Regulation of the human plasminogen activator inhibitor type 2 gene: cooperation of an upstream silencer and transactivator.

Transcriptional up-regulation of the plasminogen activator inhibitor type-2 (PAI-2) gene is a major response to cellular stress. The expression of PAI-2 is induced by a variety of cytokines and growth factors that act in a cell type- and differentiation stage-dependent manner. We previously reported that the human SERPINB2 gene promoter is controlled by three major transcription regulatory domains: an inducible proximal promoter, an upstream silencer (PAUSE-1), and a distal transactivator region between -5100 and -3300, which appears to overcome inhibition mediated by the silencer. The distal transactivator region is inducible by the phorbol ester PMA, a potent activator of the protein kinase C (PKC) pathway that is a powerful inducer of PAI-2 gene expression in monocytes, macrophages, and myelomonocytic cells as well as in epidermal keratinocytes. Here we show that a 21-bp region (-4952/-4932), containing an AP-1 element, is both necessary and sufficient for PMA-induced transactivator activity in PAI-2-expressing U937 cells. This site specifically binds FosB in PAI-2-expressing U937 cells but not in HeLa cells that do not express PAI-2, and overexpression of FosB, c-Fos, or c-Jun in HeLa cells is sufficient to cause derepression of transcription from the SERPINB2 promoter. Although FosB is likely to be involved in transactivator-mediated derepression of PAI-2 transcription in macrophage-like cells, as exemplified by the U937 cell line, c-Jun may be functional in other cell types. These data suggest a model for the transcriptional control of the human PAI-2 gene and further our understanding of the molecular basis for its tissue-specific expression.

The plasminogen activator system in fibroblasts from systemic sclerosis.

Systemic sclerosis (SSc) is characterized by excessive fibrosis throughout the body. There are two major subsets of SSc, diffuse cutaneous Systemic sclerosis (dSSc) and limited cutaneous Systemic sclerosis (lSSc). Fibroblasts play a key role in SSc. The expression and function of the urokinase (uPA)-mediated plasminogen activation (PA) system, a well-characterized system of serine-proteases involved in several pathological processes, has been investigated in SSc fibroblasts. The expression of the components of the PA system, including uPA, its type-1 and type-2 inhibitors (PAI-1 and PAI-2) and its receptor (uPAR), was examined by Western blot in fibroblasts from patients affected by limited and diffuse forms of SSc. uPA and PAI-1 secretion increased only in fibroblasts from lSSc lesions compared to normal fibroblasts. PAI-2 levels were decreased in fibroblasts from both SSc forms. Interestingly, fibroblasts from areas not adjacent to the lesions (not-affected) of the diffuse form showed reduced levels of PAI-1 and increased uPAR expression. Adhesion experiments showed reduced adherence to VN of fibroblasts from lSSc lesions and from non-affected areas of the diffuse form, as compared to normal controls. These results suggest a role for uPA and PAI-1 in the lSSc form, likely related to the activation of latent forms of cytokines and to the accumulation of ECM components, whereas a role for uPAR can be hypothesized in the evolvement of the diffuse form, based on its up-regulation in the non-affected areas.

The impact of tyrosine kinase 2 (Tyk2) on the proteome of murine macrophages and their response to lipopolysaccharide (LPS).

Tyrosine kinase 2 (Tyk2) belongs to the Janus kinase (Jak) family and is involved in signalling via a number of cytokines. Tyk2-deficient mice are highly resistant to lipopolysaccharide (LPS)-induced endotoxin shock. Macrophages are key players in the pathogenesis of endotoxin shock and, accordingly, defects in the LPS responses of Tyk2(-/-) macrophages have been reported. In the present study, the molecular role of Tyk2 is investigated in more detail using a proteomics approach. 2-D DIGE was applied to compare protein patterns from wild-type and Tyk2(-/-) macrophages and revealed significant differences in protein expression patterns between the genotypes before and after LPS treatment. Twenty-one proteins deriving from 25 differentially expressed spots were identified by MALDI/ESI MS. Among them, we show for N-myc interactor that its mRNA transcription/stability is positively influenced by Tyk2. In contrast, LPS-induced expression of plasminogen activator 2 protein but not mRNA is strongly enhanced in the absence of Tyk2. Our data furthermore suggest an influence of Tyk2 on the subcellular distribution of elongation factor 2 and on LPS-mediated changes in the peroxiredoxin 1 spot pattern. Thus, our results imply regulatory roles of Tyk2 at multiple levels and establish novel connections between Tyk2 and several cellular proteins.

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

BACKGROUND AND OBJECTIVE: 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. MATERIAL AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Angiotensin II induces plasminogen activator inhibitor-1 and -2 expression in vascular endothelial and smooth muscle cells.

Angiotensin II (AII)- and Arg8-vasopressin (AVP)-regulated gene expression in vascular cells has been reported to contribute to vascular homeostasis and hypertrophy. In this report, AVP-induced expression of plasminogen activator inhibitor (PAI)-2 mRNA in rat microvessel endothelial (RME) cells was identified using differential mRNA display. Further characterization of vasoactive peptide effects on PAI expression revealed that AII stimulated a 44.8 +/- 25.2-fold and a 12.4 +/- 3.2-fold increase in PAI-2 mRNA in RME cells and rat aortic smooth muscle cells (RASMC), respectively. AII also stimulated a 10- and 48-fold increase in PAI-1 mRNA in RME cells and RASMC, respectively. These AII effects were inhibited by either Sar1, Ile8-angiotensin or the AT1 antagonist DuP 735, but were not significantly altered in the presence of the AT2 antagonist PD123319. AII stimulation of RASMC and RME cells also significantly increased both PAI-1 protein and PAI activity released to the culture medium. Inhibition of protein kinase C completely blocked PMA-stimulated induction of PAI-2 mRNA in both cell types and inhibited the AII-stimulated increase in RASMC by 98.6 +/- 2.8%. In contrast, protein kinase C inhibition only partially decreased the AII-stimulated PAI-2 expression in RME cells by 68.8 +/- 11.1%, suggesting that a protein kinase C-independent mechanism contributes to a 6.9 +/- 1.5-fold AII induction of PAI-2 expression in endothelial cells. AII and PMA also stimulated protein tyrosine phosphorylation in RME cells, and the tyrosine kinase inhibitor genistein partially blocked their induction of PAI-2 mRNA. These findings suggest that AII may regulate plasminogen activation in the vasculature by inducing both PAI-1 and PAI-2 expression.

Characterisation of PAUSE-1, a powerful silencer in the human plasminogen activator inhibitor type 2 gene promoter.

Plasminogen activator inhibitor type 2 (PAI-2) is a serine protease inhibitor traditionally regarded as a regulator of fibrinolysis and extracellular matrix degradation. More recently, PAI-2 has been implicated in diverse processes such as keratinocyte differentiation, cell death and viral pathogenesis. The PAI-2 promoter tightly regulates PAI-2 gene expression in a cell-specific manner and this control is mediated, in part, by the upstream silencer element, PAUSE-1. Here we have defined PAUSE-1 and investigated its activity as a silencer. A series of mutations were generated within the PAUSE-1 element and analysed for transcription factor binding and transcriptional silencing activity. These studies have defined the minimal functional PAUSE-1 element as TCTN(x)AGAN(3)T(4), where x = 0, 2 or 4. Examination of related elements present in other promoters, such as the human IFNbeta promoter, suggests that PAUSE-1 is a member of a family of universal silencers with the consensus sequence TCTN(x)AGA. UV crosslinking analyses determined that the PAUSE-1 binding protein was approximately 67 kDa. Insertion of PAUSE-1 into the heterologous (SV40) or the minimal PAI-2 promoters silenced transcription by 2.5-fold. These data show that PAUSE-1 acts as a powerful silencer of PAI-2 gene transcription and is likely to be important in the silencing of other genes as well.

Inhibition of invasion of HT1080 sarcoma cells expressing recombinant plasminogen activator inhibitor 2.

Plasminogen activators (PA) elaborated by tumor cells play an important role in the complex process of tissue invasion and metastasis. In the present study the effect of the PA inhibitor type 2 (PAI-2) on tissue invasion in vitro and in vivo was investigated. Clones either expressing (B-) or not expressing the endogenous PAI-2 gene (C+) were isolated from the human HT1080 fibrosarcoma cell line and transfected with full-length PAI-2 cDNA. Recombinant PAI-2 (rPAI-2) expressed by these cells completely inhibited receptor-bound urokinase activity and partially neutralized secreted PA activity. Degradation of extracellular matrix proteins by these transfected cells was markedly decreased when compared to mock or untransfected control cells. The rPAI-2-expressing cells did not penetrate a multilayer of rat smooth muscle cells in vitro, which was readily invaded and destroyed by control cells. The PAI-2 transfectants remained tumorigenic in athymic/nude mice, but tumors originating from these cells showed the presence of a thick, collagenous capsule absent in tumors formed by control cells. Thus, expression of rPAI-2 in HT1080 cells resulted in neutralization of receptor-bound urokinase with subsequent inhibition of matrix protein degradation and invasion in vitro and induction of a thick, peritumoral capsule in vivo.

Helicobacter pylori induces plasminogen activator inhibitor 2 in gastric epithelial cells through nuclear factor-kappaB and RhoA: implications for invasion and apoptosis.

The gastric pathogen Helicobacter pylori is associated with a progression to gastric cancer. The specific targets of H. pylori that might influence this progression are still unclear. Previous studies indicated that the gastric hormone gastrin, which may be increased in H. pylori infection, stimulates gastric expression of plasminogen activator inhibitor (PAI)-2, which is an inhibitor of the urokinase plasminogen activator and has previously been shown to be increased in gastric adenocarcinoma. Here, we report that H. pylori also increases PAI-2 expression. In gastric biopsies of H. pylori-positive subjects there was increased PAI-2, including subjects with plasma gastrin concentrations in the normal range. PAI-2 was expressed mainly in chief and mucous cells. In a gastric cancer cell line (AGS), H. pylori increased PAI-2 expression, which was associated with inhibition of H. pylori-stimulated cell invasion and apoptosis. The induction of PAI-2 by H. pylori was mediated by release of interleukin-8 and activation of cyclooxygenase-2, and interestingly, gastrin stimulated PAI-2 expression by similar paracrine pathways. The activation of NFkappaB was required for interleukin-8 and cyclooxygenase-2 activation but did not occur in cells responding to these paracrine mediators. The data suggest that induction of PAI-2 is a specific target in H. pylori infection, mediated at least partly by paracrine factors; induction of PAI-2 inhibits cell invasion and apoptosis and is a candidate for influencing the progression to gastric cancer.

Overexpression of plasminogen activator inhibitor type 2 in basal keratinocytes enhances papilloma formation in transgenic mice.

The serpin plasminogen activator inhibitor (PAI) type 2 is expressed in differentiated epidermal keratinocytes. To explore its role in this tissue, we studied the impact of PAI-2 overexpression on epidermal differentiation and skin carcinogenesis. A mouse PAI-2-encoding transgene was targeted to basal epidermis and hair follicles under the control of the bovine keratin type 5 gene promoter. Two mouse lines were established, one of which strongly expressed the transgene and produced elevated levels of PAI-2 in the epidermis. Although it had no manifest impact on cellularity or differentiation of skin or hair follicles, PAI-2 overexpression rendered the mice highly susceptible to skin carcinogenesis induced by a single application of 7,12-dimethylbenz(a)anthracene (initiation) followed by twice weekly applications of 12-O-tetradecanoylphorbol-13-acetate [TPA (promotion)]. In transgenic mice, papillomas could be observed after 3 weeks of promotion; after 8 weeks, 94% (31 of 33) of transgenic mice had developed readily visible papillomas, whereas only 35% (7 of 20) of control mice (transgene-negative littermates) had barely detectable lesions. After 11 weeks, all but 1 (32 of 33) of the transgenic mice had papillomas as compared with only 65% (13 of 20) of control mice. After 11 weeks of promotion, application of TPA was terminated. In control mice, papillomas regressed and eventually disappeared; in transgenic mice, there was continued growth of papillomas, some of which further progressed to carcinomas. In contrast to massive apoptosis in regressing papillomas of control mice, only a few apoptotic cells were detected in transgenic papillomas after the cessation of TPA application. The effect of PAI-2 on papilloma formation did not appear to involve inhibition of the secreted protease urokinase-type plasminogen activator (uPA): PAI-2 accumulated predominantly in cells, and PAI-2 overexpression failed to alleviate a phenotype induced by uPA secretion, as demonstrated by a double transgenic strategy. In addition, in situ hybridization revealed that uPA mRNA is not expressed concomitantly with PAI-2 in developing papillomas. We conclude that overexpression of PAI-2 promotes the development and progression of epidermal papillomas in a manner that does not involve inhibition of its extracellular target protease, uPA, but appears to be related to an inhibition of apoptosis.

Plasminogen activator inhibitor-2: a molecular biomarker for head and neck cancer progression.

Head and neck squamous cell carcinoma (HNSCC) is an aggressive epithelial malignancy in which the early diagnosis of premalignant lesions is known to directly correlate with increased survival. However, only a portion of biopsies showing dysplasia will progress to cancer, and there are no currently accepted criteria for predicting which lesions will progress. Therefore, diagnostic protocols that can identify the lesions that are likely to become HNSCC are required. RNA was isolated from normal keratinocytes, the immortalized but nontumorigenic HaCat cell line, and the tumor cell lines SCC-4, SCC-9, SCC-25, and OSCC-3. The RNA was then labeled and used to probe nylon microarray filters that contained a total of 9184 genes (5295 named and 3889 Expression Sequence Tags). Genes whose expression demonstrated a 3-fold or greater change were considered significant. Comparison of expression profiles from normal, HaCat, and four tumor cell lines revealed changes in gene expression in a total of 508 genes. Of these, 16 genes showed a consistent loss of expression when comparing normal to immortalized keratinocytes. In addition, 10 genes demonstrated a consistent loss of expression in the tumor cell lines only. In this latter group of genes, plasminogen activator inhibitor-2 (PAI-2), a gene whose expression has been linked to cell invasion, was additionally investigated. Altered expression of PAI-2 in the different cultured cells was validated via real-time quantitative-PCR. In addition, immunohistochemical evaluation of biopsy samples revealed a high expression of PAI-2 in both normal and dysplastic epithelium with a marked decrease of expression in areas of the biopsies containing HNSCC. These data demonstrate that genomic profiling can then be used to identify potential genotypic/phenotypic biomarkers that may predict which dysplastic lesions are most likely to progress to HNSCC.

Down-regulation of SerpinB2 is associated with gefitinib resistance in non-small cell lung cancer and enhances invadopodia-like structure protrusions.

The failure of targeted therapy due to the resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), such as gefitinib, is considered a major problem in the treatment of non-small cell lung cancer (NSCLC) patients. SerpinB2, a component of the urokinase plasminogen activator (uPA) system, has been recognized as a biomarker for the progression and metastasis of lung cancer. Nevertheless, the relationship between SerpinB2 and EGFR-TKI resistance has not been elucidated. Here, we report that SerpinB2 is down-regulated in gefitinib-resistant (H292-Gef) cells compared to gefitinib-sensitive (H292) cells. The low SerpinB2 levels in H292-Gef cells were also associated with an enhancement in invasiveness and increase in the length of invadopodia-like structures in the cells. The effect on invasiveness and gefitinib sensitivity was confirmed by knockdown and overexpression of SerpinB2. In addition, the possibility to overcome the resistance through the up-regulation of SerpinB2 was supported by employing an antitumor agent yuanhuadine (YD). Treatment with YD effectively elevated SerpinB2 levels and suppressed invasive properties in H292-Gef cells. Collectively, these findings demonstrate the prospective role of SerpinB2 as a novel biomarker for acquired gefitinib resistance and a potential target for NSCLC treatment.

The novel anti-tumour agent oxamflatin differentially regulates urokinase and plasminogen activator inhibitor type 2 expression and inhibits urokinase-mediated proteolytic activity.

Cell surface, urokinase (u-PA)-mediated, plasminogen activation has recently been recognised as a process integral to extracellular matrix degradation. The primary inhibitor of u-PA activity in the extracellular matrix is plasminogen activator inhibitor type 2 (PAI-2), a serine protease inhibitor. The malignant metastatic phenotype is associated with excessive and uncontrolled, tumour cell-associated, u-PA-mediated, extracellular matrix degradation. Inhibition of the malignant metastatic phenotype via induction of PAI-2 expression and/or inhibition of u-PA expression may represent a novel means via which the metastatic phenotype can be arrested. Agents capable of inducing PAI-2 and/or inhibiting u-PA activity may restrict u-PA-mediated tumour cell proteolysis and facilitate in the development of therapeutic strategies to combat malignant disease. We have identified the hydroxamic acid derivative oxamflatin, previously noted to revert the malignant phenotype in K-ras-transformed NIH-3T3 cells, as capable of upregulating PAI-2 and simultaneously suppressing u-PA expression in two different cell systems. In addition, zymographic analysis indicated that oxamflatin treatment results in a significant reduction in u-PA proteolytic activity in both HT-1080 fibrosarcoma and U-937 histiocytic lymphoma cells. We postulate that oxamflatin represents a novel means by which induction of PAI-2 and concomitant inhibition of u-PA gene and protein expression can be achieved and may be of benefit in inhibiting the malignant metastatic phenotype.

Changes in coagulation-fibrinolysis balance in blood mononuclear cells and in gastric mucosa from patients with Helicobacter pylori infection.

INTRODUCTION: Helicobacter pylori and some of its virulence factors stimulate human blood mononuclear cells (MNC) in vitro to produce tissue factor (TF) and plasminogen activator inhibitor-2 (PAI-2). In this study we investigated the procoagulant-fibrinolytic potential of blood MNC in patients with H. pylori infection. In the same patients we also evaluated the coagulation-fibrinolysis profile in gastric tissue and in plasma. METHODS AND RESULTS: The production of TF and PAI-2 was evaluated in 61 patients with dyspepsia, 31 positive and 30 negative for H. pylori infection. TF expressed by MNC and PAI-2 accumulation in cell culture medium after incubation for 20 h at 37 degrees C were significantly higher in H. pylori(+) than in H. pylori(-) patients and were significantly correlated. TF and PAI-2 content in extracts of gastric mucosa was similar in the two groups whereas lower levels of tissue plasminogen activator (t-PA) and thrombomodulin (TM) antigens were found in the antrum of H. pylori(+) patients. No difference between the groups was observed in plasma thrombus precursor protein, prothrombin fragment 1+2, D-dimer, t-PA, PAI-1, TM and thrombin activatable fibrinolysis inhibitor. CONCLUSIONS: H. pylori infection is associated with functional abnormalities of blood MNC resulting in the coordinate expression of TF and antifibrinolytic activity. Changes in cell coagulation-fibrinolysis balance may represent a link between H. pylori infection and ischemic heart disease.

Expression of plasminogen activator inhibitors 1 and 2 in lung cancer and their role in tumor progression.

The plasminogen activator cascade initiated by urokinase type plasminogen activator (u-PA) is involved in extracellular matrix degradation during the tumor invasion process. The plasminogen activator inhibitors 1 (PAI-1) and 2 (PAI-2) are two specific inhibitors of u-PA. We hypothesized that the balance between u-PA and its two inhibitors could be disrupted to favor plasminogen activation during lung cancer progression. Using immunohistochemistry, we analyzed the pattern of expression of u-PA, PAI-1, and PAI-2 in non-small cell lung carcinomas (NSCLC) and neuroendocrine (NE) lung tumors. u-PA and PAI-1 were both detected in stromal fibroblasts and in tumor cells. In 84 NSCLCs, their epithelial expression was strongly correlated and linked to the presence of node metastasis (P = 0.008), whereas their coexpression in fibroblasts was associated with larger tumor size (P = 0.04) and advanced stages (P = 0.009). In 72 NE tumors, u-PA and PAI-1 were more frequently expressed in fibroblasts in high-grade NE tumors (SCLC and large cell NE tumors) than in low- and intermediate-grade tumors (typical and atypical carcinoids). Comparison of in situ hybridization and immunohistochemistry in 14 cases showed that PAI-1 was consistently expressed by stromal fibroblasts, although the protein was also localized in tumor cells. In contrast, the expression of PAI-2 was restricted to fibroblasts and correlated with the absence of nodal involvement (P = 0.005). Considering NE tumors, the frequency of PAI-2 expression decreased along the NE spectrum from typical carcinoids to SCLCs. These data suggest that PAI-lacts in synergy with u-PA to favor tumor invasion process and connotes aggressivity, in contrast with PAI-2, which may block u-PA-mediated proteolysis and is inversely correlated with tumor progression.

Sodium dodecyl sulfate induces plasminogen activator inhibitor type 2 expression in epidermal keratinocytes in vivo and in vitro.

The detergent sodium dodecyl sulfate is a well-known inducer of irritant contact dermatitis. In this study we show that sodium dodecyl sulfate induces the serine proteinase inhibitor, plasminogen activator inhibitor type 2, in epidermal keratinocytes. The enhancement in plasminogen activator inhibitor type 2 mRNA and antigen is observed both when sodium dodecyl sulfate is applied topically to normal human skin as well as when it is added to the growth medium of cultured human keratinocytes. In vitro, plasminogen activator inhibitor type 2 mRNA is increased within 4-8 h after addition of the detergent, and the increase in plasminogen activator inhibitor type 2 antigen occurs slightly later. The enhancing effect of sodium dodecyl sulfate on plasminogen activator inhibitor type 2 is not related to nonspecific cell lysis nor is it secondary to induction of tumor necrosis factor alpha. Similarities between our in vitro and in vivo findings lead us to hypothesize that sodium dodecyl sulfate may exert its effect on epidermal plasminogen activator inhibitor type 2 via interaction with the keratinocyte.

Short-term cytotoxic effects and long-term instability of RNAi delivered using lentiviral vectors.

BACKGROUND: RNA interference (RNAi) can potently reduce target gene expression in mammalian cells and is in wide use for loss-of-function studies. Several recent reports have demonstrated that short double-stranded RNAs (dsRNAs), used to mediate RNAi, can also induce an interferon-based response resulting in changes in the expression of many interferon-responsive genes. Off-target gene silencing has also been described, bringing into question the validity of certain RNAi-based approaches for studying gene function. We have targeted the plasminogen activator inhibitor-2 (PAI-2 or SERPINB2) mRNA using lentiviral vectors for delivery of U6 promoter-driven PAI-2-targeted short hairpin RNA (shRNA) expression. PAI-2 is reported to have anti-apoptotic activity, thus reduction of endogenous expression may be expected to make cells more sensitive to programmed cell death. RESULTS: As expected, we encountered a cytotoxic phenotype when targeting the PAI-2 mRNA with vector-derived shRNA. However, this predicted phenotype was a potent non-specific effect of shRNA expression, as functional overexpression of the target protein failed to rescue the phenotype. By decreasing the shRNA length or modifying its sequence we maintained PAI-2 silencing and reduced, but did not eliminate, cytotoxicity. ShRNA of 21 complementary nucleotides (21 mers) or more increased expression of the oligoadenylate synthase-1 (OAS1) interferon-responsive gene. 19 mer shRNA had no effect on OAS1 expression but long-term selective pressure on cell growth was observed. By lowering lentiviral vector titre we were able to reduce both expression of shRNA and induction of OAS1, without a major impact on the efficacy of gene silencing. CONCLUSIONS: Our data demonstrate a rapid cytotoxic effect of shRNAs expressed in human tumor cell lines. There appears to be a cut-off of 21 complementary nucleotides below which there is no interferon response while target gene silencing is maintained. Cytotoxicity or OAS1 induction could be reduced by changing shRNA sequence or vector titre, but stable gene silencing could not be maintained in extended cell culture despite persistent marker gene expression from the RNAi-inducing transgene cassette. These results underscore the necessity of careful controls for immediate and long-term RNAi use in mammalian cell systems.

Urokinase-mediated transactivation of the plasminogen activator inhibitor type 2 (PAI-2) gene promoter in HT-1080 cells utilises AP-1 binding sites and potentiates phorbol ester-mediated induction of endogenous PAI-2 mRNA.

Urokinase-type plasminogen activator (u-PA) bound to its receptor, u-PAR, initiates signal transduction pathways able to induce expression of the activator protein-1 (AP-1) family member c-fos [1]. Since transcription factors bound to AP-1 recognition sequences within the PAI-2 gene promoter play a role in basal and phorbol ester-mediated induction of PAI-2 gene expression, we hypothesised that u-PA/u-PAR-mediated modulation of AP-1 activity would in turn influence constitutive and inducible PAI-2 gene expression. Treatment of HT-1080 or U-937 cells with high molecular weight u-PA (HMW u-PA) resulted in induction of nuclear proteins binding to a functional AP-1 element in the proximal PAI-2 promoter. This increase in AP-1 activity correlated with a transactivation of the PAI-2 gene promoter in transiently transfected HT-1080 cells. We also demonstrate the u-PA treatment potentiated phorbol ester (PMA)-mediated induction of PAI-2 mRNA, indicating that u-PA binding produces a bone fide response in vivo.

Cultured human mesangial cells produce both type 1 and type 2 plasminogen activator inhibitors.

Cultured human mesangial cells (HMC) derived from normal kidneys have been shown to synthesize tissue-type plasminogen activator (t-PA) and excess amounts of PA inhibitor type 1 (PAI-1). Conflicting results have been obtained concerning the production of urokinase-type PA (u-PA) and efforts to show PA inhibitor 2 (PAI-2) met with failure. We evaluated the fibrinolytic profile of cultured HMC lines obtained from 12 patients with renal carcinoma and one cadaveric kidney donor. Subconfluent cells (third passage) were incubated overnight in serum-free medium. t-PA, u-PA, PAI-1 and PAI-2 antigens were assayed by ELISA methods and PA and PAI activities by amidolytic methods both in conditioned medium (CM) and cell extracts (CE). Besides PAI-1, PAI-2 antigen was detected in all but one HMC lines. At variance with the former, which was largely released in the culture medium, PAI-2 was mainly cell-associated. t-PA antigen was found in all but two cell lines while u-PA antigen was detected in relatively high concentrations in 8 cell lines. PA activity, identified as u-PA by functional and immunological criteria, was measured in CM of six of the eight u-PA producing cell lines, whereas PAI activity was undetectable or very low in CM of all cell lines, suggesting that PAI-1 was largely inactive. Functional assays of cell extracts demonstrated the presence of PA activity, again identified as u-PA, only in samples (five lines) containing u-PA antigen in excess over PAI-2. PAI activity was found instead in the extracts in which the inhibitor was higher than the activator (six lines) and was identified as PAI-2, as it inhibited u-PA but not single-chain t-PA and was neutralized by a polyclonal anti-PAI-2 antibody. The heterogeneous fibrinolytic pattern of HMC lines was confirmed by mRNA analysis of three representative lines. Results were similar when HMC lines at passage five were used, except that the u-PA content was significantly reduced both in CM and CE. These findings indicate that the fibrinolytic profile of cultured HMC is more complex than previously reported. The production of large amounts of PAI-2 may represent an additional control mechanism of proteinase activity.

Decreased expression of PAI-2 mRNA and protein in pregnancies complicated with intrauterine fetal growth retardation.

An increase in plasma plasminogen activator inhibitors (PAIs), fundamentally PAI type 2 (PAI-2), has been described in normal pregnancy probably because the placenta is the main source of the high plasma levels of this protein. Although we have previously described plasmatic alterations of these inhibitors in pregnancies complicated with intrauterine fetal growth retardation (IUGR), no reports have been published about placental PAI-2 mRNA expression. In the present study, the placental PAI-2 expression determined in pregnancies complicated with IUGR and in severe preeclamptic patients was compared with that of normal pregnancies in order to identify the placental cell types expressing PAI-2 and to determine whether the production of PAI-2 is altered in placentas from IUGR. In situ hybridization analyses show that the syncytiotrophoblasts are the cells with the greatest PAI-2 expression in placenta. We report that the significant decrease in plasma and placental PAI-2 levels in IUGR groups is fundamentally due to a diminished expression of PAI-2 mRNA in placenta.

Cytokine regulation of the synthesis of plasminogen activator inhibitor-2 by human vascular endothelial cells. Comparison with plasminogen activator inhibitor-1 synthesis.

Plasminogen activators are inhibited by plasminogen activator inhibitors-1 (PAI-1) and -2 (PAI-2). We describe the synthesis of PAI-2 by human vascular endothelial cells (EC) cultured from umbilical vein, saphenous vein and foreskin microvasculature in response to interleukin-1 alpha (IL-1 alpha) and tumour necrosis factor alpha (TNF alpha) and compare it with that of PAI-1. Both PAI-2 and PAI-1 were quantitated by ELISAs. PAI-2 was cell-associated while PAI-1 was secreted by EC. IL-1 alpha and TNF alpha increased the synthesis of PAI-2 and PAI-1 by EC in a dose-dependent manner. IL-1 alpha was a stronger stimulus for PAI-2 synthesis than TNF alpha, while both cytokines were equally effective for PAI-1. Northern blot analysis revealed similar changes in mRNA levels to those in antigen levels. PAI-2 synthesis by cytokine-stimulated EC may be important in thrombus formation and inflammation.