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PubMed2004

Integrin alpha5beta1 and ADAM-17 interact in vitro and co-localize in migrating HeLa cells.

Tumor necrosis factor (TNF) alpha-converting enzyme (TACE/ADAM-17) has diverse roles in the proteolytic processing of cell surface molecules and, due to its ability to process TNFalpha, is a validated therapeutic target for anti-inflammatory therapies. Unlike a number of other ADAM proteins, which interact with integrin receptors via their disintegrin domains, there is currently no evidence for an ADAM-17-integrin association. By analyzing the adhesion of a series of cell lines with recombinant fragments of the extracellular domain of ADAM-17, we now demonstrate a functional interaction between ADAM-17 and alpha(5)beta(1) integrin in a trans orientation. Because ADAM-17-mediated adhesion was sensitive to RGD peptides and EDTA, and the integrin-binding site within ADAM-17 was narrowed down to the disintegrin/cysteine-rich region, the two molecules appear to have a ligand-receptor relationship mediated by the alpha(5)beta(1) ligand binding pocket. Intriguingly, ADAM-17 and alpha(5)beta(1) were found to co-localize in both membrane ruffles and focal adhesions in HeLa cells. When confluent HeLa cell monolayers were wounded, ADAM-17 and alpha(5)beta(1) redistributed to the leading edge and co-localized, which is suggestive of a cis orientation. We postulate that the interaction of ADAM-17 with alpha(5)beta(1) may target or modulate its metalloproteolytic activity.

ADAM Proteins↗
PubMed2006

CD40 ligand binds to alpha5beta1 integrin and triggers cell signaling.

It was originally thought that the critical role of the CD40 ligand (CD40L) in normal and inflammatory immune responses was mainly mediated through its interaction with the classic receptor, CD40. However, data from CD40L(-/-) and CD40(-/-) mice suggest that the CD40L-induced inflammatory immune response involves at least one other receptor. This hypothesis is supported by the fact that CD40L stabilizes arterial thrombi through an alphaIIbbeta3-dependent mechanism. Here we provide evidence that soluble CD40L (sCD40L) binds to cells of the undifferentiated human monocytic U937 cell line in a CD40- and alphaIIbbeta3-independent manner. Binding of sCD40L to U937 cells was inhibited by anti-CD40L monoclonal antibody 5C8, anti-alpha5beta1 monoclonal antibody P1D6, and soluble alpha5beta1. The direct binding of sCD40L to purified alpha5beta1 was confirmed in a solid phase binding assay. Binding of sCD40L to alpha5beta1 was modulated by the form of alpha5beta1 expressed on the cell surface as the activation of alpha5beta1 by Mn(2+) or dithiothreitol resulted in the loss of sCD40L binding. Moreover, sCD40L induced the translocation of alpha5beta1 to the Triton X-100-insoluble fraction of U937 cells, the rapid activation of the MAPK pathways ERK1/2, and interleukin-8 gene expression. The binding of sCD40L to CD40 on BJAB cells, an alpha5beta1-negative B cell line, and the resulting activation of ERK1/2 was not inhibited by soluble alpha5beta1, suggesting that sCD40L can bind concomitantly to both receptors. These results document the existence of novel CD40L-dependent pathways of physiological relevance for cells expressing multiple receptors (CD40, alpha5beta1, and alphaIIbbeta3) for CD40L.

Animals↗
PubMed1997

Localization of the transmembrane 4 superfamily (TM4SF) member PETA-3 (CD151) in normal human tissues: comparison with CD9, CD63, and alpha5beta1 integrin.

It has recently been shown that several members of the tetraspan superfamily, including CD9 and CD63, associate with each other and with beta1 integrins. In this study, we examined the distribution of a recently identified tetraspan, PETA-3 (CD151), and of CD9, CD63, alpha5beta1, and the integrin beta1 chain in normal human tissues by the indirect immunoperoxidase and alkaline phosphatase-anti-alkaline phosphatase techniques. PETA-3 showed a broad distribution and was expressed by endothelium, epithelium, Schwann cells, and dendritic cells and by skeletal, smooth, and cardiac muscle. Expression in skin was mostly restricted to the basal cells of the epidermis and was downregulated on differentiation. In the small intestine, PETA-3 was expressed by crypt and villous enterocytes with a mostly basolateral distribution, but was not detectable on the brush border. CD9 was expressed on the plasma membrane of enterocytes in crypts and at the bases of the villi whereas CD63 demonstrated a unique granular appearance concentrated in the apical cytoplasm below the brush border. The findings of this study show co-localization of PETA-3 with CD9, CD63, alpha5beta1, and beta1 in particular tissues, demonstrating that tetraspan/integrin complexes may occur. However, the lack of co-localization of these antigens in other tissues also implies distinct roles for these molecules.

Adult↗
PubMed1997

Fibrinogen is a ligand for integrin alpha5beta1 on endothelial cells.

Previous studies have shown that fibrinogen can associate with endothelial cells via an Arg-Gly-Asp (RGD) recognition specificity. In the present study, we have characterized the specificity of fibrinogen binding to endothelial cells under different cation conditions. Fibrinogen binding to suspended endothelial cells was selectively supported by Mn2+ and was suppressed by Ca2+. The Mn2+-supported interaction was completely inhibited by RGD peptides but not by alphavbeta3 blocking monoclonal antibodies. In contrast, the interaction was completely blocked by two alpha5beta1 monoclonal antibodies. This interaction was not mediated by fibronectin bound to the integrin; could be demonstrated with purified alpha5beta1; and also was observed with a second alpha5beta1-bearing cell type, platelets. The binding of fibrinogen to alpha5beta1 on endothelial cells in the presence of Mn2+ was time-dependent, specific, saturable, and of high affinity (Kd = 65 nM). By employing anti-peptide monoclonal antibodies, the carboxyl-terminal RGD sequence at Aalpha 572-574 was implicated in fibrinogen recognition by alpha5beta1. Two circumstances were identified in which alpha5beta1 interacted with fibrinogen in the presence of Ca2+: when the receptor was activated with monoclonal antibody (8A2) or when the fibrinogen was presented as an immobilized substratum. These results identify fibrinogen as a ligand for alpha5beta1 on endothelial and other cells, an interaction which may have broad biological implications.

Cells, Cultured↗
PubMed2005

Mechanical properties of the interaction between fibronectin and alpha5beta1-integrin on vascular smooth muscle cells studied using atomic force microscopy.

The mechanical properties of integrin-extracellular matrix (ECM) interactions are important for the mechanotransduction of vascular smooth muscle cells (VSMC), a process that is associated with focal adhesions, and can be of particular significance in cardiovascular disease. In this study, we characterized the unbinding force and binding activity of the initial fibronectin (FN)-alpha5beta1 interaction on the surface of VSMC using atomic force microscopy (AFM). It is postulated that these initial binding events are important to the subsequent focal adhesion assembly. FN-VSMC adhesions were selectively blocked by antibodies against alpha5- and beta1-integrins as well as RGD-containing peptides but not by antibodies against alpha4- and beta3-integrins, indicating that FN primarily bound to alpha5beta1. A characteristic unbinding force of 39 +/- 8 pN was observed and interpreted to represent the FN-alpha5beta1 single-bond strength. The ability of FN to adhere to VSMC (binding probability) was significantly reduced by integrin antagonists, serum starvation, and platelet-derived growth factor (PDGF)-BB, whereas lysophosphatidic acid (LPA) increased FN binding. However, no significant change in the resolved unbinding force was observed. After engagement, the force required to dislodge the FN-coated bead from VSMC increased with increasing of contact time, suggesting a time-dependent increase in number of adhesions and/or altered binding affinity. LPA enhanced this process, whereas PDGF reduced it, suggesting that these factors also affect the multimolecular process of focal contact assembly. Thus AFM is a powerful tool for the characterization of the mechanical properties of integrin-ECM interactions and their regulation. Our results indicate that the functional activity of alpha5beta1 and focal contact assembly can be rapidly regulated.

Animals↗
PubMedDate not supplied

Regulation of alpha(v)beta3 and alpha5beta1 integrin receptors by basic fibroblast growth factor and platelet-derived growth factor-BB in intrasynovial flexor tendon cells.

Integrins are important players in soft tissue healing as molecules that mediate communication between cells and extracellular matrix. Thus, the regulation of the expression of these molecules would be important during wound repair. To explore the regulatory roles of specific growth factors on integrin expression by intrasynovial flexor tendon cells, the present study assessed the in vitro effects of basic fibroblast growth factor and platelet derived growth factor-BB on expression of the alpha5beta1 and alpha(v)beta3 integrins in these cells. Analyses were carried out at the transcriptional (reverse transcription-polymerase chain reaction) and translational (immunohistochemistry) levels of cellular metabolism. Both types of analyses revealed increased expression of alpha5beta1 and alpha(v)beta3 by tendon cells exposed to either basic fibroblast growth factor or platelet-derived growth factor-BB over a wide range of growth factor concentrations employed in the study. Semiquantitative reverse transcription-polymerase chain reaction showed that, relative to control, basic fibroblast growth factor and platelet-derived growth factor-BB increased the expression of alpha(v) mRNA by 2-and 3-fold, respectively. Alpha 5 mRNA expression was also increased 3-fold by basic fibroblast growth factor, and 2-fold by platelet-derived growth factor-BB. We believe the results of this study are significant because the specific integrins affected are intimately involved in two events that have been shown to be important to intrasynovial flexor tendon healing, namely fibronectin deposition (alpha5beta1) as part of the provisional matrix and angiogenesis/revascularization (alpha(v)beta3).

Animals↗
PubMed2000

Regulation of integrin alpha vbeta 3-mediated endothelial cell migration and angiogenesis by integrin alpha5beta1 and protein kinase A.

Recent studies indicate that angiogenesis depends, in part, on ligation of integrin alpha(5)beta(1) by fibronectin. Evidence is now provided that integrin alpha(5)beta(1) regulates the function of integrin alpha(v)beta(3) on endothelial cells during their migration in vitro or angiogenesis in vivo. Secretion of fibronectin by endothelial cells leads to the ligation of integrin alpha(5)beta(1), which potentiates alpha(v)beta(3)-mediated migration on vitronectin without influencing alpha(v)beta(3)-mediated cell adhesion. Endothelial cell attachment to vitronectin suppresses protein kinase A (PKA) activity, while addition of soluble anti-alpha(5)beta(1) restores this activity. Moreover, agents that activate intracellular PKA, such as forskolin, dibutyryl cAMP or alpha(5)beta(1) antagonists, suppress endothelial cell migration on vitronectin in vitro or angiogenesis in vivo. In contrast, inhibitors of PKA reverse the anti-migratory or anti-angiogenic effects mediated by alpha(5)beta(1) antagonists. Therefore, alpha(v)beta(3)-mediated endothelial cell migration and angiogenesis can be regulated by PKA activity, which depends on the ligation state of integrin alpha(5)beta(1).

Cell Adhesion↗
PubMed2001

The eighth FIII domain of human fibronectin promotes integrin alpha5beta1 binding via stabilization of the ninth FIII domain.

Binding of the extracellular matrix molecule fibronectin to the integrin receptor alpha(5)beta(1) elicits downstream signaling pathways that modulate cell function. Fibronectin-alpha(5)beta(1) interaction occurs via the conserved RGD sequence in the tenth FIII (FIII10) domain of fibronectin. A synergistic site containing the sequence PHSRN in the adjacent FIII9 domain has also been identified. Here we investigate the function of the eighth FIII domain in integrin-mediated cell adhesion using a wide range of methods, including biochemical, biological, and biophysical assays of integrin binding, cell adhesion, and protein denaturation. Mutation of the FIII9 synergistic site (PHSRN to PHAAA) in FIII9-10 reduced the binding activity for integrin alpha(5)beta(1) to levels observed for FIII10 alone, but the corresponding mutant in FIII8-9-10 showed no loss of binding activity. Cell adhesion assays also demonstrated enhanced functional activity of constructs containing FIII8. Equilibrium chemical denaturation studies indicated that FIII8 confers conformational stability upon FIII9, but only if the exposed loops, PHSRN and VKNEED on FIII9 and FIII8, respectively, are intact. These results demonstrate that the loss of integrin binding activity, observed upon alteration of the PHSRN synergistic site of FIII9-10, results partly from a loss of conformational stability of FIII9. Our data suggest a mechanism for integrin alpha(5)beta(1)-fibronectin interaction, which in addition to the primary RGD binding event, involves a conformation-sensitive scanning by the integrin for accessible sites on the ligand, whereupon full activation of downstream signaling occurs.

Binding Sites↗
PubMed2000

Disulphide-bond pattern and molecular modelling of the dimeric disintegrin EMF-10, a potent and selective integrin alpha5beta1 antagonist from Eristocophis macmahoni venom.

The disulphide-bond pattern of the heterodimeric disintegrin EMF-10, a potent and selective integrin alpha(5)beta(1) antagonist from Eristocophis macmahoni venom, was established by combination of amino-acid analysis, N-terminal sequencing and collision-induced dissociation by nanoelectrospray ionization quadrupole ion-trap MS of fragments isolated by reversed-phase HPLC after degradation of EMF-10 with oxalic acid. Each EMF-10 subunit contains four intrachain disulphide bonds. Two interchain cystine residues join the EMF-10 polypeptides. The intrachain linkages are conserved in monomeric disintegrins. A molecular model of EMF-10 was built using averaged NMR co-ordinates of flavoridin as a template. The active hairpin loops of the EMF-10 subunits occupy opposite locations at the ends of an elongated disulphide-bond ladder. In the EMF-10 model the N-terminal polypeptide of EMF-10B is close to the RGD-loop of the EMF-10A subunit, suggesting that the N-terminal region of the B-subunit could potentially influence the biological activity of the A-subunit.

Amino Acid Sequence↗
PubMed1996

Regulation of conformation and ligand binding function of integrin alpha5beta1 by the beta1 cytoplasmic domain.

We have studied the role of the cytoplasmic domain in the conformation and affinity modulation of the integrin beta1. Expression of a conformation-dependent anti-beta1 antibody 15/7 correlates with activation in wild-type beta1. Truncation of 16 carboxyl-terminal residues in the cytoplasmic domain (the 762t beta1 mutant) induces constitutive expression of the 15/7 epitope at a high level (which probably reflects a major conformational change of the extracellular domain) but does not activate ligand binding. The dissociation of epitope expression and affinity suggests that the epitope expression reflects the conformation of nonligand binding sites of the extracellular domain of beta1 but does not necessarily reflect that of the ligand binding sites. Indeed we discovered that the 15/7 epitope is in fact located in the nonligand binding region of beta1 (within residues 354-425). The 762t mutant has apparently normal alpha/beta association, suggesting that the overexpression of the 15/7 epitope is not due to alpha/beta dissociation. The data suggest that the carboxyl-terminal 16 residues of the beta1 cytoplasmic domain are critical for properly modulating conformation and affinity of beta1 integrins.

Amino Acid Sequence↗
PubMed1998

Chemotaxis of chinese hamster ovary cells expressing the human neutrophil formyl peptide receptor: role of signal transduction molecules and alpha5beta1 integrin.

Activation of the N-formyl peptide receptor (FPR) of human neutrophils by ligands such as N-formyl-methionine-leucine-phenylalanine (fMLP) induces mobilization of intracellular calcium, cell adhesion, chemotaxis, superoxide production and degranulation. Chinese hamster ovary (CHO) cells are normally devoid of FPR and unresponsive to fMLP, but when stably transfected with a human FPR cDNA, exhibited some of these same responses. Specifically, stimulation with fMLP resulted in release of intracellular calcium and chemotactic migration toward a gradient of fMLP. As in neutrophils, both processes were inhibited through receptor desensitization by prior exposure to a higher or equal concentration of ligand or by treatment with pertussis toxin. Soluble and membrane-bound fibronectin greatly increased fMLP-induced chemotaxis of CHO cells expressing FPR, but not of wild-type CHO cells, suggesting a role for FPR in activation of integrin function. Evidence for this hypothesis was obtained by demonstrating that CHO cells expressing FPR rapidly increased their adhesion to a fibronectin-coated surface after stimulation with fMLP. Both chemotaxis and adhesion were largely inhibited by RGDS peptide and a function-blocking antibody against alpha5 integrin. FPR-mediated chemotaxis of the CHO transfectants was partly inhibited by a tyrosine kinase inhibitor, herbimycin A, and blocked by a phosphoinositide 3-kinase inhibitor, wortmannin. These data suggest that stimulation of CHO FPR transfectants with a gradient of fMLP results in phosphoinositide 3-kinase-dependent chemotactic migration, which is enhanced by binding of activated alpha5beta1 to fibronectin. This non-myeloid, non-lymphoid fibroblastic cell line will thus serve as a useful model to investigate additional requirements of signal transduction molecules, adhesion molecules and cytoskeletal elements in FPR-mediated chemotaxis.

Animals↗
PubMed2002

The alpha5beta1 integrin provides matrix survival signals for normal and osteoarthritic human articular chondrocytes in vitro.

OBJECTIVE: Chondrocyte cell death may play an important role in the development of arthritis. The goal of the present study was to evaluate the role of the extracellular matrix (ECM) in promoting chondrocyte survival via signals through the integrin family of ECM receptors. METHODS: Chondrocytes were isolated by sequential enzymatic digestion from normal ankle cartilage of organ donors and from osteoarthritic (OA) knee tissue obtained from patients undergoing total knee replacement. Cell survival in monolayer and in suspension culture was measured using fluorescent labels after treatment with specific integrin-blocking antibodies and echistatin, a disintegrin peptide. A quantitative enzyme-linked immunosorbent assay for histone-associated DNA fragments and morphologic evaluation by electron microscopy were used to evaluate apoptosis. RESULTS: Freshly isolated chondrocytes died when plated in serum-free media at low density on poly-L-lysine, but showed >95% survival on fibronectin (FN). A monoclonal blocking antibody to the alpha5-integrin subunit (FN receptor) significantly inhibited survival on FN, whereas control antibodies had no effect. Likewise, treatment of freshly isolated chondrocytes in serum-free alginate-suspension culture with the alpha5-blocking antibody resulted in cell death in a dose-dependent manner, with 20 microg/ml of the antibody reducing normal chondrocyte survival to 20% of that in controls, and OA chondrocyte survival to 23% of that in controls. Antibody inhibition of alphav and alpha1 integrins or treatment with echistatin did not cause cell death. Addition of insulin-like growth factor 1 (IGF-1; 100 ng/ ml) was not able to improve survival of alpha5-antibody-treated cells. However, treatment with 10% fetal bovine serum improved normal chondrocyte survival to 98% (a 5.1-fold increase) and OA chondrocyte survival to 64% (a 2.8-fold increase). Cell death due to alpha5 inhibition was associated with apoptosis. CONCLUSION: These results demonstrate that chondrocyte survival signals are transmitted via the alpha5beta1 FN receptor. Inhibition of matrix survival signals mediated by alpha5beta1 also inhibits the ability of IGF-1 to promote survival, suggesting that IGF-1-mediated survival signaling may require a cosignal from alpha5beta1.

Antibodies, Monoclonal↗
PubMed2005

Transcriptional regulation of alpha5beta1 integrin, fibronectin, VCAM-1, MCSF-1/c-fms, and MCP-1 in atrioventricular valves after valvular surgery and Staphylococcus aureus bacteremia.

OBJECTIVE: The primary event in the development of infective endocarditis (IE) is bacterial adherence to damaged heart valves. This includes capture, adhesion and internalization of bacteria into host cells. METHODS: We determined in an experimental rabbit model for IE whether a transient bacteremia caused by Staphylococcus aureus and/or endothelial heart valve lesions induce transcriptional regulation of alpha(5)beta(1) integrin, fibronectin, vascular cell adhesion molecule-1 (VCAM-1), macrophage colony-stimulating factor-1 (MCSF-1), c-fms proto-oncogene (MCSF-1 receptor), and macrophage chemoattractant protein-1 (MCP-1) in mitral and tricuspid valves. RESULTS: No significant upregulation was found after isolated bacteremia. Six hours after surgical manipulation valvular endothelial lesions led to a distinct modulation in the mRNA expression of proinflammatory cytokines (MCSF-1 and MCP-1), VCAM-1 and alpha(5)beta(1) integrin. The most evident differences between the mitral and tricuspid valves were seen in the significant upregulation of VCAM-1 mRNA on the tricuspid valve in the surgical groups, whereas there was no effect on the mitral valve. MCSF-1 and MCP-1 were dramatically upregulated in both valves after surgery. CONCLUSIONS: During the host defence mechanisms in the development of IE proinflammatory cytokines, cellular adhesion molecules, and molecules of the fibronectin/integrin axis are activated, showing distinct differences in right- and left-sided heart valves.

Animals↗
PubMed2002

Central roles of alpha5beta1 integrin and fibronectin in vascular development in mouse embryos and embryoid bodies.

Vascular development and maturation are dependent on the interactions of endothelial cell integrins with surrounding extracellular matrix. Previous investigations of the primacy of certain integrins in vascular development have not addressed whether this could also be a secondary effect due to poor embryonic nutrition. Here, we show that the alpha5 integrin subunit and fibronectin have critical roles in blood vessel development in mouse embryos and in embryoid bodies (EBs) differentiated from embryonic stem cells (a situation in which there is no nutritional deficit caused by the mutations). In contrast, vascular development in vivo and in vitro is not strongly dependent on alpha(v) or beta3 integrin subunits. In mouse embryos lacking alpha5 integrin, greatly distended blood vessels are seen in the vitelline yolk sac and in the embryo itself. Additionally, overall blood vessel pattern complexity is reduced in alpha5-null tissues. This defective vascular phenotype is correlated with a decrease in the ligand for alpha5 integrin, fibronectin (FN), in the endothelial basement membranes. A striking and significant reduction in early capillary plexus formation and maturation was apparent in EBs formed from embryonic stem cells lacking alpha5 integrin or FN compared with wild-type EBs or EBs lacking alpha(v) or beta3 integrin subunits. Vessel phenotype could be partially restored to FN-null EBs by the addition of whole FN to the culture system. These findings confirm a clear role for alpha5 and FN in early blood vessel development not dependent on embryo nutrition or alpha(v) or beta3 integrin subunits. Thus, successful early vasculogenesis and angiogenesis require alpha5-FN interactions.

Animals↗
PubMed1997

Involvement of alpha5beta1 integrin in matrix interactions and proliferation of chondrocytes.

Integrins are cell surface receptors involved in cellular processes including adhesion, migration, and matrix assembly. In the present study, we analyzed the possible involvement of alpha 5 beta 1 integrin in the regulation of chondrocyte adhesion, spreading, and proliferation. We found that rabbit growth plate chondrocytes were able to attach to substrates coated with type I collagen, type II collagen, or fibronectin within 24 h of culture. During this time period, attachment to fibronectin appeared to be dependent on alpha 5 beta 1 integrin, whereas adhesion to collagens was not. By day 3 of culture, chondrocytes spread onto all the substrates tested. We found that regardless of the nature of the substrate, cell spreading was reversed by treatment with RGD peptide or antibodies against alpha 5 beta 1 or fibronectin, indicating that cell spreading involved alpha 5 beta 1 and fibronectin endogenously produced and deposited by the chondrocytes themselves. Colony formation by chondrocytes in soft agar was inhibited by treatment with RGD peptides or BIIG2, an antibody that interferes with alpha 5 beta 1 integrin-ligand interactions. Furthermore, DNA content was decreased by treatment with anti-fibronectin antibody in micromass culture of chondrocytes. Immunohistochemical analysis on tissue sections revealed that the alpha 5 subunit was particularly abundant in the proliferative and hypertrophic zones of growth plate. The results of the study indicate that alpha 5 beta 1 integrin plays multiple roles in chondrocyte behavior and function and appears to be involved in the regulation of both chondrocyte-matrix interactions and proliferation.

Animals↗
PubMed1997

Outside-in signaling in the chondrocyte. Nitric oxide disrupts fibronectin-induced assembly of a subplasmalemmal actin/rho A/focal adhesion kinase signaling complex.

Elevated levels of fibronectin (Fn) in articular cartilage have been linked to the progression of both rheumatoid and osteoarthritis. In this study, we examined intracellular events which follow ligation of Fn to its receptor, the integrin alpha5beta1. In addition, we examined the regulatory influence of nitric oxide on these events, since this free radical has been implicated in cartilage degradation. Exposure of chondrocytes to Fn-coated beads resulted in the circumferential clustering of the alpha5beta1 integrin receptor, which was accompanied by the subplasmalemmal assembly of a focal activation complex comprised of F-actin, the tyrosine kinase, focal adhesion kinase (FAK), the ras related G protein rho A, as well as tyrosine-phosphorylated proteins. Treatment with exogenous nitric oxide (NO) or catabolic cytokines which induce nitric oxide synthase blocked the assembly of F-actin, FAK, rho A and tyrosine-phosphorylated proteins while not affecting the total number of beads bound per cell nor the clustering of alpha5beta1 integrin. Use of a cGMP antagonist (Rp-8-Br cGMPS) or cGMP agonist (Sp-cGMPS) either abolished or mimicked the NO effect, respectively. Adherence of chondrocytes to fibronectin enhanced proteoglycan synthesis by twofold (vs. albumin). In addition, basic fibroblast growth factor (FGF) and insulin growth factor (IGF-1) induced proteoglycan synthesis in chondrocytes adherent to Fn but not albumin suggesting a costimulatory signal transduced by alpha5betal and the FGF receptor. Both constitutive and FGF stimulated proteoglycan synthesis were completely inhibited by nitric oxide. These data indicate that the ligation of alpha5beta1 in the chondrocyte induced the intracellular assembly of an activation complex comprised of the cytoplasmic tail of alpha5beta1 integrin, actin, and the signaling molecules rho A and FAK. We show that NO inhibits the assembly of the intracellular activation complex and the synthesis of proteoglycans, but has no effect on the extracellular aggregation of alpha5beta1 integrin. These observations provide a basis by which nitric oxide can interfere with chondrocyte functions by affecting chondrocyte-matrix interactions.

Actins↗
PubMed2000

Balancing thymocyte adhesion and motility: a functional linkage between beta1 integrins and the motility receptor RHAMM.

Thymocyte differentiation involves several processes that occur in different anatomic sites within the thymus. Therefore, thymocytes must have the ability to respond to signals received from stromal cells and adopt either adhesive or motile behavior. We will discuss our data indicating human thymocytes use alpha4beta1 integrin, alpha5beta1 integrin and RHAMM to mediate these activities. Immature multinegative (MN; CD3-4-8-19-) thymocytes use alpha4beta1 and alpha5beta1 integrins to mediate weak and strong adhesion. This subset also uses alpha4beta1 integrin to mediate motility. As thymocytes differentiate, they begin to express and use RHAMM to mediate motility in conjunction with alpha4beta1 and alpha5beta1 integrins. Motile thymocytes use beta1 integrins to maintain weakly adhesive contacts with substrate to provide traction for locomoting cells, thus weak adhesion is a requirement of motile behavior. Hyaluronan (HA) is also required by thymocytes to mediate motility. HA binding to cell surface RHAMM redistributes intracellular RHAMM to the cell surface where it functions to mediate motility. We propose that the decision to maintain adhesive or motile behavior is based on the balance between low and high avidity binding conformations of beta1 integrins on thymocytes and that RHAMM:HA interactions decrease high avidity binding conformations of integrins pushing the balance toward motile behavior.

Animals↗
PubMed1998

Force required to break alpha5beta1 integrin-fibronectin bonds in intact adherent cells is sensitive to integrin activation state.

Binding of integrin receptors to extracellular ligands is a complex process involving receptor-ligand interactions at the cell-substrate interface, signals activating the receptors, and assembly of cytoskeletal and adhesion plaque proteins at the cytoplasmic face. To analyze the contribution of these elements to overall cell adhesion, we have developed a model system that characterizes the functional binding characteristic for adhesion receptors as the force required to separate the integrin-ligand bond. A spinning disk device was used to apply a range of controlled hydrodynamic forces to adherent cells. The adhesion of K562 erythroleukemia cells, a cell line expressing a single fibronectin receptor, integrin alpha5beta1, which was uniformly activated with the monoclonal antibody TS2/16, to defined fibronectin surface densities was examined. Cell adhesion strength increased linearly with receptor and ligand densities. Based on chemical equilibrium principles, it is shown that adhesion strength is directly proportional to the number of receptor-ligand bonds. This analysis provides for the definition of a new physical parameter, the adhesion constant psi, which is related to the bond strength and binding equilibrium constant and has units of force-length2. This parameter can be measured by the experimental system presented and is governed by the activation state of integrin receptors. This simplified model isolates the integrin receptor-ligand binding parameters and provides a basis for analysis of the functions of signaling and cytoskeletal elements in the adhesion process.

Cell Adhesion↗