Activation of the alpha 4 beta 1 integrin through the beta 1 subunit induces recognition of the RGDS sequence in fibronectin

Lymphocyte attachment to fibronectin is mainly mediated by the interaction of alpha 5 beta 1 and alpha 4 beta 1 integrins with the RGD and CS-1/Hep II sites, respectively. We have recently shown that the anti-beta 1 mAb TS2/16 can convert the partly active alpha 4 beta 1 present on certain hemopoietic cells that recognizes CS-1 but not Hep II, to a high avidity form that binds both ligands. In this report we have studied whether mAb TS2/16 also affects alpha 4 beta 1 ligand specificity. Incubation of the B cell lines Ramos and Daudi (which lack alpha 5 beta 1) with mAb TS2/16 induced specific attachment to an 80-kD fragment which lacks CS-1 and Hep II and contains the RGD sequence. mAbs anti-alpha 4 and the synthetic peptides CS-1 and IDAPS inhibited adhesion to the 80-kD fragment thus implying alpha 4 beta 1 as the receptor for this fragment. Interestingly, the synthetic peptide GRGDSPC and a 15-kD peptic fibronectin fragment containing the RGD sequence also inhibited B cell adhesion to the 80-kD fragment. Because we have previously shown that RGD peptides do not affect the constitutive function of alpha 4 beta 1, we tested whether TS2/16- activated alpha 4 beta 1 acquired the capacity to recognize RGD. Indeed RGD peptides inhibited TS2/16-treated B cell adhesion to a 38-kD fragment containing CS-1 and Hep II but did not affect binding of untreated cells to this fragment. An anti-fibronectin mAb reactive with an epitope on or near the RGD sequence also efficiently inhibited cell adhesion to the 80-kD fragment, indicating that the RGD sequence is a novel adhesive ligand for activated alpha 4 beta 1. These results emphasize the role of alpha 4 beta 1 as a receptor with different ligand specificities according to the activation state, a fact that may be important for lymphocyte migration, localization, and function.     

Monoclonal antibody characterization of two distant sites required for function of the central cell-binding domain of fibronectin in cell adhesion, cell migration, and matrix assembly

Site-directed mutagenesis studies have suggested that additional peptide information in the central cell-binding domain of fibronectin besides the minimal Arg-Gly-Asp (RGD) sequence is required for its full adhesive activity. The nature of this second, synergistic site was analyzed further by protein chemical and immunological approaches using biological assays for adhesion, migration, and matrix assembly. Fragments derived from the cell-binding domain were coupled covalently to plates, and their specific molar activities in mediating BHK cell spreading were compared with that of intact fibronectin. A 37-kD fragment purified from chymotryptic digests of human plasma fibronectin had essentially the same specific molar activity as intact fibronectin. In contrast, other fragments such as an 11.5-kD fragment lacking NH2-terminal sequences of the 37-kD fragment had only poor spreading activity on a molar basis. Furthermore, in competitive inhibition assays of fibronectin-mediated cell spreading, the 37-kD fragment was approximately 325-fold more active than the GRGDS synthetic peptide on a molar basis. mAbs were produced using the 37-kD protein as an immunogen and their epitopes were characterized. Two separate mAbs, one binding close to the RGD site and the other to a site approximately 15 kD distant from the RGD site, individually inhibited BHK cell spreading on fibronectin by greater than 90%. In contrast, an antibody that bound between these two sites had minimal inhibitory activity. The antibodies found to be inhibitory in cell spreading assays for BHK cells also inhibited both fibronectin-mediated cell spreading and migration of human HT-1080 cells, functions which were also dependent on function of the alpha 5 beta 1 integrin (fibronectin receptor). Assembly of endogenously synthesized fibronectin into an extracellular matrix was not significantly inhibited by most of the anti-37-kD mAbs, but was strongly inhibited only by the antibodies binding close to the RGD site or the putative synergy site. These results indicate that a second site distant from the RGD site on fibronectin is crucial for its full biological activity in diverse functions dependent on the alpha 5 beta 1 fibronectin receptor. This site is mapped by mAbs closer to the RGD site than previously expected.     

A recombinant, arginine-glycine-aspartic acid (RGD) motif from foot-and-mouth disease virus binds mammalian cells through vitronectin and, to a lower extent, fibronectin receptors

The cell-binding abilities of a recombinant, RGD-containing peptide from foot-and-mouth disease virus (FMDV) have been characterized in HeLa and BHK cells. This peptide represents the aa sequence of the solvent-exposed G-H loop of protein VP1 which is involved in cell recognition and infection. The efficiency of the viral motif in promoting cell attachment and spreading is comparable to that shown by fibronectin or vitronectin. Cell binding is inhibited by a monoclonal antibody directed against a viral, RGD-involving B-cell epitope and also by sera against vitronectin (_Vβ₃/β₅) and fibronectin (₅β₁) receptors. In addition, a synthetic RGD peptide, which is a ligand for both integrins, prevents the cell binding mediated by the FMDV domain. These data demonstrate that the FMDV RGD motif is a potent ligand for cell-receptor integrins and sufficient to promote cell attachment to susceptible cells mainly through the vitronectin receptor.     

NCAM140 stimulates integrin-dependent cell migration by ectodomain shedding

The neural cell adhesion molecule (NCAM) plays a key role in neural development, regeneration and synaptic plasticity. This study describes a novel function of NCAM140 in stimulating integrin-dependent cell migration. Expression of NCAM140 in rat B35 neuroblastoma cells resulted in increased migration toward the extracellular matrix proteins fibronectin, collagen IV, vitronectin, and laminin. NCAM-potentiated cell migration toward fibronectin was dependent on beta1 integrins and required extracellular-regulated kinase (ERK)1/2 mitogen-activated protein kinase (MAPK) activity. NCAM140 in B35 neuroblastoma cells was subject to ectodomain cleavage resulting in a 115 kDa soluble fragment released into the media and a 30 kDa cytoplasmic domain fragment remaining in the cell membrane. NCAM140 ectodomain cleavage was stimulated by the tyrosine phosphatase inhibitor pervanadate and inhibited by the broad spectrum metalloprotease inhibitor GM6001, characteristic of a metalloprotease. Moreover, treatment of NCAM140-B35 cells with GM6001 reduced NCAM140-stimulated cell migration toward fibronectin and increased cellular attachment to fibronectin to a small but significant extent. These results suggested that metalloprotease-induced cleavage of NCAM140 from the membrane promotes integrin- and ERK1/2-dependent cell migration to extracellular matrix proteins.     

The α4β1 Fibronectin Ligands CS-19 HEP II,and RGD Induce Different Intracellular Events in B Lymphoid Cells. Comparison with the Effects of the Endothelial Ligand V CAM-1

The lymphocyte integrin α4β1 is the receptor for the Hep II domain and CS-1 site in fibronectin (Fn) as well as for VCAM-1. We recently showed that upon activation with anti-β1 mAb TS2/16, α4β1 also recognizes the RGD Fn sequence. To determine the physiological role of these multiple interactions, we have now studied some intracellular events induced by “resting” and activated α4β1 binding to its different ligands. Analyses of actin and tubulin reorganization upon adhesion of B lymphoid cells to Fn fragments or VCAM-1 showed that VCAM-1, a 38 kDa fragment (Hep II+CS-1), and the CS-1 synthetic peptide induced formation of transient cytoplasmic projections; however, cells attached to a 58 kDa (Hep II) or 80 kDa (RGD) fragments remained rounded. Using transfilter assays, we showed that VCAM-1, 38 kDa and CS-1 also induced dose-dependent B cell migration mediated by α4β1. Furthermore, these three ligands, but not the 80 kDa fragment or a synthetic peptide (H1) containing a sequence from Hep II shown to bind α4β1, induced tyrosine phosphorylation of a 110 kDa protein. Activation of α4β1 with TS2/16 inhibited the cytoplasmic protrusions and cell migration but did not affect the pattern of phosphorylation. Our results indicate that the various α4β1 ligands induce different cellular responses. Most importantly they show that α4β1 interaction with CS-1 is sufficient to trigger intracellular events in B cells. Furthermore, they suggest a regulation by the activation form of the receptor as well as by the ligand in events involving lymphocyte adhesion and migration.     

Crystal structure of α5β1 integrin ectodomain: atomic details of the fibronectin receptor

Integrin α5β1 is a major cellular receptor for the extracellular matrix protein fibronectin and plays a fundamental role during mammalian development. A crystal structure of the α5β1 integrin headpiece fragment bound by an allosteric inhibitory antibody was determined at a 2.9-Å resolution both in the absence and presence of a ligand peptide containing the Arg-Gly-Asp (RGD) sequence. The antibody-bound β1 chain accommodated the RGD ligand with very limited structural changes, which may represent the initial step of cell adhesion mediated by nonactivated integrins. Furthermore, a molecular dynamics simulation pointed to an important role for Ca²⁺ in the conformational coupling between the ligand-binding site and the rest of the molecule. The RGD-binding pocket is situated at the center of a trenchlike exposed surface on the top face of α5β1 devoid of glycosylation sites. The structure also enabled the precise prediction of the acceptor residue for the auxiliary synergy site of fibronectin on the α5 subunit, which was experimentally confirmed by mutagenesis and kinetic binding assays.     

Human B lymphocytes define an alternative mechanism of adhesion to fibronectin. The interaction of the alpha 4 beta 1 integrin with the LHGPEILDVPST sequence of the type III connecting segment is sufficient to promote cell attachment

In this report we have studied the mechanism of human B lymphocyte adhesion to fibronectin and to proteolytic fragments of this protein. B cells adhered to fibronectin and to a 38-kDa fragment, derived from the A chain, containing the Hep II domain and most of the type III connecting segment, IIICS, of fibronectin. Cells did not bind to an 80-kDa fragment containing the RGD adhesive sequence of fibronectin. Attachment to fibronectin or to the 38-kDa fragment was not affected by the 80-kDa fragment, the GRGDSPC synthetic peptide, or by a mAb specific for the alpha chain of the RGD-dependent fibronectin receptor, alpha 5 beta 1. However, B cell adhesion to fibronectin was inhibited by the synthetic peptides CS-1, comprising the first 25 amino acids of IIICS and B12, containing the sequence LHGPEILDVPST of CS-1 (residues 14-25). Moreover, this sequence was shown to be sufficient to induce stable cell adhesion when coated on plastic surfaces. A mAb specific for the alpha-subunit of the alpha 4 beta 1 integrin, completely inhibited B cell attachment to B12, CS-1, 38 kDa, and fibronectin coated substrata. These data clearly indicate that adhesion of B lymphocytes to fibronectin is exclusively mediated by the interaction of alpha 4 beta 1 with residues 14-25 of the IIICS region in fibronectin. Therefore this interaction constitutes an alternative pathway of adhesion to fibronectin, independent of RGD and alpha 5 beta 1.     

Mechanism of human keratinocyte migration on fibronectin: unique roles of RGD site and integrins.

The migration of human keratinocytes over the wound bed plays an important role in the re-epithelialization of cutaneous wounds. Fibronectin, a large glycoprotein matrix component that is abundant within cutaneous wound beds, promotes keratinocyte migration. However, the mechanisms by which keratinocytes migrate over fibronectin are unknown. In this study, we sought to identify specific sites within the fibronectin molecule that induce keratinocyte locomotion and to characterize the cell surface receptors involved. The data show that the domain within the fibronectin molecule that induces human keratinocyte migration is the 120 kD cell-binding domain close to the carboxyl terminus. The 40 kD heparin-binding domain near the carboxyl terminus and the 45 kD gelatin-binding domain near the amino terminus did not promote keratinocyte migration. In addition, keratinocyte migration on both fibronectin and the 120 kD cell-binding domain was completely inhibited by the presence of GRGDSP peptide, suggesting that keratinocyte migration on fibronectin is mediated by recognizing the RGD sequence located within the cell-binding domain of fibronectin. Furthermore, keratinocytes were able to migrate directly on immobilized RGD substratum. Cell migration on fibronectin is mediated by the alpha 5 beta 1 integrin since antibodies blocking the alpha 5 and the beta 1 subunits completely inhibited keratinocyte migration on fibronectin. In addition, we demonstrate that human keratinocytes express alpha 5 beta 1 integrin in culture by flow cytometry.     

RGD-independent cell adhesion to the carboxy-terminal heparin-binding fragment of fibronectin involves heparin-dependent and -independent activities

Cell adhesion to extracellular matrix components such as fibronectin has a complex basis, involving multiple determinants on the molecule that react with discrete cell surface macromolecules. Our previous results have demonstrated that normal and transformed cells adhere and spread on a 33-kD heparin binding fragment that originates from the carboxy-terminal end of particular isoforms (A-chains) of human fibronectin. This fragment promotes melanoma adhesion and spreading in an arginyl-glycyl-aspartyl-serine (RGDS) independent manner, suggesting that cell adhesion to this region of fibronectin is independent of the typical RGD/integrin-mediated binding. Two synthetic peptides from this region of fibronectin were recently identified that bound [3H]heparin in a solid-phase assay and promoted the adhesion and spreading of melanoma cells (McCarthy, J. B., M. K. Chelberg, D. J. Mickelson, and L. T. Furcht. 1988. Biochemistry. 27:1380-1388). The current studies further define the cell adhesion and heparin binding properties of one of these synthetic peptides. This peptide, termed peptide I, has the sequence YEKPGSP-PREVVPRPRPGV and represents residues 1906-1924 of human plasma fibronectin. In addition to promoting RGD-independent melanoma adhesion and spreading in a concentration-dependent manner, this peptide significantly inhibited cell adhesion to the 33-kD fragment or intact fibronectin. Polyclonal antibodies generated against peptide I also significantly inhibited cell adhesion to the peptide, to the 33-kD fragment, but had minimal effect on melanoma adhesion to fibronectin. Anti-peptide I antibodies also partially inhibited [3H]heparin binding to fibronectin, suggesting that peptide I represents a major heparin binding domain on the intact molecule. The cell adhesion activity of another peptide from the 33-kD fragment, termed CS1 (Humphries, M. J., A. Komoriya, S. K. Akiyama, K. Olden, and K. M. Yamada. 1987. J. Biol. Chem., 262:6886-6892) was contrasted with peptide I. Whereas both peptides promoted RGD-independent cell adhesion, peptide CS1 failed to bind heparin, and exogenous peptide CS1 failed to inhibit peptide I-mediated cell adhesion. The results demonstrate a role for distinct heparin-dependent and -independent cell adhesion determinants on the 33-kD fragment, neither of which are related to the RGD-dependent integrin interaction with fibronectin.     

Effects of inhibitors of integrin binding on cellular outgrowth from bovine inner cell masses in vitro

Summary Bovine inner cell masses (ICM) cultured on fibronectin give rise to extensive cellular outgrowths containing endoderm. Peptides with the Glu-Ile-Leu-Asp-Val (EILDV) and Arg-Gly-Asp (RGD) sequences inhibit cell migration on fibronectin by binding to the fibronectin-recognition site in several integrins. To identify integrins involved in endodermal cell outgrowth on fibronectin and vitronectin, the effects of the EILDV and RGD peptides were evaluated in vitro. In experiment 1, ICM were cultured on fibronectin in medium containing 0.5 or 1.0 mg/ml EILDV or RGD (or both). Compared with 0 mg/ml, 0.5 mg/ml EILDV suppressed (P<0.10) outgrowth area overall, and 1.0 mg/ml EILDV reduced (P<0.05) outgrowth area after 72 h of culture. Compared with 0 mg/ml, 0.5 and 1.0 mg/ml RGD reduced (P<0.05) outgrowth area after 72 h of culture. Plasminogen activator activity in conditioned medium increased (P<0.05) in 0.5 mg/ml RGD but decreased (P<0.10) in 1.0 mg/ml RGD compared with 0 mg/ml RGD. In experiment 2, bovine ICM were cultured on vitronectin in medium containing 0.5 or 1.0 mg/ml RGD. Neither concentration of RGD (P>0.10) affected the extent of cellular outgrowth on vitronectin. Bovine endodermal cell migration on fibronectin can be modulated by the RGD and EILDV peptides. Despite inhibition, neither peptide completely prevented outgrowth on fibronectin. In contrast, cellular outgrowth on vitronectin was unaffected by RGD. The persistence of cellular outgrowth on fibronectin and the absence of inhibition by RGD for ICM cultured on vitronectin suggests that bovine endodermal cells can use alternative cellular adhesion systems, such as nonintegrin receptors, during outgrowth.     

Effects of modifications of the RGD sequence and its context on recognition by the fibronectin receptor

The receptor for fibronectin is a member of the integrin superfamily of cell surface adhesion receptors, many of which recognize the sequence RGD in their ligands. We have developed sensitive enzyme-linked and radioreceptor assays to examine the ligand specificity of the fibronectin receptor. The fibronectin receptor bound only to fibronectin of the various Arg-Gly-Asp (RGD)-containing proteins tested. The smallest amount of receptor detectable in the assay was about 10 ng. Mn2+ enhanced the binding of the receptor to fibronectin 3-10-fold as compared to Ca2+ and Mg2+. Scatchard analysis of the saturation plot from the radioreceptor assay gave a dissociation constant (Kd) of 3 x 10(-8) M for the binding of fibronectin receptor to fibronectin in the presence of Mn2+. Inhibition experiments showed that the affinities of the ligands for the receptor decreased in the order of fibronectin approximately 110-kDa fibronectin fragment greater than GRGDSP peptide greater than 11.5-kDa fragment. Peptides not containing an RGD were several hundred to several thousand-fold less inhibitory than GRGDSP. These included the closely related peptides GRADSP and GRGESP, as well as three peptides containing the reverse sequence DGR. A peptide from the fibrinogen gamma-chain, KQAGDV, which had about 0.5% of the inhibitory activity of the standard GRGDSP peptide, was the most active peptide not containing an RGD. These results document the exquisite specificity of the fibronectin receptor for the RGD sequence.     

Plasminogen Activator Inhibitor-1 Represses Integrin- and Vitronectin-Mediated Cell Migration Independently of Its Function as an Inhibitor of Plasminogen Activation

Cell migration involves the integrins, their extracellular matrix ligands, and pericellular proteolytic enzyme systems. We have studied the role of plasminogen activator inhibitor-1 (PAI-1) in cell migration, using human amnion WISH cells and human epidermoid carcinoma HEp-2 cells in an assay measuring migration from microcarrier beads and a modified Boyden-chamber assay. Active, but not latent or reactive center-cleaved, PAI-1 inhibited migration. A PAI-1 mutant without ability to inhibit plasminogen activation was as active as wild-type PAI-1 as a migration inhibitor, showing that inhibition of plasminogen activation was not involved. PAI-1 specifically interfered with integrin- and vitronectin-mediated migration: Migration onto vitronectin-coated but not onto fibronectin-coated surfaces was inhibited by PAI-1, a cyclic RGD peptide inhibited migration, and both cell lines expressed vitronectin-binding α_v-integrins. In addition, active PAI-1, but not latent or reactive center-cleaved PAI-1, inhibited vitronectin binding to integrins in anin vitrobinding assay, without affecting binding of fibronectin. Monoclonal antibodies against the urokinase receptor, another vitronectin binding protein, did not affect cell migration in the beads assay, while some inhibitory effect was observed in the Boyden-chamber assay. We conclude that PAI-1, independently of its role as a proteinase inhibitor, inhibits cell migration by competing for vitronectin binding to integrins, while the interference of PAI-1 with binding of vitronectin to the urokinase receptor may play a secondary role. These data define a novel function for the serpin PAI-1, enabling it to regulate cell migration over vitronectin-rich extracellular matrix in the body.     

The RGD containing site of the mouse laminin A chain is active for cell attachment, spreading, migration and neurite outgrowth

The laminin A chain has been sequenced by cDNA cloning and was found to contain an RGD sequence. Synthetic peptides containing the RGD sequence and flanking amino acids were active in mediating cell adhesion, spreading, migration, and neurite outgrowth. Furthermore, endothelial cell attachment to a laminin substrate was inhibited by an RGD-containing synthetic peptide. Antisera against the integrin (fibronectin) receptor, and monoclonal antibody to the integrin, VLA-6, inhibited cell interaction with laminin, as well as with peptides containing an RGD sequence. These results suggest that the RGD containing site of laminin is active and interacts with the integrin family of receptors in certain cells.     

Evidence for the presence of an integrin cell adhesion receptor on the oolemma of unfertilized human oocytes.

The Arg-Gly-Asp peptide (RGD), contained in several extracellular matrix proteins such as fibronectin, laminin, vitronectin, and collagen, is a tripeptide that plays a role as a recognition sequence in many cell-to-cell and cell-to-matrix adhesion mechanisms, through its interaction with several receptors of the integrin family. We previously described the ability of the oolemma of hamster oocytes to bind GRGDTP coupled to the surface of activated immunobeads and demonstrated that RGD-containing oligopeptides inhibit the adhesion of human and hamster spermatozoa to zona-free hamster oocytes and their subsequent penetration. In the present experiments, we show, utilizing immunobeads coated with an RGD-containing peptide (PepTiteTM 2000), that the oolemma of unfertilized human eggs is also able to recognize this adhesion sequence. The binding of PepTiteTM 2000-coated immunobeads to the oolemma was inhibited by the oligopeptide GRGDTP as well as by fibronectin and laminin. When immunobeads were prepared with a PepTiteTM concentration of 10 micrograms/ml, GRGDTP 150 micrograms/ml, laminin 80 micrograms/ml, and fibronectin 60 micrograms/ml inhibited bead rosetting on the egg surface. These data suggest that a specific binding moiety for RGD is present on the human egg surface. The binding of fibronectin to the oolemma was also demonstrated by the rosetting of immunobeads coupled with antifibronectin antibody to human oocytes after their exposure to 1 mg/ml free fibronectin. Such binding of fibronectin to the oolemma could be inhibited by coincubation with a monoclonal antibody directed against the cell adhesion fragment of fibronectin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Osteopontin increases migration and MMP‐9 up‐regulation via αvβ3 integrin,FAK, ERK,and NF‐κB‐dependent pathway in human chondrosarcoma cells

Tumor malignancy is associated with several features such as proliferation ability and frequency of metastasis. Osteopontin (OPN), which abundantly expressed in bone matrix, is involved in cell adhesion, migration, invasion and proliferation via interaction with its receptor, that is, αvβ3 integrin. However, the effect of OPN on migration activity in human chondrosarcoma cells is mostly unknown. Here we found that OPN increased the migration and expression of matrix metalloproteinase (MMP)‐9 in human chondrosarcoma cells (JJ012 cells). RGD peptide, αvβ3 monoclonal antibody and MAPK kinase (MEK) inhibitors (PD98059 and U0126) but not RAD peptide inhibited the OPN‐induced increase of the migration and MMP‐9 up‐regulation of chondrosarcoma cells. OPN stimulation increased the phosphorylation of focal adhesion kinase (FAK), MEK and extracellular signal‐regulated kinase (ERK). In addition, treatment of JJ012 cells with NF‐κB inhibitor (PDTC) or IκB protease inhibitor (TPCK) inhibited OPN‐induced cell migration and MMP‐9 up‐regulation. Stimulation of JJ012 cells with OPN also induced IκB kinase α/β (IKK α/β) phosphorylation, IκBα phosphorylation, p65 Ser⁵³⁶ phosphorylation, and κB‐luciferase activity. The OPN‐mediated increases in MMP‐9 and κB‐luciferase activities were inhibited by RGD peptide, PD98059 or FAK and ERK2 mutant. Taken together, our results indicated that OPN enhances the migration of chondrosarcoma cells by increasing MMP‐9 expression through the αvβ3 integrin, FAK, MEK, ERK and NF‐κB signal transduction pathway. J. Cell. Physiol. 221: 98–108, 2009. © 2009 Wiley‐Liss, Inc     

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.     

The contribution of beta1 integrins to neuronal migration and differentiation depends on extracellular matrix molecules

The interaction of β1 integrin receptors and different extracellular matrix molecules during neuronal development was investigated by comparing both migration and morphological differentiation of D3 wild-type embryonic stem (ES) cell line-derived neural precursor cells with those of the β1 integrin knockout ES cell line G201. Analysing neurosphere explants on laminin and fibronectin as major β1 integrin ligands, the maximal spreading of outward migrating neuronal cells was determined. Compared with gelatine as a standard substrate, migration was found to be significantly increased for D3-derived neurospheres on fibronectin and laminin-1. These matrix effects were found to be even enhanced for G201 preparations. In addition, also the differentiation of wild-type and β1 integrin −/− neurones – as determined by MAP-2- and HNK-1-immunoreactive processes – was found to be increased on fibronectin and laminin when compared to gelatine standards. In the respective knockout preparations on these matrices, again perturbation effects were less pronounced than on gelatine. Our observations indicate that laminin and fibronectin are involved both in β1 integrin-dependent and -independent signalling mechanisms during neurogenesis. Upregulation of compensatory mechanisms such as β1 integrin-independent receptors for laminin and fibronectin might be responsible for the much less pronounced perturbations of G201 neural precursor migration and differentiation on these two substrates than on gelatine.     

Fucan effect on CHO cell proliferation and migration

Fucan is a term used to denominate sulfated l-fucose rich polysaccharides. Here, a heterofucan, named fucan B, was extracted from the Spatoglossum schröederi seaweed. This 21.5 kDa galactofucan inhibited CHO-K1 proliferation and migration when fibronectin was the substrate. Fucan B derivatives revealed that such effects depend on their degree of sulfation. Fucan B did not induce cell death, but promoted G₁ cell cycle arrest. Western blotting and flow cytometry analysis suggest that fucan B binds to fibronectin and activates integrin, mainly integrin α5β1, which induces FAK/RAS/MEK/ERK activation. FAK activation inhibits CHO-K1 migration on fibronectin and ERK blocks cell cycle progression. This study indicates that fucan B could be applied in developing new antitumor drugs.     

Drosophila PS2 integrin mediates RGD-dependent cell-matrix interactions.

Integrins are a family of transmembrane glycoproteins that mediate cell-matrix and cell-cell interactions. We have transfected cultured Drosophila cells with genes that express the Drosophila PS2 integrin. We demonstrate that this integrin is expressed on the surface of the cells and can mediate cell spreading on an undefined component of fetal calf serum or on the purified vertebrate matrix molecules vitronectin and fibronectin. Additionally, PS2 integrin can cause cell spreading on RGD peptide. The spreading on matrix components or RGD peptide can be inhibited by soluble RGD peptide and is dependent on divalent cations.