Triple negative breast cancer metastasis is hindered by a peptide antagonist of F11R/JAM­A protein.

BACKGROUND: The F11R/JAM-A cell adhesion protein was examined as the therapeutic target in triple negative breast cancer (TNBC) with the use of the peptide antagonist to F11R/JAM-A, that previously inhibited the early stages of breast cancer metastasis in vitro. METHODS: The online in silico analysis was performed by TNMPlot, UALCAN, and KM plotter. The in vitro experiments were performed to verify the effect of peptide 4D (P4D) on human endothelial cell lines EA.hy926 and HMEC-1 as well as on human TNBC cell line MDA-MB-231. The cell morphology upon P4D treatment was verified by light microscopy, while the cell functions were assessed by colony forming assay, MTT cell viability assay, BrdU cell proliferation assay, and Transepithelial/Endothelial Electrical Resistance measurements. The in vivo experiments on 4T1 murine breast cancer model were followed by histopathological analysis and a series of quantitative analyses of murine tissues. RESULTS: By in silico analysis we have found the elevated gene expression in breast cancer with particular emphasis on TNBC. The elevated F11R expression in TNBC was related with poorer survival prognosis. Peptide 4D has altered the morphology and increased the permeability of endothelial monolayers. The colony formation, viability, and proliferation of MDA-MB-231 cells were decreased. P4D inhibited the metastasis in 4T1 breast cancer murine model in a statistically significant manner that was demonstrated by the resampling bootstrap technique. CONCLUSIONS: The P4D peptide antagonist to F11R/JAM-A is able to hinder the metastasis in TNBC. This assumption needs to be confirmed by additional 4T1 mouse model study performed on larger group size, before making the decision on human clinical trials.

Interactions of fentanyl with blood platelets and plasma proteins: platelet sensitivity to prasugrel metabolite is not affected by fentanyl under in vitro conditions.

BACKGROUND: Clinical trials indicate that fentanyl, like morphine, may impair intestinal absorption and thus decrease the efficacy of oral P2Y12 inhibitors, such as clopidogrel, ticagrelor, and prasugrel. However, the ability of fentanyl to directly negate or reduce the inhibitory effect of P2Y12 receptor antagonists on platelet function has not been established. A series of in vitro experiments was performed to investigate the ability of fentanyl to activate platelets, potentiate platelet response to ADP, and/or diminish platelet sensitivity to prasugrel metabolite (R-138727) in agonist-stimulated platelets. The selectivity and specificity of fentanyl toward major carrier proteins has been also studied. METHODS: Blood was obtained from healthy volunteers (19 women and 12 men; mean age 40 ± 13 years). Platelet function was measured in whole blood, platelet-rich plasma and in suspensions of isolated platelets by flow cytometry, impedance and optical aggregometry. Surface plasmon resonance and molecular docking were employed to determine the binding kinetics of fentanyl to human albumin, α1-acid glycoprotein, apolipoprotein A-1 and apolipoprotein B-100. RESULTS: When applied at therapeutic and supratherapeutic concentrations under various experimental conditions, fentanyl had no potential to stimulate platelet activation and aggregation, or potentiate platelet response to ADP, nor did it affect platelet susceptibility to prasugrel metabolite in ADP-stimulated platelets. In addition, fentanyl was found to interact with all the examined carrier proteins with dissociation constants in the order of 10-4 to 10-9 M. CONCLUSIONS: It does not seem that the delayed platelet responsiveness to oral P2Y12 inhibitors, such as prasugrel, in patients undergoing percutaneous coronary intervention, results from direct interactions between fentanyl and blood platelets. Apolipoproteins, similarly to albumin and α1-acid glycoprotein, appear to be important carriers of fentanyl in blood.

In Vitro Methods for Measuring the Permeability of Cell Monolayers.

Cell monolayers, including endothelial and epithelial cells, play crucial roles in regulating the transport of biomolecules to underlying tissues and structures via intercellular junctions. Moreover, the monolayers form a semipermeable barrier across which leukocyte transmigration is tightly regulated. The inflammatory cytokines can disrupt the epithelial and endothelial permeability, thus the reduced barrier integrity is a hallmark of epithelial and endothelial dysfunction related with numerous pathological conditions, including cancer-related inflammation. Therefore, the assessment of barrier function is critical in in vitro models of barrier-forming tissues. This review summarizes the commercially available in vitro systems used to measure the permeability of cellular monolayers. The presented techniques are separated in two large groups: macromolecular tracer flux assays, and electrical impedance measurement-based permeability assays. The presented techniques are briefly described and compared.

Mycobacterium tuberculosis Binds Human Serum Amyloid A, and the Interaction Modulates the Colonization of Human Macrophages and the Transcriptional Response of the Pathogen.

As a very successful pathogen with outstanding adaptive properties, Mycobacterium tuberculosis (Mtb) has developed a plethora of sophisticated mechanisms to subvert host defenses and effectively enter and replicate in the harmful environment inside professional phagocytes, namely, macrophages. Here, we demonstrated the binding interaction of Mtb with a major human acute phase protein, namely, serum amyloid A (SAA1), and identified AtpA (Rv1308), ABC (Rv2477c), EspB (Rv3881c), TB 18.6 (Rv2140c), and ThiC (Rv0423c) membrane proteins as mycobacterial effectors responsible for the pathogen-host protein interplay. SAA1-opsonization of Mtb prior to the infection of human macrophages favored bacterial entry into target phagocytes accompanied by a substantial increase in the load of intracellularly multiplying and surviving bacteria. Furthermore, binding of human SAA1 by Mtb resulted in the up- or downregulation of the transcriptional response of tubercle bacilli. The most substantial changes were related to the increased expression level of the genes of two operons encoding mycobacterial transporter systems, namely, mmpL5/mmpS5 (rv0676c), and rv1217c, rv1218c. Therefore, we postulate that during infection, Mtb-SAA1 binding promotes the infection of host macrophages by tubercle bacilli and modulates the functional response of the pathogen.

Efficacy of a Combined Antiplatelet Therapy Is Not Affected by a Simultaneous Binding of Cangrelor and PSB 0777 to Albumin.

Concurrent administration of two drugs may complicate the management of acute coronary syndromes: competitive drug displacement diminishes drug binding and alters drug pharmacodynamics. We investigated the interaction of two antiplatelet compounds (PSB 0777 and cangrelor) with human serum albumin (HSA) to determine whether they compete with one another for the binding to albumin. Both examined compounds have been earlier claimed to bind to HSA (PSB 0777) or plasma proteins (cangrelor). Fluorescence spectroscopy, surface plasmon resonance spectroscopy and molecular modeling indicated that PSB 0777 and cangrelor interacted with HSA with moderate affinity (KD∼10-5 M). The binding of cangrelor to HSA involved primarily hydrophobic interactions, while the interaction of PSB 0777 with HSA was driven by hydrophobic and electrostatic forces. It was found that PSB 0777 and cangrelor do not share the same binding site on the protein. Our findings highlight the importance of albumin in the transport of PSB 0777 and cangrelor and suggest that the antiplatelet activity of the examined compounds used in combination is not affected by competition-induced changes in drug binding to HSA.

P2Y12 receptor antagonists and AR receptor agonists regulates Protein Disulfide Isomerase secretion from platelets and endothelial cells.

Secretion of PDI from platelets and endothelial cells is an important step of all thrombotic events. In the absence of extracellular PDI thrombus formation and fibrin generation may be impaired. Thrombin-mediated PDI secretion is regulated by the stimulation of P2Y12 receptors. This paper provides evidences that P2Y12 antagonists or AR agonists may modulate release of PDI molecules from platelets and with less efficiency from endothelial cells. Moreover P2Y12 antagonization or AR agonization modulates platelet-endothelial interaction. We prove that combinations of P2Y12 antagonists and AR agonists inhibit platelet-dependent adhesion of cancer cells to endothelium and attenuate cancer cell invasiveness, but longer exposition to AR agonists may stimulate migration of invasive breast cancer cells through endothelium thus leading to increased metastasis.

Binding of adenosine derivatives to carrier proteins may reduce their antiplatelet activity.

Adenosine analogues have high affinity and selectivity for adenosine receptors (AR), and exhibit anti-platelet activity. Plasma proteins play an important role in the regulation of platelet function and may influence the action of anti-platelet compounds. Little is known about the interactions of AR agonists with plasma proteins. This study investigates the interplay between AR agonists and plasma proteins and the consequences of those interactions. Surface plasmon resonance was employed together with molecular docking study to determine the binding kinetics of four selected ARagonists (PSB0777, Cl-Ado, MRE0094, UK432097) to several carrier proteins and to clarify the nature of these interactions. The influence of a whole plasma and of some plasma components on the effectiveness of ARagonists in the inhibition of platelet function was assessed by flow cytometry (platelet activation) and ELISA (platelet adhesion). Plasma proteins remarkably diminished the effectiveness of ARagonists in inhibiting platelet activation and adhesion in vitro. ARagonists were found to strongly bind to human serum albumin (HSA) and the protein components of lipoproteins - apolipoproteins; HSA was essential for the binding of water-soluble PSB0777, whereas apolipoproteins were needed for interactions with poorly-water soluble compounds such as UK432097 and MRE0094. In addition, HSA was shown to significantly reduce the effectiveness of PSB0777 in inhibiting ADP-induced platelet activation. In conclusion, HSA and lipoproteins are important carriers for ARagonists, which can affect pharmacodynamics of ARagonists used as platelet inhibitors.

Functional inhibition of F11 receptor (F11R/junctional adhesion molecule-A/JAM-A) activity by a F11R-derived peptide in breast cancer and its microenvironment.

PURPOSE: To examine the involvement of the F11R/JAM-A protein in breast cancer metastasis, we utilized the F11R/JAM-A antagonistic peptide 4D (P4D) in experiments of transendothelial migration (TEM) of breast cancer cells. METHODS: Experiments were conducted in the mouse 4T1 breast cancer model utilizing the human mammary epithelial cell and endothelial cell lines. The levels of soluble F11R/JAM-A (sJAM-A) in the murine plasmas were measured by ELISA. Levels of F11R/JAM-A mRNA and protein in cell lines were assessed by qRT-PCR and Western blot, respectively. Cell surface expression of F11R/JAM-A was demonstrated by flow cytometry. Functional tests included the TEM of breast cancer cells and adhesion of breast cancer cells to the endothelium. The endothelial permeability was studied by fluorescent tracer assay and by the Real-Time Cell Analysis (RTCA). RESULTS: The tumor inducers Tß4 and TGF-ß1 reduced the levels of sJAM-A in murine plasma, and reduced the F11R/JAM-A protein levels in the human microvascular endothelial cell line HMEC-1. The adhesion and TEM measured between breast cancer cells and inflamed or Tß4-treated endothelium were inhibited by P4D. The presence of P4D did not destabilize the pre-existing tight junctions in the endothelial monolayer. The barrier-protecting effect of P4D was stronger than that of forskolin, when a booster dose of P4D was applied to the inflamed endothelium. CONCLUSIONS: F11R/JAM-A protein can be considered as a novel target in the treatment of breast cancer metastasis. In vivo and clinical studies are needed to further investigate the effectiveness of F11R/JAM-A-derived peptide as a possible anti-metastatic drug.

Contribution of activated beta3 integrin in the PDI release from endothelial cells.

Protein disulfide isomerase (PDI) is an abundant reticulum endoplasmic protein but also acts as an important functional regulator of some extracellular surface proteins. Recent studies suggest that PDI plays a role in integrin activation and thrombus formation. The aim of this study was to examine whether activation of integrin is the first stage leading to release of PDI from the subcellular compartments of endothelial cells to extracellular space. Our results show that endothelial cells which adhere to fibronectin or fibrinogen release significantly more PDI than those which adhere to poly-L-lysine. Cells treated with RGD peptide, Src and FAK kinase inhibitors and anti alphaVbeta3 antibody display lower PDI secretion. The destruction of the actin cytoskeleton of endothelial cells by cytochalasin D inhibits PDI release. When the endothelial cells adhere to fibrinogen or fibronectin, PDI and alphaVbeta3 gain free thiol groups. Our data suggest that upon activation of integrins, PDI is released from endothelial cells and forms a disulfide bond complex with alphaVbeta3 integrin.

The role of Protein Disulfide Isomerase and thiol bonds modifications in activation of integrin subunit alpha11.

Integrins belong to a family of transmembrane receptors that mediate cell migration and adhesion to ECM. Extracellular domains of integrin heterodimers contain cysteine-rich regions, which are potential sites of thiol-disulfide exchanges. Rearrangements of extracellular disulfide bonds regulate activation of integrin receptors by promoting transition from an inactive state into a ligand-binding competent state. Modifications of integrin disulfide bonds dependent on oxidation-reduction can be mediated by Protein Disulfide Isomerse (PDI). This paper provides evidences that binding to integrin ligands initiate changes in free thiol pattern on cell surface and that thiol-disulfide exchange mediated by PDI leads to activation of integrin subunit α11. By employing co-immunoprecipitation and confocal microscopy analysis we showed that α11ß1 and PDI create complexes bounded by disulfide bonds. Using surface plasmon resonance we provide biochemical evidence that PDI can interact directly with integrin subunit α11.

Oxidation of C-reactive protein by hypochlorous acid leads to the formation of potent platelet activator.

We examined the structural and functional consequences of oxidative modification of C-reactive protein (CRP) by hypochlorous acid (HOCl), which can be generated in vivo via the myeloperoxidase/H2O2/Cl- system. HOCl exposure resulted in the oxidation and chlorination of CRP amino acid residues, leading to protein unfolding, greater surface hydrophobicity and the formation of aggregates. After treatment of isolated platelets with 50µg/ml HOCl-CRP, the modified CRP significantly stimulated platelet activation (over 10-fold increase in the fraction of CD62-positive platelets compared to controls, P<0.008), enhanced deposition of platelets onto immobilized fibrinogen (two-fold rise in platelet adhesion compared to controls, P<0.0001), and induced platelet aggregation by up to 79.5%. The ability of HOCl-CRP to interact with several platelet receptors (TLR-4, GPIIbIIIa) and plasma proteins (C1q, IgG) strongly indicates that HOCl-modification leads to structural changes of CRP resulting in the formation of new ligand binding sites, which is characteristic of the monomeric form of CRP exerting pro-inflammatory effects on a variety of cells. Overall, the oxidation of native CRP by HOCl seems to represent an alternative mechanism of CRP modification, by which CRP reveals its pro-inflammatory and pro-thrombotic properties, and as such, it might be of causal relevance in the pathogenesis of atherosclerosis.

Bacterial expression, purification and angiogenesis-promoting activity of human thymosin ß4.

Thymosin ß4 (Tß4) is an actin-binding peptide involved in tissue regeneration and angiogenesis. This 43-amino acid peptide is chemically synthesized for research or clinical trials. To overcome the high costs of solid phase synthesis, we developed a genetic engineering procedure of Tß4 expression in a protease-deficient host strain, Escherichia coli BL21(DE3), transformed with different expression vectors (pRSETA, pET-15b and pEcoli-Cterm6 × HN). The recombinant, non-glycosylated peptide was overexpressed in soluble form and purified by two-step immobilized metal ion affinity chromatography. Use of the pET vector expression system allowed for easy removal of the polyhistidine tag by thrombin. Functional studies revealed that recombinant Tß4 stimulated angiogenesis via activation of the endothelial proteolytic systems, inhibition of endothelial cell adhesion, promotion of migration and capillary tube formation in Matrigel, and that its activity was similar to that observed for the synthetic peptide. The presented study comprises the first evidence that recombinant Tß4 promotes angiogenesis in an in vitro endothelial cell model.

Thymosin ß4 is rapidly internalized by cells and does not induce intracellular Ca2+ elevation.

Thymosin ß4 (Tß4) is a multifunctional protein that has pleiotropic activities both intracellularly and extracellularly. The mechanisms by which it influences cellular processes such as adhesion, migration, differentiation, or apoptosis are not yet understood. Calcium is a ubiquitous signal molecule that is involved in the regulation of almost all cellular functions. Our data indicate that the release of Ca(2+) from intracellular stores following stimulation of cells with Tß4 does not occur. Interestingly, Tß4 becomes rapidly internalized, supporting the concept that it may express its activities via intracellular receptors.

Thymosin ß4 promotes the migration of endothelial cells without intracellular Ca2+ elevation.

Numerous studies have demonstrated the effects of Tß4 on cell migration, proliferation, apoptosis and inflammation after exogenous treatment, but the mechanism by which Tß4 functions is still unclear. Previously, we demonstrated that incubation of endothelial cells with Tß4 induced synthesis and secretion of various proteins, including plasminogen activator inhibitor type 1 and matrix metaloproteinases. We also showed that Tß4 interacts with Ku80, which may operate as a novel receptor for Tß4 and mediates its intracellular activity. In this paper, we provide evidence that Tß4 induces cellular processes without changes in the intracellular Ca(2+) concentration. External treatment of HUVECs with Tß4 and its mutants deprived of the N-terminal tetrapeptide AcSDKP (Tß4(AcSDKPT/4A)) or the actin-binding sequence KLKKTET (Tß4(KLKKTET/7A)) resulted in enhanced cell migration and formation of tubular structures in Matrigel. Surprisingly, the increased cell motility caused by Tß4 was not associated with the intracellular Ca(2+) elevation monitored with Fluo-4 NW or Fura-2 AM. Therefore, it is unlikely that externally added Tß4 induces HUVEC migration via the surface membrane receptors known to generate Ca(2+) influx. Our data confirm the concept that externally added Tß4 must be internalized to induce intracellular mechanisms supporting endothelial cell migration.

Lumican inhibits angiogenesis by interfering with α2ß1 receptor activity and downregulating MMP-14 expression.

INTRODUCTION: Previous studies showed that lumican, a small leucine-rich proteoglycan that binds to α2 integrin I domain, is an efficient inhibitor of cell adhesion and migration. In this report, we tested its effect on angiogenesis in vitro and in vivo. MATERIALS AND METHODS: Effect of lumican on angiogenesis was evaluated by in vitro capillary tube formation test performed between Fibrin II Gels or in Matrigel™ and in vivo by Matrigel(™) plug assay in BALB/c mice. Changes in matrix metalloproteinases expression caused by lumican were analyzed in endothelial cells by real-time PCR, Western immunoblotting and gelatin zymography. RESULTS: In unchallenged endothelial cells, Matrigel™ induced robust capillary morphogenesis. In contrast, tube formation was dramatically reduced by lumican, and by siRNA to ß1 integrin subunit mRNA but not by control siRNA. Similarly, lumican effectively inhibited neovascularization in vivo in assays using Matrigel™ plugs formed in BALB/c mice. Interestingly, lumican significantly reduced expression of matrix metalloproteinases, particularly MMP-14 that is known to activate other MMPs in close vicinity of endothelial cell membranes. CONCLUSIONS: Our results provide strong evidence that lumican affects angiogenesis both by interfering with α2ß1 receptor activity and downregulating proteolytic activity associated with surface membranes of endothelial cells.

Thymosin beta4 regulates migration of colon cancer cells by a pathway involving interaction with Ku80.

Aberrant expression of thymosin beta4 (Tbeta4) has recently been found to be associated with colorectal carcinoma (CRC) progression evidently due to an increase of the motility and invasion of tumor cells and the induction of a proangiogenic phenotype of endothelial cells. Both mechanisms depend upon matrix-degrading proteases, particularly plasmin and matrix metalloproteinases (MMPs) that are responsible for extensive tissue remodeling. Cleavage of ECM macromolecules weakens the structural integrity of tissues and exposes cryptic domains of extracellular components, which elicit biological responses distinct from intact molecules. Interestingly, signaling via integrins (alphaVbeta3, alpha5beta1) in CRC cells (HT29, CX1.1) is induced by Tbeta4 and VEGF-A only when they grow in 3D fibrin gels but not in 2D ones. The cells growing in 3D fibrin gels release upon Tbeta4 significant amounts of active MMPs (MMP-2, MMP-9, and MMP-7) that cause extensive proteolysis in their close vicinity. As evidenced by a variety of approaches (transfection experiments, coimmunoprecipitation, gene silencing with siRNA), we found that this involves interaction of Tbeta4 with Ku80, which has recently been described by us to mediate Tbeta4 intracellular activity.

Ku80 as a novel receptor for thymosin beta4 that mediates its intracellular activity different from G-actin sequestering.

Our data demonstrate that increased intracellular expression of thymosin beta4(Tbeta4) is necessary and sufficient to induce plasminogen activator inhibitor type 1 (PAI-1) gene expression in endothelial cells. To describe the mechanism of this effect, we produced Tbeta4 mutants with impaired functional motifs and tested their intracellular location and activity. Cytoplasmic distributions of Tbeta4((AcSDKPT/4A)), Tbeta4((KLKKTET/7A)), and Tbeta4((K16A)) mutants fused with green fluorescent protein did not differ significantly from those of wild-type Tbeta4. Overexpression of Tbeta4, Tbeta4((AcSDKPT/4A)), and Tbeta4((K16A)) affected intracellular formation of actin filaments. As expected, Tbeta4((K16A)) uptake by nuclei was impaired. On the other hand, overexpression of Tbeta4((KLKKTET/7A)) resulted in developing the actin filament network typical of adhering cells, indicating that the mutant lacked the actin binding site. The mechanism by which intracellular Tbeta4 induced the PAI-1 gene did not depend upon the N-terminal tetrapeptide AcSDKP and depended only partially on its ability to bind G-actin or enter the nucleus. Both Tbeta4 and Tbeta4((AcSDKPT/4A)) induced the PAI-1 gene to the same extent, whereas mutants Tbeta4((KLKKTET/7A)) and Tbeta4((K16A)) retained about 60% of the original activity. By proteomic analysis, the Ku80 subunit of ATP-dependent DNA helicase II was found to be associated with Tbeta4. Ku80 and Tbeta4 consistently co-immunoprecipitated in a complex from endothelial cells. Co-transfection of endothelial cells with the Ku80 deletion mutants and Tbeta4 showed that the C-terminal arm domain of Ku80 is directly involved in this interaction. Furthermore, down-regulation of Ku80 by specific short interference RNA resulted in dramatic reduction in PAI-1 expression at the level of both mRNA and protein synthesis. These data suggest that Ku80 functions as a novel receptor for Tbeta4 and mediates its intracellular activity.

Adhesive and proteolytic phenotype of migrating endothelial cells induced by thymosin beta-4.

The early stages of angiogenesis are usually accompanied by the occurrence of vascular leakage, and the deposition of fibrin in extravascular spaces. Initially, the fibrin network acts as a sealing matrix, but later on also as a scaffolding for invading endothelial cells. This process is induced by angiogenic growth factors, particularly by vascular endothelial growth factor (VEGF). Angiogenesis involves proteolytic activities, in particular cell-bound urokinase/plasmin and matrix metalloproteinase (MMPs) activities that modulate the fibrin structure and affect adhesion and migration of endothelial cells. Recent data show that formation of new vessels may be stimulated by thymosin beta-4 (Tbeta-4), but it is still not clear whether Tbeta-4 alone is angiogenic or the angiogenic potential of Tbeta-4 is mediated by VEGF. In this report to further characterize Tbeta-4 angiogenic activity, we produced its mutants that were deprived of the N-terminal tetrapeptide AcSDKP (Tbeta-4((AcSDKPT/4A))), the actin-binding sequence KLKKTET (Tbeta-4((KLKKTET/7A))) and with the nuclear localization sequence damaged by a point mutation Lys16Ala (Tbeta-4((K16A))). Then we tested their activity to induce expression and release of MMPs as well as plasminogen activators inhibitor type-1 (PAI-1). We also analyzed their effect on migration and proliferation of endothelial cells in three-dimensional (3D) fibrin matrix as well as on their ability to stimulate the outgrowth of human endothelial cells in capillary-like tubular structures. Our data demonstrate that increased intracellular expression of Tbeta-4 and its mutants is necessary and sufficient to induce PAI-1 gene expression in endothelial cells. Similarly, they stimulate expression and release of MMP-1, -2, and -3. As evaluated by using specific inhibitors to these MMPs, they modified specifically the structure of fibrin and thus facilitated migration of endothelial cells. To sum up, our data show that the mechanism by which Tbeta-4 induced transition of endothelial cells from quiescent to proangiogenic phenotype is characterized by increased expression of PAI-1 and MMPs did not require the presence of the N-terminal sequence AcSDKP, and depended only partially on its ability to bind G-actin or to enter the nucleus.

Thymosin beta 4 induces the synthesis of plasminogen activator inhibitor 1 in cultured endothelial cells and increases its extracellular expression.

Thymosin beta 4(T beta 4), a 4.9-kDa polypeptide primarily known as a main G-actin-sequestering peptide, is present in high concentrations in various cells and in the circulation. We have found that T beta 4 upregulates the expression of plasminogen activator inhibitor 1 (PAI-1) in endothelial cells measured both at the level of mRNA and protein synthesis. This effect seems to be cell specific and was not observed when other cells such as human fibroblasts, PC3, and U937 were tested. T beta 4 significantly activated the PAI-1 promoter in EA.hy 926 cells transiently transfected either with plasmid p800LUC containing PAI-1 promoter fragment (-800 to +71) or the PAI-1 promoter linked with green fluorescent protein. T beta 4 mediated up-regulation of PAI-1 involved activation of the mitogen-activated protein kinase cascade. Furthermore, T beta 4 enhanced c-Fos/c-Jun DNA-binding activity to the activator protein 1 (AP-1)-like element (-59 to -52). The specificity of this binding activity was demonstrated by competition electrophoretic mobility shift assay and after transfection of EA.hy 926 cells with the mutated PAI-1 promoter. Taken together, these data indicate that, in response to T beta 4 stimulation, AP-1 activity increases to enhance PAI-1 transcription through its unique AP-1-like element at -59 to -52 in the PAI-1 promoter.