Surface chemistry modulates focal adhesion composition and signaling through changes in integrin binding

Biomaterial surface properties influence protein adsorption and elicit diverse cellular responses in biomedical and biotechnological applications. However, the molecular mechanisms directing cellular activities remain poorly understood. Using a model system with well-defined chemistries (CH₃, OH, COOH, NH₂) and a fixed density of the single adhesive ligand fibronectin, we investigated the effects of surface chemistry on focal adhesion assembly and signaling. Surface chemistry strongly modulated integrin binding and specificity—₅β₁ integrin binding affinity followed the pattern OH>NH₂=COOH>CH₃, while integrin _Vβ₃ displayed the relationship COOH>NH₂OH=CH₃. Immunostaining and biochemical analyses revealed that surface chemistry modulates the structure and molecular composition of cell-matrix adhesions as well as focal adhesion kinase (FAK) signaling. The neutral hydrophilic OH functionality supported the highest levels of recruitment of talin, -actinin, paxillin, and tyrosine-phosphorylated proteins to adhesive structures. The positively charged NH₂ and negatively charged COOH surfaces exhibited intermediate levels of recruitment of focal adhesion components, while the hydrophobic CH₃ substrate displayed the lowest levels. These patterns in focal adhesion assembly correlated well with integrin ₅β₁ binding. Phosphorylation of specific tyrosine residues in FAK also showed differential sensitivity to surface chemistry. Finally, surface chemistry-dependent differences in adhesive interactions modulated osteoblastic differentiation. These differences in focal adhesion assembly and signaling provide a potential mechanism for the diverse cellular responses elicited by different material properties.     

Enhanced expression of the osteoblastic phenotype on substrates that modulate fibronectin conformation and integrin receptor binding

Integrins represent the primary mechanism of cell-extracellular matrix interactions and control cell morphology, proliferation, and differentiation. We have previously shown that substrate-dependent modulation of adsorbed fibronectin (Fn) conformation alters alpha5beta1 integrin binding to Fn and directs C2C12 myoblast proliferation and differentiation (Mol. Biol. Cell 10 (1999) 785). The model substrates used in these experiments were bacteriological (untreated) polystyrene (B), tissue culture polystyrene (T), and type-I collagen-coated T (C). In the present study, we examined MC3T3-EI osteoblast-like cell differentiation on Fn-coated B, T, and C substrates. Immunofluorescence staining revealed substrate-dependent differences in integrin alpha5beta1 binding and clustering into focal adhesions (C > T > B), consistent with our previous integrin binding analysis. Alkaline phosphatase activity and matrix mineralization showed substrate-dependent differences (C > T > B, p < 0.05). Similar trends were observed for alkaline phosphatase, osteocalcin, and bone sialoprotein gene expression. Blocking experiments with antibodies directed against Fn completely inhibited matrix mineralization on Fn-coated C, indicating that Fn is critical to expression of the osteoblastic phenotype on this extracellular matrix component. These substrate-dependent differences in osteoblast differentiation correlated with differences in alpha5beta1 binding, suggesting that these differences arise from substrate modulation of integrin-matrix interactions. Substrate-dependent modulation of cell function may provide a versatile mechanism to control cell responses in numerous biomedical applications.     

Mast cell adhesion to fibronectin.

The MCP-5 murine mast cell line, as well as primary bone marrow-derived cultured mast cells (BMCMC), are demonstrated to bind to fibronectin, a ubiquitous adhesion protein of the extracellular matrix. BMCMC required activation by phorbol myristate acetate (PMA) to adhere to fibronectin, whereas MCP-5 displayed spontaneous adherence. The binding of both MCP-5 and BMCMC was dose dependent, with maximal adhesion at a fibronectin concentration of 20 micrograms/ml. The 120,000 molecular weight (MW) proteolytic fragment of fibronectin containing the RGDS cell attachment site was able to substitute for the native fibronectin molecule in promoting mast cell attachment. Mast cell adhesion to fibronectin, in addition, could be inhibited by the RGDS peptide alone. These data suggest that, in addition to the previously described mast cell-laminin interactions, mast cells also adhere to fibronectin, thus providing further insight into their tissue localization and possible roles in processes such as wound healing and fibrosis.     

Activation-dependent changes in soluble fibronectin binding and expression of beta1 integrin activation epitopes in T cells: relationship to T cell adhesion and migration

The relationship between activation-dependent changes in beta1 integrin conformation, T cell adhesion to immobilized fibronectin, and T cell migration in vitro was analyzed in this study. Stimulation of Jurkat T cells and peripheral T cells with Mn(2+), the activating beta1 integrin-specific monoclonal antibody (mAb) TS2 /16, CD2, or CD28 stimulation led to increased adhesion, soluble fibronectin (FN) binding and expression of the activation epitope defined by the beta1 integrin mAb HUTS-21. Phorbol 12-myristate 13-acetate treatment increased adhesion, but not soluble FN binding or HUTS-21 epitope expression. In peripheral T cells, CD3 or CD7 stimulation also led to increased adhesion, soluble FN binding and HUTS-21 epitope expression. Soluble FN blocked peripheral T cell adhesion induced by Mn(2+) or TS2/16, but had no effect on adhesion induced by the other integrin-activating signals. In contrast, migration induced by TS2/16, CD2, CD3, CD7 or CD28 stimulation was blocked by excess soluble FN. Phosphoinositide 3-OH kinase (PI 3-K) inhibitors blocked receptor-mediated increases in cell adhesion, but not soluble FN binding or HUTS-21 expression. Migration was similarly unaffected by PI 3-K inhibitors, with the exception of CD7- and CD28-induced migration, which was specifically blocked by LY294,002. These results suggest that activation-dependent changes in beta1 integrin conformation are PI 3-K-independent and are involved in T cell migration but not adhesion.     

Beta1 integrin activation on human neutrophils promotes beta2 integrin-mediated adhesion to fibronectin

Although the importance of beta1 integrin-mediated binding to adhesion molecules and extracellular matrix (ECM) molecules is well established for most types of leukocytes, the expression patterns and functional importance of beta1 integrins on neutrophils have remained controversial. Using flow cytometry, we found that human neutrophils express the alpha4, alpha5, alpha9 and beta1 integrin subunits. To examine whether the integrins VLA-4 (alpha4/beta1) and VLA-5 (alpha5/beta1) have a functional role on neutrophils, we studied adhesion to their ligand fibronectin. Treatment of neutrophils with antibody 8A2, which specifically binds and activates beta1 integrins, resulted in increased binding to fibronectin. However, addition of blocking mAb revealed that 8A2-induced adhesion did not depend on beta1 integrins, but on the beta2 integrin CD11b/CD18. Similarly, activation of beta1 integrins by 8A2 resulted in CD11b-dependent binding of neutrophils to fibrinogen. 8A2 treatment increased expression of an activation epitope of CD11b/CD18, which depended on phosphoinositide 3-OH kinase activity and an adequate concentration of intracellular free Ca2+. These data suggest that engagement of beta1 integrins on neutrophils results in a cross-talk signal that leads to activation of the beta2 integrin CD11b/CD18, followed by CD11b-mediated adhesion. As transmigrated neutrophils are surrounded by both beta1 and beta2 ligands in the ECM, this integrin cross-talk could play a role in modifying migration and cellular activation in inflamed tissues.     

Distinct mechanism of human neuroblastoma cell adhesion to fibronectin

We investigated the adhesion of three morphologically distinct human neuroblastoma cell lines (NCG, GOTO and SK-N-DZ) to intact fibronectin, central cell binding domain fragment (CBF) and CS peptide-IgG conjugates in the fibronectin molecule. Each cell line was found to express different integrin fibronectin receptors ( _{3 1}, _{4 1} and ₅₁), although similarly attached on intact fibronectin. To CBF, NCG attached well, while GOTO moderately and SK-N-DZ poorly attached. Only GOTO adhered to CS1-IgG. RGDS inhibited the spreading of NCG and SK-N-DZ on intact fibronectin, but it barely inhibited that of GOTO. The analysis by fluorescence-activated cell sorting (FACS) revealed that NCG expressed abundant ₃₁ and ₅₁, but little ₄₁, while GOTO expressed a large amount of ₄₁ as well as ₅₁. SK-N-DZ was undetectable in any of these molecules, but expressed _v1, which was identified by immunoprecipitation and immunoblotting. Polyclonal antibody to _v3 inhibited the adhesion of SK-N-DZ but not that of NCG or GOTO on intact fibronectin. These results suggest the existence of a distinct mechanism of cell adhesion to fibronectin among human neuroblastoma cell lines. It remains to be determined if such heterogeneous adhesion properties are related to the unique metastatic character of human neuroblastoma.     

Integrin alpha2beta1 on rat myeloma cells modulates interaction of alpha4beta1 integrin with vascular cell adhesion molecule-1 but not fibronectin

It is well established that alpha2beta1 integrin functions as a receptor for collagen and laminin; whereas alpha4beta1 integrin binds fibronectin and vascular cell adhesion molecule-1 (VCAM-1). In the present study, we showed that rat myeloma YB2/0 cells constitutively expressed alpha4beta1 but not alpha2beta1 integrin. Transfection of cDNA of mouse a2 integrin subunit resulted in the expression of heterologous alpha2beta1 integrin on YB2/0 cells (YBmalpha2). The expression of alpha2beta1 conferred YBmalpha2 cells the ability to interact with collagen and laminin. In comparison with mock transfected YB2/0 cells (YBpF), YBmalpha2 cells exhibited increases in the binding and migration on VCAM-1; in contrast, both YBpF and YBmalpha2 were similar in their interactions with fibronectin or fibronectin fragment FN-40 that contains the binding site for alpha4beta1 integrin. The interaction of alpha4beta1 with VCAM-1 was further stimulated upon ligation with alpha2beta1-specific mAb. The use of specific inhibitory mAb demonstrated the role of alpha4beta1 in mediating the observed interactions with fibronectin and VCAM-1. Therefore, results show that expression of alpha2beta1 differentially regulated alpha4alpha1 integrin function by stimulating its interactions with VCAM-1 but not fibronectin. The in vivo significance of alpha2beta1 integrin expression was demonstrated by intravital videomicroscopy showing that ligation of alpha2beta1 enhanced alpha4beta1-mediated extravasation of YBmalpha2 cells in the liver.     

Decrease in cAMP levels modulates adhesion to fibronectin and immunostimulatory ability of human dendritic cells

The aim of the present study was to analyze the early events elicited by tumor necrosis factor alpha (TNF-alpha) on monocyte-derived dendritic cells (moDC) adhesion to fibronectin (FN) and the involvement of cAMP in the signal transduction mechanism. The intracellular concentration of cAMP and moDC adhesion to FN decreased after TNF-alpha treatment. An inverted dose-dependency for TNF-alpha effect was observed for adhesion and cAMP levels. The presence of a phosphodiesterase (PDE) inhibitor (IBMX) and cAMP analogs (8Br-cAMP, Db-cAMP) reversed the observed TNF-alpha effects. The role of cAMP was analyzed further by examining the cAMP levels in nonadhered and adhered, TNF-alpha-treated moDC. Nonadhered moDC showed lower cAMP levels compared with adhered moDC. Furthermore, nonadhered moDC showed higher IL-12 content and allostimulatory ability compared with adhered moDC. The higher allostimulatory capacity was abolished in the presence of cAMP analogs and a PDE inhibitor. These results suggest that cAMP levels correlate with TNF-alpha-induced changes of moDC adhesion and allostimulatory capacity.     

Electrospray deposited fibronectin retains the ability to promote cell adhesion

Scaffolds for tissue engineering require the correct biochemical cues if the seeded cells are to migrate into the scaffold and proliferate. For complex tissues this would require precise patterning of the scaffold structure with the particular biochemical cue required at each location on the scaffold. Electrospray enables the deposition of a wide number of biomolecules onto surfaces and can be used for precise patterning. We assessed the functionality of a key cell-adhesion molecule, fibronectin, after depositing it onto a surface using the electrospray technique. The addition of polypropylene glycol allowed a stable spray to be obtained from solutions with a range of fibronectin concentrations. Immunoassay tests showed that the amount of fibronectin retained on the surface was proportional to that sprayed from the solution. Increasing the surface density of fibronectin deposited onto silicon surfaces enhanced fibroblast attachment. The fibronectin thus appears to have retained its cell attachment functionality after undergoing the electrospray process. Since recent advances allow electrospray to pattern material from solution with micrometre accuracy this may allow materials to be biologically functionalized on a similar scale.     

Controlling integrin specificity and stem cell differentiation in 2D and 3D environments through regulation of fibronectin domain stability

The extracellular matrix (ECM) exerts powerful control over many cellular phenomena, including stem cell differentiation. As such, design and modulation of ECM analogs to ligate specific integrin is a promising approach to control cellular processes in vitro and in vivo for regenerative medicine strategies. Although fibronectin (FN), a crucial ECM protein in tissue development and repair, and its RGD peptide are widely used for cell adhesion, the promiscuity with which they engage integrins leads to difficulty in control of receptor-specific interactions. Recent simulations of force-mediated unfolding of FN domains and sequences analysis of human versus mouse FN suggest that the structural stability of the FN's central cell-binding domains (FN III9–10) affects its integrin specificity. Through production of FN III9–10 variants with variable stabilities, we obtained ligands that present different specificities for the integrin α₅β₁ and that can be covalently linked into fibrin matrices. Here, we demonstrate the capacity of α₅β₁ integrin-specific engagement to influence human mesenchymal stem cell (MSC) behavior in 2D and 3D environments. Our data indicate that α₅β₁ has an important role in the control of MSC osteogenic differentiation. FN fragments with increased specificity for α₅β₁ versus α_vβ₃ results in significantly enhanced osteogenic differentiation of MSCs in 2D and in a clinically relevant 3D fibrin matrix system, although attachment/spreading and proliferation were comparable with that on full-length FN. This work shows how integrin-dependant cellular interactions with the ECM can be engineered to control stem cell fate, within a system appropriate for both 3D cell culture and tissue engineering.     

Hypoxia modulates human mast cell adhesion to hyaluronic acid

Hypoxia is an inherent factor in the inflammatory process and is important in the regulation of some immune cell functions, including the expression of mast cell pro- and anti-inflammatory mediators. Hypoxia also influences cell adhesion to the extracellular matrix (ECM). Hyaluronic acid is one of the major components of the ECM that is involved in inflammatory and tissue regeneration processes in which mast cells play a prominent role. This prompted us to investigate the effects of hypoxia on the expression of hyaluronic acid receptors in mast cells and mast cell adhesion to this ECM component. We found that human LAD2 mast cells spontaneously adhered to hyaluronic acid in a CD44-dependent manner and that reduced oxygen concentrations inhibited or even completely abolished this adhesion process. The mechanism of hypoxia downregulation of mast cell adhesion to hyaluronic acid did not involve a decrease in CD44 expression and hyaluronidase-mediated degradation of adhesion substrates but rather conformational changes in the avidity of CD44 to hyaluronic acid. Hypoxia-mediated regulation of mast cell adhesion to extracellular matrix components might be involved in the pathogenic accumulation of mast cells observed in the course of certain diseases including rheumatoid arthritis and cancer.

     

Amino acid residues required for binding of vascular cell adhesion molecule-1 to integrin alpha 4 beta 7

The homologous Ig-like domains 1 and 4 of vascular cell adhesion molecule (VCAM)-1 present binding sites to the leukocyte integrins alpha 4 beta 1 and alpha 4 beta 7 . In the present study, amino acid substitution mutants were used to identify sequence motifs mediating binding of integrin alpha 4 beta 7 to the first domain of VCAM-1. We demonstrate that binding of integrin alpha 4 beta 7 to VCAM-1 containing the D40A mutation located in the loop between beta strands C1 and D1 was completely abrogated and was not restored by activating integrin binding functions with Mn2+. Thus, the I(39)DSP motif functions as a central recognition site for integrin alpha 4 beta 7. Analysis of the E66A mutation demonstrated that the G(64)NEH sequence, which is exposed on the loop structure between beta strands E1 and F1, represents an additional recognition site for alpha 4 beta 7 integrin. However, the inhibitory effect of the E66A mutation on cell binding was not specific for alpha 4 beta 7 but was also observed for integrin alpha 4 beta 1. In contrast to the I(39)DSP and G(64)NEH sequences, the K(79)LEK motif present in beta strand G1 was involved in binding to alpha 4 beta 1 but not alpha 4 beta 7. The function of G(64)NEH and K(79)LEK motifs in alpha 4++-integrin interactions was confirmed by divalent cation titration assays and peptide inhibition studies. Integrin binding to E66A or E81A;K82A mutants was restored by activation with saturating concentrations of Mn2+. Binding of both alpha 4 beta 1 and alpha 4 beta 7 integrins was not affected by E29A, R36A, E50A or E87A mutations. Together, these results identify the I(39)DSP and G(64)NEH motifs as common recognition sites for both alpha 4 beta 1 and alpha 4 beta 7 integrins, whereas the K(79)LEK sequence appears to confer specificity for alpha 4 beta 1 binding.      

Adsorption characteristics of human plasma fibronectin in relationship to cell adhesion

Adsorption of human plasma fibronectin (FN) on nonsulfonated and sulfonated polymer surfaces was studied, by using a polyclonal antiserum to FN and the ELISA method. ELISA signal was recorded as a function of FN concentration in solutions. The concentration dependence of FN binding shows the saturation effect in the range 5-10 microg/mL. ELISA data are discussed in the terms of a self-assembled monolayer and different conformations of the FN molecule.The early adhesion of L1210 cells to polymer surfaces after prior adsorption of FN on these surfaces was studied under static conditions. In the case of FN adsorbed on sulfonated surfaces, the relative number of adhering cells increased with the increase of the interfacial surface tension (i.e., the cell adhesion depends on the surface density of sulfonic groups). However, in the case of FN adsorbed on nonsulfonated surfaces, the relative number of adhering cells was low and independent on the interfacial surface tension.The alpha(5)beta(1)-integrin blocking by a monoclonal antibody resulted in a strong inhibition of the cell adhesion to FN adsorbed on sulfonated polymer surfaces. This indicates that cell adhesion to FN adsorbed on these surfaces is mostly mediated by the alpha(5)beta(1)-integrin. In contrast, in the case of FN adsorbed on nonsulfonated surfaces the cell adhesion was not inhibited by the alpha(5)beta(1)-integrin blocking.     

Shear stress prevents fibronectin binding protein-mediated Staphylococcus aureus adhesion to resting endothelial cells

Fibronectin binding proteins (FnBP) on the surface of Staphylococcus aureus have previously been shown to mediate adherence of the organism to resting endothelial cells in static adhesion assays. However, in this study using well-defined flow assays, we demonstrate that physiologic levels of shear stress prevent FnBP-mediated adhesion of S. aureus 8325-4 to resting endothelial cells. This result suggests that mechanical forces present in vivo may influence the ability of staphylococci to bind endothelial cell surfaces.     

Hydrophobic characterization of thermophilicCampylobacterspecies and adhesion to INT 407 cell membranes and fibronectin

Cell surface hydrophobicity ofCampylobacter jejuni,C. coli,C. lariandC. upsaliensiswas tested by hydrophobic interaction chromatography on octylsepharose CL-4B. The hydrophobicity was influenced by cultivation mode, presence or absence of intact lipopolysaccharide (LPS) and outer membrane protein structures. Species-specific differences of hydrophobic characteristics were not detected. Bacteria grown in fluid medium exhibited a high degree of hydrophobicity. Agar-grown bacteria showed hydrophobic interaction to a significant lower extent. By oxidation of LPS with sodiummeta-periodate the hydrophobicity of agar-grown bacteria was slightly increased. Bacteria pretreated with proteinase K exhibited a marked decrease of hydrophobic interaction, whereas pretreatment with trypsin did not influence the hydrophobic interaction. Live bacteria were allowed to adhere to INT 407 cell membranes. With exception of one aflagellate strain, bacteria grown in fluid medium adhered better to the cellular substrate than agar-grown bacteria. This difference was not found when adhesion to fibronectin was tested. LPS-oxidized bacteria adhered significantly better to both cell membranes and fibronectin, whereas proteinase K treated bacteria exhibited a significant loss of adhesion capacity for both substrates.     

Engineering cell adhesive surfaces that direct integrin alpha5beta1 binding using a recombinant fragment of fibronectin

Integrin receptors mediate cell adhesion to extracellular matrices and trigger signals that direct cell function. While many integrins bind to the arginine-glycine-aspartic acid (RGD) motif present in numerous extracellular proteins, integrin alpha(5)beta(1) requires both the PHSRN synergy site in the 9th and the RGD site in the 10th type III repeat of fibronectin (FN). Binding of alpha(5)beta(1) to FN is critical to many cellular processes, including osteoblast and myoblast differentiation. This work focused on engineering integrin-specific bioadhesive surfaces by immobilizing a recombinant FN fragment (FNIII(7-10)) encompassing the alpha(5)beta(1) binding domains of FN. Model hybrid surfaces were engineered by immobilizing FNIII(7-10) onto passively adsorbed, non-adhesive albumin. Homo- and hetero-bifunctional crosslinkers of varying spacer-arm length targeting either the cysteine or lysine groups on FNIII(7-10) were investigated in ELISA and cell adhesion assays to optimize immobilization densities and activity. FN-mimetic surfaces presenting controlled densities of FNIII(7-10) were generated by varying the concentration of FNIII(7-10) in the coupling solution at a constant crosslinker concentration. Cells adhered to these functionalized surfaces via integrin alpha(5)beta(1) and blocking with integrin-specific antibodies completely eliminated adhesion. In addition, adherent cells spread and assembled focal adhesions containing alpha(5)beta(1), vinculin, and talin. This biomolecular engineering strategy represents a robust approach to increase biofunctional activity and integrin specificity of biomimetic materials.     

Integrin–fibronectin interactions at the cell-material interface: initial integrin binding and signaling

Integrin receptors mediate cell adhesion to extracellular matrices and provide signals that direct proliferation and differentiation. Integrin binding involves receptor–ligand interactions at the cell-substrate interface and assembly and reorganization of structural and signaling elements at the cytoplasmic face. Using a cross-linking/extraction/reversal method to quantify bound integrins, we demonstrate that the density of α₅β₁ integrin-fibronectin bonds increases linearly with ligand density, as predicted by simple receptor–ligand equilibrium. This linear relationship is consistent with linear increases in cell adhesion strength with receptor and ligand surface densities. Furthermore, we show that phosphorylation of FAK, a tyrosine kinase involved in early integrin-mediated signaling, increases linearly with the number of integrin–Fn bonds. These linear relationships suggest the absence of cooperative effects in the initial stages of mechanical coupling and adhesion-mediated signaling.     

Rolling adhesion through an extended conformation of integrin alphaLbeta2 and relation to alpha I and beta I-like domain interaction

In vivo, beta(2) integrins and particularly alpha(L)beta(2) (LFA-1) robustly support firm adhesion of leukocytes, but can also cooperate with other molecules in supporting rolling adhesion. Strikingly, a small molecule alpha/beta I-like allosteric antagonist, XVA143, inhibits LFA-1-dependent firm adhesion, while at the same time it enhances adhesion in shear flow and rolling both in vitro and in vivo. XVA143 appears to induce the extended conformation of integrins as shown by increased activation epitope exposure. Fab to the beta(2) I-like domain converts firm adhesion to rolling adhesion, but does not enhance adhesion. Residue alpha(L)-Glu-310 in the linker following the I domain is critical for communication to the beta(2) I-like domain, rolling, integrin extension, and activation by Mn(2+) of firm adhesion. The results demonstrate the importance of integrin extension in rolling, and suggest that rolling and firm adhesion are mediated by extended conformations of alpha(L)beta(2) that differ in the affinity of the alpha(L) I domain for ICAM-1.     

Vav1 controls integrin clustering and MHC/peptide-specific cell adhesion to antigen-presenting cells

Integrin-mediated adhesion is essential for the formation of stable contacts between T cells and antigen-presenting cells (APCs). We show that Vav1 controls integrin-mediated adhesion of thymocytes and T cells to ECM proteins and ICAM1 following TCR stimulation. In a peptide-specific system, Vav1 is required for T cell adhesion to peptide-loaded APCs. Intriguingly, TCR-induced cell adhesion and aggregation of integrins occurs independent of WASP. Whereas LFA-1 and actin caps colocalize in wasp(-/-) T cells in response to TCR stimulation, loss of WASP uncouples TCR caps from actin patches. Our data reveal a novel role for Vav1 and WASP in the regulation of TCR-induced integrin clustering and cell adhesion and show that integrin and TCR clustering are controlled by distinct pathways.