytic system facilitates collagen degradation suppression. Hence, PAI-1 inhibitor could be applied as an anti-fibrotic drug in CD treatment.

The TGF-β1/p53/PAI-1 Signaling Axis in Vascular Senescence: Role of Caveolin-1

Stress-induced premature cellular senescence is a significant factor in the onset of age-dependent disease in the cardiovascular system. Plasminogen activator inhibitor-1 (PAI-1), a major TGF-β1/p53 target gene and negative regulator of the plasmin-based pericellular proteolytic cascade, is elevated in arterial plaques, vessel fibrosis, arteriosclerosis, and thrombosis, correlating with increased tissue TGF-β1 levels. Additionally, PAI-1 is necessary and sufficient for the induction of p53-dependent replicative senescence. The mechanism of PAI-1 transcription in senescent cells appears to be dependent on caveolin-1 signaling. Src kinases are upstream effectors of both FAK and caveolin-1 activation as FAKY577,Y861 and caveolin-1Y14 phosphorylation are not detected in TGF-β1-stimulated src family kinase (pp60c-src, Yes, Fyn) triple-deficient (SYF−/−/−) cells. However, restoration of pp60c-src expression in SYF-null cells rescued both caveolin-1Y14 phosphorylation and PAI-1 induction in response to TGF-β1. Furthermore, TGF-β1-initiated Src phosphorylation of caveolin-1Y14 is critical in Rho-ROCK-mediated suppression of the SMAD phosphatase PPM1A maintaining and, accordingly, SMAD2/3-dependent transcription of the PAI-1 gene. Importantly, TGF-β1 failed to induce PAI-1 expression in caveolin-1-null cells, correlating with reductions in both Rho-GTP loading and SMAD2/3 phosphorylation. These findings implicate caveolin-1 in expression controls on specific TGF-β1/p53 responsive growth arrest genes. Indeed, up-regulation of caveolin-1 appears to stall cells in G0/G1 via activation of the p53/p21 cell cycle arrest pathway and restoration of caveolin-1 in caveolin-1-deficient cells rescues TGF-β1 inducibility of the PAI-1 gene. Although the mechanism is unclear, caveolin-1 inhibits p53/MDM2 complex formation resulting in p53 stabilization, induction of p53-target cell cycle arrest genes (including PAI-1), and entrance into premature senescence while stimulating the ATM→p53→p21 pathway. Identification of molecular events underlying senescence-associated PAI-1 expression in response to TGF-β1/src kinase/p53 signaling may provide novel targets for the therapy of cardiovascular disease.

Compound Astragalus and Salvia miltiorrhiza extract suppresses hepatocellular carcinoma progression by inhibiting fibrosis and PAI-1 mRNA transcription

Ethnopharmacological relevanceAstragalus membranaceus and Salvia miltiorrhiza have been used for centuries in China to treat liver diseases. Previous studies have shown that these herbs and their extracts inhibit the development of liver fibrosis and the proliferation and invasion of human hepatoma HepG2 cells. Further study of their pharmacological effects on hepatocellular carcinoma (HCC) is needed. To investigate the effects of Compound Astragalus and Salvia miltiorrhiza Extract (CASE) on diethylinitrosamine (DEN)-induced hepatocarcinogenesis in rats. Materials and methodsMale rats were divided into five groups, with the first group serving as normal control, the second group receiving 0.2% DEN solution five times a week for 14 weeks, and the third to fifth group receiving the same DEN as in the second group together with CASE at the doses of 60, 120, and 240mg/kg per day for 16 weeks, respectively. Hepatoma incidence, serum enzymes levels, degree of fibrosis and hydroxyproline content were evaluated and compared across the five groups to determine CASE's suppression of fibrosis and HCC progression. In addition, an in vitro experiment using HepG2 cells was conduct to verify CASE's effect on the transcription of plasminogen activator inhibitor-1 (PAI-1) mRNA. ResultsCASE treatment significantly reduced the incidence and multiplicity of DEN-induced HCC development in a dose-dependent manner. It significantly suppressed the elevation of alanine transaminase, aspartate aminotransferase, gamma-glutamyl transferase, alkaline phosphatase, hyaluronic acid, direct bilirubin and total bilirubin, and significantly lessened the depression of serum total protein in DEN-induced HCC rats. CASE treatment also significantly suppressed the elevated expression of GST-P and α-SMA. The in vitro experiment confirmed that CASE inhibits the transcription of PAI-1 mRNA in HepG2 cells induced by TGF-β1 in a dose-dependent manner. ConclusionsCASE suppresses DEN-induced hepatocarcinogenesis by inhibiting fibrosis and PAI-1 mRNA transcription, suggesting its potential clinical application in preventing and treating human HCC.

Preservation of peritoneal fibrinolysis owing to decreased transcription of plasminogen activator inhibitor-1 in peritoneal mesothelial cells suppresses postoperative adhesion formation in laparoscopic surgery

Postoperative adhesion formation is regulated by peritoneal fibrinolysis, which is determined by tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1). This study compared peritoneal fibrinolysis and adhesion formation after laparoscopic surgery (LAP) and open surgery (OP). We divided 154 male rats into 3 groups after cecal cauterization: Control, no treatment; LAP, CO2 pneumoperitoneum at 5 mmHg for 60 minutes; and OP, laparotomy for 60 minutes. Adhesions were quantified at day 7. The activity and mRNA level of tPA and PAI-1 were determined by enzyme-linked immunosorbent assay in plasma and peritoneal lavage and by real-time polymerase chain reaction in peritoneal mesothelial cells from omentum. We also examined peritoneal fibrinolysis in human gastric cancer patients treated with LAP (n = 14) or OP (n = 10). In the animal study, adhesion scores, PAI-1 activity in peritoneal lavage fluid, and PAI-1 mRNA levels in peritoneal mesothelium were significantly greater in the OP group than the control and LAP groups. In the human study, postoperative PAI-1 mRNA levels were significantly greater in the OP group than the LAP group. Additionally, PAI-1 mRNA levels and subsequent adhesion formation were induced by prolonged operative time in the OP group, but not the LAP group. Preservation of peritoneal fibrinolysis owing to decreased PAI-1 expression at the transcriptional level in peritoneal mesothelial cells is associated with suppression of postoperative adhesion formation in LAP. PAI-1 mRNA levels and subsequent adhesion formation were not induced by prolonged operative time in LAP. These results highlight the less invasiveness nature of LAP.

Impact of the -675 4G/5G polymorphism of the plasminogen activator inhibitor-1 gene on childhood IgA nephropathy

Plasminogen activator inhibitor-1 (PAI-1) is an important regulator of the fibrinolytic pathway and extracellular matrix (ECM) turnover. The -675 4G/5G polymorphism in the PAI-1 promoter is associated with altered PAI-1 transcription, suggesting that this polymorphism may be a candidate risk factor for diseases characterized by ECM accumulation, such as immunoglobulin A nephropathy (IgAN) and mesangial proliferative glomerulonephritis (MesPGN). We genotyped childhood patients with biopsy-confirmed IgAN (n=111) and MesPGN (n=47), and healthy control subjects (n=230) for the -675 4G/5G PAI-1 polymorphism by polymerase chain reaction-restriction fragment length polymorphism methods. The distribution of the 4G/4G (27.9%), 4G/5G (45.1%) and 5G/5G (27.0%) genotypes in IgAN patients was significantly different from the healthy controls (32.2, 54.3 and 13.5%, respectively) (p=0.0092). There was no significant difference in the genotype distributions of the 4G/5G polymorphism between MesPGN patients and the healthy controls. Regarding the impact of the polymorphism on IgAN, the 4G/4G genotype was markedly increased in patients with proteinuria (≥1,000 mg/day) and/or hypertension when compared to patients without proteinuria and hypertension (OR=5.23, 95% CI 1.34-20.38, P=0.0183). These findings indicate that the PAI-1 gene polymorphism may affect the susceptibility of childhood IgAN.

Redox-Induced Src Kinase and Caveolin-1 Signaling in TGF-β1-Initiated SMAD2/3 Activation and PAI-1 Expression

BackgroundPlasminogen activator inhibitor-1 (PAI-1), a major regulator of the plasmin-based pericellular proteolytic cascade, is significantly increased in human arterial plaques contributing to vessel fibrosis, arteriosclerosis and thrombosis, particularly in the context of elevated tissue TGF-β1. Identification of molecular events underlying to PAI-1 induction in response to TGF-β1 may yield novel targets for the therapy of cardiovascular disease.Principal FindingsReactive oxygen species are generated within 5 minutes after addition of TGF-β1 to quiescent vascular smooth muscle cells (VSMCs) resulting in pp60c-src activation and PAI-1 expression. TGF-β1-stimulated Src kinase signaling sustained the duration (but not the initiation) of SMAD3 phosphorylation in VSMC by reducing the levels of PPM1A, a recently identified C-terminal SMAD2/3 phosphatase, thereby maintaining SMAD2/3 in an active state with retention of PAI-1 transcription. The markedly increased PPM1A levels in triple Src kinase (c-Src, Yes, Fyn)-null fibroblasts are consistent with reductions in both SMAD3 phosphorylation and PAI-1 expression in response to TGF-β1 compared to wild-type cells. Activation of the Rho-ROCK pathway was mediated by Src kinases and required for PAI-1 induction in TGF-β1-stimulated VSMCs. Inhibition of Rho-ROCK signaling blocked the TGF-β1-mediated decrease in nuclear PPM1A content and effectively attenuated PAI-1 expression. TGF-β1-induced PAI-1 expression was undetectable in caveolin-1-null cells, correlating with the reduced Rho-GTP loading and SMAD2/3 phosphorylation evident in TGF-β1-treated caveolin-1-deficient cells relative to their wild-type counterparts. Src kinases, moreover, were critical upstream effectors of caveolin-1Y14 phosphoryation and initiation of downstream signaling.ConclusionsTGF-β1-initiated Src-dependent caveolin-1Y14 phosphorylation is a critical event in Rho-ROCK-mediated suppression of nuclear PPM1A levels maintaining, thereby, SMAD2/3-dependent transcription of the PAI-1 gene.

Pyruvate promotes tumor angiogenesis through HIF-1-dependent PAI-1 expression

Cancer cells usually obtain energy from a high rate of glycolysis rather than oxidative phosphorylation under normoxia as well as hypoxia. Under these circumstances, pyruvate, the end-product of glycolysis, accumulates in cancer cells. We have previously reported that pyruvate activates endothelial cells and induces angiogenesis. Here, we examined the angiogenic activity of pyruvate in tumor cells. Plasminogen activator inhibitor-1 (PAI-1), the gene most upregulated by pyruvate, showed a pro-angiogenic activity, which was abolished by a PAI-1 neutralizing antibody. Moreover, stabilization of hypoxia-inducible factor-1α (HIF-1α) by pyruvate was required for induction of PAI-1 transcription through direct binding to hypoxia response element-2 (HRE-2) on the promoter. These results suggest that pyruvate can activate the angiogenic activity of cancer cells under normoxia and that PAI-1 may act as a pro-angiogenic factor in pyruvate-induced angiogenesis.

Leptin upregulates the expression of plasminogen activator inhibitor-1 in human vascular endothelial cells

A prothrombotic state in obesity may be partially responsible for the higher incidence of atherosclerotic complications. However the factors responsible for this prothrombotic state, linked with high levels of plasminogen activator inhibitor-1 (PAI-1), are not fully known. Leptin is elevated in obesity and studies have shown a positive correlation between leptin and PAI-1 levels in human subjects, along with a negative correlation with tissue-type plasminogen activator (tPA). We tested the hypothesis that leptin induces PAI-1 and inhibits tPA expression using human coronary artery endothelial cells (HCAEC) in culture as these cells play an important role in atherosclerosis. We demonstrate that leptin induces the transcription and translation of PAI-1 in HCAEC. The leptin dependent upregulation of PAI-1 mRNA and protein was comparable to insulin-induced PAI-1 expression. We show leptin concentration (0–150 ng/ml) dependent increases in PAI-1 mRNA and protein after 6 and 12 h of leptin administration, respectively. Increased intracellular PAI-1 expression correlates with increased PAI-1 activity in conditioned media and inhibition of specific ERK1/2 pathway by treatment with PD98059 (20–40 μM) inhibits leptin dependent PAI-1 expression. However no changes in tPA expression were seen with time or increasing concentrations of leptin. Also leptin treatment did not alter total tPA concentration or tPA activity in conditioned media. In conclusion, our study shows that leptin upregulates the expression of PAI-1 in vascular endothelial cells via activation of ERK1/2 but does not regulate tPA expression. These studies demonstrate a novel mechanism for the prothrombotic role of leptin in development of atherosclerosis.

Nox4 mediates the expression of plasminogen activator inhibitor-1 via p38 MAPK pathway in cultured human endothelial cells

Introduction Plasminogen Activator Inhibitor-1 (PAI-1) is the most potent endogenous inhibitor of fibrinolysis which is implicated in the pathogenesis of myocardial infarction and metabolic syndrome. The formation of reactive oxygen species (ROS) plays an important role in the pathology of vascular disorders and has been shown to increase PAI-1 expression by endothelial cells. Growing evidence indicates that NADPH oxidase and in particular the constitutively active Nox4-p22 phox complexes are major sources of ROS in endothelial cells. The aim of the present study was to characterize the role of NADPH oxidase and in particular Nox4 in the regulation of PAI-1 expression in cultured Human Umbilical Venous Endothelial Cells (HUVECs). Methods and Results N-acetylcysteine (NAC, scavenger of ROS), diphenylene iodonium chloride (DPI, inhibitor of flavoproteins), M40403 (superoxyde dismutase mimic) and S17834 (inhibitor of NADPH oxidase) inhibited PAI-1 release and promoter activity in HUVECs. Specific knock down of Nox4 mRNA by siRNA caused a decrease in ROS production and NADPH oxidase activity. Moreover, Nox4 silencing decreased PAI-1 expression, release and activity as well as p38 MAPK pathways and NFκB activation. These signalling pathways are also involved in PAI-1 release. Conclusions The NADPH oxidase inhibitors DPI and S 17834 as well as Nox4 silencing decreased PAI-1 synthesis in human cultured endothelial cells demonstrating the involvement of the constitutively active Nox4-containing NADPH oxidase in ROS-mediated PAI-1 transcription via p38 MAPK pathways. NADPH oxidase targeting with inhibitors such as S17834 could be an interesting strategy to decrease both oxidative stress and PAI-1 synthesis.

Cox-2 inactivates Smad signaling and enhances EMT stimulated by TGF- through a PGE2-dependent mechanisms

Although it is well established that mammary tumorigenesis converts transforming growth factor-beta (TGF-beta) from a tumor suppressor to a tumor promoter, the molecular, cellular and microenvironmental mechanisms underlying the dichotomous nature of TGF-beta in mammary epithelial cells (MECs) remains to be determined definitively. Aberrant upregulation of the inducible cyclooxygenase, Cox-2, occurs frequently in breast cancers and is associated with increasing disease severity and the acquisition of metastasis; however, the impact of Cox-2 expression on normal and malignant MEC response to TGF-beta remains unknown. We show here that TGF-beta induced Cox-2 expression in normal MECs during their acquisition of an epithelial-mesenchymal transition (EMT) phenotype. Moreover, stable Cox-2 expression in normal MECs stimulated their invasion, EMT and anchorage-independent growth and inhibited their activation of Smad2/3 by TGF-beta. Conversely, antagonizing TGF-beta signaling in malignant, metastatic MECs significantly reduced their expression of Cox-2 as well as enhanced their activation of Smad2/3 by TGF-beta. Along these lines, elevated Cox-2 expression elicited prostaglandin E(2) (PGE(2)) production and the autocrine activation of EP receptors, which antagonized Smad2/3 signaling in normal and malignant MECs. Importantly, rendering normal and malignant MECs Cox-2 deficient inhibited their production of PGE(2) and acquisition of an EMT morphology as well as potentiated their nuclear accumulation of Smad2/3 and transcription of plasminogen activator inhibitor-1 and p15 messenger RNA. Collectively, our findings establish Cox-2 as a novel antagonist of Smad2/3 signaling in normal and malignant MECs; they also suggest that chemotherapeutic targeting of Cox-2 may offer new inroads in restoring the tumor-suppressing activities of TGF-beta in malignant, metastatic breast cancers.

Transcriptional regulation of plasminogen activator inhibitor-1 in vascular endothelial cells induced by oxidized very low density lipoproteins

Elevated levels of triglycerides and very low density lipoproteins (VLDL) are biochemical markers of metabolic syndrome and diabetes. VLDL from hypertriglyceridemic or diabetic patients increased the generation of plasminogen activator inhibitor-1 (PAI-1) from cultured vascular endothelial cells (EC). Susceptibility of VLDL to peroxidation was increased in diabetic patients. Heat shock factor-1 (HSF1) is implicated in the transcriptional regulation of PAI-1 induced by glycated low density lipoprotein (LDL). The present study examined the effects of oxidized VLDL (oxVLDL) on the expression of PAI-1 and HSF1 in cultured human EC and mouse embryo fibroblasts (MEF). OxVLDL modified by copper or iron ions increased the expression of PAI-1 and HSF1 in EC compared to VLDL or LDL. Butylated hydroxytulene inhibited oxVLDL-induced expression of PAI-1 and HSF1 in EC. OxVLDL increased the binding of HSF1 to PAI-1 promoter. Short interference RNA for HSF1 inhibited oxVLDL-induced PAI-1 expression in EC. OxVLDL stimulated the expression of PAI-1 from MEF of wild-type mice, but failed to increase PAI-1 expression in MEF of HSF1-knockout mice. The results indicate that oxVLDL increased PAI-1 expression, and HSF1 mediates the transcription of PAI-1 in cultured vascular EC or fibroblasts.

TGF-β1-induced plasminogen activator inhibitor-1 expression in vascular smooth muscle cells requires pp60c-src/EGFRY845 and Rho/ROCK signaling

TGF-β1 and its target gene encoding plasminogen activator inhibitor-1 (PAI-1) are major causative factors in the pathology of tissue fibrosis and vascular disease. The increasing complexity of TGF-β1 action in the cardiovascular system requires analysis of specific TGF-β1-initiated signaling events that impact PAI-1 transcriptional regulation in a physiologically-relevant cell system. TGF-β1-induced PAI-1 expression in both primary cultures and in an established line (R22) of vascular smooth muscle cells (VSMC) was completely blocked by inhibition of epidermal growth factor receptor (EGFR) activity or adenoviral delivery of a kinase-dead EGFRK721A construct. TGF-β1-stimulated PAI-1 expression, moreover, was preceded by EGFR phosphorylation on Y845 (a src kinase target residue) and required pp60c-src activity. Infection of VSMC with an adenovirus encoding the EGFRY845F mutant or transfection with a dominant-negative pp60c-src (DN-Src) expression vector effectively decreased TGF-β1-stimulated, but not PDGF-induced, PAI-1 expression implicating the pp60c-src phosphorylation site EGFRY845 in the inductive response. Consistent with these findings, TGF-β1 failed to induce PAI-1 synthesis in src kinase-deficient (SYF−/−/−) fibroblasts and reexpression of a wild-type pp60c-src construct in SYF−/−/− cells rescued the PAI-1 response to TGF-β1. TGF-β1-induced EGFR activation, but not SMAD2 activation, moreover, was virtually undetectable in SYK−/−/− fibroblasts in comparison to wild type (SYK+/+/+) counterparts, confirming an upstream signaling role of src family kinases in EGFRY845 phosphorylation. Genetic EGFR deficiency or infection of VSMCs with EGFRK721A virtually ablated TGF-β1-stimulated ERK1/2 activation as well as PAI-1 expression but not SMAD2 phosphorylation. Transient transfection of a dominant-negative RhoA (DN-RhoA) expression construct or pretreatment of VSMC with C3 transferase (a Rho inhibitor) or Y-27632 (an inhibitor of p160ROCK, a downstream effector of Rho) also dramatically attenuated the TGF-β1-initiated PAI-1 inductive response. In contrast to EGFR pathway blockade, interference with Rho/ROCK signaling effectively inhibited TGF-βR-mediated SMAD2 phosphorylation and nuclear accumulation. TGF-β1-stimulated SMAD2 activation, moreover, was not sufficient to induce PAI-1 expression in the absence of EGFR signaling both in VSMC and mouse embryonic fibroblasts. Thus, two distinct pathways involving the EGFR/pp60c-src/MEK-ERK pathway and Rho/ROCK-dependent SMAD2 activation are required for TGF-β1-induced PAI-1 expression in VSMC. The identification of such novel interactions between two TGF-β1-activated signaling networks that specifically impact PAI-1 transcription in VSMC may provide therapeutically-relevant targets to manage the pathophysiology of PAI-1-associated cardiovascular/fibrotic diseases.

Effects of platelet‐derived growth factor isoforms on plasminogen activation by periodontal ligament and gingival fibroblasts

Platelet-derived growth factor isoforms and components of the plasminogen activator system are expressed at higher levels during periodontal regeneration. Recombinant platelet-derived growth factor-BB is approved for the treatment of periodontal defects. In the present study we investigated the effect of platelet-derived growth factor isoforms on the plasminogen activator system in periodontal fibroblasts. Human periodontal ligament fibroblasts and gingival fibroblasts were exposed to platelet-derived growth factor isoforms. Changes in urokinase-type plasminogen activator, tissue-type plasminogen activator, plasminogen activator inhibitor-1 and plasminogen activator inhibitor-2 transcript levels by platelet-derived growth factor-BB were monitored with a quantitative reverse transcription-polymerase chain reaction. Urokinase-type plasminogen activator and plasminogen activator inhibitor-1 protein levels were assessed by immunoassays. The effects of platelet-derived growth factor-BB on mitogen-activated protein kinase and phosphoinositol-3 kinase/Akt signaling were investigated by western blot and inhibitor studies. Casein zymography and kinetic assays revealed the size and activity, respectively, of the plasminogen activators. We found that incubation of periodontal ligament fibroblasts and gingival fibroblasts with platelet-derived growth factor-BB resulted in enhanced levels of urokinase-type plasminogen activator and plasminogen activator inhibitor-1 transcripts, but not of tissue-type plasminogen activator and plasminogen activator inhibitor-2. Platelet-derived growth factor-BB also increased urokinase-type plasminogen activator and plasminogen activator inhibitor-1 release into the culture medium. Phosphorylation of extracellular signal-regulated kinase, p38, c-Jun N-terminal kinase and Akt was observed in fibroblasts of both origin. Inhibition of phosphoinositol-3 kinase signaling abrogated the platelet-derived growth factor-BB effect on plasminogen activator inhibitor-1 production. Casein zymography revealed enzymatic activity of the urokinase-type plasminogen activator in cell-conditioned media and lysates of periodontal ligament fibroblasts and gingival fibroblasts. Exposure of gingival fibroblasts, but not of periodontal ligament fibroblasts, to platelet-derived growth factor isoforms moderately increased total plasminogen activation in the medium. These findings suggest that periodontal ligament fibroblasts attempt to maintain an equilibrium of the plasminogen activator system in the presence of platelet-derived growth factor isoforms.

Role of CAGA boxes in the plasminogen activator inhibitor-1 promoter in mediating oxidized low-density lipoprotein-induced transcriptional activation in mesangial cells

Oxidized low-density lipoprotein (Ox-LDL) activates transforming growth factor-beta (TGF-beta)/Smad signaling to stimulate plasminogen activator inhibitor-1 (PAI-1) expression in mesangial cells. Smad-binding sequences, termed CAGA boxes, are present in the promoter of human PAI-1 gene, and they mediate TGF-beta transcriptional induction. However, the functional role of each CAGA box in the Ox-LDL-induced PAI-1 promoter activation is unknown. In this study, mutation of 1 of the 3 CAGA boxes located at -730, -580, and -280 of the PAI-1 promoter decreased the Ox-LDL-induced luciferase activity by 40 to 58%, whereas mutations in 2 sites reduced it over 75% or completely abolished it. Overexpression of Smad3 in N-terminal tagged Smad3-transfected cells increased the Ox-LDL-induced transcriptional activation of the PAI-1 promoter, whereas mutation of Smad3 abolished it. Electrophoretic mobility shift assay showed that the labeled -280, -580, and -730 CAGA box probes detected DNA/protein complexes induced by Ox-LDL, whereas mutant probes did not. When nuclear extracts were preincubated with a 100-fold of an unlabeled -280, -580, and -730 CAGA oligonucleotide, the formation of complexes was prevented but not with mutant CAGA box competitors. The addition of anti-Smad3 to the reaction with the labeled -280 or -580 CAGA box probe resulted in a supershift, but not with the -730 CAGA box probe. These results suggest that the 3 CAGA elements in the PAI-1 promoter mediate the Ox-LDL-induced PAI-1 transcription to a different degree, of which the -280 and -580 CAGA regions directly bind to Smad3.

Regulation of PAI‐1 gene expression during adipogenesis

Obesity is characterized by elevated levels of circulating plasminogen activator inhibitor-1 (PAI-1), which contribute towards the development of secondary disorders such as type 2 diabetes mellitus and cardiovascular complications. This increase in plasma PAI-1 levels is attributed to an increase in PAI-1 derived from adipose tissue. This study shows that adipose tissue evolved into a major PAI-1 producing organ by gaining capacity during adipocyte differentiation to respond to inducers of PAI-1 transcription. This is mediated by a decrease in E2F1 protein levels, an increase in pRB levels and a decrease in pRB phosphorylation, all leading to a decrease in levels of free E2F, a known transcriptional repressor of PAI-1. Depletion of E2F1-3 was sufficient for inducers such as insulin to potently induce PAI-1 gene expression in pre-adipocytes. Conversely, forced release of pRB-bound endogenous E2F using cell-penetrating peptides can suppress PAI-1 gene expression in adipocytes. This study describes the novel paradigm of cellular differentiation-associated increase in PAI-1 gene expression which is mediated by a decrease in repressor activity, and describes a way of desensitising terminally differentiated cells to PAI-1 inducing agents by restoring endogenous repressor activity.

Mice Lacking Neutrophil Elastase Are Resistant to Bleomycin-Induced Pulmonary Fibrosis

Neutrophil elastase is a serine protease stored in the azurophilic granules of leukocytes. It has been implicated in the pathology of several lung diseases and is generally presumed to contribute to the tissue destruction and extracellular matrix damage associated with these conditions. To delineate the role of neutrophil elastase in pulmonary inflammation and fibrosis, neutrophil elastase-null mice were intratracheally instilled with bleomycin. In neutrophil elastase-null mice, biochemical and morphological characteristics of pulmonary fibrosis were attenuated for at least 60 days after bleomycin administration despite a typical response to bleomycin as evidenced by assessment of indices of DNA and cell damage. Neutrophil burden of bleomycin-treated wild-type and neutrophil elastase-null mice was comparable, and marked neutrophilic alveolitis was manifest in bleomycin-treated neutrophil elastase-null mice. An absence of immunostaining for active transforming growth factor (TGF)-beta in lung tissue from bleomycin-treated neutrophil elastase-null mice suggested a defect in TGF-beta activation, which was confirmed by biochemical assessment of TGF-beta levels in bronchoalveolar lavage fluid and lung tissue. These data point to novel and unexpected fibrogenic consequences of neutrophil elastase activity in the inflamed lung.

The alteration of plasminogen activator inhibitor-1 expression by linoleic acid and fenofibrate in HepG2 cells

The present study investigated the influence of linoleic acid and fenofibrate on plasminogen activator inhibitor-1 (PAI-1) expression in HepG2 cells and the mechanism possibly involved. Using gene recombination techniques, chloromycetin acetyltransferase (CAT) reporter gene plasmids containing nuclear factor-kappaB response element deletion (del1-PAI-pCAT) or very-low-density lipoprotein/fatty acid response element deletion (del2-PAI-pCAT) in the PAI-1 promoter were constructed and transiently transfected into HepG2 cells, respectively. Linoleic acid and fenofibrate were added to induce the transfected cells. The PAI-1 expression in mRNA and protein level was significantly induced by linoleic acid, but suppressed by fenofibrate. In the HepG2 cells transfected with PAI-pCAT plasmid, the PAI-1 transcription activity was significantly induced by linoleic acid, but suppressed by fenofibrate. Under transfection with del1-PAI-pCAT, both linoleic acid and fenofibrate increased the PAI-1 transcriptional activity; whereas in those cells transfected with del2-PAI-pCAT, fenofibrate significantly reduced PAI-1 transcriptional activity but no change was found with linoleic acid stimulation. Peroxisome proliferator-activated receptor alpha may be one of transcription factors playing a role in the upregulation of PAI-1 gene expression by linoleic acid in HepG2 cells. The inhibition of the nuclear factor-kappaB signaling pathway may be involved in the downregulation of PAI-1 gene expression by fenofibrate.