Inhibition of surgical trauma-enhanced peritoneal dissemination of tumor cells by human catalase derivatives in mice

Surgical trauma, which is inevitably associated with the surgical removal of cancer, has been reported to accelerate tumor metastasis. The close association of reactive oxygen species with the trauma and tumor metastasis supports the possibility of using antioxidants for the inhibition of metastasis. To inhibit surgical trauma-enhanced peritoneal dissemination, human catalase (hCAT) derivatives, i.e., hCAT-nona-arginine peptide (hCAT-R9) and hCAT-albumin-binding peptide (hCAT-ABP), were designed to increase the retention time of the antioxidant enzyme in the abdominal cavity after intraperitoneal administration. Both ¹²⁵I-labeled derivatives showed significantly prolonged retention in the cavity compared to ¹²⁵I-hCAT. Cauterization of the cecum of mice with a hot iron, an experimental model of surgical trauma, induced abdominal adhesions. In addition, cauterization followed by colon26 tumor cell inoculation increased lipid peroxidation in the cecum and mRNA expression of molecules associated with tissue repair/adhesion and inflammation in the peritoneum. hCAT derivatives significantly suppressed the increased mRNA expression. The cauterization also increased the number of tumor cells in the abdominal organs, and the number was significantly reduced by hCAT-R9 or hCAT-ABP. These results indicate that hCAT-R9 and hCAT-ABP, both of which have a long retention time in the peritoneal cavity, can be effective at inhibiting surgery-induced peritoneal metastasis.     

The relative importance of topography and RGD ligand density for endothelial cell adhesion

The morphology and function of endothelial cells depends on the physical and chemical characteristics of the extracellular environment. Here, we designed silicon surfaces on which topographical features and surface densities of the integrin binding peptide arginine-glycine-aspartic acid (RGD) could be independently controlled. We used these surfaces to investigate the relative importance of the surface chemistry of ligand presentation versus surface topography in endothelial cell adhesion. We compared cell adhesion, spreading and migration on surfaces with nano- to micro-scaled pyramids and average densities of 6×10(2)-6×10(11) RGD/mm(2). We found that fewer cells adhered onto rough than flat surfaces and that the optimal average RGD density for cell adhesion was 6×10(5) RGD/mm(2) on flat surfaces and substrata with nano-scaled roughness. Only on surfaces with micro-scaled pyramids did the topography hinder cell migration and a lower average RGD density was optimal for adhesion. In contrast, cell spreading was greatest on surfaces with 6×10(8) RGD/mm(2) irrespectively of presence of feature and their size. In summary, our data suggest that the size of pyramids predominately control the number of endothelial cells that adhere to the substratum but the average RGD density governs the degree of cell spreading and length of focal adhesion within adherent cells. The data points towards a two-step model of cell adhesion: the initial contact of cells with a substratum may be guided by the topography while the engagement of cell surface receptors is predominately controlled by the surface chemistry.     

Regulation of endothelial glutathione by ICAM-1 governs VEGF-A-mediated eNOS activity and angiogenesis

Previous studies suggest that inflammatory cell adhesion molecules may modulate endothelial cell migration and angiogenesis through unknown mechanisms. Using a combination of in vitro and in vivo approaches, herein we reveal a novel redox-sensitive mechanism by which ICAM-1 modulates endothelial GSH that controls VEGF-A-induced eNOS activity, endothelial chemotaxis, and angiogenesis. In vivo disk angiogenesis assays showed attenuated VEGF-A-mediated angiogenesis in ICAM-1(-/-) mice. Moreover, VEGF-A-dependent chemotaxis, eNOS phosphorylation, and nitric oxide production were impaired in ICAM-1(-/-) mouse aortic endothelial cells (MAEC) compared to WT MAEC. Decreasing intracellular GSH in ICAM-1(-/-) MAEC to levels observed in WT MAEC with 150 microM buthionine sulfoximine restored VEGF-A responses. Conversely, GSH supplementation of WT MAEC with 5 mM glutathione ethyl ester mimicked defects observed in ICAM-1(-/-) cells. Deficient angiogenic responses in ICAM-1(-/-) cells were associated with increased expression of the lipid phosphatase PTEN, consistent with antagonism of signaling pathways leading to eNOS activation. PTEN expression was also sensitive to GSH status, decreasing or increasing in proportion to intracellular GSH concentrations. These data suggest a novel role for ICAM-1 in modulating VEGF-A-induced angiogenesis and eNOS activity through regulation of PTEN expression via modulation of intracellular GSH status.     

Triflavin,an Arg-Gly-Asp-containing peptide,inhibits B16-F10 mouse melanoma cell adhesion to matrix proteins via direct binding to tumor cells

Triflavin, an Arg-Gly-Asp (RGD)-containing snake venom peptide, inhibits B16-F10 mouse melanoma cell adhesion to extracellular matrices, e.g., fibronectin, vitronectin, fibrinogen, and collagen type I. In this study, GRGDS inhibits B16-F10 mouse melanoma cell adhesion to immobilized triflavin in a dose-dependent manner. In addition, flow-cytometric analysis and the fluorescence staining method in which FITC-triflavin is utilized as a binding ligand were used. GRGDS inhibits the binding of FITC-triflavin to B16-F10 cells. Additionally, the above results suggest that triflavin directly binds to its receptors expressed on B16-F10 cell surface primarily via its RGD sequence, there-by inhibiting B16-F10 cell adhesion to extracellular matrices.     

Palmitate induces apoptosis in mouse aortic endothelial cells and endothelial dysfunction in mice fed high-calorie and high-cholesterol diets

AimsObesity is associated with hypertriglyceridemia and elevated circulating free fatty acids (FFA), resulting in endothelial dysfunction. Endoplasmic reticulum (ER) stress has been implicated in many of these processes. To determine if ER stress participates in palmitate-induced apoptosis, we investigated the effects of diet-induced obesity and palmitate on mouse aortic endothelial cells (MAEC) in vivo and in vitro.Main methodsMale C57BL/6 mice were fed standard chow diets (SCD) or high-calorie and high-cholesterol diets (HCD) for 3 months. Insulin resistance was detected, and the serum, including proinflammatory indices and markers of endothelial function, was also analyzed. The ultrastructure and apoptosis of the endothelial cells in the thoracic aorta were observed. The primary MAEC were separated and treated with palmitate at different concentrations or different times respectively to observe any changes in cellular proliferation, intracellular reactive oxygen species (ROS) levels and apoptosis. Finally, the ER stress markers C/EBP homologous protein (CHOP) and glucose-regulated protein 78 (GRP78) were analyzed.Key findingsHCD-fed obese mice became inflammation-activated and insulin-resistant. Swollen mitochondria, expanded ER and apoptosis in the endothelial cells of the thoracic aorta were observed in HCD-fed mice. Palmitate inhibited cell proliferation, increased production of ROS and induced apoptosis in MAEC. CHOP was overexpressed and shifted into the nucleus (mainly), while the expression of GRP78 was upregulated in the palmitate-treated MAEC.SignificanceOur results indicate that diet-induced obesity results in endothelial dysfunction in vivo, and that oxidative and ER stress may be involved in apoptosis induced by the palmitate in vitro.     

Constrained cell recognition peptides engineered into streptavidin.

Streptavidin is widely used as an adaptor molecule in diagnostics, separations, and laboratory assay applications. We have here engineered cell adhesive peptides into the three-dimensional scaffolding of streptavidin to convert streptavidin into a functional protein. The mutations did not alter refolding or tetramer assembly and the slow biotin dissociation rate of wild-type streptavidin was retained. The peptide targets were hexapeptide sequences derived from osteopontin and fibronectin that contain the RGD cell adhesion sequence. Cell binding assays directly demonstrated that rat aortic endothelial cells and human melanoma cells adhered to surfaces coated with either of the two RGD streptavidin mutants in a dose-dependent fashion. Wild-type streptavidin displayed no significant cell binding activity. Inhibition studies with soluble RGD peptides confirmed that the cell adhesion was RGD mediated. Further inhibition studies with antibodies directed against alphavbeta3 demonstrated that the RGD-streptavidin interaction was primarily mediated by this integrin with melanoma cells. These results demonstrate that peptide recognition sequences can be engineered into accessible surface regions of streptavidin without disrupting biotin binding properties. This approach to introducing secondary functional activities into streptavidin may improve streptavidin's utility in existing applications or provide new technology opportunities.     

Bioactive microarrays immobilized on low-fouling surfaces to study specific endothelial cell adhesion

With the aim to study how to modulate the specific endothelial cell patterning and responses on biomaterials surfaces, bioactive microarrays were developed and validated for specific cell patterning. These microarrays were made of low-fouling surfaces, that prevent nonspecific cell adhesion, bearing bioactive molecules at given known locations by presenting specific ligands to cell receptors. Arrays of bioactive molecules (RGD, REDV, and SVVYGLR sequences and vascular endothelial growth factor (VEGF)) were immobilized on a carboxy-methyl-dextran low-fouling surface and were exposed to human endothelial cells and fibroblasts to screen for the effect of bioactive spot molecular composition on cell adhesion. Endothelial cells only were sensitive to RGD peptide co-immobilized with REDV or SVVYGLR sequences: they induced a reduction in cell spreading and a loss of actin stress fibers. RGD co-immobilized with VEGF also resulted in the reorganization of actin filaments and focal points in endothelial cells. Combination of RGD with these endothelial cell-selective biomolecules did not elicit a strong adhesion phenotype but rather one characteristic of migrating cells.     

Endothelial cell migration on RGD-peptide-containing PEG hydrogels in the presence of sphingosine 1-phosphate

Sphingosine 1-phosphate (S1P) is a potent chemokinetic agent for endothelial cells that is released by activated platelets. We previously developed Arg-Gly-Asp (RGD)-containing polyethylene glycol biomaterials for the controlled delivery of S1P to promote endothelialization. Here, we studied the effects of cell adhesion strength on S1P-stimulated endothelial cell migration in the presence of arterial levels of fluid shear stress, since an upward shift in optimal cell adhesion strengths may be beneficial for promoting long-term cell adhesion to materials. Two RGD peptides with different integrin-binding specificities were added to the polyethylene glycol hydrogels. A linear RGD bound primarily to β₃ integrins, whereas a cyclic RGD bound through both β₁ and β₃ integrins. We observed increased focal adhesion formation and better long-term adhesion in flow with endothelial cells on linear RGD peptide, versus cyclic RGD, even though initial adhesion strengths were higher for cells on cyclic RGD. Addition of 100 nM S1P increased cell speed and random motility coefficients on both RGD peptides, with the largest increases found on cyclic RGD. For both peptides, much of the increase in cell migration speed was found for smaller cells (<1522 μm² projected area), although the large increases on cyclic RGD were also due to medium-sized cells (2288-3519 μm²). Overall, a compromise between high cell migration rates and long-term adhesion will be important in the design of materials that endothelialize after implantation.     

Mouse M290 is the functional homologue of the human mucosal lymphocyte integrin HML-1: antagonism between the integrin ligands E-cadherin and RGD tripeptide.

Human mucosal lymphocyte antigen-1 (HML-1, alphaEbeta7) and E-cadherin, two members of unrelated cell adhesion superfamilies, have evolved to play cooperative roles in gut mucosal immunity. Human E-cadherin is self-ligand mediating intercellular adhesion of epithelial cells, as well as adhesion of intra-epithelial lymphocytes to intestinal enterocytes via an interaction with HML-1. Herein we report that both dimeric and monomeric forms of recombinant mouse E-cadherin-human immunoglobulin Fc chimera self-associate and support attachment of E-cadherin+ mouse colon epithelial cells. Both forms also support the adhesion of mouse MTC-1 T cells via M290, thereby establishing M290 as the functional mouse homologue of HML-1 and revealing that E-cadherin homophilic and heterophilic binding sites are distinct. Adhesion of MTC-1 cells to E-cadherin-Fc was inhibited by arginine-glycine-aspartate (RGD) peptides and vice versa cells bound to immobilized RGD polymer in an M290-dependent fashion, where adhesion was inhibitable with soluble E-cadherin-Fc. Hence, E-cadherin and RGD integrin ligands antagonize cell binding by one another, either by inducing integrin cross-talk or by binding to shared or overlapping sites within M290. Binding of E-cadherin-Fc by HML-1 costimulated the CD3-induced proliferation of purified CD4+ T cells, suggesting that E-cadherin expressed on dendritic cells may play a T cell costimulatory role in addition to facilitating dendritic cell-keratinocyte adhesion.     

Plasmodium falciparum-infected erythrocytes bind to the RGD motif of fibronectin via the band 3-related adhesin

Previously it was shown that Plasmodium falciparum-infected erythrocytes bound to thrombospondin by the interaction of the peptidic sequence, HPLQKTY, of the band 3 protein of infected erythrocytes, and the RGD motif of thrombospondin. Here, we show that falciparum-parasitized erythrocytes bind to immobilized fibronectin by the RGD sequence of fibronectin. Involvement of the HPLQKTY region of band 3 in binding was demonstrated by inhibition of adhesion of parasitized erythrocytes to fibronectin by an HPLQKTY-containing peptide and the binding of the HPLQKTY peptide to the RGD sequence of immobilized fibronectin. Since fibronectin occurs on endothelial cells and platelets, this interaction may contribute to the binding of falciparum-infected erythrocytes to such host cells.     

Identification of a novel integrin alphavbeta3 binding site in CCN1 (CYR61) critical for pro-angiogenic activities in vascular endothelial cells

CCN1 (CYR61) is a matricellular inducer of angiogenesis essential for successful vascular development. Though devoid of the canonical RGD sequence motif recognized by some integrins, CCN1 binds to, and functions through integrin alphavbeta3 to promote pro-angiogenic activities in activated endothelial cells. In this study we identify a 20-residue sequence, V2 (NCKHQCTCIDGAVGCIPLCP), in domain II of CCN1 as a novel binding site for integrin alphavbeta3. Immobilized synthetic V2 peptide supports alphavbeta3-mediated cell adhesion; soluble V2 peptide inhibits endothelial cell adhesion to CCN1 and the homologous family members CCN2 (connective tissue growth factor, CTGF) or CCN3 (NOV) but not to collagen. These activities are obliterated by mutation of the aspartate residue in the V2 peptide to alanine. The corresponding D125A mutation in the context of the N-terminal half of CCN1 (domains I and II) greatly diminished direct solid phase binding to purified integrin alphavbeta3 and abolished alphavbeta3-mediated cell adhesion activity. Likewise, soluble full-length CCN1 with the D125A mutation is defective in binding purified alphavbeta3 and impaired in alphavbeta3-mediated pro-angiogenic activities in vascular endothelial cells, including stimulation of cell migration and enhancement of DNA synthesis. In contrast, immobilized full-length CCN1-D125A mutant binds alphavbeta3 and supports alphavbeta3-mediated cell adhesion similar to wild type CCN1. These results indicate that V2 is the primary alphavbeta3 binding site in soluble CCN1, whereas additional cryptic alphavbeta3 binding site(s) in the C-terminal half of CCN1 becomes exposed when the protein is immobilized. Together, these results identify a novel and functionally important binding site for integrin alphavbeta3 and provide a new approach for dissecting alphavbeta3-specific CCN1 functions both in cultured cells and in the organism.     

Activation of classical protein kinase C decreases transport via systems y+ and y+L

Activation of protein kinase C (PKC) downregulates the human cationic amino acid transporters hCAT-1 (SLC7A1) and hCAT-3 (SLC7A3) (Rotmann A, Strand D, Martiné U, Closs EI. J Biol Chem 279: 54185-54192, 2004; Rotmann A, Vekony N, Gassner D, Niegisch G, Strand D, Martine U, Closs EI. Biochem J 395: 117-123, 2006). However, others found that PKC increased arginine transport in various mammalian cell types, suggesting that the expression of different arginine transporters might be responsible for the opposite PKC effects. We thus investigated the consequence of PKC activation by phorbol-12-myristate-13-acetate (PMA) in various human cell lines expressing leucine-insensitive system y(+) [hCAT-1, hCAT-2B (SLC7A2), or hCAT-3] as well as leucine-sensitive system y(+)L [y(+)LAT1 (SLC7A7) or y(+)LAT2 (SLC7A6)] arginine transporters. PMA reduced system y(+) activity in all cell lines tested, independent of the hCAT isoform expressed, while mRNAs encoding the individual hCAT isoforms were either unchanged or increased. System y(+)L activity was also inhibited by PMA. The extent and onset of inhibition varied between cell lines; however, a PMA-induced increase in arginine transport was never observed. In addition, when expressed in Xenopus laevis oocytes, y(+)LAT1 and y(+)LAT2 activity was reduced by PMA, and this inhibition could be prevented by the PKC inhibitor bisindolylmaleimide I. In ECV304 cells, PMA-induced inhibition of systems y(+) and y(+)L could be prevented by G?6976, a specific inhibitor of conventional PKCs. Thymelea toxin, which activates preferentially classical PKC, had a similar inhibitory effect as PMA. In contrast, phosphatidylinositol-3,4,5-triphosphate-dipalmitoyl, an activator of atypical PKC, had no effect. These data demonstrate that systems y(+) and y(+)L are both downregulated by classical PKC.     

Molecular properties of poly(RGD) and its binding capacities to metastatic melanoma cells.

We examined the binding capacity of anti-metastatic polypeptide containing repetitive Arg-Gly-Asp(RGD) sequence derived from cell binding site of fibronectin, poly(RGD), to the surface of tumor cells. Poly(RGD) competitively inhibited the binding of radiolabeled fibronectin to the cell surface more potently than oligo(RGD) or RGD tripeptide on a molar basis. Compared on a weight basis to oligo(RGD) or RGD peptide, poly(RGD) was more active than the oligo- and monomeric peptide at inhibiting tumor cell adhesion to immobilized fibronectin. The secondary structure of poly(RGD) was predicted to be a beta-turn from the data of CD spectra and its amino acid sequence. These findings suggest that poly(RGD)-mediated inhibition of cell adhesion is due to its potent binding capacity to fibronectin receptors on cell surface probably through its conformational properties.     

Effect of receptor-ligand affinity on the strength of endothelial cell adhesion.

The objective of this study was to determine the effect of receptor-ligand affinity on the strength of endothelial cell adhesion. Linear and cyclic forms of the fibronectin (Fn) cell-binding domain peptide Arg-Gly-Asp (RGD) were covalently immobilized to glass, and Fn was adsorbed onto glass slides. Bovine aortic endothelial cells attached to the surfaces for 15 min. The critical wall shear stress at which 50% of the cells detached increased nonlinearly with ligand density and was greater with immobilized cyclic RGD than with immobilized linear RGD or adsorbed Fn. To directly compare results for the different ligand densities, the receptor-ligand dissociation constant and force per bond were estimated from data for the critical shear stress and contact area. Total internal reflection fluorescence microscopy was used to measure the contact area as a function of separation distance. Contact area increased with increasing ligand density. Contact areas were similar for the immobilized peptides but were greater on surfaces with adsorbed Fn. The dissociation constant was determined by nonlinear regression of the net force on the cells to models that assumed that bonds were either uniformly stressed or that only bonds on the periphery of the contact region were stressed (peeling model). Both models provided equally good fits for cells attached to immobilized peptides whereas the peeling model produced a better fit of data for cells attached to adsorbed Fn. Cyclic RGD and linear RGD both bind to the integrin alpha v beta 3, but immobilized cyclic RGD exhibited a greater affinity than did linear RGD. Receptor affinities of Fn adsorbed to glycophase glass and Fn adsorbed to glass were similar. The number of bonds was calculated assuming binding equilibrium. The peeling model produced good linear fits between bond force and number of bonds. Results of this study indicate that 1) bovine aortic endothelial cells are more adherent on immobilized cyclic RGD peptide than linear RGD or adsorbed Fn, 2) increased adhesion is due to a greater affinity between cyclic RGD and its receptor, and 3) the affinity of RGD peptides and adsorbed Fn for their receptors is increased after immobilization.     

Synthesis and Cell Adhesive Properties of Linear and Cyclic RGD Functionalized Polynorbornene Thin Films

Described herein is the efficient synthesis and evaluation of bioactive arginine-glycine-aspartic acid (RGD) functionalized polynorbornene-based materials for cell adhesion and spreading. Polynorbornenes containing either linear or cyclic RGD peptides were synthesized by ring-opening metathesis polymerization (ROMP) using the well-defined ruthenium initiator [(H(2)IMes)(pyr)(2)(Cl)(2)Ru═CHPh]. The random copolymerization of three separate norbornene monomers allowed for the incorporation of water-soluble polyethylene glycol (PEG) moieties, RGD cell recognition motifs, and primary amines for postpolymerization cross-linking. Following polymer synthesis, thin-film hydrogels were formed by cross-linking with bis(sulfosuccinimidyl) suberate (BS(3)), and the ability of these materials to support human umbilical vein endothelial cell (HUVEC) adhesion and spreading was evaluated and quantified. When compared to control polymers containing either no peptide or a scrambled RDG peptide, polymers with linear or cyclic RGD at varying concentrations displayed excellent cell adhesive properties in both serum-supplemented and serum-free media. Polymers with cyclic RGD side chains maintained cell adhesion and exhibited comparable integrin binding at a 100-fold lower concentration than those carrying linear RGD peptides. The precise control of monomer incorporation enabled by ROMP allows for quantification of the impact of RGD structure and concentration on cell adhesion and spreading. The results presented here will serve to guide future efforts for the design of RGD functionalized materials with applications in surgery, tissue engineering, and regenerative medicine.     

Endothelial cells interact with the core protein of basement membrane perlecan through beta 1 and beta 3 integrins: an adhesion modulated by glycosaminoglycan

Aortic endothelial cells adhere to the core protein of murine perlecan, a heparan sulfate proteoglycan present in endothelial basement membrane. We found that cell adhesion was partially inhibited by beta 1 integrin-specific mAb and almost completely blocked by a mixture of beta 1 and alpha v beta 3 antibodies. Furthermore, adhesion was partially inhibited by a synthetic peptide containing the perlecan domain III sequence LPASFRGDKVTSY (c-RGD) as well as by GRGDSP, but not by GRGESP. Both antibodies contributed to the inhibition of cell adhesion to immobilized c-RGD whereas only beta 1-specific antibody blocked residual cell adhesion to proteoglycan core in the presence of maximally inhibiting concentrations of soluble RGD peptide. A fraction of endothelial surface-labeled detergent lysate bound to a core affinity column and 147-, 116-, and 85-kD proteins were eluted with NaCl and EDTA. Polyclonal anti-beta 1 and anti-beta 3 integrin antibodies immunoprecipitated 116/147 and 85/147 kD surface-labeled complexes, respectively. Cell adhesion to perlecan was low compared to perlecan core, and cell adhesion to core, but not to immobilized c-RGD, was selectively inhibited by soluble heparin and heparan sulfates. This inhibition by heparin was also observed with laminin and fibronectin and, in the case of perlecan, was found to be independent of heparin binding to substrate. These data support the hypothesis that endothelial cells interact with the core protein of perlecan through beta 1 and beta 3 integrins, that this binding is partially RGD- independent, and that this interaction is selectively sensitive to a cell-mediated effect of heparin/heparan sulfates which may act as regulatory ligands.     

Expression and characterization of recombinant osteopontin peptides representing matrix metalloproteinase proteolytic fragments.

Osteopontin (OPN) is a secreted, arginine-glycine-aspartic acid (RGD)-containing phosphoprotein proteolytically modified by members of the matrix metalloproteinase (MMP) family. We previously defined the MMP-3 and MMP-7 cleavage sites in OPN and found increased adhesive and migratory activity of a pool of MMP-cleaved fragments compared to full-length OPN. In the present study, we performed mutational analysis of recombinant full-length OPN and generated recombinant OPN fragments corresponding to the MMP-cleaved fragments, which have apparent molecular weights of 40, 32, and 25 kD by SDS-PAGE. Single residue mutations in 167L and 211L do not abrogate MMP cleavage although processing of the putative C-terminal fragment appears to be affected by a 167L to 167A mutation. The N-terminal 40-kD fragment was a stronger adhesive substrate compared to full-length OPN despite the observation that full-length OPN displayed greater binding in soluble phase to endothelial cell surfaces. While the 32-kD fragment showed significant binding to endothelial cell surfaces, the C-terminal 25-kD fragment did not interact with cell surface. Our data indicate that the increased adhesive activity of MMP-cleaved OPN was accountable by the N-terminal 40-kD fragment. We further analyzed receptor binding, using competition with peptides representing the alpha4beta1 and alpha9beta1 binding sites in the 40-kD N-terminal fragment. Using Jurkat cells, we found that a peptide corresponding to 131ELVTDFPTDLPATE144 had no effect on cell adhesion, whereas the peptide SVVYGLR competitively inhibited cell adhesion. These results suggest that a shorter motif that is found in MMP-cleaved OPN, 162SVVYG166, is sufficient to mediate cell adhesion of Jurkat cells to receptors, including the beta1 integrins, which have been previously characterized to bind the SVVYGLR sequence.     

Human herpesvirus 8 envelope glycoprotein B mediates cell adhesion via its RGD sequence

Human herpesvirus 8 (HHV-8) or Kaposi's sarcoma-associated herpesvirus, implicated in the pathogenesis of Kaposi's sarcoma, utilizes heparan sulfate-like molecules to bind the target cells via its envelope-associated glycoproteins gB and gpK8.1A. HHV-8-gB possesses the Arg-Gly-Asp (RGD) motif, the minimal peptide region of many proteins known to interact with subsets of host cell surface integrins. HHV-8 utilizes alpha3beta1 integrin as one of the receptors for its entry into the target cells via its gB interaction and induces the activation of focal adhesion kinase (FAK) (S. M. Akula, N. P. Pramod, F.-Z. Wang, and B. Chandran, Cell 108:407-419, 2002). Since FAK activation is the first step in the outside-in signaling necessary for integrin-mediated cytoskeletal rearrangements, cell adhesions, motility, and proliferation, the ability of HHV-8-gB to mediate the target cell adhesion was examined. A truncated form of gB without the transmembrane and carboxyl domains (gBdeltaTM) and a gBdeltaTM mutant (gBdeltaTM-RGA) with a single amino acid mutation (RGD to RGA) were expressed in a baculovirus system and purified. Radiolabeled HHV-8-gBdeltaTM, gBdeltaTM-RGA, and deltaTMgpK8.1A proteins bound to the human foreskin fibroblasts (HFFs), human dermal microvascular endothelial (HMVEC-d) cells, human B (BJAB) cells, and Chinese hamster ovary (CHO-K1) cells with equal efficiency, which was blocked by preincubation of proteins with soluble heparin. Maxisorp plate-bound gBdeltaTM protein induced the adhesion of HFFs and HMVEC-d and monkey kidney epithelial (CV-1) cells in a dose-dependent manner. In contrast, the gBdeltaTM-RGA and DeltaTMgpK8.1A proteins did not mediate adhesion. Adhesion mediated by gBdeltaTM was blocked by the preincubation of target cells with RGD-containing peptides or by the preincubation of plate-bound gBdeltaTM protein with rabbit antibodies against gB peptide containing the RGD sequence. In contrast, adhesion was not blocked by the preincubation of plate-bound gBdeltaTM protein with heparin, suggesting that the adhesion is mediated by the RGD amino acids of gB, which is independent of the heparin-binding domain of gB. Integrin-ligand interaction is dependent on divalent cations. Adhesion induced by the gBdeltaTM was blocked by EDTA, thus suggesting the role of integrins in the observed adhesions. Focal adhesion components such as FAK and paxillin were activated by the binding of gBdeltaTM protein to the target cells but not by gBdeltaTM-RGA protein binding. Inhibition of FAK phosphorylation by genistein blocked gBdeltaTM-induced FAK activation and cell adhesion. These findings suggest that HHV-8-gB could mediate cell adhesion via its RGD motif interaction with the cell surface integrin molecules and indicate the induction of cellular signaling pathways, which may play roles in the infection of target cells and in Kaposi's sarcoma pathogenesis.     

Identification of cell-binding site of angiomodulin (AGM/TAF/Mac25) that interacts with heparan sulfates on cell surface.

Angiomodulin (AGM/TAF/mac25) is a 30-kDa glycoprotein that was identified as an integrin-independent cell adhesion protein secreted by human bladder carcinoma cells. AGM is highly accumulated in small blood vessels of tumor tissues. In the present study, we attempted to identify the cell surface receptor and the cell-binding site of AGM using ECV-304 human vascular endothelial cells and BALB/c3T3 mouse fibroblasts. Heparin, heparan sulfate, and dextran sulfate, but not chondroitin sulfate, inhibited both adhesion of the two cell lines to AGM-coated plates and binding of AGM to these cells. Treatment of cells with heparinase, but not chondroitinase, inhibited both cell adhesion to AGM and AGM binding to cells. These results strongly suggested that heparan sulfates are the major receptor for AGM. Furthermore, we determined a 20-amino acid sequence within AGM molecule as its major cell-binding site. The synthetic peptide for the cell-binding sequence showed cell adhesion activity comparable to that of AGM, and the activity was inhibited by heparin and heparan sulfate. The peptide competitively inhibited cell adhesion to AGM and the binding of AGM to cells. These results indicated that AGM binds to cells through interaction of the identified cell-binding sequence with heparan sulfates on cell surface. It was also found that the heparan sulfate-binding peptide inhibited the formation of capillary tube-like structures of vascular endothelial cells in culture.     

Site-directed mutagenesis of the arginine-glycine-aspartic acid sequence in osteopontin destroys cell adhesion and migration functions

Osteopontin (OPN) is a secreted calcium-binding phosphoprotein produced in a variety of normal and pathological contexts, including tissue mineralization and cancer. OPN contains a conserved RGD (arg-gly-asp) amino acid sequence that has been implicated in binding of OPN to cell surface integrins. To determine whether the RGD sequence in OPN is required for adhesive and chemotactic functions, we have introduced two site-directed mutations in the RGD site of the mouse OPN cDNA, in which the RGD sequence was either deleted or mutated to RGE (arg-gly-glu). In order to test the effect of these mutations on OPN function, we expressed control and mutated mouse OPN in E. coli as recombinant glutathione-S-transferase (GST)-OPN fusion proteins. Control mouse GST-OPN was functional in cell adhesion assays, supporting attachment and spreading of mouse (malignant PAP2 ras-transformed NIH 3T3, and, to a lesser extent, normal NIH 3T3 fibroblasts) and human (MDA-MB-435 breast cancer, and normal gingival fibroblast) cells. In contrast, neither of the RGD-mutated OPN proteins (“delRGD” or “RGE”) supported adhesion of any of the cell lines, even when used at high concentrations or for long assay times. GRGDS (gly-arg-gly-asp-ser) peptides inhibited cell adhesion to intact GST-OPN, as well as to fibronectin and vitronectin. In chemotaxis assays, GST-OPN promoted directed cell migration of both malignant (PAP2, MDA-MB-435) and normal (gingival fibroblast, and NIH 3T3) cells, while RGD-mutated OPN proteins did not. Together these results suggest that the conserved RGD sequence in OPN is required for the majority of the protein's cell attachment and migration-stimulating functions.