Plasminogen Activator Inhibitor-1 Knockdown Research Articles

PAI-1 mediates acquired resistance to MET-targeted therapy in non-small cell lung cancer.

Mechanisms underlying primary and acquired resistance to MET tyrosine kinase inhibitors (TKIs) in managing non-small cell lung cancer remain unclear. In this study, we investigated the possible mechanisms acquired for crizotinib in MET-amplified lung carcinoma cell lines. Two MET-amplified lung cancer cell lines, EBC-1 and H1993, were established for acquired resistance to MET-TKI crizotinib and were functionally elucidated. Genomic and transcriptomic data were used to assess the factors contributing to the resistance mechanism, and the alterations hypothesized to confer resistance were validated. Multiple mechanisms underlie acquired resistance to crizotinib in MET-amplified lung cancer cell lines. In EBC-1-derived resistant cells, the overexpression of SERPINE1, the gene encoding plasminogen activator inhibitor-1 (PAI-1), mediated the drug resistance mechanism. Crizotinib resistance was addressed by combination therapy with a PAI-1 inhibitor and PAI-1 knockdown. Another mechanism of resistance in different subline cells of EBC-1 was evaluated as epithelial-to-mesenchymal transition with the upregulation of antiapoptotic proteins. In H1993-derived resistant cells, MEK inhibitors could be a potential therapeutic strategy for overcoming resistance with downstream mitogen-activated protein kinase pathway activation. In this study, we revealed the different mechanisms of acquired resistance to the MET inhibitor crizotinib with potential therapeutic application in patients with MET-amplified lung carcinoma.

Abstract 3277: PAI-1 mediates resistance to MET-targeted therapy in non-small cell lung cancer

Abstract [Purpose] The mesenchymal epithelial transition factor receptor (MET) is a potential therapeutic target in various cancers, including non-small cell lung cancer (NSCLC). In NSCLC, the activation of MET pathway is thought to occur through diverse mechanisms that influence properties affecting cancer cell survival, growth, and invasiveness. These tumors show oncogene addiction to MET and exhibit remarkable responses to MET-Tyrosine Kinase Inhibitors (TKIs). However, the long-term effectiveness of MET-TKIs is usually limited due to acquired drug resistance. The mechanisms underlying primary and acquired resistance to MET-TKIs in the management of NSCLC remain unclear. In this study, we investigated the mechanisms acquired for crizotinib in MET-amplified lung carcinoma cell lines. [Experimental Designs] Two MET-amplified lung cancer cell lines, EBC-1 and H1993, were established for acquired resistance to MET-TKI crizotinib and were functionally elucidated. The factors contributing to the resistance mechanism were assessed by genomic and transcriptomic data, and the alterations hypothesized to confer resistance were validated. [Results] Multiple mechanisms underlay acquired resistance to crizotinib in MET-amplified lung cancer cell lines. In EBC-1-derived resistant cells, the drug resistance mechanism was mediated by the overexpression of SERPINE1, the gene encoding plasminogen activator inhibitor-1 (PAI-1). Crizotinib resistance was overcome by combination therapy with PAI-1 inhibitor and PAI-1 knockdown. Another mechanism of resistance in different subline cells of EBC-1 was evaluated as epithelial to mesenchymal transition (EMT) with the upregulation of antiapoptotic proteins. In the H1993-derived resistant cells, the phosphorylated forms of MEK and ERK were observed to be upregulated and MEK inhibitors could be a potential therapeutic strategy for overcoming the resistance. [Conclusions] In this study, we revealed the different mechanisms of drug resistance to MET-TKI treatment and presented some possible strategies to overcome the resistance. Citation Format: Tomoaki Higashihara, Yin Min Thu, Ken Suzawa, Keiichi Date, Atsushi Matsuoka, Daisuke Mizuno, Ryo Yoshichika, Naohiro Hayashi, Fumiaki Mukohara, Mao Yoshikawa, Kazuhiko Shien, Hiromasa Yamamoto, Shinichi Toyooka. PAI-1 mediates resistance to MET-targeted therapy in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3277.

Suppression of antitumor cytokine IL‑24 by PRG4 and PAI‑1 may promote myxoid liposarcoma cell survival.

Suppression of the antitumor cytokine interleukin-24 (IL-24) is critical for the survival of myxoid liposarcoma (MLS) cells. It has been previously demonstrated by the authors that an MLS-specific chimeric oncoprotein, translocated in liposarcoma-CCAAT/enhancer-binding protein homologous protein (TLS-CHOP), supresses IL24 mRNA expression via induction of proteoglycan 4 (PRG4) to sustain MLS cell proliferation. However, IL-24 has also been revealed to be suppressed by the ubiquitin-proteasome system in human ovarian and lung cancer cells. Therefore, the aim of the present study was to elucidate the mechanism of IL-24 suppression in MLS cells. The results revealed that the proteasome inhibitor, MG-132, induced cell death in MLS cells in vitro; this effect was reduced following IL-24 knockdown. This indicated that proteasomal degradation of IL-24 may be an important process for MLS cell survival. In addition, it was also previously revealed by the authors that knockdown of plasminogen activator inhibitor-1 (PAI-1), a TLS-CHOP downstream molecule, suppressed the growth of MLS cells, thus instigating the investigation of the effect of PAI-1 on IL-24 expression in MLS cells. Double knockdown of PAI-1 and IL-24 negated the growth-suppressive effect of PAI-1 single knockdown in MLS cells. Interestingly, PAI-1 single knockdown did not increase the mRNA expression of IL24, but it did increase the protein abundance of IL-24, indicating that PAI-1 suppressed IL-24 expression by promoting its proteasomal degradation. Moreover, treatment of MLS cells with a PAI-1 inhibitor, TM5275, induced IL-24 protein expression and apoptosis. Collectively, the results of the present as well as previous studies indicated that IL-24 expression may be suppressed at the transcriptional level by PRG4 and at the protein level by PAI-1 in MLS cells. Accordingly, PAI-1 may represent an effective therapeutic target for MLS treatment.

Eupafolin suppresses TGF-β1-induced human lung fibroblasts differentiation by downregulating β-catenin/plasminogen activator inhibitor-1 signaling

The anti-fibrotic activity of flavonoids from Eupatorium lindleyanum DC (EL) remains unclear. Our results showed that flavones from the plant exerted a stronger inhibitory effect than flavonols, and eupafolin presented the highest activity on lung fibroblast differentiation. Eupafolin weakened the levels of α-smooth muscle actin, the production of type I collagen and fibronectin, fibroblast-containing gel contractility and microfilament formation in transforming growth factor-β1-induced lung fibroblasts. Interestingly, eupafolin did not affect the canonical TGF-β/Smad2/3 pathway. Eupafolin reduced the levels of plasminogen activator inhibitor-1 (PAI-1), and pharmacological inhibition and knockdown of PAI-1 mitigated the differentiation of lung fibroblasts. Finally, β-catenin agonist, SKL2001, reversed the suppressive effect of eupafolin on PAI-1 expression and lung fibroblast differentiation. Therefore, flavones have higher anti-fibrotic activity than flavonols obtained from EL, and eupafolin suppressed lung fibroblast differentiation by downregulating β-catenin/PAI-1 signaling rather than the canonical TGF-β/Smad2/3 pathway.

Lupeol suppresses plasminogen activator inhibitor-1-mediated macrophage recruitment and attenuates M2 macrophage polarization

Tumor-associated macrophages (TAMs) are closely related with poor prognosis of cancers. The current study investigated whether lupeol regulates TAMs by focusing on the recruitment and polarization of macrophages. We found that lupeol suppressed the recruitment of THP-1 macrophages (THP-1 cells differentiated into macrophages) towards H1299 lung carcinoma cells by inhibiting plasminogen activator inhibitor-1 (PAI-1) production from H1299 cells. The reduced migration of THP-1 macrophages by lupeol was recovered by adding recombinant human PAI-1 as a chemoattractant. Knockdown of PAI-1 or treatment of tiplaxtinin, a PAI-1 inhibitor, in H1299 cells abrogated the chemotaxis of macrophages. Furthermore, lupeol suppressed the interleukin (IL)-4- and IL-13-induced M2 macrophage polarization. The mRNA expression of M2 macrophage markers and the phosphorylation of signal transducer and activator of transcription 6 (STAT6) were commonly decreased by lupeol in RAW264.7 cells. In addition, lupeol-suppressed M2 macrophage polarization led to the reduced migration of Lewis lung carcinoma (LLC) cells. Taken together, our results suggest that lupeol attenuates PAI-1-mediated macrophage recruitment towards cancer cells and inhibits M2 macrophage polarization.

Combined treatment with benzo[a]pyrene and 1α,25-dihydroxyvitamin D3 induces expression of plasminogen activator inhibitor 1 in monocyte/macrophage-derived cells

Benzo[a]pyrene (BaP) is an environmental pollutant found in cigarette smoke and is implicated as a causative agent of tobacco-related diseases, such as arteriosclerosis. In contrast, vitamin D signaling, which is principally mediated by conversion of vitamin D to the active form, 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3], decreases cardiovascular disease risk. However, combined treatment with BaP and 1,25(OH)2D3 enhances BaP toxicity, including BaP-DNA adduct formation. We further investigated the cross-talk between BaP and 1,25(OH)2D3 signaling pathways, and found that combined treatment with these compounds induces mRNA and protein expression of plasminogen activator inhibitor 1 (PAI-1) in monocyte/macrophage-derived THP-1 and U937 cells. Protein synthesis inhibitor treatment did not inhibit induction of the PAI-1 gene (SERPINE1) in these cells. BaP plus 1,25(OH)2D3 induced differentiation markers, inhibited cellular proliferation, and induced apoptosis and oxidative stress in these cells. Reactive oxygen species scavenger treatment suppressed apoptosis but not SERPINE1 induction in cells treated with BaP plus 1,25(OH)2D3. Thus, combined treatment with BaP and 1,25(OH)2D3 induced SERPINE1 mRNA expression in these cells through a mechanism that does not require de novo protein synthesis or reactive oxygen species production. These findings suggest that induction of the proinflammatory factor PAI-1 plays a role in BaP toxicity. Interestingly, PAI-1 knockdown decreased expression of the cell surface antigen CD14, a monocytic differentiation marker, in THP-1 cells treated with BaP plus 1,25(OH)2D3. PAI-1 induction may also be related to a function of monocytes/macrophages in response to xenobiotic and vitamin D signaling.

Silencing of plasminogen activator inhibitor-1 suppresses colorectal cancer progression and liver metastasis

Plasminogen activator inhibitor-1 (PAI-1) is reported to be expressed in many cancer cell types and regarded as one of the most informative biochemical markers for poor prognosis. However, no previous study has evaluated whether PAI-1 could serve as a target in antitumor and antimetastasis therapies of colorectal cancer (CRC). The plasma level of PAI-1 in CRC patients was detected and its correlation with the clinicopathologic features was evaluated. PAI-1 protein expression was assessed by Western blot assay and immunohistochemistry. The biologic consequences of PAI-1 silencing in colon cancer cell lines and CRC bearing nude mice were also investigated. Plasma PAI-1 level was higher in CRC patients with liver metastasis and correlated with liver metastasis, tumor size, differentiation, serosa infiltration, Duke's stage, and lymphatic metastasis. PAI-1 protein expression in the CRC tissue of patients with liver metastasis was significantly greater than that in those without liver metastasis. In addition, the abilities of proliferation, invasion, and migration of CRC cells transfected with lentivirus expressing PAI-1 small interfering RNA were reduced significantly. Nude mice inoculated with PAI-1 knockdown cells also had fewer metastatic nodules in the liver and smaller tumor volumes. Plasma PAI-1 level was increased in CRC patients with liver metastasis, and PAI-1 silencing may significantly compromise the malignant behaviors of CRC cells in vitro and in vivo. These findings may provide evidence for PAI-1 targeted therapy of CRC.

Krüppel-Like Factor 4 Inhibits the Transforming Growth Factor-β1-Promoted Epithelial-to-Mesenchymal Transition via Downregulating Plasminogen Activator Inhibitor-1 in Lung Epithelial Cells.

Transforming growth factor-β (TGF-β) signaling and TGF-β-promoted epithelial-to-mesenchymal transition (EMT) have been postulated to be the common pathway causing pulmonary fibrosis. However, the up- or downstreaming markers of TGF-β-induced EMT still need to be further recognized. In the present study, we investigated the regulation on Krüppel-like factor 4 (KLF-4) and plasminogen activator inhibitor-1 (PAI-1) by TGF-β in the murine lung epithelial LA-4 cells and then examined the regulation of both markers in the TGF-β-induced EMT by the PAI-1 knockdown or the KLF-4 overexpression. Our study indicated that TGF-β induced EMT in mouse LA-4 lung epithelial cells via reducing E-cadherin, while promoting Collagen I and α-SMA. And PAI-1 was upregulated, whereas KLF-4 was downregulated in the TGF-β-induced EMT model in LA-4 cells. Moreover, the siRNA-mediated PAI-1 knockdown inhibited the TGF-β-induced EMT, whereas the adenovirus-medicated KLF-4 overexpression markedly reduced the PAI-1 expression and inhibited the TGF-β-induced EMT in LA-4 cells. In conclusion, our study confirmed the downregulation of KLF-4 in the TGF-β-induced EMT in LA-4 cells. And the KLF-4 overexpression significantly reduced the TGF-β-induced PAI-1 and thus inhibited the TGF-β-induced EMT in mouse lung epithelial LA-4 cells. It implies that KLF-4 might be a promising target for effective control of the pulmonary fibrosis.

HIF-2alpha-dependent PAI-1 induction contributes to angiogenesis in hepatocellular carcinoma

Hypoxia promotes progression of hepatocellular carcinoma (HCC), not only affecting tumor cell proliferation and invasion, but also angiogenesis and thus, increasing the risk of metastasis. Hypoxia inducible factors (HIF)-1α and -2α cause adaptation of tumors to hypoxia, still with uncertainties towards the angiogenic switch. We created a stable knockdown of HIF-1α and HIF-2α in HepG2 cells and generated cocultures of HepG2 spheroids with embryonic bodies as an in vitro tumor model mimicking the cancer microenvironment. The naturally occuring oxygen and nutrient gradients within the cocultures allow us to question the role of distinct HIF isoforms in regulating HCC angiogenesis. In cocultures with a HIF-2α knockdown, angiogenesis was attenuated, while the knockdown of HIF-1α was without effect. Microarray analysis identified plasminogen activator inhibitor 1 (PAI-1) as a HIF-2α target gene in HepG2 cells. The knockdown of PAI-1 in HepG2 cells also lowered angiogenesis. Blocking plasmin, the downstream target of PAI-1, with aprotinin in HIF-2α knockdown (k/d) cells proved a cause–effect relation and restored angiogenesis, with no effect on control cocultures. Suggestively, HIF-2α increases PAI-1 to lower concentrations of active plasmin, thereby supporting angiogenesis. We conclude that the HIF-2α target gene PAI-1 favors the angiogenic switch in HCC.

Plasminogen activator inhibitor-1 regulates LPS induced inflammation in rat macrophages through autophagy activation.

Background. The mechanisms by which plasminogen activator inhibitor-1 (PAI-1) regulates inflammation, especially in acute respiratory distress syndrome (ARDS), are largely unknown. Objective. To assess the relationship between PAI-1 and autophagy in inflammatory reactions induced by LPS in rat NR8383 cells. Methods. ELISA was used to assess the amounts of TNF-α, IL-1β, and PAI-1 in cell culture supernatants; TLR4, MyD88, PAI-1, LC3, Beclin1, and mTOR protein and mRNA levels were determined by western blot and quantitative RT-PCR, respectively; western blot was used to determine NF-κB protein levels. To further evaluate the role of PAI-1, the PAI-1 gene was downregulated and overexpressed using the siRNA transfection technology and the pCDH-PAI-1, respectively. Finally, the GFP Positive Expression Rate Method was used to determine the rate of GFP-LC3 positive NR8383 cells. Results. In LPS-induced NR8383 cells, TNF-α, IL-1β, and PAI-1 expression levels increased remarkably. Upon PAI-1 knockdown, TNF-α, IL-1β, PAI-1, TLR4, MyD88, NF-κB, LC3, and Beclin1 levels were decreased, while mTOR increased. Conversely, overexpression of PAI-1 resulted in increased amounts of TNF-α, IL-1β, PAI-1, TLR4, MyD88, NF-κB, LC3, and Beclin1. However, no significant change was observed in mTOR expression. Conclusions. In NR8383 cells, PAI-1 contributes in the regulation of LPS-induced inflammation, likely by promoting autophagy.

Functional relevance of d,l-sulforaphane-mediated induction of vimentin and plasminogen activator inhibitor-1 in human prostate cancer cells

D,L-Sulforaphane (SFN) is a promising chemopreventive agent with in vivo efficacy against prostate cancer in experimental rodents. This study was undertaken to determine the role of vimentin and plasminogen activator inhibitor-1 (PAI-1) in anticancer effects of SFN. Effect of SFN on levels of different proteins was determined by Western blotting or immunofluorescence microscopy. RNA interference of vimentin and PAI-1 was achieved by transient transfection. Apoptosis was quantified by flow cytometry. Transwell chambers were used to determine cell migration. Exposure of PC-3 and DU145 human prostate cancer cells to SFN resulted in induction of vimentin protein, which was accompanied by down-regulation of E-cadherin protein expression. The SFN-mediated induction of vimentin was also observed in a normal human prostate epithelial cell line. RNA interference of vimentin did not have any appreciable effect on early or late apoptosis resulting from SFN exposure. On the other hand, SFN-mediated inhibition of PC-3 and DU145 cell migration was significantly augmented by knockdown of the vimentin protein. Knockdown of vimentin itself was inhibitory against cell migration. The SFN-treated cells also exhibited induction of PAI-1, which is an endogenous inhibitor of urokinase-type plasminogen activator system. Similar to vimentin, PAI-1 knockdown resulted in a modest augmentation of PC-3 cell migration inhibition by SFN. Tumors from SFN-treated transgenic adenocarcinoma of mouse prostate mice showed a 1.7-fold increase in vimentin protein level compared with control tumors. The present study indicates that vimentin and PAI-1 inductions confer modest protection against SFN-mediated inhibition of prostate cancer cell migration.

Ex vivo evaluation of antifibrotic compounds in skin scarring: EGCG and silencing of PAI-1 independently inhibit growth and induce keloid shrinkage

Keloid disease (KD) is a common fibroproliferative disorder of unknown etiopathogenesis. Its unique occurrence in human skin and lack of animal models pose challenges for KD research. The lack of a suitable model in KD and over-reliance on cell culture has hampered the progress in developing new treatments. Therefore, we evaluated the effect of two promising candidate antifibrotic therapies: (−)-epigallocatechin-3-gallate (EGCG) and plasminogen activator inhibitor-1 (PAI-1) silencing in a long-term human keloid organ culture (OC). Four millimeters of air–liquid interface (ALI) keloid explants on collagen gel matrix in serum-free medium (n=8 cases) were treated with different modalities (EGCG treatment; PAI-1 knockdown by short interfering RNA (siRNA) and application of dexamethasone (DEX) as control). Normal skin (n=6) was used as control (only for D0 keloid-untreated comparison). Besides routine histology and quantitative (immuno-) histomorphometry, the key phenotypic and growth parameters of KD were assessed. Results demonstrated that EGCG reduced keloid volume significantly (40% by week 4), increased apoptosis (≥40% from weeks 1 to 4), and decreased proliferation (≤17% in week 2). EGCG induced epidermal shrinkage, reduced collagen-I and -III at mRNA and protein levels, depleted 98% of keloid-associated mast cells, and reduced the percentage of both cellularity and blood vessel count by week 4. Knockdown of PAI-1 significantly reduced keloid volume by 28% in week 4, respectively, and reduced collagen-I and -III at both mRNA and protein levels. As expected, DEX increased keloid apoptosis, decreased keloid proliferation, and collagen synthesis, but induced connective tissue growth factor overexpression. In conclusion, using keloid OC model, we provide the first functional evidence for testing candidate antifibrotic compounds in KD. We show that EGCG and PAI-1 silencing effectively inhibits growth and induces shrinkage of human keloid tissue in situ. Therefore, the application of EGCG, PAI-1 silencing, and other emerging compounds tested using this model may provide effective treatment and potentially aid in the prevention of recurrence of KD following surgery.

TLS-CHOP represses miR-486 expression, inducing upregulation of a metastasis regulator PAI-1 in human myxoid liposarcoma

Myxoid liposarcomas (MLSs) are characterized by t(12;16)(q13;p11) translocation and expression of TLS-CHOP chimeric oncoprotein. However, the molecular functions of TLS-CHOP have not been fully understood. On the other hand, microRNAs (miRNAs) comprise an abundant class of endogenous small non-coding RNAs that negatively regulate the expression of their target genes, and are involved in many biological processes. It is now evident that dysregulation of miRNAs is an important step in the development of many cancers. To our knowledge, however, there have been no reports of the miRNAs involved in MLS tumorigenesis and development. In this study, we have found that miR-486 expression was repressed in TLS-CHOP-expressed NIH3T3 fibroblasts and MLS tissues, and exogenous overexpression of miR-486 repressed growth of MLS cells. Thus, downregulation of miR-486 may be an important process for MLS. In addition, we have identified plasminogen activator inhibitor-1 (PAI-1) as a novel target gene of miR-486. PAI-1 is a unique type of serine protease inhibitor and is known to be one of the key regulators of tumor invasion and metastasis. Furthermore, knockdown of PAI-1 by a specific small interfering RNA (siRNA) inhibited growth of MLS cells, suggesting that increased expression of PAI-1 by miR-486 repression is critical for survival of MLS cells. Collectively, these results suggest a novel essential molecular mechanism that TLS-CHOP activates PAI-1 expression by repression of miR-486 expression in MLS tumorigenesis and development.

Protumorigenic activity of plasminogen activator inhibitor-1 through an antiapoptotic function.

Plasminogen activator inhibitor-1 (PAI-1) is a protease inhibitor but is paradoxically associated with poor outcomes in cancer patients. However, the mechanisms of its effects on tumor cells have not been explored. Endogenous PAI-1 in human tumor cell lines (HT-1080, A549, HCT-116, and MDA-MB-231) was suppressed by small interfering RNAs (siRNAs) and PAI-039, a small molecule inhibitor of PAI-1, and the effects on apoptosis were examined. Tumorigenicity of PAI-1 knockdown (KD) tumor cells was examined in immunodeficient PAI-1 wild-type and knockout (KO) mice (9-15 per group), and event-free survival was analyzed by the Kaplan-Meier method. The effect of PAI-1 suppression on HT-1080 xenotransplanted tumors was evaluated for cell proliferation, apoptosis, and angiogenesis. All statistical tests were two-sided. Genetic and pharmacological inhibition of PAI-1 in the four tumor cell lines increased spontaneous apoptosis (mean fold increase relative to control: HT-1080, siRNA#1, mean = 4.0, 95% CI = 2.6 to 5.3, P < .001; siRNA#2, mean = 2.6, 95% CI = 2.4 to 2.9, P < .001, Student t test), which was blocked in the presence of recombinant PAI-1, a caspase-8 inhibitor, or Fas/FasL neutralizing antibodies and was partially attenuated by a plasmin inhibitor-aprotinin. PAI-1 KO mice implanted with PAI-1 KD HT-1080 cells had decreased tumorigenesis and prolonged survival compared with control mice (P = .002, log-rank test), and their tumors exhibited decreased cell proliferation and angiogenesis and increased apoptosis. Furthermore, five of 15 PAI-1 KO mice implanted with PAI-1 KD HT-1080 cells never developed tumors. These data suggest that PAI-1 exerts a protective effect against tumor cell apoptosis by a mechanism that, in part, involves plasmin activation and inhibition of Fas/Fas-L-mediated apoptosis and may be a promising therapeutic target.

Adenoviral Overexpression and Small Interfering RNA Suppression Demonstrate That Plasminogen Activator Inhibitor-1 Produces Elevated Collagen Accumulation in Normal and Keloid Fibroblasts

Keloids are tumor-like skin scars that grow as a result of the aberrant healing of skin injuries, with no effective treatment. We provide new evidence that both overexpression of plasminogen activator inhibitor-1 (PAI-1) and elevated collagen accumulation are intrinsic features of keloid fibroblasts and that these characteristics are causally linked. Using seven strains each of early passage normal and keloid fibroblasts, the keloid strains exhibited inherently elevated collagen accumulation and PAI-1 expression in serum-free, 0.1% ITS+ culture; larger increases in these parameters occurred when cells were cultured in 3% serum. To demonstrate a causal relationship between PAI-1 overexpression and collagen accumulation, normal fibroblasts were infected with PAI-1-expressing adenovirus. Such cells exhibited a two- to fourfold increase in the accumulation of newly synthesized collagen in a viral dose-dependent fashion in both monolayers and fibrin gel, provisional matrix-like cultures. Three different PAI-1-targeted small interfering RNAs, alone or in combination, produced greater than an 80% PAI-1 knockdown and reduced collagen accumulation in PAI-1-overexpressing normal or keloid fibroblasts. A vitronectin-binding mutant of PAI-1 was equipotent with wild-type PAI-1 in inducing collagen accumulation, whereas a complete protease inhibitor mutant retained approximately 50% activity. Thus, PAI-1 may use more than its protease inhibitory activity to control keloid collagen accumulation. PAI-1-targeted interventions, such as small interfering RNA and lentiviral short hairpin RNA-containing microRNA sequence suppression reported here, may have therapeutic utility in the prevention of keloid scarring.

Plasminogen Activator Inhibitor-1 Protects Endothelial Cells from FasL-Mediated Apoptosis

Plasminogen activator inhibitor-1 (PAI-1) paradoxically enhances tumor progression and angiogenesis; however, the mechanism supporting this role is not known. Here we provide evidence that PAI-1 is essential to protect endothelial cells (ECs) from FasL-mediated apoptosis. In the absence of host-derived PAI-1, human neuroblastoma cells implanted in PAI-1-deficient mice form smaller and poorly vascularized tumors containing an increased number of apoptotic ECs. We observed that knockdown of PAI-1 in ECs enhances cell-associated plasmin activity and increases spontaneous apoptosis in vitro. We further demonstrate that plasmin cleaves FasL at Arg144-Lys145, releasing a soluble proapoptotic FasL fragment from the surface of ECs. The data provide a mechanism explaining the proangiogenic activity of PAI-1.

Transforming Growth Factor-β Requires Its Target Plasminogen Activator Inhibitor-1 for Cytostatic Activity

The cytokine transforming growth factor beta (TGFbeta) has strong antiproliferative activity in most normal cells but contributes to tumor progression in the later stages of oncogenesis. It is not fully understood which TGFbeta target genes are causally involved in mediating its cytostatic activity. We report here that suppression of the TGFbeta target gene encoding plasminogen activator inhibitor-1 (PAI-1) by RNA interference leads to escape from the cytostatic activity of TGFbeta both in human keratinocytes (HaCaTs) and primary mouse embryo fibroblasts. Consistent with this, PAI-1 knock-out mouse embryo fibroblasts are also resistant to TGFbeta growth arrest. Conversely, we show that ectopic expression of PAI-1 in proliferating HaCaT cells induces a growth arrest. PAI-1 knockdown does not interfere with canonical TGFbeta signaling as judged by SMAD phosphorylation and induction of bona fide TGFbeta target genes. Instead, knockdown of PAI-1 results in sustained activation of protein kinase B. Significantly, we find that constitutive protein kinase B activity leads to evasion of the growth-inhibitory action of TGFbeta. Our data are consistent with a model in which induction of PAI-1 by TGFbeta is critical for the induction of proliferation arrest.

Plasminogen activator inhibitor-1 is a critical downstream target of p53 in the induction of replicative senescence

p53 limits the proliferation of primary diploid fibroblasts by inducing a state of growth arrest named replicative senescence - a process which protects against oncogenic transformation and requires integrity of the p53 tumour suppressor pathway. However, little is known about the downstream target genes of p53 in this growth-limiting response. Here, we report that suppression of the p53 target gene encoding plasminogen activator inhibitor-1 (PAI-1) by RNA interference (RNAi) leads to escape from replicative senescence both in primary mouse embryo fibroblasts and primary human BJ fibroblasts. PAI-1 knockdown results in sustained activation of the PI(3)K-PKB-GSK3beta pathway and nuclear retention of cyclin D1, consistent with a role for PAI-1 in regulating growth factor signalling. In agreement with this, we find that the PI(3)K-PKB-GSK3beta-cyclin D1 pathway is also causally involved in cellular senescence. Conversely, ectopic expression of PAI-1 in proliferating p53-deficient murine or human fibroblasts induces a phenotype displaying all the hallmarks of replicative senescence. Our data indicate that PAI-1 is not merely a marker of senescence, but is both necessary and sufficient for the induction of replicative senescence downstream of p53.