Role of IIb-IIIa-like glycoproteins in cell-substratum adhesion of human melanoma cells

The platelet fibrinogen receptor, glycoprotein complex IIb-IIIa, was isolated from human platelets by lectin and monoclonal antibody affinity chromatography and a polyclonal antiserum (anti-IIb-IIIa) was generated and used to probe for the presence and function of IIb-IIIa-like molecules in two adherent human cell lines. Both C32 melanoma cells and WI38 fibroblasts expressed a IIb-IIIa-like complex on their surface as indicated by immunoprecipitation of detergent extracts of surface radiolabeled cells. When added to cells plated in medium containing 10% serum, the anti-IIb-IIIa antiserum perturbed the adhesion of C32 melanoma cells, but not of WI38 fibroblasts. In a serum-free system, anti-IIb-IIIa antibodies inhibited attachment and spreading of C32 cells to fibrinogen, vitronectin, and fibronectin adsorbed to glass. Anti-IIb-IIIa had no effect on the attachment and spreading of WI38 cells to the extracellular matrix proteins, however. Thus, the IIb-IIIa-like complex appears to play a predominant role in cell-substratum adhesion of C32 cells, but not WI38 cells, and may result from the fact that, on a protein basis, the C32 melanoma cells express approximately 3 times more complex on their surface than do WI38 fibroblasts. The results suggest that the relative abundance of a particular adhesion receptor on the cell surface may govern its importance to cell-substratum adhesion.     

Extracellular Alix regulates integrin-mediated cell adhesions and extracellular matrix assembly

Alix (ALG-2-interacting protein X), a cytoplasmic adaptor protein involved in endosomal sorting and actin cytoskeleton assembly, is required for the maintenance of fibroblast morphology. As Alix has sequence similarity to adhesin in Entamoeba histolytica, and we observed that Alix is secreted, we determined whether extracellular Alix affects fibroblast morphology. Here, we demonstrate that secreted Alix is deposited on the substratum of non-immortalized WI38 fibroblasts. Antibody binding to extracellular Alix retards WI38 cell adhesion and spreading on fibronectin and vitronectin. Alix knockdown in WI38 cells reduces spreading and fibronectin assembly, and the effect is partially complemented by coating recombinant Alix on the cell substratum. Immortalized NIH/3T3 fibroblasts deposit less Alix on the substratum and have defects in α₅β₁-integrin functions. Coating recombinant Alix on the culture substratum for NIH/3T3 cells promotes α₅β₁-integrin-mediated cell adhesions and fibronectin assembly, and these effects require the aa 605–709 region of Alix. These findings demonstrate that a sub-population of Alix localizes extracellularly and regulates integrin-mediated cell adhesions and fibronectin matrix assembly.     

Adhesion, growth, and matrix production by fibroblasts on laminin substrates

Human embryonic skin fibroblasts have been shown to attach and spread on laminin substrates in the absence of protein synthesis and presence of fibronectin-depleted serum and anti-fibronectin antibodies. Rates of attachment and the type of spreading are virtually identical on fibronectin and laminin-coated substrates with the development of microfilament bundles and focal adhesions. Antibodies to laminin, but not fibronectin, will prevent or reverse fibroblast adhesion to laminin, whereas antibodies to fibronectin but not laminin will give similar results on fibronectin-coated substrates. These and other results indicate that fibroblasts possess distinct receptors for laminin and fibronectin which on contact with suitable substrates promote adhesion through interaction with common intermediates. This type of adhesion is compatible with subsequent growth and extracellular matrix production.     

Integrin-mediated migration of murine B82L fibroblasts is dependent on the expression of an intact epidermal growth factor receptor

To evaluate the mechanisms by which epidermal growth factor (EGF) regulates actin-based cellular processes such as cell migration, we first examined the effects of EGF on cell adhesion, which is essential for cell migration. In mouse B82L fibroblasts transfected with the full-length EGF receptor, EGF promotes cell rounding and attenuates cell spreading on fibronectin, laminin, and vitronectin, and thus appears to reduce the strength of cell adhesion. Moreover, EGF synergizes with multiple extracellular matrix (ECM) components in the promotion of integrin-mediated cell migration of several different cell types, including fibroblasts and various carcinoma and osteosarcoma cell lines. Interestingly, co-presentation (co-positioning) of EGF with laminin or fibronectin is essential for EGF-stimulated migration. When EGF is mixed with the cells instead of the ECM components, it has little effect on cell migration. These results suggest that co-presentation of EGF with ECM components can enhance the polarization events required for directional cell movement. To identify the EGF receptor elements critical for the EGF stimulation of cell migration, B82L fibroblasts were transfected with either mutated or wild-type EGF receptors. Surprisingly, we found that B82L-Parental cells that lack the EGF receptor are not able to migrate to fibronectin, even though they can adhere to fibronectin. However, the introduction of wild-type EGF receptors into these fibroblasts enables them to migrate toward fibronectin even in the absence of EGF. The requirement of the EGF receptor for cell migration does not appear to result from the secretion of EGF or TGF-alpha by the cells transfected with the EGF receptor. Furthermore, cells expressing EGF receptors that are kinase-inactive, or C-terminally truncated, exhibit little migration toward fibronectin, indicating that an intact EGF receptor kinase is required for fibronectin-induced cell migration. In addition, neutralizing anti-EGF receptor antibodies attenuate cell migration in the presence of EGF, and inhibit migration to fibronectin or laminin alone. These results further suggest that the EGF receptor is downstream of integrin activation in the signal transduction pathways leading to fibroblast migration.     

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.     

Identification and characterization of the T lymphocyte adhesion receptor for an alternative cell attachment domain (CS-1) in plasma fibronectin

Using mAb technology (Wayner, E. A., W. G. Carter, R. Piotrowicz, and T. J. Kunicki. 1988. J. Cell Biol. 107:1881-1891), we have identified a new fibronectin receptor that is identical to the integrin receptor alpha 4 beta 1. mAbs P3E3, P4C2, and P4G9 recognized epitopes on the alpha 4 subunit and completely inhibited the adhesion of peripheral blood and cultured T lymphocytes to a 38-kD tryptic fragment of plasma fibronectin containing the carboxy-terminal Heparin II domain and part of the type III connecting segment (IIICS). The ligand in IIICS for alpha 4 beta 1 was the CS-1 region previously defined as an adhesion site for melanoma cells. The functionally defined mAbs to alpha 4 partially inhibited T lymphocyte adhesion to intact plasma fibronectin and had no effect on their attachment to an 80-kD tryptic fragment containing the RGD (arg-gly-asp) adhesion sequence. mAbs (P1D6 and P1F8) to the previously described fibronectin receptor, alpha 5 beta 1, completely inhibited T lymphocyte adhesion to the 80-kD fragment but had no effect on their attachment to the 38-kD fragment or to CS-1. Both alpha 4 beta 1 and alpha 5 beta 1 localized to focal adhesions when fibroblasts that express these receptors were grown on fibronectin-coated surfaces. These findings demonstrated a specific interaction of both receptors with fibronectin at focal contacts. In conclusion, these findings show clearly that cultured T lymphocytes use two independent receptors during attachment to fibronectin and that (a) alpha 5 beta 1 is the receptor for the RGD containing cell adhesion domain, and (b) alpha 4 beta 1 is the receptor for a carboxy-terminal cell adhesion region containing the Heparin II and IIICS domains. Furthermore, these data also show that T lymphocytes express a clear preference for a region of molecular heterogeneity in IIICS (CS-1) generated by alternative splicing of fibronectin pre-mRNA and that alpha 4 beta 1 is the receptor for this adhesion site.     

The effect of a collagen tripeptide fragment (GER) on fibroblast adhesion and spreading depends on properties of an adhesive surface

The effect of collagen tripeptide fragment GER on the adhesion and spreading of mouse embryonic fibroblasts STO to different substrates (polystyrene plastic, poly-L-lysine, fibronectin, gelatin) has been studied. It was found that tripeptide GER was involved in fibroblast adhesion and spreading. The cell response depended both on the mode of tripeptide addition to culture medium and the substrate type. Coincubation of fibroblasts with tripeptide stimulated the cell attachment and spreading to untreated plastic and plastic coated with fibronectin or gelatin but did not change cell adhesion to immobilized poly-L-lysine. Preincubation of cells with tripeptide resulted in partial inhibition of fibroblast adhesion and spreading on fibronectin- and gelatin-coated substrata. It was shown that activation and inhibition of adhesive processes after tripeptide treating was higher on fibronectin than gelatin. The data obtained support the assumption about concerted action of tripeptide GER (activity was dependent both on the used concentration of the tripeptide and the mode of tripeptide addition to culture medium) and chemical characteristics of substrate (polymers of styrene and L-lysine, ECM proteins in native (fibronectin) or partly denatured (gelatin) form) on the cell adhesion and spreading. The main targets that GER peptide may affect during the formation of cell-substrate interactions are discussed.     

Inhibition of binding of fibronectin to matrix assembly sites by anti- integrin (alpha 5 beta 1) antibodies

Fibroblasts have cell surface sites that mediate assembly of plasma and cellular fibronectin into the extracellular matrix. Cell adhesion to fibronectin can be mediated by the interaction of an integrin (alpha 5 beta 1) with the Arg-Gly-Asp-Ser (RGDS)-containing cell adhesion region of fibronectin. We have attempted to elucidate the role of the alpha 5 beta 1 fibronectin receptor in assembly of fibronectin in matrices. Rat monoclonal antibody mAb 13, which recognizes the integrin beta 1 subunit, completely blocked binding and matrix assembly of 125I-fibronectin as well as binding of the 125I-70-kD amino-terminal fragment of fibronectin (70 kD) to fibroblast cell layers. Fab fragments of the anti-beta 1 antibody were also inhibitory. Antibody mAb 16, which recognizes the integrin alpha 5 subunit, partially blocked binding of 125I-fibronectin and 125I-70-kD. When cell layers were coincubated with fluoresceinated fibronectin and either anti-beta 1 or anti-alpha 5, anti-beta 1 was a more effective inhibitor than anti-alpha 5 of binding of labeled fibronectin to the cell layer. Inhibition of 125I-fibronectin binding by anti-beta 1 IgG occurred within 20 min. Inhibition of 125I-fibronectin binding by anti-beta 1 Fab fragments or IgG could not be overcome with increasing concentrations of fibronectin, suggesting that anti-beta 1 and exogenous fibronectin may not compete for the same binding site. No beta 1-containing integrin bound to immobilized 70 kD. These data indicate that the beta 1 subunit plays an important role in binding and assembly of exogenous fibronectin, perhaps by participation in the organization, regeneration, or cycling of the assembly site rather than by a direct interaction with fibronectin.     

Initial adhesion of human fibroblasts in serum-free medium: possible role of secreted fibronectin.

Experiments were carried out to test the hypothesis that the initial attachment and spreading of human fibroblasts in serum-free medium occurs to cell fibronectin which has been secretd spread on tissue culture substrata in serum-free medium in 60 min. When potential protein adsorption sites on the substratum were covered with bovine serum albumin before initial human fibroblasts attachment, their subsequent attachment to the substratum was prevented. When substratum adsorption sites were covered immediately after initial attachment, subsequent cell spreading was prevented. The distribution of fibronectin on human fibroblast surfaces during initial attachment and spreading was studied by indirect immunofluorescence analysis using a monospecific anti-cold-insoluble globulin antiserum. The initial appearance (10 min) of fibronectin was in spots over the entire cell surface. Concomitant with human fibroblast spreading, the random distribution of sites disappeared, and most fibronectin was subsequently observed in spots at the cell substratum interface (60 min). A fibrillar pattern of fibronectin appeared later (2-8 hr). The sites beneath the cells could be visualized as footprints on the substratum following treatment of the attached human fibroblasts with 0.1 M NaOH. A second fluorescence pattern of fibronectin secreted on the substratum was characterized by a diffuse halo around the cells and a very faint, diffuse staining elsewhere on the substratum. Another cell type (baby hamster kideny cells) was used to assay biologically for the presence or absence of the factor secreted by human fibroblasts on the substratum. Human fibroblasts were found to secrete an adhesion factor for baby hamster kidney cells into the substratum in a time- and temperature-dependent fashion, and immunological studies indicated that the factor secreted by human fibroblasts was cross-reactive with cold-in-soluble globulin, the plasma form of fibronectin. The conditioning factor secreted by the human fibroblasts was also found to be an attachment and spreading factor for human fibroblasts in experiments measuring human fibroblast adhesion to fibronectin footprints of human fibroblasts. Substratum-adsorbed cold-insoluble globulin was also found to be an attachment and spreading factor for human fibroblasts. Based upon the timing of appearance of conditioning factors on the substratum and the immunofluorescence patterns, it seems that the diffusely organized fibronectin on the substratum constitutes the sites to which cell attachment occurs. The bright spots of fibronectin that appear beneath the cells may represent fibronectin reorganization during cell spreading.     

Localization of urokinase type plasminogen activator to focal adhesions requires ligation of vitronectin integrin receptors

Previous studies have shown that the adhesion protein, vitronectin, directs the localization of urokinase-type plasminogen activator (uPA) to areas of cell-substrate adhesion, where uPA is thought to regulate cell migration as well as pericellular proteolysis. In the present study, HT-1080 cell lines expressing either wild-type vitronectin or vitronectin containing a single amino-acid substitution in the integrin binding domain were used to assess whether ligation of the alphavbeta5 integrin was required for uPA localization to focal adhesions. The synthesis of wild-type vitronectin by HT-1080 cells adherent to either collagen or fibronectin resulted in the redistribution of both the alphavbeta5 integrin as well as uPA to focal adhesion structures. In contrast, cells synthesizing mutant vitronectin, containing the amino-acid substitution in the integrin binding domain, were unable to direct the redistribution of either alphavbeta5 or uPA to focal adhesions. Recombinant forms of wild-type and mutant vitronectin were prepared in a baculovirus system and compared for their ability to direct the redistribution of vitronectin integrin receptors as well as uPA on human skin fibroblasts. In the absence of vitronectin, fibroblast cells adherent to fibronectin assemble focal adhesions which contain the beta1 integrin but do not contain uPA. Addition of recombinant wild-type, but not mutant, vitronectin to fibroblasts adherent to fibronectin resulted in the redistribution of alphavbeta3, alphavbeta5, and uPA into focal adhesions. However, when cells were plated directly onto antibodies directed against either the alphavbeta3 or alphavbeta5 integrins, uPA was not localized on the cell surface. These data indicate that ligation of vitronectin integrin receptors is necessary but not sufficient for the localization of uPA to areas of cell matrix adhesion, and suggest that vitronectin may promote cell migration by recruiting vitronectin integrin receptors and components of the plasminogen activator system to areas of cell matrix contact.     

Opposing effects of collagen I and vitronectin on fibronectin fibril structure and function

Extracellular matrix fibronectin fibrils serve as passive structural supports for the organization of cells into tissues, yet can also actively stimulate a variety of cell and tissue functions, including cell proliferation. Factors that control and coordinate the functional activities of fibronectin fibrils are not known. Here, we compared effects of cell adhesion to vitronectin versus type I collagen on the assembly of and response to, extracellular matrix fibronectin fibrils. The amount of insoluble fibronectin matrix fibrils assembled by fibronectin-null mouse embryonic fibroblasts adherent to collagen- or vitronectin-coated substrates was not significantly different 20 h after fibronectin addition. However, the fibronectin matrix produced by vitronectin-adherent cells was ~ 10-fold less effective at enhancing cell proliferation than that of collagen-adherent cells. Increasing insoluble fibronectin levels with the fibronectin fragment, anastellin did not increase cell proliferation. Rather, native fibronectin fibrils polymerized by collagen- and vitronectin-adherent cells exhibited conformational differences in the growth-promoting, III-1 region of fibronectin, with collagen-adherent cells producing fibronectin fibrils in a more extended conformation. Fibronectin matrix assembly on either substrate was mediated by α5β1 integrins. However, on vitronectin-adherent cells, α5β1 integrins functioned in a lower activation state, characterized by reduced 9EG7 binding and decreased talin association. The inhibitory effect of vitronectin on fibronectin-mediated cell proliferation was localized to the cell-binding domain, but was not a general property of αvβ3 integrin-binding substrates. These data suggest that adhesion to vitronectin allows for the uncoupling of fibronectin fibril formation from downstream signaling events by reducing α5β1 integrin activation and fibronectin fibril extension.     

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.     

Cell surface distribution of fibronectin and vitronectin receptors depends on substrate composition and extracellular matrix accumulation

We used antibodies against the alpha subunits of the human fibronectin receptor (FNR) and vitronectin receptor (VNR) to localize simultaneously FNR and VNR at major substrate adhesion sites of fibroblasts and melanoma cells with double-label immunofluorescence microscopy. In early (2-6-h) serum-containing cultures, both FNR and VNR coaccumulated in focal contacts detected by interference reflection microscopy. Under higher resolution immunoscanning electron microscopy, FNR and VNR were also observed to be distributed randomly on the dorsal cell surface. As fibronectin-containing extracellular matrix fibers accumulated beneath the cells at 24 h, FNR became concentrated at contacts with these fibers and was no longer detected at focal contacts. VNR was not observed at matrix contacts but remained strikingly localized in focal contacts of the 24-h cells. Since focal contacts represent the sites of strongest cell-to-substrate adhesion, these results suggest that FNR and VNR together play critical roles in the maintenance of stable contacts between the cell and its substrate. In addition, the accumulation of FNR at extracellular matrix contacts implies that this receptor might also function in the process of cellular migration along fibronectin-containing matrix cables. To define the factors governing accumulation of FNR and VNR at focal contacts, fibroblasts in serum-free media were plated on substrates coated with purified ligands. Fibronectin-coated surfaces fostered accumulation of FNR but not VNR at focal contacts. On vitronectin-coated surfaces, or substrata derivatized with a tridecapeptide containing the cell attachment sequence Arg-Gly-Asp, both FNR and VNR became concentrated at focal contacts. These observations suggest that the availability of ligand is critical to the accumulation of FNR and VNR at focal contacts, and that FNR might also recognize substrate-bound vitronectin.     

Malignant astrocytoma cell attachment and migration to various matrix proteins is differentially sensitive to phosphoinositide 3-OH kinase inhibitors

Phosphoinositide 3-OH kinase (PI3-K) has been shown to play an important role in the signaling pathway necessary for cytoskeletal reorganization in non-astrocytic cells. We investigated the role of PI3-K in U-251 MG human malignant astrocytoma cell adhesion and migration. Attachment of U-251 MG cells to vitronectin, fibronectin, laminin, and collagen was inhibited in a concentration-dependent manner by two specific inhibitors of PI3-K (Wortmannin and LY294002). Attachment to vitronectin, fibronectin, and laminin was more sensitive to inhibition of PI3-K (45% inhibition at 10 nM Wortmannin) than attachment to collagen (25% inhibition at 100 nM Wortmannin). Similarly, migration toward these substrates showed differential sensitivity to inhibition. Attachment of the cells to these matrix proteins resulted in an increase in PI3-K activity, as compared to that of cells in suspension, with attachment to vitronectin resulting in the greatest increase in PI3-K activity. p125 focal adhesion kinase (p125FAK) was found to co-immunoprecipitate with PI3-K from the NP40-soluble cell fraction of a 1% NP40 detergent lysate of cells in the early stages of adhesion to vitronectin and fibronectin, but not during adhesion to collagen. The expression of p125FAK protein and level of phosphorylation were similar on adherence to all three substrates. These data indicate that the sensitivity of U-251MG cell attachment and migration to PI3-K inhibitors is substrate-dependent, and that complex formation of PI3-K and p125FAK correlates with this sensitivity to PI3-K inhibitors. Our data suggest a role for PI3-K and p125FAK complex formation in PI3-K activation.     

Syndecan-1 regulates cell migration and fibronectin fibril assembly

Corneal scarring is a major cause of blindness worldwide and can result from the deposition of abnormal amounts of collagen fibers lacking the correct size and spacing required to produce a clear cornea. Collagen fiber formation requires a preformed fibronectin (FN) matrix. We demonstrate that the loss of syndecan1 (sdc1) in corneal stromal cells (CSC) impacts cell migration rates, the sizes and composition of focal and fibrillar adhesions, the activation of integrins, and the assembly of fibronectin into fibrils. Integrin and fibronectin expression are not altered on sdc1-null CSCs. Cell adhesion, spreading, and migration studies using low compared to high concentrations of FN and collagen I (CNI) or vitronectin (VN) with and without activation of integrins by manganese chloride show that the impact of sdc1 depletion on integrin activation varies depending on the integrin-mediated activity evaluated. Differences in FN fibrillogenesis and migration in sdc1-null CSCs are reversed by addition of manganese chloride but cell spreading differences remain. To determine if our findings on sdc1 were specific to the cornea, we compared the phenotypes of sdc1-null dermal fibroblasts with those of CSCs. We found that without sdc1, both cell types migrate faster; however, cell-type-specific differences in FN expression and its assembly into fibrils exist between these two cell types. Together, our data demonstrate that sdc1 functions to regulate integrin activity in multiple cell types. Loss of sdc1-mediated integrin function results in cell-type specific differences in matrix assembly. A better understanding of how different cell types regulate FN fibril formation via syndecans and integrins will lead to better treatments for scarring and fibrosis.     

Differential properties of attachment of human fibroblasts to various extracellular matrix proteins

Human diploid fibroblasts (TIG-3) were shown to attach and spread onto substrata coated with collagen, fibronectin, laminin and vitronectin. The cell attachment to these proteins required divalent cations. Mg2+ stimulated the cell attachment to all the proteins, while Ca2+ alone was not effective for the attachment to collagen and laminin. A mild trypsin treatment had prevented cells from attaching to the laminin, while it had no effect on the attachment to the other proteins. The fibronectin fragment, which retained cell binding activity, inhibited the cells from attaching and spreading onto fibronectin, but it did not cause any inhibition on the other proteins. The synthetic peptide GRGDSP inhibited the cells from attaching and spreading onto fibronectin and vitronectin, while it did not cause any inhibition on collagen and laminin. In attempts to isolate distinct receptors for these proteins, we were able to purify proteins very similar to the fibronectin and vitronectin receptors of human placenta. Based on the differential properties of the attachment of TIG-3 cells to these proteins and biochemical data, we indicate that human diploid fibroblasts have distinctive binding sites (receptors) for collagen, fibronectin, laminin and vitronectin.     

Cell adhesion strengthening: contributions of adhesive area, integrin binding, and focal adhesion assembly

Mechanical interactions between a cell and its environment regulate migration, contractility, gene expression, and cell fate. We integrated micropatterned substrates to engineer adhesive area and a hydrodynamic assay to analyze fibroblast adhesion strengthening on fibronectin. Independently of cell spreading, integrin binding and focal adhesion assembly resulted in rapid sevenfold increases in adhesion strength to steady-state levels. Adhesive area strongly modulated adhesion strength, integrin binding, and vinculin and talin recruitment, exhibiting linear increases for small areas. However, above a threshold area, adhesion strength and focal adhesion assembly reached a saturation limit, whereas integrin binding transitioned from a uniform distribution to discrete complexes. Adhesion strength exhibited exponential increases with bound integrin numbers as well as vinculin and talin recruitment, and the relationship between adhesion strength and these biochemical events was accurately described by a simple mechanical model. Furthermore, adhesion strength was regulated by the position of an adhesive patch, comprised of bound integrins and cytoskeletal elements, which generated a constant 200-nN adhesive force. Unexpectedly, focal adhesion assembly, in particular vinculin recruitment, contributed only 30% of the adhesion strength. This work elucidates the roles of adhesive complex size and position in the generation of cell-extracellular matrix forces.     

14-3-3beta-Rac1-p21 activated kinase signaling regulates Akt1-mediated cytoskeletal organization, lamellipodia formation and fibronectin matrix assembly

Akt1 belongs to the three-gene Akt family and functions as a serine-threonine kinase regulating phosphorylation of an array of substrates and mediating cellular processes such as cell migration, proliferation, survival, and cell cycle. Our previous studies have established the importance of Akt1 in angiogenesis and absence of Akt1 resulted in impaired integrin activation, adhesion, migration, and extracellular matrix assembly by endothelial cells and fibroblasts. In this study, we identify the downstream signaling pathways activated by Akt1 in the regulation of these cellular events. We demonstrate here that Akt1 is necessary for the growth factor stimulated activation of 14-3-3beta-Rac1-p21 activated kinase (Pak) pathway in endothelial cells and fibroblasts. While activation of Akt1 resulted in translocation of Rac1 to membrane ruffles, enhanced Rac1 activity, Pak1 phosphorylation, and lamellipodia formation, resulting in enhanced adhesion and assembly of fibronectin, inhibition of Akt1 resulted in inhibition of these processes due to impaired Rac1-Pak signaling. Formation of lamellipodia, adhesion, and fibronectin assembly by myristoylated Akt1 expression in NIH 3T3 fibroblasts was inhibited by co-expression with either dominant negative Rac1 or dominant negative Pak1. In contrast, impaired lamellipodia formation, adhesion, and fibronectin assembly by dominant negative-Akt1 expression was rescued by co-expression with either constitutively active-Rac1 or -Pak1. Moreover, previously reported defects in adhesion and extracellular matrix assembly by Akt1(-/-) fibroblasts could be rescued by expression with either active-Rac1 or -Pak1, implying the importance of Rac1-Pak signaling in growth factor stimulated cytoskeletal assembly, lamellipodia formation and cell migration in endothelial cells and fibroblasts downstream of Akt1 activation.     

Mena binds α5 integrin directly and modulates α5β1 function

Mena is an Ena/VASP family actin regulator with roles in cell migration, chemotaxis, cell-cell adhesion, tumor cell invasion, and metastasis. Although enriched in focal adhesions, Mena has no established function within these structures. We find that Mena forms an adhesion-regulated complex with α5β1 integrin, a fibronectin receptor involved in cell adhesion, motility, fibronectin fibrillogenesis, signaling, and growth factor receptor trafficking. Mena bound directly to the carboxy-terminal portion of the α5 cytoplasmic tail via a 91-residue region containing 13 five-residue "LERER" repeats. In fibroblasts, the Mena-α5 complex was required for "outside-in" α5β1 functions, including normal phosphorylation of FAK and paxillin and formation of fibrillar adhesions. It also supported fibrillogenesis and cell spreading and controlled cell migration speed. Thus, fibroblasts require Mena for multiple α5β1-dependent processes involving bidirectional interactions between the extracellular matrix and cytoplasmic focal adhesion proteins.     

Influence of stereochemistry of the sequence Arg-Gly-Asp-Xaa on binding specificity in cell adhesion

Peptides containing the tripeptide sequence Arg-Gly-Asp can duplicate or inhibit the cell attachment-promoting effects of fibronectin and vitronectin. Peptides analogous to a prototype peptide, Gly-Arg-Gly-Asp-Ser-Pro-Cys, the sequence of which was taken from the cell attachment site of fibronectin, were assayed for their relative abilities to inhibit the attachment of cells to a fibronectin or vitronectin substrate. A peptide having the L-Arg residue replaced with D-Arg showed no difference in this capacity, whereas substituting Gly with D-Ala or L-Asp with D-Asp resulted in completely inactive peptides. Replacement of L-Ser with D-Ser drastically reduced the influence that the resulting peptide had on the vitronectin interaction, but this peptide showed little difference in its effect on the binding of cells to fibronectin when compared with the prototype peptide. Furthermore, substitution of the Ser with L-Asn resulted in a peptide that had an apparent increased preference for the fibronectin receptor and decreased preference for the vitronectin receptor. Conversely, threonine in this position gave a peptide with increased preference for the vitronectin receptor, whereas L-Pro in this position gave a completely inactive peptide. Finally, by cyclicizing the prototype peptide to restrict its conformational flexibility, a peptide was obtained that was a much improved inhibitor of attachment of cells to vitronectin and yet nearly inactive with respect to the interactions of cells with fibronectin substrates. These studies lend support to the hypothesis that different Arg-Gly-Asp-directed adhesion receptors can recognize differences in the conformation and environment of the Arg-Gly-Asp tripeptide, and they establish the feasibility of obtaining synthetic probes that are more selective for individual receptors than are the peptides modeled after the natural sequences of adhesive extracellular matrix molecules.