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PloS One 2015The DNA-binding protein AT-Rich Interactive Domain 3B (ARID3B) is elevated in ovarian cancer and increases tumor growth in a xenograft model of ovarian cancer. However,...
The DNA-binding protein AT-Rich Interactive Domain 3B (ARID3B) is elevated in ovarian cancer and increases tumor growth in a xenograft model of ovarian cancer. However, relatively little is known about ARID3B's function. In this study we perform the first genome wide screen for ARID3B direct target genes and ARID3B regulated pathways. We identified and confirmed numerous ARID3B target genes by chromatin immunoprecipitation (ChIP) followed by microarray and quantitative RT-PCR. Using motif-finding algorithms, we characterized a binding site for ARID3B, which is similar to the previously known site for the ARID3B paralogue ARID3A. Functionality of this predicted site was demonstrated by ChIP analysis. We next demonstrated that ARID3B induces expression of its targets in ovarian cancer cell lines. We validated that ARID3B binds to an epidermal growth factor receptor (EGFR) enhancer and increases mRNA expression. ARID3B also binds to the promoter of Wnt5A and its receptor FZD5. FZD5 is highly expressed in ovarian cancer cell lines, and is upregulated by exogenous ARID3B. Both ARID3B and FZD5 expression increase adhesion to extracellular matrix (ECM) components including collagen IV, fibronectin and vitronectin. ARID3B-increased adhesion to collagens II and IV require FZD5. This study directly demonstrates that ARID3B binds target genes in a sequence-specific manner, resulting in increased gene expression. Furthermore, our data indicate that ARID3B regulation of direct target genes in the Wnt pathway promotes adhesion of ovarian cancer cells.
Topics: Algorithms; Animals; Cell Line, Tumor; Chromatin Immunoprecipitation; DNA-Binding Proteins; Extracellular Matrix; Female; Fibronectins; Frizzled Receptors; Humans; Mice; Mice, Nude; Ovarian Neoplasms; RNA, Messenger; Reverse Transcriptase Polymerase Chain Reaction; Vitronectin
PubMed: 26121572
DOI: 10.1371/journal.pone.0131961 -
The Biochemical Journal Aug 1998Several bacterial pathogens including Neisseria gonorrhoeae bind the human serum glycoprotein vitronectin. We aimed at defining the gonococcal receptor for vitronectin....
Several bacterial pathogens including Neisseria gonorrhoeae bind the human serum glycoprotein vitronectin. We aimed at defining the gonococcal receptor for vitronectin. Ligand blots demonstrated that vitronectin bound specifically to the heparin-binding outer-membrane protein OpaA, but that coating OpaA with the sulphated polysaccharide heparin was required for the interaction to occur. Bound vitronectin could be dissociated from OpaA-heparin-vitronectin complexes by the addition of excess heparin, indicating that sulphated polysaccharides provided the main linkage between the two proteins. Binding assays with intact micro-organisms substantiated the requirement of sulphated polysaccharides such as heparin and dextran sulphate for the efficient binding of vitronectin to OpaA+ gonococci. This was underscored by the increased binding of vitronectin to gonococci that had been preincubated with saturating concentrations of dextran sulphate, as opposed to the inhibition of vitronectin binding observed when bacteria were incubated simultaneously with vitronectin and saturating concentrations of dextran sulphate. Binding assays with dextran sulphates of various sizes indicated that vitronectin binding correlated with the size of the polysaccharide rather than with the amount of OpaA produced by the bacteria. The inability of zero-length cross-linking agents to couple vitronectin to OpaA provided further evidence that sulphated polysaccharides formed the linkage between vitronectin and OpaA. Infection experiments demonstrated that proteoglycan-deficient Chinese hamster ovary cells efficiently internalized dextran sulphate/vitronectin-coated gonococci, suggesting that soluble sulphated polysaccharides could substitute for cell surface glycosaminoglycans in the internalization process. On the basis of our results, we propose a novel mechanism of vitronectin binding in which sulphated polysaccharides act as molecular bridges, linking the glycosaminoglycan-binding sites of vitronectin and gonococcal OpaA.
Topics: Animals; Antigens, Bacterial; Bacterial Outer Membrane Proteins; Binding Sites; CHO Cells; Cricetinae; Dextran Sulfate; Glycosaminoglycans; Heparin; Humans; Kinetics; Ligands; Models, Molecular; Neisseria gonorrhoeae; Vitronectin
PubMed: 9693112
DOI: 10.1042/bj3340133 -
The Journal of Biological Chemistry Jan 1998The initial site of melanoma cell metastasis is frequently the regional lymph nodes, and the appearance of lymph node metastasis correlates with poor prognosis. Lymph...
The initial site of melanoma cell metastasis is frequently the regional lymph nodes, and the appearance of lymph node metastasis correlates with poor prognosis. Lymph node adhesion is mediated by an interaction between the tumor cell integrin alphavbeta3 and lymph node vitronectin. In this study, we explored the relationship between adhesion and proteolysis by examining the direct effect of vitronectin receptor ligation on matrix metalloproteinase-2 (MMP-2) production by B16F1 and B16F10 melanoma cells. We report a dose-dependent increase in secretion of both MMP-2 and tissue inhibitor of metalloproteinases-2 (TIMP-2) in response to vitronectin. Cellular invasiveness was also enhanced by vitronectin, as shown by the increased ability of vitronectin-treated cells to invade a synthetic basement membrane (Matrigel). Both the vitronectin-induced MMP-2 production and vitronectin-enhanced invasion were blocked by the peptide ligand Arg-Gly-Asp-Ser (RGDS). Furthermore, neither plasmin-degraded vitronectin nor the peptide ligand RGDS stimulated MMP-2 secretion or invasiveness, indicating that a multivalent ligand-receptor interaction rather than simple receptor occupancy was required for MMP-2 induction. MMP-2 and MMP-2/TIMP-2 interaction with the plasma membrane of melanoma cells resulted in enhanced catalytic activity against 14C-labeled gelatin, suggesting that membrane association may function in posttranslational regulation of MMP-2 activity. This is supported by data showing increased cellular invasion by cells containing membrane-bound MMP-2. Binding of proMMP-2 and proMMP-2/TIMP-2 to melanoma cells was not inhibited by RGDS, and melanoma cell adhesion to vitronectin was unaffected by pro- or active MMP-2, indicating that MMP-2 did not interact with the murine vitronectin receptor. Together, these data provide evidence for a functional link between adhesion and proteolysis and suggest a potential mechanism whereby adhesion of an invasive cell to the extracellular matrix regulates subsequent invasive behavior.
Topics: Animals; Enzyme Induction; Gelatinases; Matrix Metalloproteinase 2; Melanoma, Experimental; Metalloendopeptidases; Mice; Neoplasm Invasiveness; Tissue Inhibitor of Metalloproteinase-2; Tumor Cells, Cultured; Vitronectin
PubMed: 9417058
DOI: 10.1074/jbc.273.1.143 -
Oligonucleotides Jun 2009The serine protease inhibitor plasminogen activator inhibitor-1 (PAI-1) is increased in several cancers, including breast, where it is associated with a poor outcome....
The serine protease inhibitor plasminogen activator inhibitor-1 (PAI-1) is increased in several cancers, including breast, where it is associated with a poor outcome. Metastatic breast cancer has a dismal prognosis, as evidenced by treatment goals that are no longer curative but are largely palliative in nature. PAI-1 competes with integrins and the urokinase plasminogen activator receptor on the surface of breast cancer cells for binding to vitronectin. This results in the detachment of tumor cells from the extracellular matrix, which is critical to the metastatic process. For this reason, we sought to isolate RNA aptamers that disrupt the interaction between PAI-1 and vitronectin. Through utilization of combinatorial chemistry techniques, aptamers have been selected that bind to PAI-1 with high affinity and specificity. We identified two aptamers, WT-15 and SM-20, that disrupt the interactions between PAI-1 and heparin, as well as PAI-1 and vitronectin, without affecting the antiprotease activity of PAI-1. Furthermore, SM-20 prevented the detachment of breast cancer cells (MDA-MB-231) from vitronectin in the presence of PAI-1, resulting in an increase in cellular adhesion. Therefore, the PAI-1 aptamer SM-20 demonstrates therapeutic potential as an antimetastatic agent and could possibly be used as an adjuvant to traditional chemotherapy for breast cancer.
Topics: Aptamers, Nucleotide; Breast Neoplasms; Cell Adhesion; Heparin; Humans; Neoplasm Metastasis; Plasminogen Activator Inhibitor 1; RNA; SELEX Aptamer Technique; Thrombin; Vitronectin
PubMed: 19284310
DOI: 10.1089/oli.2008.0177 -
BMC Evolutionary Biology Jan 2019The plasminogen (PLG) activation system is composed by a series of serine proteases, inhibitors and several binding proteins, which together control the temporal and...
BACKGROUND
The plasminogen (PLG) activation system is composed by a series of serine proteases, inhibitors and several binding proteins, which together control the temporal and spatial generation of the active serine protease plasmin. As this proteolytic system plays a central role in human physiology and pathophysiology it has been extensively studied in mammals. The serine proteases of this system are believed to originate from an ancestral gene by gene duplications followed by domain gains and deletions. However, the identification of ancestral forms in primitive chordates supporting these theories remains elusive. In addition, evolutionary studies of the non-proteolytic members of this system are scarce.
RESULTS
Our phylogenetic analyses place lamprey PLG at the root of the vertebrate PLG-group, while lamprey PLG-related growth factors represent the ancestral forms of the jawed-vertebrate orthologues. Furthermore, we find that the earliest putative orthologue of the PLG activator group is the hyaluronan binding protein 2 (HABP2) gene found in lampreys. The prime plasminogen activators (tissue- and urokinase-type plasminogen activator, tPA and uPA) first occur in cartilaginous fish and phylogenetic analyses confirm that all orthologues identified compose monophyletic groups to their mammalian counterparts. Cartilaginous fishes exhibit the most ancient vitronectin of all vertebrates, while plasminogen activator inhibitor 1 (PAI-1) appears for the first time in cartilaginous fishes and is conserved in the rest of jawed vertebrate clades. PAI-2 appears for the first time in the common ancestor of reptiles and mammals, and represents the latest appearing plasminogen activator inhibitor. Finally, we noted that the urokinase-type plasminogen activator receptor (uPAR)-and three-LU domain containing genes in general-occurred later in evolution and was first detectable after coelacanths.
CONCLUSIONS
This study identifies several primitive orthologues of the mammalian plasminogen activation system. These ancestral forms provide clues to the origin and diversification of this enzyme system. Further, the discovery of several members-hitherto unknown in mammals-provide new perspectives on the evolution of this important enzyme system.
Topics: Amino Acid Sequence; Animals; Chordata; Databases, Protein; Genetic Variation; Humans; Likelihood Functions; Phylogeny; Plasminogen; Plasminogen Activator Inhibitor 1; Protein Domains; Sequence Analysis, RNA; Transcriptome; Urokinase-Type Plasminogen Activator; Vitronectin
PubMed: 30654737
DOI: 10.1186/s12862-019-1353-z -
Laboratory Investigation; a Journal of... Jan 2002Different extracellular matrix proteins have been described as binding proteins for growth factors, influencing their storage or presentation towards cellular receptors....
Different extracellular matrix proteins have been described as binding proteins for growth factors, influencing their storage or presentation towards cellular receptors. The multifunctional adhesive glycoprotein vitronectin (VN), which is found in the circulation and widely distributed throughout different tissues, has been implicated in the regulation of vascular cell functions, and these activities could be related to interactions with various growth factors. In vitro, soluble VN interfered with transforming growth factor-beta (TGF-beta) binding to isolated extracellular matrix and was found to associate with TGF-beta1 and TGF-beta2 as well as with other growth factors such as vascular endothelial growth factor, epidermal growth factor, or basic fibroblast growth factor in a saturable manner. In particular, binding of TGF-beta was maximal for the heparin-binding multimeric isoform of VN, whereas VN in a ternary complex with thrombin and antithrombin or plasma VN exhibited weaker binding. Plasminogen activator inhibitor-1 (PAI-1) or heparin, but not desulfated glycosaminoglycans, interfered with binding of VN to TGF-beta, and soluble PAI-1 was able to dissociate VN-bound TGF-beta. Upon limited plasmin proteolysis of VN, only the fragments comprising the intact aminoterminal portion of VN bound to TGF-beta as did a synthetic peptide (amino acids 43 to 62), indicating that TGF-beta and PAI-1 share common binding site(s) on VN. Although VN did not influence TGF-beta bioactivity for mink lung epithelial cells, TGF-beta dose dependently inhibited both urokinase-receptor as well as alpha(v)-integrin-dependent adhesion to VN. This activity of TGF-beta was reminiscent of the antiadhesive function of PAI-1. In atherosclerotic tissue sections, staining patterns of VN and TGF-beta indicated their colocalization. These findings describe VN as a new binding protein for TGF-beta, whereby specific functions of both factors become modulated by this interaction.
Topics: Animals; B-Lymphocytes; Cell Line; Electrophoresis, Polyacrylamide Gel; Extracellular Matrix; Fibronectins; Humans; Kinetics; Mice; Protein Binding; Protein Isoforms; Transforming Growth Factor beta; Transforming Growth Factor beta1; Transforming Growth Factor beta2; Vitronectin
PubMed: 11796824
DOI: 10.1038/labinvest.3780393 -
Tissue Engineering. Part A Apr 2016Soft tissue defects are relatively common, yet currently used reconstructive treatments have varying success rates, and serious potential complications such as...
Soft tissue defects are relatively common, yet currently used reconstructive treatments have varying success rates, and serious potential complications such as unpredictable volume loss and reabsorption. Human adipose-derived stem cells (ASCs), isolated from liposuction aspirate have great potential for use in soft tissue regeneration, especially when combined with a supportive scaffold. To design scaffolds that promote differentiation of these cells down an adipogenic lineage, we characterized changes in the surrounding extracellular environment during adipogenic differentiation. We found expression changes in both extracellular matrix proteins, including increases in expression of collagen-IV and vitronectin, as well as changes in the integrin expression profile, with an increase in expression of integrins such as αVβ5 and α1β1. These integrins are known to specifically interact with vitronectin and collagen-IV, respectively, through binding to an Arg-Gly-Asp (RGD) sequence. When three different short RGD-containing peptides were incorporated into three-dimensional (3D) hydrogel cultures, it was found that an RGD-containing peptide derived from vitronectin provided strong initial attachment, maintained the desired morphology, and created optimal conditions for in vitro 3D adipogenic differentiation of ASCs. These results describe a simple, nontoxic encapsulating scaffold, capable of supporting the survival and desired differentiation of ASCs for the treatment of soft tissue defects.
Topics: Adipogenesis; Adipose Tissue; Amino Acid Sequence; Biomimetic Materials; Cell Adhesion; Cells, Immobilized; Extracellular Matrix; Humans; Hydrogel, Polyethylene Glycol Dimethacrylate; Integrins; Oligopeptides; Polyethylene Glycols; Stem Cells; Tissue Scaffolds; Vitronectin
PubMed: 26956095
DOI: 10.1089/ten.TEA.2015.0550 -
Journal of Thrombosis and Haemostasis :... Mar 2011A growing body of experimental evidence supports broad inhibitory and regulatory activity of plasminogen activator inhibitor 1 (PAI-1). The present study was designed to...
BACKGROUND AND OBJECTIVE
A growing body of experimental evidence supports broad inhibitory and regulatory activity of plasminogen activator inhibitor 1 (PAI-1). The present study was designed to investigate whether PAI-1 inhibits factor (F) VIIa complexed with tissue factor (TF), a well-known procoagulant risk factor.
METHODS AND RESULTS
The ability of PAI-1 to inhibit FVIIa-TF activity was evaluated in both clotting and factor X (FX) activation assays. PAI-1 and its complex with vitronectin inhibit: (i) clotting activity of FVIIa-TF (PAI-1(IC50) , 817 and 125 nm, respectively); (ii) FVIIa-TF-mediated FX activation (PAI-1(IC50) , 260 and 50 nm, respectively); and (iii) FVIIa bound to TF expressed on the surface of stimulated endothelial cells (PAI-1(IC50) , 260 and 120 nm, respectively). The association rate constant (k(a)) for PAI-1 inhibition of FVIIa-TF was determined using a chromogenic assay. K(a) for PAI-1 inhibition of FVIIa bound to relipidated TF is 3.3-fold higher than that for FVIIa bound to soluble TF (k(a) = 0.09 ± 0.01 and 0.027 ± 0.03 μm(-1) min(-1), respectively). Vitronectin increases k(a) for both soluble and relipidated TF by 3.5- and 30-fold, respectively (to 0.094 ± 0.020 and 2.7 ± 0.2 μm(-1) min(-1)). However, only a 3.5- to 5.0-fold increase in the acylated FVIIa was observed on SDS PAGE in the presence of vitronectin for both relipidated and soluble TF, indicating fast formation of PAI-1/vitronectin/FVIIa/relipidated TF non-covalent complex.
CONCLUSIONS
Our results demonstrate potential anticoagulant activity of PAI-1 in the presence of vitronectin, which could contribute to regulation of hemostasis under pathological conditions such as severe sepsis, acute lung injury and pleural injury, where PAI-1 and TF are overexpressed.
Topics: Cell Membrane; Cells, Cultured; Factor VIIa; Hemostasis; Humans; In Vitro Techniques; Kinetics; Multiprotein Complexes; Plasminogen Activator Inhibitor 1; Protein Binding; Recombinant Proteins; Thromboplastin; Vitronectin
PubMed: 21143380
DOI: 10.1111/j.1538-7836.2010.04167.x -
Proceedings of the National Academy of... Aug 2020The human blood protein vitronectin (Vn) is a major component of the abnormal deposits associated with age-related macular degeneration, Alzheimer's disease, and many...
The human blood protein vitronectin (Vn) is a major component of the abnormal deposits associated with age-related macular degeneration, Alzheimer's disease, and many other age-related disorders. Its accumulation with lipids and hydroxyapatite (HAP) has been demonstrated, but the precise mechanism for deposit formation remains unknown. Using a combination of solution and solid-state NMR experiments, cosedimentation assays, differential scanning fluorimetry (DSF), and binding energy calculations, we demonstrate that Vn is capable of binding both soluble ionic calcium and crystalline HAP, with high affinity and chemical specificity. Calcium ions bind preferentially at an external site, at the top of the hemopexin-like (HX) domain, with a group of four Asp carboxylate groups. The same external site is also implicated in HAP binding. Moreover, Vn acquires thermal stability upon association with either calcium ions or crystalline HAP. The data point to a mechanism whereby Vn plays an active role in orchestrating calcified deposit formation. They provide a platform for understanding the pathogenesis of macular degeneration and other related degenerative disorders, and the normal functions of Vn, especially those related to bone resorption.
Topics: Binding Sites; Calcium; Durapatite; Humans; Macular Degeneration; Protein Binding; Vitronectin
PubMed: 32699145
DOI: 10.1073/pnas.2007699117 -
Neurochemistry International Jul 2013Damage to the central nervous system (CNS) leads to increased production of TNF-α and TGF-β1 cytokines that have pro- or anti-inflammatory actions, respectively. To...
Damage to the central nervous system (CNS) leads to increased production of TNF-α and TGF-β1 cytokines that have pro- or anti-inflammatory actions, respectively. To define whether astrocytes or microglia express these cytokines, prior studies have used mixed glial cultures (MGC) to represent astrocytes, thought these results are inevitably complicated by the presence of contaminating microglia within MGC. To clarify the cellular source of these cytokines, here we employed a recently described method of preparing microglia-free astrocyte cultures, in which neural stem cells (NSC) are differentiated into astrocytes. Using ELISA to quantify cytokine production in three types of glial culture: MGC, pure microglia or pure astrocytes, this showed that microglia but not astrocytes, produce TNF-α, and that this expression is increased by LPS, IFN-γ, and to a lesser extent by vitronectin, but decreased by TGF-β1. In contrast, TGF-β1 was produced by microglia and astrocytes, though at 10-fold higher levels by microglia. TGF-β1 expression in microglia was increased by vitronectin and to a lesser extent by TNF-α and LPS, but astrocyte TGF-β1 expression was not regulated by any factor tested. In summary, our data reveal that microglia, not astrocytes are the major source of TNF-α and TGF-β1 in postnatal glial cultures, and that microglial production of these antagonistic cytokines is tightly regulated by cytokines, LPS, and vitronectin.
Topics: Animals; Cells, Cultured; Enzyme-Linked Immunosorbent Assay; Lipopolysaccharides; Mice; Mice, Inbred C57BL; Microglia; Transforming Growth Factor beta1; Tumor Necrosis Factor-alpha; Vitronectin
PubMed: 23619393
DOI: 10.1016/j.neuint.2013.04.007