Plasminogen Activator Inhibitor Type-1 Gene Research Articles

Attenuation by Fibrates of Plasminogen Activator Inhibitor Type-1 Expression in Human Arterial Smooth Muscle Cells

Human atherosclerotic lesions exhibit increased expression of plasminogen activator inhibitor type-1 (PAI-1) that has been implicated in atherogenesis. Although vascular smooth muscle cells are a predominant source of PAI-1 expression potentially favorable modulation of PAI-1 expression by fibrates has not yet been characterized in these cells. Human aortic smooth muscle cells were exposed to selected growth factors. PAI-1 expression was stimulated most powerfully by TGF-beta (EC50 = 0.2 ng/ml, up to 12-fold increase). Gemfibrozil inhibited basal PAI-1 expression by 23% (p = ns) and TGF-beta-induced PAI-1 expression by 52% (p = 0.017) whereas t-PA and total protein synthesis was not affected. Changes in PAI-1 protein accumulation reflected PAI-1 gene expression attributable to modulation of half-life of PAI-1 mRNA by gemfibrozil. Inhibition by other fibrates was less. Gemfibrozil specifically attenuates TGF-beta-induced PAI-1 expression in human arterial smooth muscle cells. Thus, fibrates are promising agents for normalizing increased PAI-1 expression in arterial walls in patients in whom PAI-1 expression is increased.

Overexpression of a dominant negative CREB protein in HT-1080 cells selectively disrupts plasminogen activator inhibitor type 2 but not tissue-type plasminogen activator gene expression

The tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor type 2 (PAI-2) genes are differentially regulated by 12-phorbol 13-myristate acetate (PMA) in HT-1080 fibrosarcoma cells. PMA transcriptionally down-regulates the t-PA gene in HT-1080 cells, while the PAI-2 gene is simultaneously induced by this agonist. The t-PA and PAI-2 gene promoters harbour a cAMP-response element (CRE) which influences the expression of both genes. We have compared the binding activity of nuclear factors that recognise these CRE sites. We show that CREB (CRE binding protein) recognises each CRE and that the degree of constitutive Ser119-phosphorylated t-PA CRE-bound CREB was greater than for PAI-2 CRE bound CREB. Stable transfection of HT-1080 cells with a plasmid containing a CREB that could not be phosphorylated on Ser119 (pCI-CREB ala119) did not influence PMA-mediated suppression of t-PA mRNA, but markedly impaired PMA-mediated induction of PAI-2 mRNA. Our results demonstrate that the Ser119 residue of CREB plays a crucial role in PMA-mediated induction of PAI-2 gene expression, whereas PMA-mediated suppression of t-PA in HT-1080 cells requires a different process.

Production of plasminogen activator inhibitor-1 by human mast cells and its possible role in asthma.

The plasminogen activator inhibitor type 1 (PAI-1) has an essential role in tissue remodeling. The PAI-1 gene was induced by a combination of phorbol ester and calcium ionophore at the highest level among the inducible human mast cell genes that we have analyzed on a DNA microarray. PAI-1 was secreted by both a human mast cell line (HMC)-1 and primary cultured human mast cells upon stimulation, whereas PAI-1 was undetectable in either group of unstimulated cells. The secretion of PAI-1 was due to de novo synthesis of PAI-1 rather than secretion of preformed PAI-1. The functional significance of PAI-1 secretion was demonstrated by complete inhibition of tissue-type plasminogen activator activity with supernatants of stimulated HMC-1 cells. Furthermore, we were able to regulate PAI-1 gene expression in HMC-1 cells by known therapeutic agents. High-dose (1 microM) dexamethasone induced PAI-1 mRNA expression. Cyclosporin down-regulated the expression of the PAI-1 gene. Cycloheximide abrogated PAI-1 mRNA expression, suggesting that transcription of the PAI-1 gene requires de novo synthesis of early gene products, including transcription factors. Finally, we demonstrated PAI-1 in lung mast cells from a patient with asthmatic attack by double-immunofluorescence study. This is the first report demonstrating that activated human mast cells release a striking amount of functionally active PAI-1. These results suggest that PAI-1 could play an important role in airway remodeling of asthma, and inhibition of PAI-1 activity could represent a novel therapeutic approach in the management of airway remodeling.

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.

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.

The 4G/5G polymorphism of the plasminogen activator inhibitor-1 gene is associated with severe preeclampsia.

Preeclampsia is associated with thrombosis of the intervillous or spiral artery. A deletion/insertion polymorphism (4G or 5G) in the promoter of the plasminogen activator inhibitor type 1 (PAI-1) gene is suggested to be involved in regulating the synthesis of the inhibitor, 4G allele, being associated with the enhanced gene expression and plasma PAI-1 levels. We assessed the association between preeclampsia and the 4G/5G polymorphism of the PAI-1 gene in 115 preeclamptic patients, 210 pregnant controls, and 298 healthy volunteer controls. The frequency of the homozygotes for the 4G allele was significantly higher in the patients than in the control pregnant women (P = 0.04) or in the healthy volunteers (P = 0.02). The 4G allele frequency was also significantly higher in the patients than in the control group of pregnant women (P = 0.03) and in the healthy volunteers (P = 0.02). These results suggest that the presence of the 4G/4G genotype of the PAI-1 gene is one of the risk factors for preeclampsia.

Atherosclerosis progression in LDL receptor-deficient and apolipoprotein E-deficient mice is independent of genetic alterations in plasminogen activator inhibitor-1.

Impaired fibrinolysis has been linked to atherosclerosis in a number of experimental and clinical studies. Plasminogen activator inhibitor type 1 (PAI-1) is the primary inhibitor of plasminogen activation and has been proposed to promote atherosclerosis by facilitating fibrin deposition within developing lesions. We examined the contribution of PAI-1 to disease progression in 2 established mouse models of atherosclerosis. Mice lacking apolipoprotein E (apoE-/-) and mice lacking the low density lipoprotein receptor (LDLR-/-) were crossbred with transgenic mice overexpressing PAI-1 (resulting in PAI-1 Tg(+)/apoE-/- and PAI-1 Tg(+)/LDLR-/-, respectively) or were crossbred with mice completely deficient in PAI-1 gene expression (resulting in PAI-1-/-/apoE-/- and PAI-1-/-/LDLR-/-, respectively). All animals were placed on a western diet (21% fat and 0.15% cholesterol) at 4 weeks of age and analyzed for the extent of atherosclerosis after an additional 6, 15, or 30 weeks. Intimal and medial areas were determined by computer-assisted morphometric analysis of standardized microscopic sections from the base of the aorta. Atherosclerotic lesions were also characterized by histochemical analyses with the use of markers for smooth muscle cells, macrophages, and fibrin deposition. Typical atherosclerotic lesions were observed in all experimental animals, with greater severity at the later time points and generally more extensive lesions in apoE-/- than in comparable LDLR-/- mice. No significant differences in lesion size or histological appearance were observed among PAI-1-/-, PAI-1 Tg(+), or PAI-1 wild-type mice at any of the time points on either the apoE-/- or LDLR-/- genetic background. We conclude that genetic modification of PAI-1 expression does not significantly alter the progression of atherosclerosis in either of these well-established mouse models. These results suggest that fibrinolytic balance (as well as the potential contribution of PAI-1 to the regulation of cell migration) plays only a limited role in the pathogenesis of the simple atherosclerotic lesions observed in the mouse.

PAI-1 gene expression is regionally induced in wounded epithelial cell monolayers and required for injury repair.

Induced expression of plasminogen activator inhibitor type-1 (PAI-1), a major negative regulator of pericellular plasmin generation, accompanies wound repair in vitro and in vivo. Since transcriptional control of the PAI-1 gene is superimposed on a growth state-dependent program of cell activation (Kutz et al., 1997, J Cell Physiol 170:8-18), it was important to define potentially functional relationships between PAI-1 synthesis and subpopulations of cells that emerge during the process of injury repair in T2 renal epithelial cells. Specific cohorts of migratory and proliferating cells induced in response to monolayer trauma were spatially as well as temporally distinct. Migrating cells did not divide in the initial 12 to 20 h postinjury. After 24 h, S-phase cells were generally restricted to a region 1 to 2 mm from, and parallel to, the wound edge. Proliferation of wound bed cells occurred subsequent to wound closure, whereas the distal contact-inhibited monolayer remained generally quiescent. Hydroxyurea blockade indicated, however, that proliferation (most likely of cells immediately behind the motile "tongue") was necessary for maintenance of cell-to-cell cohesiveness in the advancing front, although the ability to migrate was independent of proliferation. PAI-1 mRNA expression was rapidly up-regulated in response to wounding with inductive kinetics approximating that of serum-stimulated cultures. Differential harvesting of T2 cell subpopulations, based on proximity to the injury site, prior to Northern assessments of PAI-1 mRNA abundance indicated that PAI-1 transcripts were restricted to cells immediately bordering the wound or actively migrating and not expressed by cells in the distal contact-inhibited monolayer regions. Such cell location-specific distribution of PAI-1-producing cells was confirmed by immunocytochemistry. PAI-1 synthesis in cells that locomoted into the wound field continued until injury closure. Down-regulation of PAI-1 synthesis and matrix deposition in renal epithelial cells, stably transfected with a PAI-1 antisense expression vector, significantly impaired wound closure. Transfection of the wound repair-deficient R/A epithelial line with a sense PAI-1 expression construct restored both approximately normal levels of PAI-1 synthesis and repair ability. These data indicate that PAI-1 induction is an early event in creation of the wound-activated phenotype and appears to participate in the regulation of renal epithelial cell motility during in vitro injury resolution.

Tumor Necrosis Factor α Up-regulates in an Autocrine Manner the Synthesis of Plasminogen Activator Inhibitor Type-1 during Induction of Monocytic Differentiation of Human HL-60 Leukemia Cells

Tumor necrosis factor-alpha (TNFalpha) critically regulates several cellular functions during monocyte/macrophage differentiation. We therefore investigated during the phorbol ester (phorbol 12-myristate 13-acetate (PMA))-induced monocyte/macrophage differentiation of the human HL-60 leukemia cells, if TNFalpha contributed to plasminogen activator inhibitor type-1 (PAI-1) synthesis that is initiated by a protein kinase Cbeta-extracellular signal-regulated kinase 2-dependent pathway (Lopez, S., Peiretti, F., Morange, P., Laouar, A., Fossat, C., Bonardo, B., Huberman, E., Juhan-Vague, I., and Nalbone, G. (1999) Thromb. Haemostasis 81, 415-422). Following PMA treatment, the level of TNFalpha mRNA strongly increased and appeared earlier than PAI-1 mRNA. An anti-TNFalpha antibody significantly inhibited the PMA-induced PAI-1 mRNA and protein levels. The recombinant human TNFalpha, which is inactive on native HL-60 cells in terms of PAI-1 synthesis, optimally potentiates it once HL-60 cells are committed into the differentiation process. The use of 1) the HL-525 cell line, a clone issued from HL-60 cells rendered resistant to PMA-induced differentiation, and 2) the transforming growth factorbeta-1/vitamin D3 differentiative mixture confirmed the relationships between the induction of differentiation and the potency of TNFalpha to up-regulate PAI-1 synthesis. In conclusion, we showed that during the induction of monocyte/macrophage differentiation, TNFalpha and PAI-1 gene expressions are activated and that synthesized TNFalpha up-regulates and prolongs, in an autocrine manner, the synthesis of PAI-1.

Ethanol Downregulates Transcription of the PAI-1 Gene in Cultured Human Endothelial Cells

Human endothelial cells are a major site of synthesis for plasminogen activator inhibitor type-1. Elevated plasminogen activator inhibitor type-1 levels in young survivors of myocardial infarction [1] suggest that plasminogen activator inhibitor type-1 may have an important pathologic role in the development of coronary artery disease. Epidemiological studies indicate that moderate alcohol consumption (1–2 drinks/day) reduces the risk for cardiovascular mortality. This cardioprotective benefit has been attributed in part to an increase in fibrinolysis, which decreases fibrin-based thrombosis. The studies described herein were performed to determine whether moderate levels of ethanol affect plasminogen activator inhibitor type-1 gene expression. Cultured human endothelial cells were exposed to 0.1% v/v ethanol for 1 hour. Following incubation in the absence of ethanol plasminogen activator inhibitor type-1, mRNA levels were decreased in a time- and dose-dependent manner, reaching a maximum decrease of 3- to 4-fold at 2 to 4 hours following ethanol challenge. This decline in mRNA occurs at the transcription level; therefore, nuclear transcription run-on assays were performed. A 2.5- to 5-fold decrease in the rate of plasminogen activator inhibitor type-1 gene transcription was measured at 2 and 4 hours following ethanol challenge. Next, a 3.4- and a 1.1-kb fragment from the plasminogen activator inhibitor type-1 promoter region were linked to a luciferase reporter gene, and these constructs were transfected into human endothelial cells. Treatment of these transiently transfected human endothelial cells with ethanol showed a 2- to 3.5-fold decrease in promoter activity, respectively. These results indicate that low doses of ethanol downregulate transcription of the plasminogen activator inhibitor type-1 gene in cultured human endothelial cells. However, the mechanism(s) for this transcriptional decrease is currently unknown.

Growth state-dependent regulation of plasminogen activator inhibitor type-1 gene expression during epithelial cell stimulation by serum and transforming growth factor-beta1.

Transcription of the plasminogen activator inhibitor type-1 (PAI-1) gene appears to be growth state regulated in several cell types (e.g. , Ryan and Higgins, 1993, J Cell Physiol 155:376-384; Mu et al., 1998, J Cell Physiol 174:90-98). Transit of serum-stimulated normal rat kidney (NRK) epthelial cells through the first division cycle after release from quiescence (G(0)) provided a model system to assess the kinetics and mechanisms underlying PAI-1 expression in a growth "activated" phenotype. PAI-1 mRNA transcripts increased by more than 20-fold during the G(0)-->G(1) transition; induced expression had immediate-early response characteristics and abruptly declined prior to the onset of DNA synthesis. Transcriptional activity of the PAI-1 gene paralleled the steady-state mRNA abundance profile during this first synchronized growth cycle after release from quiescence. Although PAI-1 mRNA levels were up-regulated (approximately threefold) upon exposure to several different growth factors, neutralizing antibodies to transforming growth factor-beta1 (TGF-beta1) effectively attenuated the more than ninefold serum-associated PAI-1 inductive response by more than 70% (at both the mRNA transcript and protein levels). Similar to the metabolic requirements for serum-mediated PAI-1 transcription, PAI-1 induction upon addition of TGF-beta1 to quiescent NRK cell cultures was actinomycin D sensitive and resistant to cyclohexamide and puromycin, suggesting a primary mode of transcript control. The response to protein synthesis inhibitors, however, was complex. While cyclohexamide appeared to stabilize, or at least maintain, fetal bovine serum (FBS)- or TGF-beta1-stimulated PAI-1 mRNA levels, puromycin had no such affect. The amplitude and duration of induced PAI-1 expression were the same in either the presence or absence of puromycin. Cyclohexamide when used alone (i.e., in non-FBS- or TGF-beta1-treated cultures), moreover, effectively stimulated PAI-1 induction whereas puromycin was ineffective. Although TGF-beta1 was not a complete mitogen in the NRK cell system, incubation of quiescent renal cell cultures with TGF-beta1, prior to serum stimulation, resulted in a 10- to 12-fold increase in PAI-1 expression coincident with exit out of G(0). These data support a model in which PAI-1 gene expression is closely associated with creation of the growth-activated state and that cell cycle controls appear to be superimposed on the time course of the serum-induced expression of the PAI-1 gene.

Fibrin Fragment Induction of Plasminogen Activator Inhibitor Transcription Is Mediated by Activator Protein-1 Through a Highly Conserved Element

Plasminogen activator inhibitor type-1 (PAI-1), a serine protease inhibitor, affects the processes of fibrinolysis, wound healing, and vascular remodeling. We have demonstrated that PAI-1 transcription is induced by D dimer, a plasmin proteolytic fragment of fibrin, supporting its role in negative feedback on peri-cellular proteolysis. The focus of this study was to define the mechanism of D dimer's effects on PAI-1 transcription. D dimer increased the binding activity of the transcription factor activator protein-1 components c-fos/junD and c-fos mRNA levels in a time- and concentration-dependent manner to a greater extent than fibrinogen. Both basal and D dimer-induced PAI-1 transcriptional activity were entirely dependent on elements within the -161 to -48 bp region of the PAI-1 gene in fibroblasts. Mutations within the AP-1-like element (-59 to -52 bp) in the PAI-1 gene affected D dimer-induced transcriptional activity, c-fos/junD DNA binding, and basal and c-fos inducible PAI-1 transcriptional activity. Furthermore, expression of either wild-type or mutant c-fos proteins augmented or diminished the response of the PAI-1 promoter (-161 to +26 bp) to D dimer, respectively. D dimer-induced binding of c-fos/junD to the highly conserved and unique AP-1 like element in the PAI-1 gene provides a mechanism whereby specific fibrin fragments control fibrin persistence at sites of inflammation, fibrosis, and neoplasia.

Plasminogen activator inhibitor type-2 (PAI-2) gene transcription requires a novel NF-kappaB-like transcriptional regulatory motif.

Induction of human plasminogen activator inhibitor type-2 (PAI-2) gene transcription is the response of macrophages to inflammatory stimuli, such as the pleiotropic cytokine, tumour necrosis factor-alpha (TNFalpha). Here we have examined whether PAI-2 gene transcription in response to TNFalpha may be mediated through a regulatory pathway involving the transcription factor, NF-kappaB. We have tested the function of two potential NF-kappaB-like sites present in the PAI-2 proximal promoter for responsiveness to TNFalpha using chloramphenicol acetyl transferase reporter gene deletion and mutation analyses. While no evidence was found for TNFalpha regulation of the PAI-2 gene through either of these two sites, one of the NF-kappaB-like motifs, transcriptional regulatory motif (TRM), present at position -400 was found to be essential for constitutive PAI-2 transcription, as mutation of this motif abolished basal PAI-2 promoter activity in both monocyte-like U937 cells and HT1080 fibrosarcoma cells. Competition electrophoretic mobility shift assays identified four TRM-binding proteins present in U937, HT1080 and HeLa cell extracts, which bound to this motif but were not components of the NF-kappaB regulatory complex. Expression screening of a HeLa cell cDNA library using the -400 TRM as a probe identified two cDNAs encoding partial peptides which specifically bound the TRM motif. DNA sequence analysis revealed that one cDNA was novel, and the second cDNA encoded exon 5 of the nephroblastoma overexpressed (novH) proto-oncogene, suggesting a new role for this peptide in gene regulation. Taken together, these findings identify a new regulatory element required for constitutive PAI-2 transcription, and identify potential DNA-binding proteins associated with this element that may play a role in PAI-2 gene regulation.

Plasminogen activator inhibitor type 1 in ischemic cardiomyopathy.

Plasminogen activator inhibitor type 1 (PAI-1) is a proteinase inhibitor1 constituting the key regulator2 of the activity of the fibrinolytic system, an important protective mechanism against thrombosis. Because a reduced fibrinolytic activity, mainly caused by increased plasma levels of PAI-1, was a common finding in cross-sectional studies of patients with coronary artery disease (CAD),3 high PAI-1 levels have been regarded as a risk factor for recurrent myocardial infarction (MI).4 5 6 It has also been proposed that a reduced fibrinolytic activity may have a role in atherosclerotic plaque formation and in a prothrombotic state, carrying an increased risk of arterial occlusion. Accordingly, over the last few years, several studies have been performed to investigate the role of plasma PAI-1 levels in the development of CAD and MI. Moreover, the observation that polymorphisms of the PAI-1 gene exist, some of which apparently associated with increased plasma PAI-1 levels, stimulated studies aiming at investigating the potential relation between PAI-1 genotypes and ischemic cardiomyopathy. Thus, we shall focus on recent advances in the knowledge of the role of PAI-1 and its gene in the development of ischemic cardiomyopathy. Intravascular fibrinolytic activity results from a balance between plasminogen activators, such as the tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA), and inhibitors, such as PAI-1 and α2-antiplasmin. PAI-1 is a glycoprotein with a molecular mass of ≈54 kDa that belongs to the serine protease inhibitor superfamily (serpins). Its primary structure was deduced from the sequence of its cDNA.7 It is considered the major physiological inhibitor of t-PA and urokinase, because plasmin formation and fibrinolysis, as well as formation of other extracellular proteases, strongly depend on PAI-1 levels.8 Although the principal source of plasma PAI-1 is unknown, available evidence indicates that several cell types, including endothelial and vascular …

Differential Regulation of PAI-1 Gene Expression in Human Fibroblasts Predisposed to a Fibrotic Phenotype

Synthesis of the major negative physiologic regulator of plasmin activation [plasminogen activator inhibitor type-1 (PAI-1)] is elevated during progressive cellular senescence, in premature aging disorders (e.g., Werner's syndrome), and in conditions associated with tissue fibrosis and excessive fibrin accumulation (e.g., sclerosis, keloid formation). Dermal fibroblasts derived from Werner's patients as well as from keloid lesions markedly overexpress PAI-1 mRNA transcripts compared to normal skin fibroblasts. Such cell type-related differences in steady-state PAI-1 mRNA content, and variances in the relative abundance of the 3.0- compared to the 2.2-kb PAI-1 mRNA species, served to discriminate normal from Werner's and keloid fibroblasts. This disparity in PAI-1 mRNA levels paralleled transcriptional activities of the PAI-1 gene;de novosynthesis of PAI-1 protein among the three cell types, moreover, closely approximated the respective differences in total PAI-1 mRNA content. Despite the markedly elevated levels of PAI-1 mRNA and protein evident in newly confluent keloid fibroblasts, these cells effectively suppressed PAI-1 synthesis (as did normal dermal fibroblasts) upon culture in serum-free medium. Werner's syndrome skin fibroblasts, in contrast, continued to maintain high-level PAI-1 expression even after 3 days of growth arrest. Adhesion-mediated attenuation of serum-stimulated PAI-1 expression, a characteristic of normal cells involving sequences which mapped to the distal 5′ flanking region of the PAI-1 gene, was retained in keloid but not Werner's fibroblasts. Collectively, these data suggest that (1) specific controls on PAI-1 gene expression are fundamentally different between these two clinically significant high PAI-1-synthesizing cell types and (2) the localized keloid may define the emergence of a distinct profibrotic dermal fibroblastoid phenotype in genetically predisposed individuals.

Cooperative Binding of Smad Proteins to Two Adjacent DNA Elements in the Plasminogen Activator Inhibitor-1 Promoter Mediates Transforming Growth Factor β-induced Smad-dependent Transcriptional Activation

Transforming growth factor beta (TGFbeta) activates transcription of the plasminogen activator inhibitor type-1 (PAI-1) gene through a major TGFbeta-responsive region (-740 and -647) in the PAI-1 promoter. This process requires the Smad family of signaling molecules. Upon phosphorylation by the TGFbeta receptors, Smad2 and Smad3 homoligomerize and heteroligomerize with Smad4, translocate to the nucleus and activate transcription of TGFbeta responsive genes. Smad3 and Smad4 have been shown to bind to various sites in the PAI-1 promoter. To determine the number of Smad-binding sites within the 94-base pair major TGFbeta-responsive region and the mechanism of Smad-mediated transactivation, we systematically mapped the Smad-binding sites and show that Smad4 and Smad3 bind cooperatively to two adjacent DNA elements in this region. Both elements were required for TGFbeta-induced, Smad3- and Smad4-dependent activation of PAI-1 transcription. Contrary to previous reports, transactivation of the PAI-1 promoter was mediated by the amino- but not carboxyl-terminal domains of the Smads. Furthermore, oligomerization of Smad3 markedly enhanced its binding to the two binding sites. Finally, a Smad4 mutation identified in a human pancreatic carcinoma that inactivates Smad4 signaling abolished Smad4 DNA binding activity, hence preventing transactivation of TGFbeta-responsive genes. These results underscore the importance of the Smad4 DNA binding activity in controlling cell growth and carcinogenesis.

Perturbation of the actin cytoskeleton induces PAI-1 gene expression in cultured epithelial cells independent of substrate anchorage.

Perturbation of cellular architecture with agents that alter cytoskeletal organization provides a means to assess the relationship between cell shape and gene expression. Induced transcription of the plasminogen activator inhibitor type-1 (PAI-1) gene in serum-free cultures of normal rat kidney (NRK-52E) cells following disruption of actin microfilament structures with cytochalasin D (CD) provides a simple model to probe mechanisms underlying shape-related expression control. Transition from the typical flat epithelial cell shape to an "arborized" phenotype was a concomitant of the PAI-1 inductive response. Stimulated expression occurred rapidly (i.e., within 2 h of CD addition), involved increases in both PAI-1 mRNA abundance and de novo protein synthesis, and was dependent upon the concentration of CD used. A series of culture conditions were designed (e.g., use of bacteriological surfaces, poly-HEMA coated surfaces, maintenance in suspension on agarose) to discriminate cell shape from adhesive influences on CD-stimulated PAI-1 expression. Cytoskeletal disruption, and not simply changes in cell shape, was a critical aspect of CD-mediated PAI-1 expression in NRK cells cultured under serum-free conditions; induced expression was independent of substrate anchorage. Low concentrations of CD (1-2 microM) failed to cause cell arborization or increase either relative PAI-1 mRNA/protein abundance levels suggesting, however, that cell rounding may be a necessary but not sufficient aspect in CD-mediated PAI-1 induction. Transfection of PAI-1 promoter-CAT reporter constructs into NRK cells followed by stimulation with CD or serum additionally indicated that CD-induced PAI-1 expression did not utilize the same functional complement of serum-responsive promoter sequences, thus, further defining differences in the growth factor- and cytoskeletal-mediated pathways of PAI-1 gene regulation.

Perturbation of the actin cytoskeleton induces PAI‐1 gene expression in cultured epithelial cells independent of substrate anchorage

Perturbation of cellular architecture with agents that alter cytoskeletal organization provides a means to assess the relationship between cell shape and gene expression. Induced transcription of the plasminogen activator inhibitor type-1 (PAI-1) gene in serum-free cultures of normal rat kidney (NRK-52E) cells following disruption of actin microfilament structures with cytochalasin D (CD) provides a simple model to probe mechanisms underlying shape-related expression control. Transition from the typical flat epithelial cell shape to an “arborized” phenotype was a concomitant of the PAI-1 inductive response. Stimulated expression occurred rapidly (i.e., within 2 h of CD addition), involved increases in both PAI-1 mRNA abundance and de novo protein synthesis, and was dependent upon the concentration of CD used. A series of culture conditions were designed (e.g., use of bacteriological surfaces, poly-HEMA coated surfaces, maintenance in suspension on agarose) to discriminate cell shape from adhesive influences on CD-stimulated PAI-1 expression. Cytoskeletal disruption, and not simply changes in cell shape, was a critical aspect of CD-mediated PAI-1 expression in NRK cells cultured under serum-free conditions; induced expression was independent of substrate anchorage. Low concentrations of CD (1–2 μM) failed to cause cell arborization or increase either relative PAI-1 mRNA/protein abundance levels suggesting, however, that cell rounding may be a necessary but not sufficient aspect in CD-mediated PAI-1 induction. Transfection of PAI-1 promoter-CAT reporter constructs into NRK cells followed by stimulation with CD or serum additionally indicated that CD-induced PAI-1 expression did not utilize the same functional complement of serum-responsive promoter sequences, thus, further defining differences in the growth factor- and cytoskeletal-mediated pathways of PAI-1 gene regulation. Cell Motil. Cytoskeleton 42:218–229, 1999. © 1999 Wiley-Liss, Inc.

DNase I hypersensitive sites in the 5' flanking region of the human plasminogen activator inhibitor type 2 (PAI-2) gene are associated with basal and tumor necrosis factor-alpha-induced transcription in monocytes.

The plasminogen activator inhibitor type 2 (PAI-2) gene encodes a serine proteinase inhibitor (serpin) which is rapidly induced in response to the inflammatory cytokine, tumour necrosis factor-alpha (TNFalpha) in monocytes and macrophages. As an initial step towards understanding the molecular mechanisms underlying PAI-2 gene regulation in monocytes, we report here the analysis of the chromatin structure of 9.6 kb of 5' flanking region of the human PAI-2 gene for potential cis-acting regulatory regions using DNase I hypersensitivity mapping. Sites sensitive to DNase I were mapped in two monocytic cell lines representative of early monocytic differentiation; U937 cells, which synthesise low constitutive levels of PAI-2 that were induced following treatment with TNFalpha, and a MonoMac6 cell line which did not synthesise PAI-2 even after treatment with TNFalpha. Six DNase I hypersensitive sites (DHS) were identified; three upstream of the transcription initiation site (DH1, DH2, DH3) and three downstream of the transcription initiation site which were contained within intron A (DH4, DH5) and the exon 2/intron B junction (DH6). Among these, one distally located DH site (DH2) disappeared in both cell lines following treatment with TNFalpha. Two DH sites (DH1, DH6) were absent in PAI-2-producing U937 cells, but were present in MonoMac6 cells, which did not produce PAI-2, indicating the possible involvement of negative regulatory elements in the suppression of PAI-2 gene expression. The results demonstrate the involvement of chromatin structure in transcriptional responsiveness of the PAI-2 gene promoter and identify several loci which may be key control regions for PAI-2 gene transcription.

Cell shape‐dependent pathway of plasminogen activator inhibitor type‐1 gene expression requires cytoskeletal reorganization

Synthesis of plasminogen activator inhibitor type-1 (PAI-1), a major physiological modulator of plasmin generation, is regulated by growth factors and changes in cell shape. To evaluate the specific relationship between PAI-1 gene expression and cytoarchitecture, serum-free cultures of quiescent rat kidney (NRK) cells were exposed to cytochalasin D (CD) at concentrations that disrupt microfilament structure. Treatment with 1–10 μM CD resulted in an increased (1) incidence of rounded cells, (2) relative PAI-1 mRNA content, and (3) fraction of PAI-1 protein-expressing cells. Abrupt increases in each response were evident at a final concentration of 5 μM CD. Maximal levels of induced PAI-1 transcripts (18-fold that of control) occurred 4 hours post-CD addition and declined thereafter but remained elevated (by at least tenfold) for 24 hours. Assessment of the metabolic requirements for CD-induced PAI-1 expression by using the protein synthesis inhibitors puromycin and cycloheximide indicated that PAI-1 transcripts were regulated in a complex manner in response to CD. The predominant mode of induction reflected secondary (protein synthesis-dependent) metabolic processes, although a minor, albeit significant, primary (protein synthesis-independent) pathway was also evident. PAI-1 mRNA levels in NRK cells maintained in serum- and CD-free agarose suspension culture were low or undetectable. Relative abundance of PAI-1 transcripts in suspended cells cultured in the presence of CD, however, closely approximated that of plastic-adherent, CD-treated cells (13-fold over control). NRK cells in suspension culture with or without CD were morphologically identical, remaining spherical and unattached. It appears, therefore, that cell rounding alone is not a sufficient stimulus to induce PAI-1 expression in quiescent NRK cells and that perturbation of the actin skeleton as a consequence of CD treatment is a critical event in the inductive response. A protein tyrosine kinase is likely involved in the CD-mediated signal-transduction cascade, since induced PAI-1 expression can be down-regulated by genistein and herbimycin A but not by calphostin C or tyrphostin B46. J. Cell. Physiol. 176:293–302, 1998. © 1998 Wiley-Liss, Inc.