ß-III tubulin modulates the behavior of Snail overexpressed during the epithelial-to-mesenchymal transition in colon cancer cells.

Class III ß-tubulin (TUBB3) is a marker of drug resistance expressed in a variety of solid tumors. Originally, it was described as an important element of chemoresistance to taxanes. Recent studies have revealed that TUBB3 is also involved in an adaptive response to a microenvironmental stressor, e.g. low oxygen levels and poor nutrient supply in some solid tumors, independently of the microtubule targeting agent. Furthermore, it has been demonstrated that TUBB3 is a marker of biological aggressiveness associated with modulation of metastatic abilities in colon cancer. The epithelial-to-mesenchymal transition (EMT) is a basic cellular process by which epithelial cells lose their epithelial behavior and become invasive cells involved in cancer metastasis. Snail is a zinc-finger transcription factor which is able to induce EMT through the repression of E-cadherin expression. In the presented studies we focused on the analysis of the TUBB3 role in EMT-induced colon adenocarcinoma cell lines HT-29 and LS180. We observed a positive correlation between Snail presence and TUBB3 upregulation in tested adenocarcinoma cell lines. The cellular and behavioral analysis revealed for the first time that elevated TUBB3 level is functionally linked to increased cell migration and invasive capability of EMT induced cells. Additionally, the post-transcriptional modifications (phosphorylation, glycosylation) appear to regulate the cellular localization of TUBB3 and its phosphorylation, observed in cytoskeleton, is probably involved in cell motility modulation.

Protein disulfide isomerase directly interacts with ß-actin Cys374 and regulates cytoskeleton reorganization.

Recent studies support the role of cysteine oxidation in actin cytoskeleton reorganization during cell adhesion. The aim of this study was to explain whether protein disulfide isomerase (PDI) is responsible for the thiol-disulfide rearrangement in the ß-actin molecule of adhering cells. First, we showed that PDI forms a disulfide-bonded complex with ß-actin with a molecular mass of 110 kDa. Specific interaction of both proteins was demonstrated by a solid phase binding assay, surface plasmon resonance analysis, and immunoprecipitation experiments. Second, using confocal microscopy, we found that both proteins colocalized when spreading MEG-01 cells on fibronectin. Colocalization of PDI and ß-actin could be abolished by the membrane-permeable sulfhydryl blocker, N-ethylmaleimide, by the RGD peptide, and by anti-αIIbß3 antibodies. Consequently, down-regulation of PDI expression by antisense oligonucleotides impaired the spreading of cells and initiated reorganization of the cytoskeleton. Third, because of transfection experiments followed by immunoprecipitation and confocal analysis, we provided evidence that PDI binds to the ß-actin Cys(374) thiol. Formation of the ß-actin-PDI complex was mediated by integrin-dependent signaling in response to the adhesion of cells to the extracellular matrix. Our data suggest that PDI is released from subcellular compartments to the cytosol and translocated toward the periphery of the cell, where it forms a disulfide bond with ß-actin when MEG-01 cells adhere via the αIIbß3 integrin to fibronectin. Thus, PDI appears to regulate cytoskeletal reorganization by the thiol-disulfide exchange in ß-actin via a redox-dependent mechanism.

NFAT2 regulates COX-2 expression and modulates the integrin repertoire in endothelial cells at the crossroads of angiogenesis and inflammation.

The mechanisms controlling the switch between the pro-angiogenic and pro-inflammatory states of endothelial cells are still poorly understood. In this paper, we show that: (a) COX-2 expression induced by VEGF-A is NFAT2-dependent; and (b) the integrin profile in endothelial cells induced by the pro-angiogenic VEGF-A is distinct from that brought on by the inflammatory cytokine TNF-α. Two groups of integrin subunits specifically upregulated over time by both cytokines were identified using RT-PCR and Western Immunoblotting. The first group included α4, α5, α6, and ß5 subunits that were upregulated by VEGF-A; the second group consisted of αV and ß3 induced by TNF-α. Both cytokines significantly enhanced the expression of ß1 and modulated α2 mRNA. In contrast to TNF-α, VEGF-A induction of integrin subunits depended on the activation of the calcineurin/NFAT pathway. Both calcineurin inhibitors (cyclosporineA and 11R-VIVIT) and downregulation of NFAT2 with specific siRNA decreased induction of integrin subunits. This process of induction could be increased by upregulation of NFAT2 by pBJ5-NFAT2 transfection. This suggests that NFAT2 mediates VEGF-induced upregulation of integrin subunit synthesis by providing a constant supply of newly synthesized "refreshed" mature integrin receptors, particularly α2ß1, α5ß1, α4ß1, α6ß1 and αVß5, which are involved at different stages of angiogenesis.

Secretion of SerpinB2 from endothelial cells activated with inflammatory stimuli.

Due to the lack of an N-terminal signal peptide, SerpinB2 (plasminogen activator inhibitor type 2) accumulates in cells and only a small percentage of it is secreted. The extracellular concentration of SerpinB2 significantly increases during inflammation. In the present study we investigated the mechanism with which SerpinB2 can be secreted from endothelial cells activated with LPS. We evaluated the intracellular distribution of SerpinB2 by double immunogold labeling followed by a high resolution electron microscopy analysis. We found that SerpinB2 gathers in the vesicular structures and in the endothelial cell periphery. These vesicles stained positive for the trans-Golgi network marker TGN46, which is consistent with their formation by the endoplasmatic reticulum (ER) and Golgi-dependent pathways. SerpinB2 was delivered to the plasma membrane, apparently together with TGN46 in the same vesicles, which after fusion with the membranes released cargo. Secretion of SerpinB2 was partially inhibited by brefeldin A. The secreted SerpinB2 was predominantly in its nonglycosylated 43kDa form as evaluated by Western immunoblotting. Our data suggest that increased expression of SerpinB2 by an inflammatory stimulus is sufficient to generate structures that resemble secretory vesicles. These vesicles may represent the mechanism by which high local concentrations of SerpinB2 are released at inflammation sites from endothelial cells.

Matrix metalloproteinase-2 cleavage of the ß1 integrin ectodomain facilitates colon cancer cell motility.

Cancer cell invasion is a key element in metastasis that requires integrins for adhesion/de-adhesion, as well as matrix metalloproteinases (MMPs) for focalized proteolysis. Herein we show that MMP-2 is up-regulated in resected colorectal tumors and degrades ß1 integrins with the release of fragments containing the ß1 I-domain. The ß1 cleavage pattern is similar to that produced by digestion of α5ß1 and α2ß1 with MMP-2. Two such fragments, at 25 and 75 kDa, were identified after immunoprecipitation, with monoclonal antibody BD610468 reacting with the NH(2)-terminal I-like ectodomain followed by SDS-PAGE and microsequencing using electrospray (ISI-Q-TOF-Micromass) spectrometry. Cleavage of the ß1 integrin can be abolished by inhibition of MMP-2 activity; it can be induced by up-regulation of MMP-2 expression, as exemplified by HT29 colon cancer cells transfected with pCMV6-XL5-MMP-2. Co-immunoprecipitation studies of colon cancer cells showed that the ß1 integrin subunit is associated with MMP-2. The MMP-2-mediated shedding of the I-like domain from ß1 integrins resulted in decreased adhesion of colon cancer cells to collagen and fibronectin, thus abolishing their receptivity. Furthermore, such cells showed enhanced motility as evaluated by a "wound healing-like" assay and time-lapse microscopy, indicating their increased invasiveness. Altogether, our data demonstrate that MMP-2 amplifies the motility of colon cancer cells, not only by digesting the extracellular matrix components in the vicinity of cancer cells but also by inactivating their major ß1 integrin 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.

Association of plasminogen activator inhibitor type 2 (PAI-2) with proteasome within endothelial cells activated with inflammatory stimuli.

Quiescent endothelial cells contain low concentrations of plasminogen activator inhibitor type 2 (PAI-2). However, its synthesis can be rapidly stimulated by a variety of inflammatory mediators. In this study, we provide evidence that PAI-2 interacts with proteasome and affects its activity in endothelial cells. To ensure that the PAI-2·proteasome complex is formed in vivo, both proteins were coimmunoprecipitated from endothelial cells and identified with specific antibodies. The specificity of this interaction was evidenced after (a) transfection of HeLa cells with pCMV-PAI-2 and coimmunoprecipitation of both proteins with anti-PAI-2 antibodies and (b) silencing of the PAI-2 gene using specific small interfering RNA (siRNA). Subsequently, cellular distribution of the PAI-2·proteasome complexes was established by immunogold staining and electron microscopy analyses. As judged by confocal microscopy, both proteins appeared in a diffuse cytosolic pattern, but they also could be found in a dense perinuclear and nuclear location. PAI-2 was not polyubiquitinated, suggesting that it bound to proteasome not as the substrate but rather as its inhibitor. Consistently, increased PAI-2 expression (a) abrogated degradation of degron analyzed after cotransfection of HeLa cells with pCMV-PAI-2 and pd2EGFP-N1, (b) prevented degradation of p53, as evidenced both by confocal microscopy and Western immunoblotting, and (c) inhibited proteasome cleavage of specific fluorogenic substrate. This suggests that PAI-2, in endothelial cells induced with inflammatory stimuli, can inhibit proteasome and thus tilt the balance favoring proapoptotic signaling.

Plasminogen activator inhibitor type 1 interacts with alpha3 subunit of proteasome and modulates its activity.

Plasminogen activator inhibitor type-1 (PAI-1), a multifunctional protein, is an important physiological regulator of fibrinolysis, extracellular matrix homeostasis, and cell motility. Recent observations show that PAI-1 may also be implicated in maintaining integrity of cells, especially with respect to cellular proliferation or apoptosis. In the present study we provide evidence that PAI-1 interacts with proteasome and affects its activity. First, by using the yeast two-hybrid system, we found that the α3 subunit of proteasome directly interacts with PAI-1. Then, to ensure that the PAI-1-proteasome complex is formed in vivo, both proteins were coimmunoprecipitated from endothelial cells and identified with specific antibodies. The specificity of this interaction was evidenced after transfection of HeLa cells with pCMV-PAI-1 and coimmunoprecipitation of both proteins with anti-PAI-1 antibodies. Subsequently, cellular distribution of the PAI-1-proteasome complexes was established by immunogold staining and electron microscopy analyses. Both proteins appeared in a diffuse cytosolic pattern but also could be found in a dense perinuclear and nuclear location. Furthermore, PAI-1 induced formation of aggresomes freely located in endothelial cytoplasm. Increased PAI-1 expression abrogated degradation of degron analyzed after cotransfection of HeLa cells with pCMV-PAI-1 and pd2EGFP-N1 and prevented degradation of p53 as well as IκBα, as evidenced both by confocal microscopy and Western immunoblotting.

Matrin 3 as a key regulator of endothelial cell survival.

Matrin 3 is an integral component of nuclear matrix architecture that has been implicated in interacting with other nuclear proteins and thus modulating the activity of proximal promoters. In this study, we evaluated the contribution of this protein to proliferation of endothelial cells. To selectively modulate matrin 3 expression, we used siRNA oligonucleotides and transfection of cells with a pEGFP-N1-Mtr3. Our data indicate that downregulation of matrin 3 is responsible for reduced proliferation and leads to necrosis of endothelial cells. This conclusion is supported by observations that reducing matrin 3 expression results in (a) producing signs of necrosis detected by PI staining, LDH release, and scatter parameters in flow cytometry, (b) affecting cell cycle progression. It does not cause (c) membrane asymmetry of cells as indicated by lack of Annexin V binding as well as (d) activation of caspase 3 and cleavage of PARP. We conclude that matrin 3 plays a significant role in controlling cell growth and proliferation, probably via formation of complexes with nuclear proteins that modulate pro- and antiapoptotic signaling pathways. Thus, degradation of matrin 3 may be a switching event that induces a shift from apoptotic to necrotic death of cells.

Ero1alpha is expressed on blood platelets in association with protein-disulfide isomerase and contributes to redox-controlled remodeling of alphaIIbbeta3.

Recent evidence supports a role of protein-disulfide isomerase (PDI) in redox-controlled remodeling of the exofacial domains of α(IIb)ß(3) in blood platelets. The aim of this study was to explain whether Ero1α can be responsible for extracellular reoxidation of the PDI active site. We showed that Ero1α can be found on platelets and is rapidly recruited to the cell surface in response to platelet agonists. It is physically associated with PDI and α(IIb)ß(3), as suggested by colocalization analysis in confocal microscopy and confirmed by immunoprecipitation experiments. Apart from monomeric oxidized Ero1α, anti-α(IIb)ß(3) immunoprecipitates showed the presence of several Ero1α-positive bands that corresponded to the complexes α(IIb)ß(3)-PDI-Ero1α, PDI-Ero1α, and Ero1α-Ero1α dimers. It binds more efficiently to the activated α(IIb)ß(3) conformer, and its interaction is inhibited by RGD peptides. Ero1α appears to be involved in the regulation of α(IIb)ß(3) receptor activity because of the following: (a) blocking the cell surface Ero1α by antibodies leads to a decrease in platelet aggregation in response to agonists and a decrease in fibrinogen and PAC-1 binding, and (b) transfection of MEG01 with Ero1α increases α(IIb)ß(3) receptor activity, as indicated by increased binding of fibrinogen.

Autocrine effects of VEGF-D on endothelial cells after transduction with AD-VEGF-D(DeltaNDeltaC).

Endothelial cells in tumor vessels display unusual characteristics in terms of survival and angiogenic properties which result from the increased expression of VEGF-D and its autocrine effect. To evaluate mechanisms by which VEGF-D leads to such abnormal phenotype, we searched for proteins with modified expression in HUVECs enriched in the recombinant mature VEGF-D (VEGFD(DeltaNDeltaC)) delivered by adenovirus. Expression of membrane proteins in endothelial cells was characterized by FACS using anti-human IT-Box-135 antibodies. HUVECs transduced with Ad-VEGF-D(DeltaNDeltaC) revealed markedly increased expression of proteins involved in adhesion and migration such as (a) integrins (alphaVbeta5, alpha2beta1, alpha5beta1, alphaMbeta2, alphaLbeta2), (b) matrix metalloproteinases (MMP-2, MMP-9, and MMP-14), (c) components of fibrinolytic system (PAI-1, u-PAR), and (d) CD45, CD98, CD147. Interestingly, there also were numerous proteins with significantly reduced expression, particularly among surface exposed membrane proteins. Thus, it can be concluded that to induce proangiogenic phenotype and facilitate migration of HUVECs, VEGF-D(DeltaNDeltaC) not only upregulates expression of proteins known to participate in the cell-matrix interactions but also silences some membrane proteins which could interfere with this process.

Interaction and functional association of protein disulfide isomerase with alphaVbeta3 integrin on endothelial cells.

Adhesive properties of endothelial cells are influenced by the thioldisulfide balance. However, the molecular mechanism of this effect is unclear, although recent observations indicate that integrin receptors may be direct targets for redox modulation. The purpose of this study was to examine whether protein disulfide isomerase (PDI) is directly involved in this process. As manganese ions are known to affect the thioldisulfide balance and activate integrins to maximal affinity, we searched for PDI interactions with integrins, particularly with alpha(V)beta(3), in Mn(2+)-treated endothelial cells. By employing confocal microscopy, flow cytometry and coimmunoprecipitation experiments, we showed that exposure of endothelial cells to Mn(2+) resulted in: (a) the appearance of surface protein thiol groups, which can be found in PDI and alpha(V)beta(3), and both proteins colocalizing on the cellular surface; and (b) the formation of the PDI-alpha(V)beta(3) complex, which dissociates upon reduction. In addition, PDI in a complex with alpha(V)beta(3) induces conversion of the integrin to the ligand-competent high-affinity state, as evidenced by increased binding of vitronectin. The membrane-impermeable sulfhydryl blockers 3-N-maleimidylpropionyl biocytin 3-N-maleimidylpropionyl biocytin and p-chloromercuriphenyl sulfonate, as well as the PDI inhibitors bacitracin, MA3 018, and MA3 019, abolished the binding of vitronectin and LM609 to endothelial cells that is activated by Mn(2+). Consistently, LM609 almost completely blocked binding of vitronectin to such cells. The formation of the PDI-alpha(V)beta(3) stoichiometric complex was further demonstrated by surface plasmon resonance analysis, which showed that the initial reversible binding of PDI becomes irreversible in the presence of Mn(2+), probably mediated by disulfide bonds. Thus, we show that Mn(2+) simultaneously modulates the thiol isomerase activity of PDI that is bound to alpha(V)beta(3) and induces its transition to the ligand-competent state, suggesting an alternative mechanism of integrin regulation.

Corpora amylacea from multiple sclerosis brain tissue consists of aggregated neuronal cells.

In this report, we describe proteomic analysis of corpora amylacea collected by postmortem laser microdissection from multiple sclerosis (MS) brain lesions. Using low level protein loads (about 30 microg), a combination of two-dimensional electrophoresis with matrix-assisted laser desorption/ionization-time of flight mass spectrometry and database interrogations we identified 24 proteins of suspected neuronal origin. In addition to major cytoskeletal proteins like actin, tubulin, and vimentin, we identified a variety of proteins implicated specifically in cellular motility and plasticity (F-actin capping protein), regulation of apoptosis and senescence (tumor rejection antigen-1, heat shock proteins, valosin-containing protein, and ubiquitin-activating enzyme E1), and enzymatic pathways (glyceraldehyde-3-dehydrogenase, protein disulfide isomerase, protein disulfide isomerase related protein 5, lactate dehydrogenase). Samples taken from regions in the vicinity of corpora amylacea showed only traces of cellular proteins suggesting that these bodies may represent remnants of neuronal aggregates with highly polymerized cytoskeletal material. Our data provide evidence supporting the concept that biogenesis of corpora amylacea involves degeneration and aggregation of cells of neuronal origin.

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.

Mast cell-derived exosomes activate endothelial cells to secrete plasminogen activator inhibitor type 1.

OBJECTIVE: Previous studies supported the contribution of exosomes to an acellular mode of communication, leading to intercellular transfer of molecules. In this study we provide evidence that mast cell-derived exosomes induce plasminogen activator inhibitor type 1 (PAI-1) expression in endothelial cells, detectable at the level of PAI-1 mRNA and protein synthesis. The stimulating effect was also measured at the level of PAI-1 promoter activity. METHODS AND RESULTS: To identify components responsible for this activity, exosome proteins were separated by 2-dimensional PAGE, and protein spots were identified by microsequencing using electrospray (ISI-Q-TOF-Micromass) spectrometer. Components of 3 independent systems that can be involved in activation of endothelial cells, namely the prothrombinase complex, tumor necrosis factor-alpha, and angiotensinogen precursors were identified. Procoagulant activity of exosomes was confirmed by a thrombin generation assay using a specific chromogenic substrate. Because the potential of mast cell-derived exosomes to induce PAI-1 expression was completely abolished by hirudin, thrombin generated on exosomes seems to be responsible for this activity. CONCLUSIONS: It can be concluded that mast cell-derived exosomes via significant upregulation of PAI-1 secretion from endothelial cells may provide feedback between the characteristically increased PAI-1 levels and procoagulant states, both observed in diverse syndromes manifesting as endothelial cell dysfunction.

Fibrinogen contains cryptic PAI-1 binding sites that are exposed on binding to solid surfaces or limited proteolysis.

OBJECTIVE: In this work, we identified the fibrinogen sequence that on exposure serves as the primary binding site for functionally active PAI-1 and to a lesser extent for its latent form. In contrast, this site only weakly interacts with PAI-1 substrate. METHODS AND RESULTS: The binding site is located in the N-terminal alpha (20-88) segment of fibrinogen, in the region exposed on (1) adsorption of fibrinogen to solid surfaces; (2) the release of fibrinopeptide A during thrombin conversion of fibrinogen to fibrin; and (3) plasmin degradation of fibrinogen. This region was first identified by the yeast 2-hybrid system, then its binding characteristics were evaluated using the recombinant alpha(16-120) fragment and its shorter version, the alpha(20-88) fragment, in a solid phase binding assay and plasmon surface resonance measurements. Because fibrinogen fragment E does not bind PAI-1, it suggests that sequences of Aalpha chain interacting with PAI-1 are located in the N-terminal part of the alpha(20-88) segment. CONCLUSIONS: Therefore, PAI-1 directly bound to the alpha(20-88) and thus concentrated in fibrinogen/fibrin, particularly at sites of injury and inflammation, may account for the recent observations that both its active and latent forms stimulate cell migration and wound healing.

Induction of PAI-1 expression by tumor necrosis factor alpha in endothelial cells is mediated by its responsive element located in the 4G/5G site.

Plasminogen activator inhibitor type 1 (PAI-1) is induced by many proinflammatory and pro-oxidant factors. Among them, tumor necrosis factor alpha (TNFalpha), a pivotal early mediator that regulates and amplifies the development of inflammation, is one of the strongest PAI-1 synthesis activators. Location of the TNFalpha response element in the PAI-1 promoter is still ambiguous. In this study, we attempted to evaluate the significance of the element located in the 4G/5G site of the PAI-1 promoter in the TNFalpha stimulation of PAI-1 expression in endothelial cells. PAI-1 expression was monitored at: (a) the level of mRNA using real-time PCR, (b) PAI-1 gene transcription by transfection reporter assays, and (c) protein synthesis using the enzyme immunoassay. NF-kappaB activity was monitored using the electrophoretic mobility shift assay. Its activity was modified by either antisense oligonucleotides or transfection of endothelial cells with the wild-type or mutated IkappaBalpha. We have shown that TNFalpha-induced expression and gene transcription of PAI-1 involves a regulatory region present in segment -664/-680 of the PAI-1 promoter. This reaction involves the TNFalpha-induced generation of superoxide leading to activation of NF-kappaB, and can be abolished by antioxidants and by overexpression of a super-suppressor phosphorylation-resistant IkappaBalpha. Stimulation of PAI-1 under these conditions involves the motif of the PAI-1 promoter adjacent to the 4G/5G site, which can directly interact with NF-kappaB. We show that activation of PAI-1 gene by TNFalpha and reactive oxygen species is mediated by interaction of NF-kappaB with the cis-acting element located in the -675 4G/5G insertion/deletion in the PAI-1 promoter.

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.

Pathogenic activity of circulating anti-desmoglein-3 autoantibodies isolated from pemphigus vulgaris patients.

INTRODUCTION: There are scarce data on immunochemical properties of pemphigus antibodies detected in clinical remission in pemphigus vulgaris (PV) patients. The aim of the study was to compare biological activity of anti-Dsg3 autoantibodies purified from the sera of PV patients in active stage and in clinical remission. MATERIAL AND METHODS: The effect of purified antibodies on expression of procaspase-3, Bax, Bcl-2, uPAR, IL-1ß, IL-6, and TNF-α mRNAs in the HaCaT keratinocytes was evaluated by Western blot and RT-PCR method. RESULTS: Incubation of HaCaT cells with anti-Dsg-3 autoantibodies caused their binding to cell membranes surfaces. Anti-Dsg3 autoantibodies isolated from the patients in active stage and clinical remission showed proapoptotic effect, caused enhanced expression of analyzed proinflammatory cytokines' mRNAs and uPAR mRNA. CONCLUSIONS: Our data revealed similar pathogenic activity of anti Dsg-3 autoantibodies isolated from active and clinical remission PV patients.

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.