Plasminogen Activator Inhibitor-1 mRNA Levels Research Articles

Dysregulation of matrix metalloproteinases and their tissue inhibitors is related to abnormality of left ventricular geometry and function in streptozotocin‐induced diabetic minipigs

This study aimed to characterize matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in relation to changes in left ventricle (LV) geometry and function in a porcine model with streptozotocin (STZ)-induced diabetes. In 15 Chinese Guizhou minipigs with STZ-induced diabetes (diabetic group) and 15 age-matched normal controls (control group), Doppler tissue imaging was performed at 6 months of diabetes. Serum MMP-2, -9, TIMP-1, -4 and B-type natriuretic peptide (BNP) were determined. Expression of MMPs, TIMPs, urokinase type-plasminogen activator (uPA), its receptor (uPAR) and plasminogen activator inhibitor-1 (PAI-1) in aortic intima and LV myocardium was evaluated, with gelatinolytic activities of tissue MMP-2, -9 accessed by zymography. Left ventricle end-diastolic septum thickness (P < 0.05) and mass (P < 0.05) were increased, whereas peak systolic mitral annulus velocity (Sm, P < 0.001), LV systolic (P = 0.01) and diastolic strain (P < 0.001) were significantly decreased in diabetic group than in controls. Diabetic group showed higher expression of TIMP-1, -4 in aortic intima and LV myocardium (P < 0.01 or P < 0.05), with increased collagen content and elevated serum BNP level (P = 0.004) and lower gelatinolytic activities of tissue MMP-2, -9 (all P < 0.05). Semi-quantitative RT-PCR of those diabetic tissues revealed elevated mRNA levels of major TIMPs, uPA, uPAR and PAI-1. Reduction of serum MMP-2 and -9 levels was observed in diabetic group vs. control group (both P < 0.05). This study features elevated levels of TIMP-1, -4, uPA, uPAR and PAI-1, and decreased activities of MMP-2, -9 in aorta and myocardium in STZ-induced diabetic minipigs, indicating that MMP-TIMP dysregulation is associated with LV hypertrophy, cardiac dysfunction and increased cardiovascular fibrosis in diabetes.

Plasminogen activator inhibitor-1 (PAI-1) 4G/5G polymorphism and endometriosis. Influence of PAI-1 polymorphism on PAI-1 antigen and mRNA expression

Introduction Endometriosis is a benign gynecologic disease with a high prevalence. It is a multifactorial and polygenic entity in which the fibrinolytic system may be implicated. The objective of this study was to evaluate the plasminogen activator inhibitor-1 (PAI-1) 4G/5G polymorphism in a group of women with and without endometriosis and to analyze the influence of this polymorphism in PAI-1 expression in endometrial tissue and peritoneal fluid. Material and methods In 389 women (170 patients with endometriosis and 219 controls) PAI-1 4G/5G polymorphism was determined by PCR amplification using allele–specific primers. Quantitative real–time RT-PCR assay was used to quantify PAI-1 mRNA and PAI-1 antigen (ag) levels were quantified by ELISA. Results The genotype and allele frequencies of PAI-1 4G/5G polymorphism did not differ significantly between patients and controls. Control women with the 4G/4G genotype had higher endometrial PAI-1ag ( P = 0.026) and mRNA ( P = 0.014) levels than those with the 5G/5G genotype. Control carrying the 4G/4G genotype tended to have higher peritoneal fluid PAI-1ag levels than those carrying the 5G/5G genotype. Moreover, PAI-1ag levels in peritoneal fluid were higher in patients than in controls (P = 0.003). Conclusions The PAI-1 genotype distribution was similar in patients and controls. PAI-1 levels in endometrial tissue and peritoneal fluid seem to be associated with PAI-1 4G/5G polymorphism in controls. The increased PAI-1ag levels observed in peritoneal fluid from patients could contribute to increase the peritoneal adhesions observed in endometriosis.

Effect of the luteinising hormone surge on regulation of vascular endothelial growth factor and extracellular matrix-degrading proteinases and their inhibitors in bovine follicles

The aim of the present study was to evaluate the pattern of regulation of vascular endothelial growth factor (VEGF)-A (isoforms 121, 165, 189), VEGF receptor tyrosine kinases (VEGF-R1 and VEGF-R2), matrix metalloproteinase (MMP)-1, MMP-2, MMP-14, MMP-19, tissue-specific inhibitor of metalloproteinases (TIMP)-1, TIMP-2, tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), urokinase plasminogen activator receptor (uPAR) and plasminogen activator inhibitor-1 (PAI-1) in time-defined follicle classes before (0 h) and after the application of gonadotrophin-releasing hormone (GnRH). Bovine ovaries containing periovulatory follicles or new corpora lutea (CL; Days 1-2) were collected 0, 4, 10, 20 and 25 h (follicles) or 60 h (CL) after the injection of GnRH. Transcripts of VEGF isoforms (VEGF(121), VEGF(165), VEGF(189)) were upregulated 4 h after GnRH injection (during the luteinising hormone (LH) surge) and decreased thereafter to lowest levels around ovulation. All VEGF isoforms and their receptors were upregulated again after ovulation. The VEGF peptide concentration in follicular fluid decreased 20 h after GnRH injection, followed by an increase in follicles 25 h after GnRH. Expression of MMP-1 mRNA increased rapidly 4 h after GnRH injection and remained high during the entire experimental period. In contrast, MMP-19 mRNA increased significantly only after ovulation. Expression of TIMP-1 mRNA increased 4 h after GnRH and again after ovulation. Expression of tPA mRNA increased 4 h after GnRH and remained high during the entire experimental period, whereas expression of uPA transcripts increased significantly only after ovulation. Both uPAR and PAI-1 mRNA levels increased in follicles 4 h after GnRH and again after ovulation. The amount of MMP-1 protein (immunolocalisation) increased in follicles 10 h after GnRH: additional staining was observed in the granulosa cell layer. In conclusion, the temporal and spatial pattern of regulation of VEGF and extracellular matrix-degrading proteinases during periovulation suggests they are important mediators of the LH-dependent rupture of bovine follicles and for early CL formation (angiogenesis).

Genes overexpressed in cerebral arteries following salt-induced hypertensive disease are regulated by angiotensin II, JunB, and CREB

Although changes in gene expression are necessary for arterial remodeling during hypertension, the genes altered and their mechanisms of regulation remain uncertain. The goal of this study was to identify cerebral artery genes altered by hypertension and define signaling pathways important in their regulation. Intact cerebral arteries from Dahl salt-sensitive normotensive and hypertensive high-salt (HS) rats were examined by immunostaining, revealing an increased phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and expression of the proliferative marker Ki-67 in arteries from hypertensive animals. Arterial RNA analyzed by microarray and validated with RT-quantitative PCR revealed that jun family member junB and matricellular genes plasminogen activator inhibitor-1 (PAI-1) and osteopontin (OPN) were significantly overexpressed in HS arteries. Fisher exact test and annotation-based gene subsets showed that genes upregulated by Jun and Ca(2+)/cAMP-response element-binding protein (CREB) were overrepresented. A model of cultured rat cerebrovascular smooth muscle cells was used to test the hypothesis that angiotensin II (ANG II), JunB, and CREB are important in the regulation of genes identified in the rat hypertension model. ANG II induced a transient induction of junB and a delayed induction of PAI-1 and OPN mRNA levels, which were reduced by ERK inhibition with U-0126. Silencing junB using small-interfering RNA reduced mRNA levels of OPN but not PAI-1. The silencing of CREB reduced PAI-1 induction by ANG II but enhanced the transcription of OPN. Together, these results suggest that salt-induced hypertensive disease promotes changes in matricellular genes that are stimulated by ANG II, regulated by ERK, and selectively regulated by JunB and CREB.

C-reactive protein increases plasminogen activator inhibitor-1 expression in human endothelial cells

C-reactive protein (CRP) is an inflammatory marker which predicts cardiovascular disease. However, it is not fully understood whether CRP has direct effects on endothelial functions and gene expression. The purpose of current study was to determine the effects and molecular mechanisms of CRP on the expression of plasminogen activator inhibitor-1 (PAI-1) in human endothelial cells. Human coronary artery endothelial cells (HCAEC) were treated with CRP at clinically relevant concentrations for different durations. PAI-1 mRNA, protein and enzyme activities were studied. The effects of CRP on MAPK p38 phosphorylation was also studied by Bio-Plex luminex immunoassay. In addition, other types of human endothelial cells isolated from umbilical vein, skin, and lung microvessels were tested. CRP significantly increased PAI-1 mRNA levels in a time- and concentration-dependent manner. The protein level and enzyme activity of PAI-1 in the supernatant of CRP-treated HCAEC cultures were significantly increased. Anti-CD32 antibody effectively blocked CRP-induced PAI-1 mRNA expression. In addition, CRP significantly increased CD32 mRNA levels and enhanced phosphorylation of MAPK p38. Furthermore, antioxidant curcumin dramatically inhibited CRP-induced PAI-1 mRNA expression. The effect of CRP on PAI-1 expression was also confirmed in other types of human endothelial cells. In conclusion, CRP significantly increased the expression of PAI-1 in HCAEC and other human endothelial cells. CRP also increased its receptor CD32 expression which may further enhance its action. CRP-induced PAI-1 expression may be mediated by oxidative stress and p38 signal pathway as antioxidant effectively blocks the effect of CRP on HCAEC.

Effect of aldosterone on the expression of plasminogen activator inhibitor-1 in renal mesangial cells: experiment with rat renal mesangial cells

To evaluate the effects of aldosterone (ald) on the expression of plasminogen activator inhibitor-1 (PAI-1) in renal mesangial cells (MCs) and the changes after aldosterone was blocked with spironolactone. Rat MCs of the line HBZY-1 were cultured and divided into 5 groups: ald group, treated with aldosterone (1 micromol/L or 100 nmol/L), ald combined with spironolactone 1 nmol/L for 24 hours, and control group. Anotfher cells were cultured and treated with ald 100 nmol/L for 0, 0.5, 1, 2, 4, 6, 8, and 24 h respectively or treated with ald of the concentrations of 10(-5), 10(-6), 10(-7), 10(-8), 10(-9), 10(-10), pr 10(-11) mol/L. RT-PCR and Western blotting were used to detect the mRNA and protein expression of PAI-1. Confocal laser scanning microscopy was used to detect the ROS level in the MCs. The transforming growth factor-beta1 (TGF-beta1) level in the medium was detected by ELISA. The PAI-1 mRNA and protein levels of the ald 100 nmol/L and 1 micromol/L groups became 2 times and 1.8 times, and 1.7 times and 1.9 times respectively those of the control group (all P < 0.01). The PAI-1 mRNA and protein levels of the ald + spirolactone group were not significantly different from those of the control group (all P > 0.05). The TGF-beta1 levels of the 100 nmol/L and 1 micromol/L aldosterone groups were 147 pg/ml +/- 27 pg/ml and 183 pg/ml +/- 25 pg/ml respectively, both significantly higher than that of the control group (75 pg/ml +/- 23 pg/ml, both P < 0.01). The TGF-beta1 levels of the groups with spirolactone were not significantly different from that of the control group (both P > 0.05). The ROS levels of the 1 micromol/L and 100 nmol/L ald groups were 4.87 times and 4.77 times that of the control group (both P < 0.01), and the ROS levels of the groups with spirolactone were 1.95 time and 1.66 times that of the control group (both P > 0.05). 100 nmol/L ald time-dependently increased the PAI-1 level since 4 hours after exposure to ald (all P < 0.05). Aldosterone dose-dependently increased the PAI-1 level (all P < 0.05). Aldosterone upregulates the production of PAI-1 by MCs and increases the expression of TGF-beta1 and cellular ROS in the MCs significantly, which is mediated through mineralocorticoid receptor.

PAI-1 Plays an Important Role in Liver Failure After Excessive Hepatectomy in the Rat

Well-organized turnover of the extracellular matrix is important in liver regeneration, which is regulated by the plasminogen activating system. The aim of this study was to investigate the role of plasminogen activator inhibitor-1 (PAI-1) after excessive hepatectomy and to ascertain whether angiotensin-converting enzyme (ACE) inhibitors, which are PAI-1 inhibitors as well, successfully improve the survival rate of rats that have undergone 95% partial hepatectomy (PHx). Using liver tissues sampled after 90% or 95% PHx, the expression of PAI-1 mRNA was evaluated using reverse transcription polymerase chain reaction. Hepatic PAI-1 protein and urokinase-type plasminogen activator (uPA) levels were determined by enzyme-linked immunosorbent assay. Survival study and cytodynamic analysis by 5-bromo-2'-deoxyuridine staining were performed to evaluate the effects of ACE inhibition. The levels of PAI-1 mRNA and hepatic PAI-1 protein in the 95% PHx group peaked and were then maintained. By contrast, the uPA level fell relative to the 90% PHx group. Additionally, the hepatic PAI-1 protein level decreased and the survival rate improved in the 95% PHx rats that had undergone ACE inhibition. Our experimental results suggest that PAI-1 plays a role in the occurrence of liver failure after excessive hepatectomy via accelerated maturation of pro-uPA and fibrinolytic factors. These are potential molecular therapeutic targets for liver failure after excessive hepatectomy.

Progestin inhibits and thrombin stimulates the plasminogen activator/inhibitor system in term decidual stromal cells: implications for parturition

Labor is associated with 'decidual activation' with increased proteolysis and extracellular matrix degradation. The balance between plasminogen activator inhibitor-1 (PAI-1) and urokinase (uPA) and tissue-type plasminogen activator (tPA) is an important determinant of proteolytic activity at the maternal-fetal interface. Thrombin released at the time of placental abruption (decidual hemorrhage) is known to promote decidual proteolysis and uterine contractions. This study investigates the separate and interactive effects of steroid hormones and thrombin on PAI-1, uPA, and tPA expression by term decidual cells (DCs). Term DCs were isolated by enzymatic digestion, purified, and depleted of leukocytes. Cells were treated with estradiol (10(-8) mol [E2]), medroxyprogesterone acetate (10(-7) mol [MPA]), both, or vehicle for 7 days. After 24-hour incubation with or without thrombin (0.1-2.5 U/mL), levels of PAI-1, uPA, and tPA in conditioned supernatant were measured by specific ELISA and Western blotting. Levels of PAI-1 and uPA mRNA were measured by quantitative RT-PCR. In the cultured term DCs, ELISA measurements indicated that basal output of PAI-1 was about 2 logs higher than that of either uPA or tPA (2.5 +/- 0.7 ng/mL per microg protein, 13.4 +/- 6.3 pg/mL per microg protein, and 25.4 +/- 10.8 pg/mL per microg protein, respectively). Although E2 alone did not affect PAI-1 output, MPA and E2+MPA significantly enhanced PAI-1 production (2.5 +/- 0.7 vs 8.2 +/- 2.0 ng/mL per microg protein for E2+MPA [3.3-fold]; P < .01). By contrast, uPA output was inhibited by exposure to MPA (13.4 +/- 6.3 vs 2.6 +/- 1.1 pg/mL per microg protein [0.2-fold]; P < .05), whereas tPA production was not affected by MPA. Thrombin did not significantly affect uPA and tPA production by term DCs. In contrast, in E2+MPA-treated term DCs, thrombin, a hemostatic proinflammatory cytokine, selectively increased PAI-1 output in a dose-dependent fashion, which could be blocked by the selective thrombin inhibitor, hirudin. Western blotting confirmed the effects of MPA and thrombin in elevating secreted levels of PAI-1. Unlike the increase in PAI-1 output elicited by thrombin, term DCs were unresponsive to either of the classic proinflammatory cytokines, TNFalpha or IL-1beta. Corresponding effects on PAI-1 mRNA levels were elicited by MPA and thrombin as seen for PAI-1 protein expression, suggesting that these up-regulatory effects are transcriptionally mediated. Progestin enhanced PAI-1 and inhibited uPA expression by term DCs, which may explain in part the pregnancy-prolonging properties of progesterone as a consequence of inhibited proteolytic activity at the maternal-fetal interface. Thrombin augmented PAI-1 expression in the absence of increased uPA or tPA expression by term DCs, suggesting that abruption-associated decidual proteolysis and preterm labor is mediated primarily by thrombin-enhanced matrix metalloproteinase expression rather than an indirect effect on the plasminogen activator/inhibitor system.

Food deprivation induces adipose plasminogen activator inhibitor-1 (PAI-1) expression without accumulation of plasma PAI-1 in genetically obese and diabetic db/db mice

Relationships between energy intake and fibrinolytic functions have been documented in detail. We evaluated food deprivation (FD) as a means of modulating fibrinolytic activity in genetically obese and diabetic db/db mice and in their lean counterparts. Twelve hours of FD induced considerable gene expression of plasminogen activator inhibitor-1 (PAI-1) in both epididymal (3.8-fold, p < 0.05) and intestinal (2.4-fold, p < 0.05) adipose tissues without affecting plasma PAI-1 levels in db/db mice, whereas the FD did not affect these parameters in wild-type mice. Importantly, 24 hours of FD increased the plasma PAI-1 content in wild-type (1.9-fold, p < 0.01) but not in db/db mice, although adipose PAI-1 mRNA levels were significantly increased in db/db mice. The plasma PAI-1 content significantly correlated with hepatic PAI-1 mRNA levels in wild-type (r = 0.84, p < 0.01) and in db/db (r = 0.63, p < 0.01) mice. However, plasma PAI-1 did not correlate with adipose PAI-1 expression in db/db mice, although adipose tissue in general is thought to be the principal site of PAI-1 production in obesity. Hepatic PAI-1 expression was closely correlated with serum levels of free fatty acids in wild-type (r = 0.72, p < 0.01), but not in db/db mice. Adipose PAI-1 expression significantly correlated with serum corticosterone levels in both genotypes (wild-type, r = 0.52, p < 0.05; db/db, r = 0.51, p < 0.01), suggesting that adipose PAI-1 expression is up-regulated by fasting-induced glucocorticoids. The present findings suggested that fasting differentially affects fibrinolytic activity in obese and lean subjects and that PAI-1 expression in the liver as well as in adipose tissues comprises an important determinant of increased risk for cardiovascular disease in obesity.

Orphan Nuclear Receptor Small Heterodimer Partner Inhibits Transforming Growth Factor-β Signaling by Repressing SMAD3 Transactivation

Orphan nuclear receptor small heterodimer partner (SHP) is an atypical member of the nuclear receptor superfamily; SHP regulates the nuclear receptor-mediated transcription of target genes but lacks a conventional DNA binding domain. In this study, we demonstrate that SHP represses transforming growth factor-beta (TGF-beta)-induced gene expression through a direct interaction with Smad, a transducer of TGF-beta signaling. Transient transfection studies demonstrate that SHP represses Smad3-induced transcription. In vivo and in vitro protein interaction assays revealed that SHP directly interacts with Smad2 and Smad3 but not with Smad4. Mapping of domains mediating the interaction between SHP and Smad3 showed that the entire N-terminal domain (1-159 amino acids) of SHP and the linker domain of Smad3 are involved in this interaction. In vitro glutathione S-transferase pulldown competition experiments revealed the SHP-mediated repression of Smad3 transactivation through competition with its co-activator p300. SHP also inhibits the activation of endogenous TGF-beta-responsive gene promoters, the p21, Smad7, and plasminogen activator inhibitor-1 (PAI-1) promoters. Moreover, adenovirus-mediated overexpression of SHP decreases PAI-1 mRNA levels, and down-regulation of SHP by a small interfering RNA increases both the transactivation of Smad3 and the PAI-1 mRNA levels. Finally, the PAI-1 gene is expressed in SHP(-/-) mouse hepatocytes at a higher level than in normal hepatocytes. Taken together, these data indicate that SHP is a novel co-regulator of Smad3, and this study provides new insights into regulation of TGF-beta signaling.

Prognostic significance of urokinase plasminogen activator and plasminogen activator inhibitor-1 mRNA expression in lymph node- and hormone receptor-positive breast cancer.

BackgroundOne of the most thoroughly studied systems in relation to its prognostic relevance in patients with breast cancer, is the plasminogen activation system that comprises of, among others, the urokinase Plasminogen Activator (uPA) and its main inhibitor, the Plasminogen Activator Inhibitor-1 (PAI-1). In this study, we investigated the prognostic value of uPA and PAI-1 at the mRNA level in lymph node- and hormone receptor-positive breast cancer.MethodsThe study included a retrospective series of 87 patients with hormone-receptor positive and axillary lymph node-positive breast cancer. All patients received radiotherapy, adjuvant anthracycline-based chemotherapy and five years of tamoxifen treatment. The median patient age was 54 and the median follow-up time was 79 months. Distant relapse occurred in 30 patients and 22 patients died from breast cancer during follow-up. We investigated the prognostic value of uPA and PAI-1 at the mRNA level as measured by real-time quantitative RT-PCR.ResultsuPA and PAI-1 gene expression was not found to be correlated with any of the established clinical and pathological factors. Metastasis-free Survival (MFS) and Breast Cancer specific Survival (BCS) were significantly shorter in patients expressing high levels of PAI-1 mRNA (p < 0.0001; p < 0.0001; respectively). In Cox multivariate analysis, the level of PAI-1 mRNA appeared to be the strongest prognostic factor for MFS (Hazard Ratio (HR) = 10.12; p = 0.0002) and for BCS (HR = 13.17; p = 0.0003). Furthermore, uPA gene expression was not significantly associated neither with MFS (p = 0.41) nor with BCS (p = 0.19). In a Cox-multivariate regression analysis, uPA expression did not demonstrate significant independent prognostic value.ConclusionThese findings indicate that high PAI-1 mRNA expression represents a strong and independent unfavorable prognostic factor for the development of metastases and for breast cancer specific survival in a population of hormone receptor- and lymph node-positive breast cancer patients.

Inhibitory effects of fenofibrate on plasminogen activator inhibitor-1 expression in human endothelial cells

The effects of fenofibrate on plasminogen activator inhibitor-1 (PAI-1) expression in human umbilical endothelial cell-derived transformed cell line--ECV 304 cells were investigated. ECV 304 cells were incubated with different concentrations of fenofibrate (0, 10, 50, 100 micromol/L) for 24 h. PAI-1 mRNA and protein was detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot respectively. PAI-1 antigenic content of endothelial cells was measured by using ELISA. Fenofibrate could inhibit the PAI-1 mRNA and protein expression and reduce PAI-1 antigenic content dependently. After treatment with fenofibrate (10 micromol/L), the expression levels of PAI-1 mRNA and protein were 0.65 +/- 0.05 and 0.96 +/- 0.11 respectively, significantly lower than in the control group (0.78 +/- 0.03 and 1.21 +/- 0.15, respectively, P<0.05). PAI-1 antigenic contents (24.52 +/- 8.39) in ECV304 cells treated with 10 micromol/L fenofibrate were significantly lower than those in the control group (6.98 +/- 5.12, P<0.05). It was concluded that fenofibrate inhibited the expression of PAI-1 mRNA in ECV304 cells, and reduce the protein expression and the antigenic content of PAI-1, suggesting that fenofibrate may have an antiatherosclerotic effect on endothelial cells by PAI-1 pathway.

Shear stress induces hepatocytePAI-1gene expression through cooperative Sp1/Ets-1 activation of transcription

Partial hepatectomy causes hemodynamic changes that increase portal blood flow in the remaining lobe, where the expression of immediate-early genes, including plasminogen activator inhibitor-1 (PAI-1), is induced. We hypothesized that a hyperdynamic circulatory state occurring in the remaining lobe induces immediate-early gene expression. In this study, we investigated whether the mechanical force generated by flowing blood, shear stress, induces PAI-1 expression in hepatocytes. When cultured rat hepatocytes were exposed to flow, PAI-1 mRNA levels began to increase within 3 h, peaked at levels significantly higher than the static control levels, and then gradually decreased. The flow-induced PAI-1 expression was shear stress dependent rather than shear rate dependent and accompanied by increased hepatocyte production of PAI-1 protein. Shear stress increased PAI-1 transcription but did not affect PAI-1 mRNA stability. Functional analysis of the 2.1-kb PAI-1 5'-promoter indicated that a 278-bp segment containing transcription factor Sp1 and Ets-1 consensus sequences was critical to the shear stress-dependent increase of PAI-1 transcription. Mutations of both the Sp1 and Ets-1 consensus sequences, but not of either one alone, markedly prevented basal PAI-1 transcription and abolished the response of the PAI-1 promoter to shear stress. EMSA and chromatin immunoprecipitation assays showed binding of Sp1 and Ets-1 to each consensus sequence under static conditions, which increased in response to shear stress. In conclusion, hepatocyte PAI-1 expression is flow sensitive and transcriptionally regulated by shear stress via cooperative interactions between Sp1 and Ets-1.

Notoginsenoside R1 from Panax notoginseng inhibits TNF-α-induced PAI-1 production in human aortic smooth muscle cells

Notoginsenoside R1 is the main ingredient with cardiovascular activity in Panax notoginseng. We reported that notoginsenoside R1 significantly decreased tumor necrosis factor-α (TNF-α)-induced plasminogen activator inhibitor-1 (PAI-1) mRNA, protein level and secretion in human aortic smooth muscle cells (HASMCs) in a dose-dependent manner. TNF-α significantly increased extracellular signal-related kinases (ERK) and protein kinase B (PKB) activation and, which was blocked by notoginsenoside R1, PD098059, U0126 or wortmannin. Our data demonstrates that TNF-α-induced up-regulation of PAI-1 mRNA and protein levels and secretion occur via activation of ERK and PKB, which was prevented by treatment of notoginsenoside R1, PD098059, U0126 or wortmannin. These results suggest that notoginsenoside R1 inhibits TNF-α-induced PAI-1 overexpression in HASMCs by suppressing ERK and PKB signaling pathways.

Chronic blockade of the angiotensin II receptor has a differential effect on adipose and vascular PAI-1 in OLETF rats

Angiotensinogen (AGT) and plasminogen activator inhibitor-1 (PAI-1) are expressed in both vascular and adipose tissues. Angiotensin II (AG II) has an adipogenic effect and increases PAI-1 expression. To evaluate the chronic effects of AG II type 1 receptor (AT 1R) antagonism on adipose mass and PAI-1 expression in vascular and adipose tissues, losartan (30 mg/kg/day) was administered to Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a model of type 2 diabetes, for 20 weeks. Adipose mass and regional fat distribution in the abdomen did not change after chronic AT 1R antagonism in OLETF rats. AGT and PAI-1 mRNA expressions in adipose tissue of OLETF rats were significantly increased compared with Long-Evans Tokushima Otsuka (LETO) rats, the normal control. Chronic losartan therapy further increased the level of adipose AGT in OLETF rats, but did not affect the level of adipose PAI-1 mRNA. In contrast, aortic PAI-1 expression in OLETF rats was attenuated by chronic losartan therapy. Our results have two implications. First, adipose tissue may be an important source of AG II in metabolic syndrome even after chronic losartan therapy. Second, chronic AT 1R antagonism with losartan causes differential effects on vascular and adipose PAI-1 expression.

Posttranslational, Reversible O-Glycosylation Is Stimulated by High Glucose and Mediates Plasminogen Activator Inhibitor-1 Gene Expression and Sp1 Transcriptional Activity in Glomerular Mesangial Cells

Metabolic flux through the hexosamine biosynthetic pathway (HBP) is increased in the presence of high glucose (HG) and potentially stimulates the expression of genes associated with the development of diabetic nephropathy. A number of synthetic processes are coupled to the HBP, including enzymatic intracellular O-glycosylation (O-GlcNAcylation), the addition of single O-linked N-acetylglucosamine monosaccharides to serine or threonine residues. Despite much data linking flow through the HBP and gene expression, the exact contribution of O-GlcNAcylation to HG-stimulated gene expression remains unclear. In glomerular mesangial cells, HG-stimulated plasminogen activator inhibitor-1 (PAI-1) gene expression requires the HBP and the transcription factor, Sp1. In this study, the specific role of O-GlcNAcylation in HG-induced PAI-1 expression was tested by limiting this modification with a dominant-negative O-linked N-acetylglucosamine transferase, by overexpression of neutral beta-N-acetylglucosaminidase, and by knockdown of O-linked beta-N-acetylglucosamine transferase expression by RNA interference. Decreasing O-GlcNAcylation by these means inhibited the ability of HG to increase endogenous PAI-1 mRNA and protein levels, the activity of a PAI-1 promoter-luciferase reporter gene, and Sp1 transcriptional activation. Conversely, treatment with the beta-N-acetylglucosaminidase inhibitor, O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate, in the presence of normal glucose increased Sp1 O-GlcNAcylation and PAI-1 mRNA and protein levels. These findings demonstrate for the first time that among the pathways served by the HBP, O-GlcNAcylation, is obligatory for HG-induced PAI-1 gene expression and Sp1 transcriptional activation in mesangial cells.

Effects of silica on the expression of plasminogen activator inhibitor-1 and activator protein-1 in human alveolar epithelial cells type II

To investigate the mechanism of silicosis by observing the effects of silica on the expression of plasminogen activator inhibitor-1 (PAI-1) and activator protein-1 (AP-1) in human alveolar epithelial cells type II (A549). A549 cell and SiO(2) (200 microg /ml) were co-cultured for 0, 4, 8, 16 and 24 h respectively. The reverse transcriptase polymerase chain reaction (RT-PCR), Western blotting and SP immunocytochemistry were used for detections of the PAI-1 mRNA and protein expression. The nucleoprotein and total protein expression of AP-1 were investigated by Western blotting. The expression levels of PAI-1 mRNA and protein were increased in a time-dependent manner(r(mRNA) = 0.911, r(protein) = 0.902, P < 0.05). The expressions of PAI-1 mRNA and protein in experimental groups were higher than that in control group (P < 0.05) and was the highest in 24 h group [(0.73 +/- 0.01) vs (0.36 +/- .03)]. The nucleoprotein expressions of c-jun/c-fos in experimental groups were also higher than in control group (P < 0.05), and the nucleoprotein expression level of c-jun was the highest in 4 h group [(1.54 +/- 0.02) vs (0.56 +/- 0.03)]; the nucleoprotein expression level of c-fos was the highest in 8 h group [(0.36 +/- 0.01) vs (0.15 +/- 0.01)]. Both c-jun and c-fos expression were decreased after 16 h, but the total protein expression of c-jun/c-fos had no difference in all experimental groups. The positive signal of PAI-1 was located in cytoplasm and nucleus. SiO(2) could induce PAI-1 expression of A549 in a time-dependent manner, and AP-1 activation can be observed in early time.

Radiation and Transforming Growth Factor-β Cooperate in Transcriptional Activation of the Profibrotic Plasminogen Activator Inhibitor-1 Gene

Radiation-induced fibrosis is an important side effect in the treatment of cancer. Profibrotic proteins, such as plasminogen activator inhibitor-1 (PAI-1), transforming growth factor-beta (TGF-beta), and tissue type inhibitor of metalloproteinases-1 (Timp-1), are thought to play major roles in the development of fibrosis via the modulation of extracellular matrix integrity. We did a detailed analysis of transcriptional activation of these profibrotic genes by radiation and TGF-beta. Irradiation of HepG2 cells led to a high increase in PAI-1 mRNA levels and a mild increase in Timp-1 mRNA levels. In contrast, TGF-beta1 and Smad7 were not increased. Radiation and TGF-beta showed strong cooperative effects in transcription of the PAI-1 gene. The TGF-beta1 gene showed a mild cooperative activation, whereas Timp-1 and Smad7 were not cooperatively activated by radiation and TGF-beta. Analysis using the proximal 800 bp of the human PAI-1 promoter revealed a dose-dependent increase of PAI-1 levels between 2 and 32 Gy gamma-rays that was independent of latent TGF-beta activation. Subsequent site-directed mutagenesis of the PAI-1 promoter revealed that mutation of a p53-binding element abolished radiation-induced PAI-1 transcription. In line with this, PAI-1 was not activated in p53-null Hep3B cells, indicating that p53 underlies the radiation-induced PAI-1 activation and the cooperativity with the TGF-beta/Smad pathway. Together, these data show that radiation and TGF-beta activate PAI-1 via partially nonoverlapping signaling cascades that in concert synergize on PAI-1 transcription. This may play a role in patient-to-patient variations in susceptibility toward fibrosis after radiotherapy.

Synergistic effect of hypoxia and TNF-α on production of PAI-1 in human proximal renal tubular cells

Chronic hypoxia has been newly proposed as a common mechanism of tubulointerstitial fibrosis in the progression of various chronic inflammatory renal diseases, where plasminogen activator inhibitor-1 (PAI-1) plays an important role in the accumulation of extracellular matrix (ECM) through inhibition of plasmin-dependent ECM degradation. In the present study, we investigated the presence of PAI-1 in renal tubular cells by immunostaining renal biopsy samples. We also closely examined the effects of hypoxia and tumor necrosis factor-alpha (TNF-alpha) on PAI-1 expression in cultured human proximal renal tubular cells (HPTECs). Confluent cells growth-arrested in Dulbecco's modified Eagle's medium (DMEM) for 24 hours were exposed to hypoxia (1% O(2)) and/or TNF-alpha at 10 ng/mL for up to 48 hours. Amounts of PAI-1 protein and mRNA after stimulation were measured by enzyme-linked immunosorbent assay (ELISA) and TaqMan quantitative polymerase chain reaction (PCR) or cDNA array analysis, respectively, and compared to those in cells incubated under control conditions (18% O(2) without TNF-alpha). Hypoxia-inducible factor-1alpha (HIF-1alpha) was demonstrated by immunoblot and immunofluorescence analyses. Human PAI-1 promoter activity was estimated by luciferase reporter gene assay. In crescentic glomerulonephritis, clusters of proximal tubules were specifically stained for PAI-1. cDNA array analysis identified PAI-1 as a major gene highly induced by hypoxia in HPTECs. Treatment of 24 hours with hypoxia, TNF-alpha, and their combination induced a 2.8-fold, a 1.8-fold, and a 4.6-fold increase in PAI-1 protein secretion, and produced a 3.6-fold, a 3.3-fold, and a 12.1-fold increase at the PAI-1 mRNA level, respectively. Immunoblot analysis and immunocytochemistry revealed that hypoxia-inducible factor-1alpha (HIF-1alpha) was markedly accumulated in the cell lysates and exclusively translocated to nuclei after 16 hours' exposure of HPTECs to hypoxia but not to TNF-alpha. Luciferase reporter gene assay showed that hypoxia, TNF-alpha, and their combination increased PAI-1 transcription activity by 1.8-fold, 1.4-fold, and 2.2-fold, respectively. A dominant-negative form of HIF-1alpha significantly suppressed PAI-1 transcription activity induced by hypoxia. Inhibition of nuclear factor-kappaB (NF-kappaB) caused a moderate decrease in PAI-1 production under hypoxia. Hypoxia induces PAI-1 expression via remarkable nuclear accumulation of HIF-1alpha and partially via NF-kappaB activation in HPTECs. TNF-alpha can synergistically enhance this hypoxia-induced PAI-1 expression.

Transcriptional regulation of plasminogen activator inhibitor-1 by transforming growth factor-β, activin A and microphthalmia-associated transcription factor

Plasminogen activator inhibitor-1 ( PAI-1) is a key molecule that regulates turnover of the extracellular matrix. In the present study, we characterized PAI-1 gene expression in mast cells and melanocytes. In bone marrow-derived cultured mast cells, up-regulation of the PAI-1 gene was observed upon treatment with TGF-β 1, and was regulated at the transcriptional level. Microphthalmia-associated transcription factor (MITF), a member of the basic helix–loop–helix leucine zipper family of tissue-specific transcription factors predominantly expressed in mast cells, melanocytes and osteoclasts, also stimulated PAI-1 gene transcription, and TGF-β 1 did not increase PAI-1 mRNA levels in mast cells from mi/ mi mice expressing dominant-negative MITF. MITF isoforms regulated TGF-β 1-induced transcription of PAI-1 differently; MITF-E-mediated transcription was further increased by TGF-β 1, whereas transcriptional activation by TGF-β 1 was blocked by MITF-M or MITF-mc expression. In contrast, activin A, another member of the TGF-β family, enhanced transcription induced by MITF-M, as well as by MITF-E, although MITF-mc blocked activin A-induced transcription of PAI-1. Different regulation of PAI-1 gene expression upon TGF-β 1 and activin A treatment was also detected in B16 melanocytes; TGF-β 1 transiently increased the PAI-1 mRNA level, whereas activin A had prolonged effects on up-regulation of PAI-1. Our results on the control of PAI-1 gene expression by MITF isoforms, TGF-β 1 and activin A suggest that discrete regulation of the plasminogen activator system occurs in a cell type-specific manner.