Joanna Boncela

Acute Phase Protein α1-Acid Glycoprotein Interacts with Plasminogen Activator Inhibitor Type 1 and Stabilizes Its Inhibitory Activity

α1-Acid glycoprotein, one of the major acute phase proteins, was found to interact with plasminogen activator inhibitor type 1 (PAI-1) and to stabilize its inhibitory activity toward plasminogen activators. This conclusion is based on the following observations: (a) α1-acid glycoprotein was identified to bind PAI-1 by a yeast two-hybrid system. Three of 10 positive clones identified by this method to interact with PAI-1 contained almost the entire sequence of α1-acid glycoprotein; (b) this protein formed complexes with PAI-1 that could be immunoprecipitated from both the incubation mixtures and blood plasma by specific antibodies to either PAI-1 or α1-acid glycoprotein. Such complexes could be also detected by a solid phase binding assay; and (c) the real-time bimolecular interactions monitored by surface plasmon resonance indicated that the complex of α1-acid glycoprotein with PAI-1 is less stable than that formed by vitronectin with PAI-1, but in both cases, the apparentK D values were in the range of strong interactions (4.51 + 1.33 and 0.58 + 0.07 nm, respectively). The on rate for binding of PAI-1 to α1-glycoprotein or vitronectin differed by 2-fold, indicating much faster complex formation by vitronectin than by α1-acid glycoprotein. On the other hand, dissociation of PAI-1 bound to vitronectin was much slower than that from the α1-acid glycoprotein, as indicated by 4-fold lower k off values. Furthermore, the PAI-1 activity toward urokinase-type plasminogen activator and tissue-type plasminogen activator was significantly prolonged in the presence of α1-acid glycoprotein. These observations suggest that the complex of PAI-1 with α1-acid glycoprotein can play a role as an alternative reservoir of the physiologically active form of the inhibitor, particularly during inflammation or other acute phase reactions.

Ku80 as a Novel Receptor for Thymosin β4 That Mediates Its Intracellular Activity Different from G-actin Sequestering

Our data demonstrate that increased intracellular expression of thymosin β4(Tβ4) 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 Tβ4 mutants with impaired functional motifs and tested their intracellular location and activity. Cytoplasmic distributions of Tβ4(AcSDKPT/4A), Tβ4(KLKKTET/7A), and Tβ4(K16A) mutants fused with green fluorescent protein did not differ significantly from those of wild-type Tβ4. Overexpression of Tβ4, Tβ4(AcSDKPT/4A), and Tβ4(K16A) affected intracellular formation of actin filaments. As expected, Tβ4(K16A) uptake by nuclei was impaired. On the other hand, overexpression of Tβ4(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 Tβ4 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 Tβ4 and Tβ4(AcSDKPT/4A) induced the PAI-1 gene to the same extent, whereas mutants Tβ4(KLKKTET/7A) and Tβ4(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 Tβ4. Ku80 and Tβ4 consistently co-immunoprecipitated in a complex from endothelial cells. Co-transfection of endothelial cells with the Ku80 deletion mutants and Tβ4 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 Tβ4 and mediates its intracellular activity.

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 α (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 α(16-120) fragment and its shorter version, the α(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 Aα chain interacting with PAI-1 are located in the N-terminal part of the α(20-88) segment. Conclusions—Therefore, PAI-1 directly bound to the α(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.

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.

Lumican Inhibits SNAIL-Induced Melanoma Cell Migration Specifically by Blocking MMP-14 Activity

Lumican, a small leucine rich proteoglycan, inhibits MMP-14 activity and melanoma cell migration in vitro and in vivo. Snail triggers epithelial-mesenchymal transitions endowing epithelial cells with migratory and invasive properties during tumor progression. The aim of this work was to investigate lumican effects on MMP-14 activity and migration of Snail overexpressing B16F1 (Snail-B16F1) melanoma cells and HT-29 colon adenocarcinoma cells. Lumican inhibits the Snail induced MMP-14 activity in B16F1 but not in HT-29 cells. In Snail-B16F1 cells, lumican inhibits migration, growth, and melanoma primary tumor development. A lumican-based strategy targeting Snail-induced MMP-14 activity might be useful for melanoma treatment.

Binding of PAI-1 to Endothelial Cells Stimulated by Thymosin β4 and Modulation of Their Fibrinolytic Potential

Our previous studies showed that thymosin β4 (Tβ4) induced the synthesis of plasminogen activator inhibitor-1 (PAI-1) in cultured human umbilical vein endothelial cells (HUVECs) via the AP-1 dependent mechanism and its enhanced secretion. In this work we provide evidence that the released PAI-1 is accumulated on the surface of HUVECs, exclusively in its active form, in a complex with α1-acid glycoprotein (AGP) that is also up-regulated and released from the cells. This mechanism is supported by several lines of experiments, in which expression of both proteins was analyzed by flow cytometry and their colocalization supported by confocal microscopy. PAI-1 did not bind to quiescent cells but only to the Tβ4-activated endothelial cells. In contrast, significant amounts of AGP were found to be associated with the cells overexpressing enhanced green fluorescent protein (EGFP)-α1-acid glycoprotein (AGP) without Tβ4 treatment. The AGP·PAI-1 complex was accumulated essentially at the basal surface of endothelial cells, and such cells showed (a) morphology characteristic for strongly adhered and spread cells and (b) significantly reduced plasmin formation. Taken together, these results provide the evidence supporting a novel mechanism by which active PAI-1 can be bound to the Tβ4-activated endothelial cells, thus influencing their adhesive properties as well as their ability to generate plasmin.

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.

Plasminogen Activator Inhibitor Type 1 Interacts with α3 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.

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

Class III β-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.

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.