Importance of the Heparin-binding Domain of Fibronectin for Enhancing Cell Adhesion Activity of the Recombinant Fibronectin

We have previously shown that the recombinant human fibronectin (FN) fragment composed of central cell binding domains (CCBD) spanning the ninth and tenth type III domains promotes cell adhesion and proliferation of osteoblasts. In the present study, we investigated the biological potency of heparin-binding domain (HBD) of FN spanning the twelfth and fourteenth type III domains. The HBD of FN significantly enhances the RGD-containing CCBD-mediated cell adhesion and proliferation in HOS cells (P < 0.05).     

IGF2BP1

The oncofetal RNA-binding protein IGF2BP1 (IGF2 mRNA binding protein 1) controls the cytoplasmic fate of specific target mRNAs including ACTB and CD44. During neural development, IGF2BPs promote neurite protrusion and the migration of neuronal crest cells. In tumor-derived cells, IGF2BP1 enhances the formation of lamellipodia and invadopodia. Accordingly, the de novo synthesis of IGF2BP1 observed in primary malignancies was reported to correlate with increased metastasis and an overall poor prognosis. However, if and how the protein enhances metastasis remains controversial. In recent studies, we reveal that IGF2BP1 promotes the directed migration of tumor-derived cells in vitro by controlling the expression of MAPK4 and PTEN. The IGF2BP1-facilitated inhibition of MAPK4 mRNA translation interferes with MK5-directed phosphorylation of the heat shock protein 27 (HSP27). This limits G-actin sequestering by phosphorylated HSP27, enhances cell adhesion and elevates the velocity of tumor cell migration. Concomitantly, IGF2BP1 promotes the expression of PTEN by interfering with PTEN mRNA turnover. This results in a shift of cellular PtdIns(3,4,5)P₃/PtdIns(4,5)P₂ ratios and enhances RAC1-dependent cell polarization which finally promotes the directionality of tumor cell migration. These findings identify IGF2BP1 as a potent oncogenic factor that regulates the adhesion, migration and invasiveness of tumor cells by modulating intracellular signaling.     

Identification of binding peptides of the ADAM15 disintegrin domain using phage display

ADAM15 plays an important role in tumour development by interacting with integrins. In this study, we investigated the target peptides of the ADAM15 disintegrin domain. First, we successfully produced the recombinant human ADAM15 disintegrin domain (RADD) that could inhibit melanoma cell adhesion by using Escherichia coli. Second, four specific binding peptides (peptides A, B, C, and D) were selected using a phage display 12-mer peptide library. The screening protocol involved 4 rounds of positive panning on RADD and 2 rounds of subtractive selection with streptavidin. By using the BLAST software and a relevant protein database, integrin α _ν β ₃ was found to be homologous to peptide A. Synthetic peptide A had a highly inhibitory effect on RADD-integrin α _v β ₃ binding. The results demonstrate the potential application of short peptides for disrupting high-affinity ADAM-integrin interactions.     

VE-Cadherin Mediates Endothelial Cell Capillary Tube Formation in Fibrin and Collagen Gels

Various cell adhesion molecules mediate the diverse functions of the vascular endothelium, such as cell adhesion, neutrophil migration, and angiogenesis. In order to identify cell adhesion molecules important for angiogenesis, we used anin vitromodel (Chalupowicz, Chowdhury, Bach, Barsigian, and Martinez,J. Cell Biol.130, 207–215, 1995) in which human umbilical vein endothelial cell monolayers are induced to form capillary-like tubes when a second gel, composed of either fibrin or collagen, is formed overlying the apical surface. In the present investigation, we observed that a monoclonal antibody directed against the first extracellular domain of human vascular endothelial cadherin (VE-cadherin, cadherin 5) inhibited the formation of capillary tubes formed between either fibrin or collagen gels. Moreover, when added to preformed capillary tubes, this antibody disrupted the capillary network. In contrast, monoclonal antibodies directed against the extracellular domain of N-cadherin, the α_vβ₃integrin, and PECAM-1 failed to inhibit capillary tube formation. During capillary tube formation, Western blot and RT-PCR analysis revealed no marked change in VE-cadherin expression. Immunocytochemical studies demonstrated that VE-cadherin was concentrated at intercellular junctions in multicellular capillary tubes. Thus, VE-cadherin plays a specific role in fibrin-induced or collagen-induced capillary tube formation and is localized at areas of intercellular contact where it functions to maintain the tubular architecture. Moreover, its function at tubular intercellular junctions is distinct from that at intercellular junctions present in confluent monolayers, since only the former was inhibited by monoclonal antibodies.     

Transmembrane and Extracellular Domains of Syndecan-1 Have Distinct Functions in Regulating Lung Epithelial Migration and Adhesion

Syndecan-1 is a cell surface proteoglycan that can organize co-receptors into a multimeric complex to transduce intracellular signals. The syndecan-1 core protein has multiple domains that confer distinct cell- and tissue-specific functions. Indeed, the extracellular, transmembrane, and cytoplasmic domains have all been found to regulate specific cellular processes. Our previous work demonstrated that syndecan-1 controls lung epithelial migration and adhesion. Here, we identified the necessary domains of the syndecan-1 core protein that modulate its function in lung epithelial repair. We found that the syndecan-1 transmembrane domain has a regulatory function in controlling focal adhesion disassembly, which in turn controls cell migration speed. In contrast, the extracellular domain facilitates cell adhesion through affinity modulation of α₂β₁ integrin. These findings highlight the fact that syndecan-1 is a multidimensional cell surface receptor that has several regulatory domains to control various biological processes. In particular, the lung epithelium requires the syndecan-1 transmembrane domain to govern cell migration and is independent from its ability to control cell adhesion via the extracellular domain.     

Focal adhesion kinase stabilizes the cytoskeleton

Focal adhesion kinase (FAK) is a central focal adhesion protein that promotes focal adhesion turnover, but the role of FAK for cell mechanical stability is unknown. We measured the mechanical properties of wild-type (FAKwt), FAK-deficient (FAK−/−), FAK-silenced (siFAK), and siControl mouse embryonic fibroblasts by magnetic tweezer, atomic force microscopy, traction microscopy, and nanoscale particle tracking microrheology. FAK-deficient cells showed lower cell stiffness, reduced adhesion strength, and increased cytoskeletal dynamics compared to wild-type cells. These observations imply a reduced stability of the cytoskeleton in FAK-deficient cells. We attribute the reduced cytoskeletal stability to rho-kinase activation in FAK-deficient cells that suppresses the formation of ordered stress fiber bundles, enhances cortical actin distribution, and reduces cell spreading. In agreement with this interpretation is that cell stiffness and cytoskeletal stability in FAK−/− cells is partially restored to wild-type level after rho-kinase inhibition with Y27632.     

Focal adhesion kinase regulates tractional collagen remodeling,matrix metalloproteinase expression,and collagen structure,which in turn affects matrix-induced signaling

Focal adhesion kinase (FAK) is critical for collagen expression but its regulation of collagen remodeling is not defined. We examined the role of FAK in the degradation and reorganization of fibrillar collagen. Compared with wild-type (WT) mouse embryonic fibroblasts, FAK null (FAK^−/−) fibroblasts generated twofold (p < .0001) higher levels of ¾ collagen I fragment and expressed up to fivefold more membrane-type matrix metalloproteinase (MMP). When plated on stiff collagen substrates, compared with WT, FAK^−/− cells were smaller (threefold reduced cell surface area; p < .0001) and produced fivefold fewer cell extensions (p < .0001) that were 40% shorter (p < .001). When cultured on soft collagen gels (stiffness of ~100 Pa) for 6–48 hr, cell spreading and cell extension formation were reduced by greater than twofold (p < .05 and p < .0001, respectively) while collagen compaction and alignment were reduced by approximately 30% (p < .0001) in FAK^−/− cells. Similar results were found after treatment with PF573228, a FAK inhibitor. Reconstitution of FAK^−/− cells with FAK mutants showed that compared with WT, cell extension formation was reduced twofold (p < .0001) in the absence of the kinase domain and sixfold (p < .0001) with a Y397F mutant. Enhanced collagen degradation was exhibited by the mutants (~threefold increase; p < .0001 of ¾ collagen fragments without kinase domain or Y397F mutant; p < .01). Compared with FAK^+/+ cells, matrices produced by FAK^−/− cells generated higher levels of β₁ integrin activation (p < 0.05), extracellular-signal-regulated kinase (ERK) phosphorylation, and production of ¾ collagen I fragment by human gingival fibroblasts. Collectively these data indicate that (a) the kinase activity of FAK enhances collagen remodeling by tractional forces but inhibits collagen degradation by MMPs; (b) FAK influences the biological activity of fibroblast-secreted extracellular matrices, which in turn impacts β1 integrin and ERK signaling, and collagen degradation.     

Neurite Fasciculation Mediated by Complexes of Axonin-1 and Ng Cell Adhesion Molecule

Neural cell adhesion molecules composed of immunoglobulin and fibronectin type III-like domains have been implicated in cell adhesion, neurite outgrowth, and fasciculation. Axonin-1 and Ng cell adhesion molecule (NgCAM), two molecules with predominantly axonal expression exhibit homophilic interactions across the extracellular space (axonin- 1/axonin-1 and NgCAM/NgCAM) and a heterophilic interaction (axonin-1–NgCAM) that occurs exclusively in the plane of the same membrane (cis-interaction). Using domain deletion mutants we localized the NgCAM homophilic binding in the Ig domains 1-4 whereas heterophilic binding to axonin-1 was localized in the Ig domains 2-4 and the third FnIII domain. The NgCAM–NgCAM interaction could be established simultaneously with the axonin-1–NgCAM interaction. In contrast, the axonin-1–NgCAM interaction excluded axonin-1/axonin-1 binding. These results and the examination of the coclustering of axonin-1 and NgCAM at cell contacts, suggest that intercellular contact is mediated by a symmetric axonin-1₂/NgCAM₂ tetramer, in which homophilic NgCAM binding across the extracellular space occurs simultaneously with a cis-heterophilic interaction of axonin-1 and NgCAM. The enhanced neurite fasciculation after overexpression of NgCAM by adenoviral vectors indicates that NgCAM is the limiting component for the formation of the axonin-1₂/NgCAM₂ complexes and, thus, neurite fasciculation in DRG neurons.     

Intermediate monomer-dimer equilibrium structure of native ICAM-1: implication for enhanced cell adhesion

Dimeric intercellular adhesion molecule-1 (ICAM-1) has been known to more efficiently mediate cell adhesion than monomeric ICAM-1. Here, we found that truncation of the intracellular domain of ICAM-1 significantly enhances surface dimerization based on the two criteria: 1) the binding degree of monomer-specific antibody CA-7 and 2) the ratio of dimer/monomer when a mutation (L42 → C42) was introduced in the interface of domain 1. Mutation analysis revealed that the positively charged amino acids, including very membrane-proximal ⁵⁰⁵R, are essential for maintaining the structural transition between the monomer and dimer. Despite a strong dimer presentation, the ICAM-1 mutants lacking an intracellular domain (IC1ΔCTD) or containing R to A substitution in position 505 (⁵⁰⁵R/A) supported a lower degree of cell adhesion than did wild-type ICAM-1. Collectively, these results demonstrate that the native structure of surface ICAM-1 is not a dimer, but is an intermediate monomer–dimer equilibrium structure by which the effectiveness of ICAM-1 can be fully achieved.     

Enhanced osteoblast adhesion on transglutaminase 2-crosslinked fibronectin

Fibronectin (FN) is a cell adhesion protein that binds integrins in a process also involving the protein-crosslinking enzyme transglutaminase 2 (TG2) as a co-receptor. The cell-adhesive property of TG2 has been linked to a complex formation with FN and to its ability to crosslink and polymerize FN on the cell surface. We tested here the effects of extracellular FN, before and after in vitro crosslinking and polymerization by TG2, on MC3T3-E1 osteoblast adhesion. We show that TG2-mediated crosslinking creates large, compacted chain-like protein clusters that include both TG2 and FN molecules as analyzed by Western blotting and atomic force microscopy. Crosslinking of FN significantly promotes osteoblast adhesion as measured by crystal violet staining, and enhances β₁-integrin clustering on the cell surface as visualized by immunofluorescence microscopy. We hypothesize that TG2-mediated crosslinking enhances the cell-adhesive properties of FN by increasing the molecular rigidity of FN in the extracellular matrix.     

Molecular characterization of a new member of the immunoglobulin superfamily that potentially functions in T-cell activation and development

 Differential hybridization cloning has been used to isolate a novel chicken thymic activation and developmental sequence (cTADS). The nucleotide sequence of the cTADS cDNA predicts an open reading frame of 439 amino acids. The inferred cTADS protein possesses a hydrophobic membrane-spanning domain and putative intracellular kinase activation domains. Its extracellular domain shares similarities with the immunoglobulin protein superfamily, featuring two conserved immunoglobulin folds that resemble C1 and C2 constant regions. The cTADS sequence shows similarity to a subfamily of proteins involved in cellular adhesion: chicken neural cell adhesion molecule and human opioid-binding adhesion molecule, and to proteins that have a biological role in intracellular signaling: mouse platelet-derived growth factor receptor and human fibroblast growth factor receptor. cTADS is differentially expressed in chicken thymic cells during embryonic development and during activation through the T-cell receptor. Sequence similarities and expression patterns suggest that cTADS could be involved in cell recognition and adhesion, and/or peptide ligand binding. Received: 1 May 1999 / Revised: 1 October 1999     

sFRP-1 binds via its netrin-related motif to the N-module of thrombospondin-1 and blocks thrombospondin-1 stimulation of MDA-MB-231 breast carcinoma cell adhesion and migration

Secreted frizzled-related protein (sFRP)-1 is a Wnt antagonist that inhibits breast carcinoma cell motility, whereas the secreted glycoprotein thrombospondin-1 stimulates adhesion and motility of the same cells. We examined whether thrombospondin-1 and sFRP-1 interact directly or indirectly to modulate cell behavior. Thrombospondin-1 bound sFRP-1 with an apparent K_d = 48 nM and the related sFRP-2 with a K_d = 95 nM. Thrombospondin-1 did not bind to the more distantly related sFRP-3. The association of thrombospondin-1 and sFRP-1 is primarily mediated by the amino-terminal N-module of thrombospondin-1 and the netrin domain of sFRP-1. sFRP-1 inhibited α3β1 integrin-mediated adhesion of MDA-MB-231 breast carcinoma cells to a surface coated with thrombospondin-1 or recombinant N-module, but not adhesion of the cells on immobilized fibronectin or type I collagen. sFRP-1 also inhibited thrombospondin-1-mediated migration of MDA-MB-231 and MDA-MB-468 breast carcinoma cells. Although sFRP-2 binds similarly to thrombospondin-1, it did not inhibit thrombospondin-1-stimulated adhesion. Thus, sFRP-1 binds to thrombospondin-1 and antagonizes stimulatory effects of thrombospondin-1 on breast carcinoma cell adhesion and motility. These results demonstrate that sFRP-1 can modulate breast cancer cell responses by interacting with thrombospondin-1 in addition to its known effects on Wnt signaling.     

Butanol-extracted lipoteichoic acid induces in vivo leukocyte adhesion

Lipoteichoic acid (LTA), an immunostimulatory component of the cell walls of Gram positive bacteria, has pro-inflammatory effects in vitro and in vivo. However, one in vivo study concluded that LTA had no noticeable effects on leukocyte recruitment. In this study we investigated the effects of highly purified LTA, prepared by butanol extraction (Bu-LTA) at room temperature, on in vivo leukocyte adhesion. Using intravital microscopy we measured adhesion of leukocytes in mesenteric post-capillary venules of rats and mice. Topical superfusion of Bu-LTA (1 μg/ml) in rats significantly (p < 0.05) increased adhesion within 30 min. By contrast, hot phenol-extracted LTA did not increase adhesion. Alkaline hydrolysis of Bu-LTA removed alanine residues and prevented adhesion. Also, pre-administration of anti-rat β₂-integrin antibody abolished Bu-LTA-induced adhesion. Finally, intraperitoneal injection of Bu-LTA (100 μg/ml) into mice also significantly (p < 0.01) increased leukocyte adhesion measured at 60 min. In conclusion, Bu-LTA with intact alanine residues promotes β₂-integrin-dependent leukocyte adhesion in vivo.     

αvβ3 Integrin Mediates the Cell-adhesive Capacity and Biological Activity of Basic Fibroblast Growth Factor (FGF-2) in Cultured Endothelial Cells

Fibroblast growth factor-2 (FGF-2) immobilized on non-tissue culture plastic promotes adhesion and spreading of bovine and human endothelial cells that are inhibited by anti-FGF-2 antibody. Heat-inactivated FGF-2 retains its cell-adhesive activity despite its incapacity to bind to tyrosine-kinase FGF receptors or to cell-surface heparan sulfate proteoglycans. Recombinant glutathione-S-transferase-FGF-2 chimeras and synthetic FGF-2 fragments identify two cell-adhesive domains in FGF-2 corresponding to amino acid sequences 38–61 and 82–101. Both regions are distinct from the FGF-receptor-binding domain of FGF-2 and contain a DGR sequence that is the inverse of the RGD cell-recognition sequence. Calcium deprivation, RGD-containing eptapeptides, soluble vitronectin (VN), but not fibronectin (FN), inhibit cell adhesion to FGF-2. Conversely, soluble FGF-2 prevents cell adhesion to VN but not FN, thus implicating VN receptor in the cell-adhesive activity of FGF-2. Accordingly, monoclonal and polyclonal anti-α_vβ₃ antibodies prevent cell adhesion to FGF-2. Also, purified human α_vβ₃ binds to immobilized FGF-2 in a cation-dependent manner, and this interaction is competed by soluble VN but not by soluble FN. Finally, anti-α_vβ₃ monoclonal and polyclonal antibodies specifically inhibit mitogenesis and urokinase-type plasminogen activator (uPA) up-regulation induced by free FGF-2 in endothelial cells adherent to tissue culture plastic. These data demonstrate that FGF-2 interacts with α_vβ₃ integrin and that this interaction mediates the capacity of the angiogenic growth factor to induce cell adhesion, mitogenesis, and uPA up-regulation in endothelial cells.     

Adhesion of a <Emphasis Type="Italic">Pseudomonas putida</Emphasis> strain isolated from a paper machine to cellulose fibres

The adhesion to cellulose fibres of a strain of Pseudomonas putida isolated from a paper machine was studied under different environmental conditions. The physicochemical properties of both P. putida cells and cellulose fibres were also determined to better understand the adhesion phenomenon. Adhesion was rapid (1 min) and increased with time, cell concentration and temperature (from 25 to 40°C), indicating that bacterial adhesion to cellulose fibres is essentially governed by a physicochemical process. The P. putida cell surface was negatively charged, as shown by electrophoretic mobility measurements, and was hydrophilic due to a strong electron-donor character, as shown by the microbial adhesion to solvents method. Cellulose fibres were shown to be hydrophilic by contact angle measurements using the capillary rise method. These results suggest the importance of Lewis acid-base interactions in the adhesion process. In various ionic solutions (NaCl, KCl, CaCl₂ and MgCl₂), adhesion increased with increasing ionic strength up to 10–100 mM, indicating that, at low ionic strength, electrostatic interactions were involved in the adhesion process. An increase in the C/N ratio of the growth medium (from 5 to 90) decreased adhesion but this could not be related to changes in physicochemical properties, suggesting that other factors may be involved. In practice, temperature, ionic strength and nitrogen concentration must be taken into consideration to reduce bacterial contamination in the paper industry.     

The influence of topographic microstructures on the initial adhesion of L929 fibroblasts studied by single-cell force spectroscopy

Single-cell force spectroscopy was used to investigate the initial adhesion of L929 fibroblasts onto periodically grooved titanium microstructures (height ~6 μm, groove width 20 μm). The position-dependent local adhesion strength of the cells was correlated with their rheological behavior. Spherical cells exhibited a significantly lower Young’s modulus (<1 kPa) than that reported for spread cells, and their elastic properties can roughly be explained by the Hertz model for an elastic sphere. While in contact with the planar regions of the substrate, the cells started to adapt their shape through slight ventral flattening. The process was found to be independent of the applied contact force for values between 100 and 1,000 pN. The degree of flattening correlated with the adhesion strength during the first 60 s. Adhesion strength can be described by fast exponential kinetics as C₁ [ 1 - exp( - C₂ ·t ) ] C_{1} \left[ {1 - \exp \left( { - C_{2} \cdot t} \right)} \right] with C ₁ = 2.34 ± 0.19 nN and C ₂ = 0.09 ± 0.02 s⁻¹. A significant drop in the adhesion strength of up to 50% was found near the groove edges. The effect can be interpreted by the geometric decrease of the contact area, which indicates the inability of the fibroblasts to adapt to the shape of the substrate. Our results explain the role of the substrate’s topography in contact guidance and suggest that rheological cell properties must be considered in cell adhesion modeling.     

ORP2 couples LDL‐cholesterol transport to FAK activation by endosomal cholesterol/PI(4,5)P2 exchange

Low‐density lipoprotein (LDL)‐cholesterol delivery from late endosomes to the plasma membrane regulates focal adhesion dynamics and cell migration, but the mechanisms controlling it are poorly characterized. Here, we employed auxin‐inducible rapid degradation of oxysterol‐binding protein‐related protein 2 (ORP2/OSBPL2) to show that endogenous ORP2 mediates the transfer of LDL‐derived cholesterol from late endosomes to focal adhesion kinase (FAK)‐/integrin‐positive recycling endosomes in human cells. In vitro, cholesterol enhances membrane association of FAK to PI(4,5)P₂‐containing lipid bilayers. In cells, ORP2 stimulates FAK activation and PI(4,5)P₂ generation in endomembranes, enhancing cell adhesion. Moreover, ORP2 increases PI(4,5)P₂ in NPC1‐containing late endosomes in a FAK‐dependent manner, controlling their tubulovesicular trafficking. Together, these results provide evidence that ORP2 controls FAK activation and LDL‐cholesterol plasma membrane delivery by promoting bidirectional cholesterol/PI(4,5)P₂ exchange between late and recycling endosomes.     

Structure of the Y. pseudotuberculosis adhesin InvasinE

Enteropathogenic Yersinia expresses several invasins that are fundamental virulence factors required for adherence and colonization of tissues in the host. Within the invasin‐family of Yersinia adhesins, to date only Invasin has been extensively studied at both structural and functional levels. In this work, we structurally characterize the recently identified inverse autotransporter InvasinE from Yersinia pseudotuberculosis (formerly InvasinD from Yersinia pseudotuberculosis strain IP31758) that belongs to the invasin‐family of proteins. The sequence of the C‐terminal adhesion domain of InvasinE differs significantly from that of other members of the Yersinia invasin‐family and its detailed cellular and molecular function remains elusive. In this work, we present the 1.7 Å crystal structure of the adhesion domain of InvasinE along with two Immunoglobulin‐like domains. The structure reveals a rod shaped architecture, confirmed by small angle X‐ray scattering in solution. The adhesion domain exhibits strong structural similarities to the C‐type lectin‐like domain of Yersinia pseudotuberculosis Invasin and enteropathogenic/enterohemorrhagic E. coli Intimin. However, despite the overall structural similarity, the C‐type lectin‐like domain in InvasinE lacks motifs required for Ca²⁺/carbohydrate binding as well as sequence or structural features critical for Tir binding in Intimin and β₁‐integrin binding in Invasin, suggesting that InvasinE targets a distinct, yet unidentified molecule on the host‐cell surface. Although the biological role and target molecule of InvasinE remain to be elucidated, our structural data provide novel insights into the architecture of invasin‐family proteins and a platform for further studies towards unraveling the function of InvasinE in the context of infection and host colonization.     

Effects on in vitro and in vivo angiogenesis induced by small peptides carrying adhesion sequences

It is well known that tumor growth is strictly dependent on neo‐vessel formation inside the tumor mass and that cell adhesion is required to allow EC proliferation and migration inside the tumor. In this work, we have evaluated the in vitro and in vivo effects on angiogenesis of some peptides, originally designed to promote cell adhesion on biomaterials, containing RGD motif mediating cell adhesion via integrin receptors [RGD, GRGDSPK, and (GRGDSP)₄K] or the heparin‐binding sequence of human vitronectin that interacts with HSPGs [HVP(351–359)]. Cell adhesion, proliferation, migration, and capillary‐like tube formation in Matrigel were determined on HUVECs, whereas the effects on in vivo angiogenesis were evaluated using the CAM assay. (GRGDSP)₄K linear sequence inhibited cell adhesion, decreased cell proliferation, migration and morphogenesis in Matrigel, and induced anti‐angiogenic responses on CAM at higher degree than that determined after incubation with RGD or GRGDSPK. Moreover, it counteracted both in vitro and in vivo the pro‐angiogenic effects induced by the Fibroblast growth factor (FGF‐2). On the other hand, HVP was not able to affect cell adhesion and appeared less effective than (GRGDSP)₄K. Our data indicate that the activity of RGD‐containing peptides is related to their adhesive properties, and their effects are modulated by the number of cell adhesion motifs and the aminoacidic residues next to these sequences. The anti‐angiogenic properties of (GRGDSP)₄K seem to depend on its interaction with integrins, whereas the effects of HVP may be partially due to an impairment of HSPGs/FGF‐2. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Peptide array-based screening of human mesenchymal stem cell-adhesive peptides derived from fibronectin type III domain

Human mesenchymal stem cell-adhesive peptides were screened based on the amino acid sequence of fibronectin type III domain 8-11 (FN-III_8-11) using a peptide array synthesized by the Fmoc-chemistry. Using hexameric peptide library of FN-III_8-11 scan, we identified the ALNGR (Ala-Leu-Asn-Gly-Arg) peptide that induced cell adhesion as well as RGDS (Arg-Gly-Asp-Ser) peptide. After incubation for 2 h, approximately 68% of inoculated cells adhere to the ALNGR peptide disk. Adhesion inhibition assay with integrin antibodies showed that the ALNGR peptide interacts with integrin β1 but not with αvβ3, indicating that the receptors for ALNGR are different from RGDS. Additionally, the ALNGR peptide expressed cell specificities for adhesion: cell adhesion was promoted for fibroblasts but not for keratinocytes or endotherial cells. The ALNGR peptide induced cell adhesion and promoted cell proliferation without changing its property. It is therefore useful for the construction of functional biomaterials.