Novel Poly-Dopamine Adhesive for a Halloysite Nanotube-Ru(bpy)3 2+ Electrochemiluminescent Sensor

Herein, for the first time, the electrochemiluminescent sensor based on Ru(bpy)₃ ²⁺-modified electrode using dopamine as an adhesive was successfully developed. After halloysite nanotube slurry was cast on a glassy carbon electrode and dried, an alkaline dopamine solution was added on the electrode surface. Initially, polydopamine belts with dimensions of tens to hundreds of nanometers formed via oxidization of the dopamine by ambient oxygen. As the incubation time increased, the nanobelts became broader and then united with each other to form a polydopamine film. The halloysite nanotubes were embedded within the polydopamine film. The above electrode was soaked in Ru(bpy)₃ ²⁺ aqueous solution to adsorb Ru(bpy)₃ ²⁺ into the active sites of the halloysite nanotubes via cation-exchange procedure. Through this simple procedure, a Ru(bpy)₃ ²⁺-modified electrode was obtained using only 6.25 µg Ru(bpy)₃ ²⁺, 15.0 µg dopamine, and 9.0 µg halloysite nanotubes. The electrochemistry and electrochemiluminescence (ECL) of the modified electrode was investigated using tripropylamine (TPA) and nitrilotriacetic acid (NTA) as co-reactants. The different ECL behaviors of the modified electrode using NTA and TPA as well as the contact angle measurements reflected the hydrophilic character of the electrode. The results indicate that halloysite nanotubes have a high loading capacity for Ru(bpy)₃ ²⁺ and that dopamine is suitable for the preparation of modified electrodes.     

c-Abl Kinase Is a Regulator of αvβ3 Integrin Mediated Melanoma A375 Cell Migration

Integrins are heterodimeric transmembrane receptors that physically link the extracellular matrix (ECM) to the intracellular actin cytoskeleton, and are also signaling molecules that transduce signals bi-directionally across the plasma membrane. Integrin regulation is essential for tumor cell migration in response to growth factors. c-Abl kinase is a nonreceptor tyrosine kinase and is critical for signaling transduction from various receptors. Here we show that c-Abl kinase is involved in A375 cell migration mediated by α_vβ₃ integrin in response to PDGF stimulation. c-Abl kinase colocalizes with α_vβ₃ integrin dynamically and affects α_vβ₃ integrin affinity by regulating its cluster. The interaction between c-Abl kinase and α_vβ₃ integrin was dependent on the activity of c-Abl kinase induced by PDGF stimulation, but was not dependent on the binding of α_vβ₃ integrin with its ligands, suggesting that c-Abl kinase is not involved in the outside-in signaling of α_vβ₃ integrin. Talin head domain was required for the interaction between c-Abl kinase and α_vβ₃ integrin, and the SH3 domain of c-Abl kinase was involved in its interaction with talin and α_vβ₃ integrin. Taken together, we have uncovered a novel and critical role of c-Abl kinase in α_vβ₃ integrin mediated melanoma cell migration.     

Mitochondrially Mediated Integrin αIIbβ3 Protein Inactivation Limits Thrombus Growth

When platelets are strongly stimulated, a procoagulant platelet subpopulation is formed that is characterized by phosphatidylserine (PS) exposure and epitope modulation of integrin α_IIbβ₃ or a loss of binding of activation-dependent antibodies. Mitochondrial permeability transition pore (mPTP) formation, which is essential for the formation of procoagulant platelets, is impaired in the absence of cyclophilin D (CypD). Here we investigate the mechanisms responsible for these procoagulant platelet-associated changes in integrin α_IIbβ₃ and the physiologic role of procoagulant platelet formation in the regulation of platelet aggregation. Among strongly stimulated adherent platelets, integrin α_IIbβ₃ epitope changes, mPTP formation, PS exposure, and platelet rounding were closely associated. Furthermore, platelet mPTP formation resulted in a decreased ability to recruit additional platelets. In the absence of CypD, integrin α_IIbβ₃ function was accentuated in both static and flow conditions, and, in vivo, a prothrombotic phenotype occurred in mice with a platelet-specific deficiency of CypD. CypD-dependent proteolytic events, including cleavage of the integrin β₃ cytoplasmic domain, coincided closely with integrin α_IIbβ₃ inactivation. Calpain inhibition blocked integrin β₃ cleavage and inactivation but not mPTP formation or PS exposure, indicating that integrin inactivation and PS exposure are mediated by distinct pathways subsequent to mPTP formation. mPTP-dependent alkalinization occurred in procoagulant platelets, suggesting a possible alternative mechanism for enhancement of calpain activity in procoagulant platelets. Together, these results indicate that, in strongly stimulated platelets, mPTP formation initiates the calpain-dependent cleavage of integrin β₃ and associated regulatory proteins, resulting in integrin α_IIbβ₃ inactivation, and demonstrate a novel CypD-dependent negative feedback mechanism that limits platelet aggregation and thrombotic occlusion.     

Secreted Heat Shock Protein 90α Induces Colorectal Cancer Cell Invasion through CD91/LRP-1 and NF-κB-mediated Integrin αV Expression

HCT-8 colon cancer cells secreted heat shock protein 90α (HSP90α) and had increased invasiveness upon serum starvation. The concentrated conditioned medium of serum-starved HCT-8 cells was able to stimulate the migration and invasion of non-serum-starved cells, which could be prevented by treatment with an anti-HSP90α antibody. Recombinant HSP90α (rHSP90α) also enhanced HCT-8 cell migration and invasion, suggesting a stimulatory role of secreted HSP90α in cancer malignancy. HSP90α binding to CD91α and Neu was evidenced by the proximity ligation assay, and rHSP90α-induced HCT-8 cell invasion could be suppressed by the addition of anti-CD91α or anti-Neu antibodies. Via CD91α and Neu, rHSP90α selectively induced integrin α_V expression, and knockdown of integrin α_V efficiently blocked rHSP90α-induced HCT-8 cell invasion. rHSP90α induced the activities of ERK, PI3K/Akt, and NF-κB p65, but only NF-κB activation was involved in HSP90α-induced integrin α_V expression. Additionally, we investigated the serum levels of HSP90α and the expression status of tumor integrin α_V mRNA in colorectal cancer patients. Serum HSP90α levels of colorectal cancer patients were significantly higher than those of normal volunteers (p < 0.001). Patients with higher serum HSP90α levels significantly exhibited elevated levels of integrin α_V mRNA in tumor tissues as compared with adjacent non-tumor tissues (p < 0.001). Furthermore, tumor integrin α_V overexpression was significantly correlated with TNM (Tumor, Node, Metastasis) staging (p = 0.001).     

Direct and Regulated Interaction of Integrin αEβ7 with E-Cadherin

The cadherins are a family of homophilic adhesion molecules that play a vital role in the formation of cellular junctions and in tissue morphogenesis. Members of the integrin family are also involved in cell to cell adhesion, but bind heterophilically to immunoglobulin superfamily molecules such as intracellular adhesion molecule (ICAM)–1, vascular cell adhesion molecule (VCAM)–1, or mucosal addressin cell adhesion molecule (MadCAM)–1. Recently, an interaction between epithelial (E-) cadherin and the mucosal lymphocyte integrin, α_Eβ₇, has been proposed. Here, we demonstrate that a human E-cadherin–Fc fusion protein binds directly to soluble recombinant α_Eβ₇, and to α_Eβ₇ solubilized from intraepithelial T lymphocytes. Furthermore, intraepithelial lymphocytes or transfected JY′ cells expressing the α_Eβ₇ integrin adhere strongly to purified E-cadherin–Fc coated on plastic, and the adhesion can be inhibited by antibodies to α_Eβ₇ or E-cadherin.

The binding of α_Eβ₇ integrin to cadherins is selective since cell adhesion to P-cadherin–Fc through α_Eβ₇ requires >100-fold more fusion protein than to E-cadherin–Fc. Although the structure of the α_E-chain is unique among integrins, the avidity of α_Eβ₇ for E-cadherin can be regulated by divalent cations or phorbol myristate acetate. Cross-linking of the T cell receptor complex on intraepithelial lymphocytes increases the avidity of α_Eβ₇ for E-cadherin, and may provide a mechanism for the adherence and activation of lymphocytes within the epithelium in the presence of specific foreign antigen. Thus, despite its dissimilarity to known integrin ligands, the specific molecular interaction demonstrated here indicates that E-cadherin is a direct counter receptor for the α_Eβ₇ integrin.

     

Structural determinants of the integrin transmembrane domain required for bidirectional signal transmission across the cell membrane

Studying the tight activity regulation of platelet-specific integrin α_IIbβ₃ is foundational and paramount to our understanding of integrin structure and activation. α_IIbβ₃ is essential for the aggregation and adhesion function of platelets in hemostasis and thrombosis. Structural and mutagenesis studies have previously revealed the critical role of α_IIbβ₃ transmembrane (TM) association in maintaining the inactive state. Gain-of-function TM mutations were identified and shown to destabilize the TM association leading to integrin activation. Studies using isolated TM peptides have suggested an altered membrane embedding of the β₃ TM α-helix coupled with α_IIbβ₃ activation. However, controversies remain as to whether and how the TM α-helices change their topologies in the context of full-length integrin in native cell membrane. In this study, we utilized proline scanning mutagenesis and cysteine scanning accessibility assays to analyze the structure and function correlation of the α_IIbβ₃ TM domain. Our identification of loss-of-function proline mutations in the TM domain suggests the requirement of a continuous TM α-helical structure in transmitting activation signals bidirectionally across the cell membrane, characterized by the inside-out activation for ligand binding and the outside-in signaling for cell spreading. Similar results were found for α_Lβ₂ and α₅β₁ TM domains, suggesting a generalizable mechanism. We also detected a topology change of β₃ TM α-helix within the cell membrane, but only under conditions of cell adhesion and the absence of α_IIb association. Our data demonstrate the importance of studying the structure and function of the integrin TM domain in the native cell membrane.     

The syndecan-1 ectodomain regulates alphavbeta3 integrin activity in human mammary carcinoma cells

The α_vβ₃ integrin participates in cell morphogenesis, growth factor signaling, and cell survival. Activation of the integrin is central to these processes and is influenced by specific ECM components, which engage both integrins and syndecans. This paper demonstrates that the α_vβ₃ integrin and syndecan-1 (S1) are functionally coupled. The integrin is dependent on the syndecan to become activated and to mediate signals required for MDA-MB-231 and MDA-MB-435 human mammary carcinoma cell spreading on vitronectin or S1-specific antibody. Coupling of the syndecan to α_vβ₃ requires the S1 ectodomain (ED), as ectopic expression of glycosylphosphatidylinositol-linked S1ED enhances α_vβ₃ recognition of vitronectin; and treatments that target this domain, including competition with recombinant S1ED protein or anti-S1ED antibodies, mutation of the S1ED, or down-regulation of S1 expression by small-interfering RNAs, disrupt α_vβ₃-dependent cell spreading and migration. Thus, S1 is likely to be a critical regulator of many cellular behaviors that depend on activated α_vβ₃ integrins.     

Modulation of Integrin Activity is Vital for Morphogenesis

Cells can vary their adhesive properties by modulating the affinity of integrin receptors. The activation and inactivation of integrins by inside-out mechanisms acting on the cytoplasmic domains of the integrin subunits has been demonstrated in platelets, lymphocytes, and keratinocytes. We show that in the embryo, normal morphogenesis requires the α subunit cytoplasmic domain to control integrin adhesion at the right times and places. PS2 integrin (α_PS2β_PS) adhesion is normally restricted to the muscle termini, where it is required for attaching the muscles to the ends of other muscles and to specialized epidermal cells. Replacing the wild-type α_PS2 with mutant forms containing cytoplasmic domain deletions results in the rescue of the majority of defects associated with the absence of the α_PS2 subunit, however, the mutant PS2 integrins are excessively active. Muscles containing these mutant integrins make extra muscle attachments at aberrant positions on the muscle surface, disrupting the muscle pattern and causing embryonic lethality. A gain- of-function phenotype is not observed in the visceral mesoderm, showing that regulation of integrin activity is tissue-specific. These results suggest that the α_PS2 subunit cytoplasmic domain is required for inside-out regulation of integrin affinity, as has been seen with the integrin α_IIbβ₃.     

A Novel Cell Adhesion Region in Tropoelastin Mediates Attachment to Integrin αVβ5

Tropoelastin protein monomers assemble to form elastin. Cellular integrin α_Vβ₃ binds RKRK at the C-terminal tail of tropoelastin. We probed cell interactions with tropoelastin by deleting the RKRK sequence to identify other cell-binding interactions within tropoelastin. We found a novel human dermal fibroblast attachment and spreading site on tropoelastin that is located centrally in the molecule. Inhibition studies demonstrated that this cell adhesion was not mediated by either elastin-binding protein or glycosaminoglycans. Cell interactions were divalent cation-dependent, indicating integrin dependence. Function-blocking monoclonal antibodies revealed that α_V integrin(s) and integrin α_Vβ₅ specifically were critical for cell adhesion to this part of tropoelastin. These data reveal a common α_V integrin-binding theme for tropoelastin: α_Vβ₃ at the C terminus and α_Vβ₅ at the central region of tropoelastin. Each α_V region contributes to fibroblast attachment and spreading, but they differ in their effects on cytoskeletal assembly.     

Activation of Distinct α5β1-mediated Signaling Pathways by Fibronectin's Cell Adhesion and Matrix Assembly Domains

The interaction of cells with fibronectin generates a series of complex signaling events that serve to regulate several aspects of cell behavior, including growth, differentiation, adhesion, and motility. The formation of a fibronectin matrix is a dynamic, cell-mediated process that involves both ligation of the α₅β₁ integrin with the Arg-Gly-Asp (RGD) sequence in fibronectin and binding of the amino terminus of fibronectin to cell surface receptors, termed “matrix assembly sites,” which mediate the assembly of soluble fibronectin into insoluble fibrils. Our data demonstrate that the amino-terminal type I repeats of fibronectin bind to the α₅β₁ integrin and support cell adhesion. Furthermore, the amino terminus of fibronectin modulates actin assembly, focal contact formation, tyrosine kinase activity, and cell migration. Amino-terminal fibronectin fragments and RGD peptides were able to cross-compete for binding to the α₅β₁ integrin, suggesting that these two domains of fibronectin cannot bind to the α₅β₁ integrin simultaneously. Cell adhesion to the amino-terminal domain of fibronectin was enhanced by cytochalasin D, suggesting that the ligand specificity of the α₅β₁ integrin is regulated by the cytoskeleton. These data suggest a new paradigm for integrin-mediated signaling, where distinct regions within one ligand can modulate outside-in signaling through the same integrin.     

An internal ligand-bound,metastable state of a leukocyte integrin, αXβ2

How is massive conformational change in integrins achieved on a rapid timescale? We report crystal structures of a metastable, putative transition state of integrin α_Xβ₂. The α_Xβ₂ ectodomain is bent; however, a lattice contact stabilizes its ligand-binding αI domain in a high affinity, open conformation. Much of the αI α7 helix unwinds, loses contact with the αI domain, and reshapes to form an internal ligand that binds to the interface between the β propeller and βI domains. Lift-off of the αI domain above this platform enables a range of extensional and rotational motions without precedent in allosteric machines. Movements of secondary structure elements in the β₂ βI domain occur in an order different than in β₃ integrins, showing that integrin β subunits can be specialized to assume different intermediate states between closed and open. Mutations demonstrate that the structure trapped here is metastable and can enable rapid equilibration between bent and extended-open integrin conformations and up-regulation of leukocyte adhesiveness.     

Laminin 5 Binds the NC-1 Domain of Type VII Collagen

Mutational analyses of genes that encode components of the anchoring complex underlying the basolateral surface of external epithelia indicate that this structure is the major element providing for resistance to external friction. Ultrastructurally, laminin 5 (α3β3γ2; a component of the anchoring filament) appears as a thin filament bridging the hemidesmosome with the anchoring fibrils. Laminin 5 binds the cell surface through hemidesmosomal integrin α6β4. However, the interaction of laminin 5 with the anchoring fibril (type VII collagen) has not been elucidated. In this study we demonstrate that monomeric laminin 5 binds the NH₂-terminal NC-1 domain of type VII collagen. The binding is dependent upon the native conformation of both laminin 5 and type VII collagen NC-1. Laminin 6 (α3β1γ1) has no detectable affinity for type VII collagen NC-1, indicating that the binding is mediated by the β3 and/or γ2 chains of laminin 5. Approximately half of the laminin 5 solubilized from human amnion or skin is covalently complexed with laminins 6 or 7 (α3β2γ1). The adduction occurs between the NH₂ terminus of laminin 5 and the branch point of the short arms of laminins 6 or 7. The results are consistent with the presumed orientation of laminin 5, having the COOH-terminal G domain apposed to the hemidesmosomal integrin, and the NH₂-terminal domains within the lamina densa. The results also support a model predicting that monomeric laminin 5 constitutes the anchoring filaments and bridges integrin α6β4 with type VII collagen, and the laminin 5–6/7 complexes are present within the interhemidesmosomal spaces bound at least by integrin α3β1 where they may mediate basement membrane assembly or stability, but contribute less significantly to epithelial friction resistance.     

The angiogenic response is dictated by β3 integrin on bone marrow–derived cells

Angiogenesis is dependent on the coordinated action of numerous cell types. A key adhesion molecule expressed by these cells is the α_vβ₃ integrin. Here, we show that although this receptor is present on most vascular and blood cells, the key regulatory function in tumor and wound angiogenesis is performed by β₃ integrin on bone marrow–derived cells (BMDCs) recruited to sites of neovascularization. Using knockin mice expressing functionally stunted β₃ integrin, we show that bone marrow transplantation rescues impaired angiogenesis in these mice by normalizing BMDC recruitment. We demonstrate that α_vβ₃ integrin enhances BMDC recruitment and retention at angiogenic sites by mediating cellular adhesion and transmigration of BMDCs through the endothelial monolayer but not their release from the bone niche. Thus, β₃ integrin has the potential to control processes such as tumor growth and wound healing by regulating BMDC recruitment to sites undergoing pathological and adaptive angiogenesis.     

Thy-1-Integrin αvβ5 Interactions Inhibit Lung Fibroblast Contraction-induced Latent Transforming Growth Factor-β1 Activation and Myofibroblast Differentiation

Myofibroblasts, key effector cells in tissue fibrosis, are specialized contractile cells. Lung myofibroblast contraction induces integrin α_vβ₅-dependent latent transforming growth factor (TGF)-β1 activation suggests that myofibroblast contractility may be a driving force for the persistent myofibroblast differentiation observed in fibrotic lungs. Understanding the mechanisms that regulate fibroblast contraction and mechanotransduction will add new insights into the pathogenesis of lung fibrosis and may lead to new therapeutic approaches for treating fibrotic lung diseases. We and others previously demonstrated that lung fibroblast expression of Thy-1 prevents lung fibrosis. The mechanisms underlying the anti-fibrotic effect of Thy-1 are not well understood. In this study, we showed that Thy-1 interacts with integrin α_vβ₅, both in a cell-free system and on the cell surface of rat lung fibroblasts. Thy-1-integrin α_vβ₅ interactions are RLD-dependent because mutated Thy-1, in which RLD is replaced by RLE, loses the ability to bind the integrin. Furthermore, Thy-1 expression prevents fibroblast contraction-induced, integrin α_vβ₅-dependent latent TGF-β1 activation and TGF-β1-dependent lung myofibroblast differentiation. In contrast, lack of Thy-1 expression or disruption of Thy-1-α_vβ₅ interactions renders lung fibroblasts susceptible to contraction-induced latent TGF-β1 activation and myofibroblast differentiation. These data suggest that Thy-1-integrin α_vβ₅ interactions inhibit contraction-induced latent TGF-β1 activation, presumably by blocking the binding of extracellular matrix-bound latent TGF-β1 with integrin α_vβ₅. Our studies suggest that targeting key mechanotransducers to inhibit mechanotransduction might be an effective approach to inhibit the deleterious effects of myofibroblast contraction on lung fibrogenesis.