Concurrent signaling from Gαq- and Gαi-coupled pathways is essential for agonist-induced αvβ3 activation on human platelets

Summary.  The integrin αvβ3 mediates platelet adhesion to the matrix protein osteopontin and likely is the predominant integrin mediating platelet adhesion to the matrix protein vitronectin. To address the mechanism that regulates αvβ3 activity in platelets, we measured the effect of the P2Y₁ antagonist adenosine 3'-phosphate-5'-phosphate (A3P5P) and the P2Y₁₂ antagonist AR-C66096 on ADP-stimulated platelet adhesion to osteopontin and vitronectin. Each antagonist completely inhibited platelet adhesion, implying that concurrent stimulation of P2Y₁ and P2Y₁₂ was required to activate αvβ3. The reducing agent dithiothreitol and Mn²⁺ also induced platelet adhesion to osteopontin, but did so without stimulating platelet activation. Thus, these data suggest that ADP stimulation regulates αvβ3 activity by perturbing the conformation of its extracellular domain. The actin polymerization inhibitors cytochalasin D and latrunculin A also induced platelet adhesion to osteopontin and vitronectin. Thus, αvβ3 activity in resting platelets appears to be constrained by the platelet cytoskeleton. Moreover, the effect of these agents was inhibited by A3P5P and AR-C66096 at micromolar and subnanomolar concentrations, respectively, suggesting that subthreshold platelet stimulation by ADP was required. Our data suggest that signals from both Gα_q- and Gα_i-coupled receptors converge to release cytoskeletal constraints on αvβ3. We propose that the release of cytoskeletal constraints and a concurrent increase in affinity for ligands is responsible for αvβ3-mediated platelet adhesion.     

Molecular and biological analysis of echovirus 9 strain isolated from a diabetic child

The full-length infectious cDNA clone was constructed and sequenced from the strain DM of echovirus 9, which was recently isolated from a 6-week-old child at the clinical onset of type 1 diabetes. Parallel with the isolate DM, the full-length infectious cDNA clone of the prototype strain echovirus 9 Barty (Barty-INF), was constructed and sequenced. Genetic relationships of the sequenced echo 9 viruses to the other members of the human enterovirus type B species were studied by phylogenetic analyses. Comparison of capsid protein sequences showed that the isolate DM was closely related to both prototype strains: Hill and Barty-INF. The only exception was the inner capsid protein VP4 where serotype specificity was not evident and the isolate DM clustered with the strain Hill and the strain Barty-INF with echovirus 30 Bastianni. Likewise, the nonstructural protein coding region, P2P3, of isolate DM was more similar to strain Hill than to strain Barty-INF. However, like echovirus 9 Barty, the isolate DM contained the RGD-motif in the carboxy terminus of capsid protein VP1. By blocking experiments using an RGD-containing peptide and a polyclonal rabbit antiserum to the alpha(v)beta(3)-integrin, it was shown that this molecule works as a cellular receptor for isolate DM. By using primary human islets, it was shown that the isolate DM is capable of infecting insulin-producing beta-cells like the corresponding prototype strains did. However, only isolate DM was clearly cytolytic for beta-cells. The infectious clones that were made allow further investigations of the molecular features responsible for the diabetogenicity of the isolate DM.     

Vitronectin-mediated inhibition of complement: evidence for different binding sites for C5b-7 and C9.

In the activated complement system, vitronectin (complement S-protein) occupies the metastable membrane binding site of the nascent precursor complex C5b-7, so that the newly formed SC5b-7 is unable to insert into cell membranes. Some evidence also indicates that vitronectin limits on-going membrane-associated pore formation by inhibiting C9 polymerization. It has been assumed that these two stages of terminal complement complex (TCC) inhibition take place through charge interactions between the heparin-binding region of vitronectin and homologous cysteine-rich sequences of the late complement proteins C6, C7, C8 and C9. We examined SC5b-7 formation and inhibition of C9 binding in the TCC using separate haemolytic assays. The mode of action of vitronectin in these assays was compared with two 15mer peptides which span residues 348-379 of the heparin-binding region, and a heparin-affinity polypeptide, protamine sulphate. The results showed that vitronectin acts predominantly through SC5b-7 production with a lesser effect on the inhibition of C9 lytic pore formation. In contrast, protamine sulphate did not prevent C5b-7 membrane attachment, but was a potent inhibitor of C9-mediated lysis. The peptides did not inhibit C5b-7 membrane insertion and only one affected C9 binding. These data suggest that the two stages of TCC inhibition involve separate binding sites on the vitronectin molecule. The site for association with nascent C5b-7 is unknown, whereas inhibition of C9 binding and pore formation takes place through the heparin-binding region.     

Plasminogen activator inhibitor-1 is an aggregate response factor with pleiotropic effects on cell signaling in vascular disease and the tumor microenvironment

In hemostasis, the serine protease inhibitor (serpin) plasminogen activator inhibitor-1 (PAI-1) functions to stabilize clots via inhibition of tissue plasminogen activator (tPA) with subsequent inhibition of fibrinolysis. In tissues, PAI-1 functions to inhibit extracellular matrix degradation via inhibition of urokinase plasminogen activator (uPA). Elevated levels of PAI-1 in the vasculature and in tissues have long been known to be associated with thrombosis and fibrosis, respectively. However, there is emerging evidence that PAI-1 may participate in the pathophysiology of a number of diseases such as atherosclerosis, restenosis, and cancer. In many of these disease states, the canonical view of PAI-1 as an inhibitor of tPA and uPA cannot fully account for a mechanism whereby PAI-1 contributes to the disease. In these cases, one must consider recent data, which indicates PAI-1 can directly promote pro-proliferative and anti-apoptotic signaling in a variety of cell types. Given the wide variety of inflammatory, hormonal, and metabolic signals that increase PAI-1 expression, it is important to consider mechanisms by which PAI-1 can directly participate in disease etiology.     

[Online First]Improvement of human embryonic stem cell-derived retinal pigment epithelium cell adhesion, maturation， and function through coating with truncated recombinant human vitronectin

AIM: To explore an xeno-free and defined coating substrate suitable for the culture of H9 human embryonic stem cell-derived retinal pigment epithelial (hES-RPE) cells in vitro, and compare the behaviors and functions of hES-RPE cells on two culture substrates, laminin521 (LN-521) and truncated recombinant human vitronectin (VTN-N). METHODS: hES-RPE cells were used in the experiment. The abilities of LN-521 and VTN-N at different concentrations to adhere to hES-RPE cells were compared with a high-content imaging system. Quantitative real-time polymerase chain reaction was used to evaluate RPE-specific gene expression levels midway (day 10) and at the end (day 20) of the time course. Cell polarity was observed by immunofluorescent staining for apical and basal markers of the RPE. The phagocytic ability of hES-RPE cells was identified by flow cytometry and immunofluorescence. RESULTS: The cell adhesion assay showed that the ability of LN-521 to adhere to hES-RPE cells was dose-dependent. With increasing coating concentration, an increasing number of cells attached to the surface of LN-521-coated wells. In contrast, VTN-N presented a strong adhesive ability even at a low concentration. The optimal concentration of LN-521 and VTN-N required to coat and adhesion to hES-RPE cells were 2 and 0.25 µg/cm2, respectively. Furthermore, both LN-521 and VTN-N could facilitate adoption of the desired cobblestone cellular morphology with tight junction and showed polarity by the hES-RPE cells. However, hES-RPE cells cultivated in VTN-N had a greater phagocytic ability, and it took less time for these hES-RPE cells to mature. CONCLUSION: VTN-N is a more suitable coating substrate for cultivating hES-RPE cells.     

The soluble form of urokinase receptor promotes angiogenesis through its Ser88‐Arg‐Ser‐Arg‐Tyr92 chemotactic sequence

Summary. Background: The urokinase plasminogen activator receptor (u‐PAR) focuses the proteolytic activity of the urokinase plasminogen activator (u‐PA) on the endothelial cell surface, thus promoting angiogenesis in a protease‐dependent manner. The u‐PAR may exist in a glycophosphatidylinositol‐anchored and in a soluble form (soluble u‐PAR [Su‐PAR]), both including the chemotactic Ser⁸⁸‐Arg‐Ser‐Arg‐Tyr⁹² internal sequence. Objective: To investigate whether Su‐PAR may trigger endothelial cell signaling leading to new vessel formation through its chemotactic Ser⁸⁸‐Arg‐Ser‐Arg‐Tyr⁹² sequence. Methods and Results: In this study, the formation of vascular‐like structures by human umbilical vein endothelial cells was assessed by using a matrigel basement membrane preparation. First, we found that Su‐PAR protein promotes the formation of cord‐like structures, and that this ability is retained by the isolated Ser⁸⁸‐Arg‐Ser‐Arg‐Tyr⁹² chemotactic sequence, the maximal effect being reached at 10 nmol L^?1 SRSRY peptide (SRSRY). This effect is mediated by the α_vβ₃ vitronectin receptor, is independent of u‐PA proteolytic activity, and involves the internalization of the G‐protein‐coupled formyl‐peptide receptor in endothelial cells. Furthermore, exposure of human saphenous vein rings to Su‐PAR or SRSRY leads to a remarkable degree of sprouting. Finally, we show that Su‐PAR and SRSRY promote a marked response in angioreactors implanted into the dorsal flank of nude mice, retaining 91% and 66%, respectively, of the angiogenic response generated by a mixture of vascular endothelial growth factor and fibroblast growth factor type 2. Conclusions: Our results show a new protease‐independent activity of Su‐PAR that stimulates in vivo angiogenesis through its Ser⁸⁸‐Arg‐Ser‐Arg‐Tyr⁹² chemotactic sequence.     

Early vitronectin receptor downregulation in a melanoma cell line during all-trans retinoic acid-induced apoptosis

BACKGROUND: Recent evidence assigns the vitronectin receptors (VnRs) an important role in regulating tumour cell invasion and dissemination. In vivo and in vitro studies document that all trans-retinoid acids (ATRAs) inhibit growth-inducing apoptosis in melanomas. OBJECTIVES: We have analysed the effects of ATRA treatment on melanoma cell adhesion and motility. METHODS: Human M14 melanoma cells were treated with 10 micromol L-1 ATRA for different times and stained with rhodamine-phalloidin to analyse the effect of treatment on cytoskeleton organization. Cell adhesion and cell migration assays were performed to analyse the role of VnRs in the ATRA-induced early stages of apoptosis. VnR expression was evaluated by Western blot, immunoprecipitation and immunocytochemistry assays. RESULTS: First, using an annexin V assay, we found that apoptosis was triggered by 48 h with 10 micromol L-1 ATRA exposure. At this time point, decrease in the F-actin polymerization as well as inhibition of cell adhesive ability to vitronectin (Vn) was exerted by ATRA treatment. In the presence of serum, exposure to 10 micromol L-1 ATRA for 48 h produced a dramatic inhibition of the cell adhesion ability that was comparable with that exerted by untreated cells preincubated with anti-alpha(v)beta(3) or anti-alpha(v)beta(5) VnR monoclonal antibodies. Functionally, the treatment of melanoma cells with 10 micromol L-1 ATRA for 48 h causes an inhibition of directional cell migration towards Vn-coated filters. Therefore, we analysed the effect of ATRA on the VnR expression. Both alpha(v)beta(3) and alpha(v)beta(5) VnR levels were reduced upon exposure to 10 micromol L-1 ATRA for 48 h as shown by Western blot, immunoprecipitation and immunocytochemistry assays. CONCLUSIONS: Altogether, our data indicate that treatment of M14 melanoma cells with ATRA downregulates VnR expression and that this reduction is closely correlated with the ATRA-dependent inhibition of actin-fibre organization, cell adhesion and migration. Although the mechanism by which ATRA regulates the expression of VnR in M14 melanoma cells needs further elucidation, this system may represent a model for understanding the molecular basis of ATRA therapy in melanoma.     

Engaged urokinase receptors enhance tumor breast cell migration and invasion by upregulating alpha(v)beta5 vitronectin receptor cell surface expression

We have previously shown that urokinase receptor physically and functionally interacts with alpha(v)beta5 vitronectin receptor, leading to tumor breast cell migration and invasion. Here, the link between these 2 receptors was further investigated by analyzing the expression levels of urokinase receptor and alpha(v)beta5 integrin in 35 human breast carcinomas and 5 benign breast lesions. The occurrence of a positive correlation between urokinase receptor and alpha(v)beta5 protein levels in benign and malignant tumor specimens prompted us to investigate whether engaged urokinase receptors might modulate alpha(v)beta5 expression. Here, we report the receptor-dependent ability of catalytically inactive urokinase to upregulate the alpha(v) and beta5 chains in MDA-MB-231 and MCF-7 breast carcinoma cell lines in a time- and concentration-dependent manner. This effect is dependent on protein kinase C activity and requires new protein synthesis. Accordingly, the availability of assembled alpha(v)beta5 receptors on the cell surface increases upon urokinase treatment, as shown by immunoprecipitation and immunocytochemical analyses. Exposure to urokinase leads to enhanced tumor cell migration and invasion, which is prevented by the "phosphorylation-like" urokinase receptor antagonist His-uPA(138E/303E), the DNA-binding drug mithramycin, the protein kinase C inhibitor calphostin C and anti-alpha(v)beta5 antibodies. Finally, urokinase enables benign breast MCF-10A cells to cross Matrigel in a alpha(v)beta5- and urokinase receptor-dependent manner, indicating that urokinase controls a regulatory circuitry crucial to breast tumor progression.     

Matrix metalloproteinase expression in breast cancer

BACKGROUND: Matrix metalloproteinases (MMPs) have been implicated as possible mediators of invasion and metastasis in some cancers. Our objective was to investigate which MMPs were constitutively expressed in breast tumor cells versus those that could be up-regulated by a number of agents known to affect MMP expression in other cell systems. METHODS: We evaluated expression of MMPs 1-16 in breast tumor cell lines MDA-MB-231, T47D, and MCF-7 using semiquantitative RT-PCR and gelatin zymography. Exposure to 12-O-tetradecanoylphorbal-3-acetate (TPA), concanavalin-A (Con-A), the fibronectin-mimetic peptide GRGDSP (RGD), extracellular matrix (ECM) components, and anti-integrin antibodies was used to test for possible MMP up-regulation. Mitogen-activated protein kinase inhibitors (MAPK-I) were used to evaluate signal transduction pathways and regulation of MMP expression. RESULTS: MMPs 1, 2, 7-11, 13, 14, and 16 were constitutively expressed in some tumor cell lines but not in normal breast epithelial cells. Administration of TPA, Con-A, and RGD increased the expression of MMPs 1, 2, 9, and 10. No MMP up-regulation was seen in MDA-MB-231 or MCF-7 after exposure to ECM components or after exposure to anti-integrin antibodies. MAPK-I had no effect on constitutive MMP expression but reduced or abolished the TPA up-regulation of MMP-9 in MDA-MB-231 and MCF-7. CONCLUSIONS: Breast tumor cell lines constitutively express a number of MMPs. Because MMP expression can be up-regulated by Con-A, the fibronectin-mimetic peptide RGD, and TPA while being susceptible to inhibition by MAPK antagonists, MAPK signaling appears to play a role in this expression.