RGD-dependent mechanotransduction of suspension cultured Taxus cell in response to shear stress

Plant cells cultured in bioreactors are strongly influenced by mechanical forces. However, the molecular mechanism of plant cell mechanoreception has maintained unclear. In animal cells, the Arg-Gly-Asp (RGD) motif can be found in proteins of the extracellular matrix. Integrins link the intracellular cytoskeleton of cells with the extracellular matrix by recognizing this RGD motif. Integrin has been demonstrated to function as an apparatus not only for adhesion but also for mechanotransduction. In plant cells, the molecules that mediate the structural continuity between wall and membrane are unknown. Here, we found that synthetic RGD peptide could dramatically reduce the level of phosphorylation of MAPK-like cascades that are activated by shear stress and reduce the alkalinization response, production of reactive oxygen species (ROS) and accumulation of phenolics by Taxus cuspidata cells during shear stress. These results implicate that a RGD recognition system may exist in Taxus cells and play an important role in signal transduction of shear stress. Although the Arabidopsis genome database shows that the plant seems to lack a homologue of animal integrin, plant cells may use other RGD-binding proteins to recognize the RGD motif. The correlative mechanism is discussed.     

Arginyl-glycyl-aspartic acid (RGD): a cell adhesion motif

The tripeptide Arg-Gly-Asp (RGD) was originally identified as the sequence within fibronectin that mediates cell attachment. The RGD motif has now been found in numerous other proteins and supports cell adhesion in many, but not all, of these. The integrins, a family of cell-surface proteins, act as receptors for cell adhesion molecules. A subset of the integrins recognize the RGD motif within their ligands, the binding of which mediates both cell-substratum and cell-cell interactions. RGD peptides and mimetics, in addition to providing insights into the fundamental mechanisms of cell adhesion, are potential therapeutic agents for the treatment of diseases such as thrombosis and cancer.     

The Inhibitory Effect of an RGD-Human Chitin-Binding Domain Fusion Protein on the Adhesion of Fibroblasts to Reacetylated Chitosan Films

Biomaterials used for tissue engineering applications must provide a structural support for the tissue development and also actively interact with cells, promoting adhesion, proliferation, and differentiation. To achieve this goal, adhesion molecules may be used, such as the tripeptide Arg-Gly-Asp (RGD). A method based on the use of a carbohydrate-binding module, with affinity for chitin, was tested as an alternative approach to the chemical grafting of bioactive peptides. This approach would simultaneously allow the production of recombinant peptides (alternatively to peptide synthesis) and provide a simple way for the specific and strong adsorption of the peptides to the biomaterial. A fusion recombinant protein, containing the RGD sequence fused to a human chitin-binding module (ChBM), was expressed in E. coli. The adhesion of fibroblasts to reacetylated chitosan (RC) films was the model system selected to analyze the properties of the obtained proteins. Thus, the evaluation of cell attachment and proliferation on polystyrene surfaces and reacetylated chitosan films, coated with the recombinant proteins, was performed using mouse embryo fibroblasts 3T3. The results show that the recombinant proteins affect negatively fibroblasts anchorage to the materials surface, inhibiting its adhesion and proliferation. We also conclude that this negative effect is fundamentally due to the human chitin-binding domain.     

Micromechanical coupling between cell surface receptors and RGD peptides

Contact between an adherent cell and the extracellular matrix (ECM) promotes the recruitment of structural and signaling molecules to the cytoplasmic domain of integrins, which mediate cell adhesion, cell migration, and cell growth. It is unclear whether the intracellular recruitment of these cytoplasmic molecules enhances the affinity between the ECM and the extracellular domain of the cell surface receptors (integrins). Using soft microneedles coated with Arg-Gly-Asp (RGD) peptides, a sequence commonly shared by ECM proteins, we apply a localized ramp shear stress to the surface of a HeLa cell and measure the cell stiffness and the collective (or apparent) unbinding lifetime of its surface receptors to RGD. These measurements demonstrate that both cell stiffness and the collective cell surface receptor-RGD unbinding lifetime increase with the duration of the pre-shear cell-microneedle contact and with the rate of shear applied to the cell membrane. These parameters are also crucially dependent on the integrity of the actin filament network. Our results are consistent with a model of positive feedback signaling where RGD-mediated initial recruitment of cytoskeletal proteins to the cytoplasmic domain of integrins directly enhances the interaction between the extracellular domain of integrins and the RGD sequence of ECM molecules.     

Enhanced cell adhesion and mature intracellular structure promoted by squaramide-based RGD mimics on bioinert surfaces

Highly selective molecular binding and the subsequent dynamic protein assemblies control the adhesion of mammalian cells. Molecules that inhibit cell adhesion have the therapeutic potential for a wide range of diseases. Here, we report an efficient synthesis (2–4 steps) of a class of squaramide molecules that mimics the natural tripeptide ligand Arg-Gly-Asp (RGD) that mediates mammalian cell adhesion through binding with membrane protein integrin. In solution, this class of squaramides exhibits a higher potency at inhibiting mammalian cell adhesion than RGD tripeptides. When immobilized on a bio-inert background formed by self-assembled monolayers of alkanethiols on gold films, squaramide ligands mediate vastly different intracellular structures than RGD ligands. Immunostaining revealed that the focal adhesions are smaller, but with a larger quantity, for cells adhered on squaramides than that on RGD ligands. Furthermore, the actin filaments are also more fibrous and well distributed for cell adhesion mediated by squaramide than that by RGD ligands. Quantification reveal that squaramide ligands mediate about 1.5 times more total focal adhesion (measured by the summation of the area of all focal adhesions) than that by natural RGD ligands. This result suggests that cell adhesion inhibitors, while blocking the attachment of cells to surfaces, may induce more focal adhesion proteins. Finally, this work demonstrates that immobilizing new ligands on bioinert surfaces provide a powerful tool to study mammalian cell adhesion.     

(Arg-Gly-Asp)n-albumin conjugates as a model substratum for integrin-mediated cell adhesion

We have prepared protein-peptide conjugates composed of bovine serum albumin (BSA) derivatized with short peptides containing the Arg-Gly-Asp (RGD) sequence derived from the adhesion site of fibronectin. The RGD-BSA conjugates were used to coat tissue culture plastic surfaces which then served as substrata in cell adhesion experiments. Our results indicate that the efficiency of adhesion to RGD-BSA-coated surfaces is highly dependent on the valency of the (RGD)n-BSA conjugates. For example, on surfaces with approximately equal amounts of RGD ligand, CHO cells adhered virtually 100% to the (RGD)n-BSA (n = 20.8) conjugate and not at all to the (RGD)n-BSA (n = 3.5) conjugate. Adhesion on (RGD)n-BSA-coated substrata and on fibronectin- or vitronectin-coated substrata was also examined in terms of the relationship between cell adhesion and the intermolecular distances of adsorbed proteins. It was observed that for substrata coated with relatively compact, symmetric molecules, such as RGD-BSA or vitronectin, adhesion dropped off sharply as intermolecular distances increased; by contrast, for fibronectin, a large asymmetric molecule, adhesion declined more gradually as intermolecular distances increased. Finally, we have examined the role of different cell-surface receptors in the process of adhesion to RGD-BSA substrata. Interestingly, competition and blocking experiments with antibodies and with soluble competing proteins suggest that it is the vitronectin receptor rather than the fibronectin receptor which mediates adhesion to RGD-BSA.     

植物细胞中的RGD模体及受体

Arg-Gly-Asp(RGD)模体是动物细胞底物黏附分子的基本识别结构,许多胞外黏附蛋白是通过RGD模体与质膜受体整合素结合的,它参与细胞的跨膜信号转导,介导多种生物学过程。越来越多的实验表明植物细胞中也存在RGD结合模体,现就近年来植物细胞在这方面的研究进展进行综述。     

Interaction of human lactoferrin with cell adhesion molecules through RGD motif elucidated by lactoferrin-binding epitopes

Lactoferrin (LF) is an iron-binding secretory protein, which is distributed in the secondary granules of polynuclear lymphocytes as well as in the milk produced by female mammals. Although it has multiple functions, for example antimicrobial, immunomodulatory, antiviral, and anti-tumor metastasis activities, the receptors responsible for these activities are not fully understood. In this study, the binding epitopes for human LF were first isolated from a hexameric random peptide library displayed on T7 phage. Interestingly, two of the four isolated peptides had a representative cell adhesion motif, Arg-Gly-Asp (RGD), implying that human LF interacts with proteins with the RGD motif. We found that human LF bound to the RGD-containing human extracellular matrix proteins, fibronectin and vitronectin. Furthermore, human LF inhibited cell adhesion to these matrix proteins in a concentration-dependent manner but not to the RGD-independent cell adhesion molecule like laminin or collagen. These results indicate that a function of human LF is to block the various interactions between the cell surface and adhesion molecules. This may explain the multifunctionality of LF.     

以整合素αvβ3为靶点的RGD配体在抗肿瘤血管新生中的应用

整合素αvβ3是一种能特异性识别RGD序列的膜受体蛋白,其与含RGD（Arg-Gly-Asp）模体的蛋白质结合的特异性在肿瘤细胞的粘附、迁移、浸润及肿瘤血管新生中起重要作用.由于整合素αvβ3在多种肿瘤细胞表面高表达而在正常细胞中低表达或不表达,因此其成为肿瘤治疗的理想靶点.肿瘤新生血管为肿瘤的生长提供营养,因此近年来抑制肿瘤血管新生也成为肿瘤治疗的重要途径.有研究显示,几种RGD毒素蛋白不但以整合素αvβ3为靶点靶向结合到肿瘤部位从而具有直接抗肿瘤细胞增殖、黏附、迁移及浸润功能,而且它们还具有抗肿瘤血管新生的作用,因此RGD毒素蛋白可从上述两方面抑制肿瘤生长与转移.本文就整合素αvβ3为靶向的RGD配体结构特点及其在肿瘤治疗中的靶向治疗和抗血管新生应用及前景加以综述.     

Integrin-like substrate adhesion in RTG-2 cells, a fibroblastic cell line derived from rainbow trout

Fluorometric cell attachment assays together with competitive inhibitors of adhesion were used to probe for the presence of integrins, a diverse family of heterodimeric cell-surface glycoproteins involved in cell-cell and cell-extracellular matrix adhesion, in the fibroblastic rainbow trout cell line, RTG-2. The adhesive properties of this cell line were evaluated. RTG-2 cells adhered poorly to TC plastic in the absence of serum but as little as 2.5% fetal bovine serum allowed over 75% of the cells to attach after 5 h. Surfaces coated with the extracellular matrix proteins collagen I, collagen IV, fibrin, fibrinogen, or fibronectin were able to support attachment of RTG-2 cells. Adhesion of RTG-2 cells to fibronectin varied linearly with fibronectin coating densities in the range 0 to 65 ng/mm(2). Oligopeptides containing the sequence Arg-Gly-Asp (RGD) caused dose-dependent inhibition of adhesion to microtiter plates coated with fibrin, fibrinogen, and fibronectin, whereas attachment to collagen I and collagen IV was less severely affected. In all cases, peptides containing Arg-Gly-Glu (RGE) or Asp-Gly-Arg (DGR) sequences caused no reduction of cell attachment. Since many integrins mediate adhesion by binding to RGD sequences in their target ligands, these results suggest the presence of integrin-like adhesion molecules on the surface of RTG-2 cells.     

重组七鳃鳗富含半胱氨酸RGD蛋白rLj-RGD1对人肝癌HepG2细胞的抑制作用

rLj-RGD1为源于日本七鳃鳗口腔腺的基因重组蛋白,其富含半胱氨酸并具有一个RGD(Arg-Gly-Asp)模体.前期工作表明,rLj-RGD1具有抑制血小板聚集及血管新生的RGD毒素蛋白典型功能.为了研究rLj RGD1是否具有RGD毒素蛋白的另一典型抗肿瘤功能,以人肝癌HepG2细胞为模型,对rLj RGD1进行了活性研究.MTT法结果显示,rLj-RGD1呈剂量依赖方式抑制HepG2细胞的增殖,其半抑制浓度(IC50)为36 μmol/L;细胞迁移与浸润实验结果显示,rLj-RGD1能以剂量依赖方式抑制HepG2细胞碱性成纤维生长因子（bFGF）诱导的迁移与浸润.Hoechst染色和DNA Ladder等细胞凋亡实验表明,rLj-RGD1能够以剂量依赖方式诱导HepG2细胞发生凋亡.细胞黏附实验表明,rLj-RGD1以剂量依赖方式抑制HepG2细胞与玻连蛋白(vitronectin, VN)的黏附.上述结果表明,rLj-RGD1具有抑制人肝癌HepG2细胞增殖、迁移和浸润的功能,并可诱导其发生失巢凋亡. 本研究结果提示,rLj-RGD1具有典型的RGD毒素蛋白抗肿瘤功能,其未来具有成为抗肿瘤药物的潜力.     

Lectin receptor kinases participate in protein-protein interactions to mediate plasma membrane-cell wall adhesions in Arabidopsis

Interactions between plant cell walls and plasma membranes are essential for cells to function properly, but the molecules that mediate the structural continuity between wall and membrane are unknown. Some of these interactions, which are visualized upon tissue plasmolysis in Arabidopsis (Arabidopsis thaliana), are disrupted by the RGD (arginine-glycine-aspartic acid) tripeptide sequence, a characteristic cell adhesion motif in mammals. In planta induced-O (IPI-O) is an RGD-containing protein from the plant pathogen Phytophthora infestans that can disrupt cell wall-plasma membrane adhesions through its RGD motif. To identify peptide sequences that specifically bind the RGD motif of the IPI-O protein and potentially play a role in receptor recognition, we screened a heptamer peptide library displayed in a filamentous phage and selected two peptides acting as inhibitors of the plasma membrane RGD-binding activity of Arabidopsis. Moreover, the two peptides also disrupted cell wall-plasma membrane adhesions. Sequence comparison of the RGD-binding peptides with the Arabidopsis proteome revealed 12 proteins containing amino acid sequences in their extracellular domains common with the two RGD-binding peptides. Eight belong to the receptor-like kinase family, four of which have a lectin-like extracellular domain. The lectin domain of one of these, At5g60300, recognized the RGD motif both in peptides and proteins. These results imply that lectin receptor kinases are involved in protein-protein interactions with RGD-containing proteins as potential ligands, and play a structural and signaling role at the plant cell surfaces.     

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.     

Computerized analysis of tumor cell interactions with extracellular matrix proteins, peptides, and endothelial cells under laminar flow

Arrest and formation of stable adhesive interactions between circulating cells and the endothelium or exposed subendothelial matrix are important processes in many biological situations. We have developed a highly sensitive hydrodynamic assay that utilizes a parallel-plate flow chamber, video microscopy, and digital image processing to separate and measure the primary arrest and adhesion stabilization of flowing cells. Our data indicate that primary cell contact triggers secondary adhesion stabilization, and the secondary events are likely to be critical to metastasis formation. To study the relationship between tumor cell adhesion stabilization and organ-specific blood-borne metastasis, we investigated the adhesion stabilization of metastatic murine RAW117 large-cell lymphoma cells to the extracellular matrix proteins fibronectin and vitronectin, several Arg-Gly-Asp (RGD) containing peptides, and microvascular endothelial cells from the liver or lung. The highly liver metastatic RAW117-H10 subline showed the fastest stabilization to fibronectin, vitronectin, and RGD peptides. Poorly metastatic RAW117-P cells had stabilization times 3-10 times longer than for RAW117-H10 cells, while the lung- and liver-metastatic RAW117-L17 subline failed to stabilize at all. The adhesion stabilization of the RAW117-H10 cells to the extracellular matrix proteins and RGD peptides was inhibited by anti-beta(3) integrin monoclonal antibodies and RGD peptides. In contrast, the RAW117-L17 subline had the shortest stabilization time to unstimulated microvascular endothelial cells of the lung and hepatic sinusoids, followed by RAW117-H10 cells and RAW117-P cells. Monoclonal antibodies against the beta(3) integrin subunit and RGD peptides did not inhibit adhesion stabilization of RAW117-H10 cells to endothelial cells, suggesting that different metastatic variants of large-cell lymphoma cells use differing mechanisms to adhere to organ-specific endothelial cells. (c) 1996 John Wiley & Sons, Inc.     

αvβ3 and α5β1 integrin-specific ligands: From tumor angiogenesis inhibitors to vascularization promoters in regenerative medicine?

Integrins are cell adhesion receptors predominantly important during normal and tumor angiogenesis. A sequence present on several extracellular matrix proteins composed of Arg-Gly-Asp (RGD) has attracted attention due to its role in cell adhesion mediated by integrins. The development of ligands that can bind to integrins involved in tumor angiogenesis and brake disease progression has resulted in new investigational drug entities reaching the clinical trial phase in humans. The use of integrin-specific ligands can be useful for the vascularization of regenerative medicine constructs, which remains a major limitation for translation into clinical practice. In order to enhance vascularization, immobilization of integrin-specific RGD peptidomimetics within constructs is a recommended approach, due to their high specificity and selectivity towards certain desired integrins. This review endeavours to address the potential of peptidomimetic-coated biomaterials as vascular network promoters for regenerative medicine purposes. Clinical studies involving molecules tracking active integrins in cancer angiogenesis and reasons for their failure are also addressed.     

Mutations that alter an Arg-Gly-Asp (RGD) sequence in the adenovirus type 2 penton base protein abolish its cell-rounding activity and delay virus reproduction in flat cells.

The adenovirus penton base protein has a cell rounding activity and may lyse endosomes during virus entry into the cytoplasm. We found that penton base that was expressed in Escherichia coli also caused cell rounding and that cells adhered to polystyrene wells that were coated with the protein. Mutant analysis showed that both properties required an Arg-Gly-Asp (RGD) sequence at residues 340 to 342 of penton base. In flat adherent cells, virus mutants with amino acid substitutions in the RGD sequence were delayed in virus reproduction and in the onset of viral DNA synthesis. In nonadherent or poorly spread cells, the kinetics of mutant virus reproduction were similar to those of wild-type adenovirus type 2. Expression of the mutant phenotype exclusively in the flat cells that we tested supports a model in which penton base interacts with an RGD-directed cell adhesion molecule during adenovirus uptake or uncoating.     

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.     

Construction of a thermostable cell adhesion protein for reverse transfection

Transfection arrays are useful to analyze multiple genes at one time. In order to carry out gene transfection, cells are cultured on a plate on which genes are spotted to make extracellular matrix. However, this method is limited by low cell adhesion and transfection efficiency. To overcome these problems, we attempted to construct a novel extracellular matrix protein consisting of a variety of functional peptides. Here we fused the elastin derived peptide Ala-Pro-Gly-Val-Gly-Val (APGVGV) with the cell adhesive peptides, Pro-His-Ser-Arg-Asn (PHSRN) and Arg-Gly-Asp (RGD). The resulting fusion proteins, E12PSGR, had high cell adhesive activity, transfection efficiency, and thermal stability.     

Liposomes modified with a synthetic Arg-Gly-Asp mimetic inhibit lung metastasis of B16BL6 melanoma cells

Administration of large amounts of synthetic peptides based on the Arg-Gly-Asp (RGD) sequence has been shown to suppress tumor metastasis. To overcome the rapid degradation of peptides in the circulation, an RGD mimetic, L-arginyl-6-aminohexanoic acid (NOK), was synthesized and conjugated with phosphatidylethanolamine (PE) (NOK-PE) for liposomalization. Cell adhesion assays revealed that B16BL6 murine melanoma cells adhered to immobilized NOK-PE. This adhesion was inhibited by addition of either soluble RGDS or NOK at similar concentration in a dose-dependent manner. Administration of NOK-PE liposomes (equivalent to ca. 500 microg RGD peptides) via the tail vein completely inhibited lung colonization of B 16BL6 cells. The same dose of soluble NOK was not effective in inhibition of the tumor metastasis. In addition, injection of NOK-PE liposomes via the tail vein inhibited spontaneous lung metastasis of B16BL6 cells from the primary tumor site in the hind footpad. These results suggest that NOK, a structural mimetic of RGD, is capable of suppressing metastasis by blockade of the binding of the integrins present on tumor cells to the RGD-containing extracellular matrix.