Impaired integrin α5/β1‐mediated hepatocyte growth factor release by stellate cells of the aged liver

Hepatic blood flow and sinusoidal endothelial fenestration decrease during aging. Consequently, fluid mechanical forces are reduced in the space of Disse where hepatic stellate cells (HSC) have their niche. We provide evidence that integrin α₅/β₁ is an important mechanosensor in HSC involved in shear stress‐induced release of hepatocyte growth factor (HGF), an essential inductor of liver regeneration which is impaired during aging. The expression of the integrin subunits α₅ and β₁ decreases in liver and HSC from aged rats. CRISPR/Cas9‐mediated integrin α₅ and β₁ knockouts in isolated HSC lead to lowered HGF release and impaired cellular adhesion. Fluid mechanical forces increase integrin α₅ and laminin gene expression whereas integrin β₁ remains unaffected. In the aged liver, laminin β2 and γ1 protein chains as components of laminin‐521 are lowered. The integrin α₅ knockout in HSC reduces laminin expression via mechanosensory mechanisms. Culture of HSC on nanostructured surfaces functionalized with laminin‐521 enhances Hgf expression in HSC, demonstrating that these ECM proteins are critically involved in HSC function. During aging, HSC acquire a senescence‐associated secretory phenotype and lower their growth factor expression essential for tissue repair. Our findings suggest that impaired mechanosensing via integrin α₅/β₁ in HSC contributes to age‐related reduction of ECM and HGF release that could affect liver regeneration.     

Engineered cystine knot peptides that bind αvβ3, αvβ5, and α5β1 integrins with low‐nanomolar affinity

There is a critical need for compounds that target cell surface integrin receptors for applications in cancer therapy and diagnosis. We used directed evolution to engineer the Ecballium elaterium trypsin inhibitor (EETI‐II), a knottin peptide from the squash family of protease inhibitors, as a new class of integrin‐binding agents. We generated yeast‐displayed libraries of EETI‐II by substituting its 6‐amino acid trypsin binding loop with 11‐amino acid loops containing the Arg‐Gly‐Asp integrin binding motif and randomized flanking residues. These libraries were screened in a high‐throughput manner by fluorescence‐activated cell sorting to identify mutants that bound to α_vβ₃ integrin. Select peptides were synthesized and were shown to compete for natural ligand binding to integrin receptors expressed on the surface of U87MG glioblastoma cells with half‐maximal inhibitory concentration values of 10–30 nM. Receptor specificity assays demonstrated that engineered knottin peptides bind to both α_vβ₃ and α_vβ₅ integrins with high affinity. Interestingly, we also discovered a peptide that binds with high affinity to α_vβ₃, α_vβ₅, and α₅β₁ integrins. This finding has important clinical implications because all three of these receptors can be coexpressed on tumors. In addition, we showed that engineered knottin peptides inhibit tumor cell adhesion to the extracellular matrix protein vitronectin, and in some cases fibronectin, depending on their integrin binding specificity. Collectively, these data validate EETI‐II as a scaffold for protein engineering, and highlight the development of unique integrin‐binding peptides with potential for translational applications in cancer. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

The expression of α2β1 integrin and α smooth muscle actin in fibroblasts grown on collagen

The maturation of connective tissue involves the organization of collagen fibres by resident fibroblasts. Fibroblast attachment to collagen has been demonstrated to involve cell surface receptors, integrins of the β₁ family. Integrins are associated with cytoplasmic actin of microfilaments either directly or through focal adhesions. The major actin isoform of fibroblast microfilaments is β actin and to a lesser extent α smooth muscle (α SM) actin. Cultured human dermal fibroblasts derived from adult dermis, newborn foreskin or keloid scar were grown on either uncoated or collagen-coated surfaces. The expression and synthesis of both α₂β₁ integrin and α SM actin were followed by immunohistology and immunoprecipitation. Fibroblasts on uncoated surfaces expressed little α₂β₁ integrin on their surface, while 20 per cent of them demonstrated α SM actin within microfilaments. Fibroblasts grown on a collagen-coated surface minimally expressed α SM actin in microfilament structures and a majority of the cells were positive for α₂β₁ integrin on their membranes. Using [³⁵S]-methionine incorporation and immunoprecipitation, it was shown that fibroblasts grown in uncoated dishes synthesized more α SM actin than fibroblasts grown on collagen-coated dishes. In contrast, fibroblasts grown on collagen coated dishes synthesized more α₂β₁ integrin compared to the same cells grown on uncoated dishes. Fibroblasts maintained on a type I collagen upregulate the expression and synthesis of α₂β₁ integrin, and downregulate the expression and synthesis of α SM actin. © 1998 John Wiley & Sons, Ltd.     

Cyclic strain induces reorganization of integrin α5β1 and α2β1 in human umbilical vein endothelial cells

Cyclic strain has been shown to modulate endothelial cell (EC) morphology, proliferation, and function. We have recently reported that the focal adhesion proteins focal adhesion kinase (pp125^FAK) and paxillin, are tyrosine phosphorylated in EC exposed to strain and these events regulate the morphological change and migration induced by cyclic strain. Integrins are also localized on focal adhesion sites and have been reported to induce tyrosine phosphorylation of pp125^FAK under a variety of stimuli. To study the involvement of different integrins in signaling induced by cyclic strain, we first observed the redistribution of α and β integrins in EC subjected to 4 h cyclic strain. Human umbilical vein endothelial cells (HUVEC) seeded on either fibronectin or collagen surfaces were subjected to 10% average strain at a frequency 60 cycles/min. Confocal microscopy revealed that β₁ integrin reorganized in a linear pattern parallel with the long axis of the elongated cells creating a fusion of focal adhesion plaques in EC plated on either fibronectin (a ligand for α₅β₁) or collagen (a ligand for α₂β₁) coated plates after 4 h exposure to cyclic strain. β₃ integrin, which is a vitronectin receptor, did not redistribute in EC exposed to cyclic strain. Cyclic strain also led to a reorganization of α₅ and α₂ integrins in a linear pattern in HUVEC seeded on fibronectin or collagen, respectively. The expression of integrins α₅, α₂, and β₁ did not change even after 24 h exposure to strain when assessed by immunoprecipitation of these integrins. Cyclic strain-induced tyrosine phosphorylation of pp125^FAK occurred concomitant with the reorganization of β₁ integrin. We concluded that α₅β₁ and α₂β₁ integrins play an important role in transducing mechanical stimuli into intracellular signals. J. Cell. Biochem. 64:505–513. © 1997 Wiley-Liss, Inc.     

Activation mechanisms of αVβ3 integrin by binding to fibronectin: A computational study

Integrin αVβ3 plays an important role in regulating cellular activities and in human diseases. Although the structure of αVβ3 has been studied by crystallography and electron microscopy, the detailed activation mechanism of integrin αVβ3 induced by fibronectin remains unclear. In this study, we investigated the conformational and dynamical motion changes of Mn²⁺‐bound integrin αVβ3 by binding to fibronectin with molecular dynamics simulations. Results showed that fibronectin binding to integrin αVβ3 caused the changes of the conformational flexibility of αVβ3 domains, the essential mode of motion for the domains of αV subunit and β3 subunit and the degrees of correlated motion of residues between the domains of αV subunit and β3 subunit of integrin αVβ3. The angle of Propeller domain with respect to the Calf‐2 domain of αV subunit and the angle of Hybrid domain with respect to βA domain of β3 subunit significantly increased when integrin αVβ3 was bound to fibronectin. These changes could result in the conformational change tendency of αVβ3 from a bend conformation to an extended conformation and lead to the open swing of Hybrid domain relative to βA domain of β3 subunit, which have demonstrated their importance for αVβ3 activation. Fibronectin binding to integrin αVβ3 significantly decreased the relative position of α1 helix to βA domain and that to metal ion‐dependent adhesion site, stabilized Mn²⁺ ions binding in integrin αVβ3 and changed fibronectin conformation, which are important for αVβ3 activation. Results from this study provide important molecular insight into the “outside‐in” activation mechanism of integrin αVβ3 by binding to fibronectin.     

Binding of fibrinogen to platelet integrin αIIbβ3 in solution as monitored by tracer sedimentation equilibrium

Fibrinogen showed essentially no binding (K_D>1 mM ) to platelet αIIbβ3 integrin in solution in the presence of Triton or octylglucoside above critical micellar concentrations. Under these conditions the integrin was an αβ monomer. After removal of the detergent from the Triton containing buffer (25 mM Tris/HCl;, 150 mM NaCl, 1 mM CaCl₂, 1 mM MgCl₂, pH 7.4) the integrin formed aggregates with hexamers as the most prominent species, as demonstrated by analytical ultracentrifugation and electron microscopy. Tracer sedimentation equilibrium experiments indicate that fibrinogen binds to the integrin aggregates, but with a surprisingly large K_D (at least 3 μM ). This value is 10- to 100-fold higher than values determined by solid phase assays or with integrins reconstituted onto lipid bilayers.     

Expression and regulation of α1β1 integrin in Schwann cells

The interaction of cells with the extracellular matrix plays a critical role in morphogenesis and cell differentiation. To define how Schwann cells might interact with the extracellular matrix, we chose to study the expression of the laminin/collagen receptor α1β1 integrin during nerve development in the rat from embryonic day 14 to maturity. We found that this integrin is expressed predominantly on mature non-myelin-forming cells and only at very low levels on myelin-forming cells. Significant levels of this integrin were not detected on Schwann cell precursors or embryonic Schwann cells in vivo. Experiments using transected and crushed sciatic nerve showed that α1β1 integrin expression is regulated at least in part by axonal contact. Furthermore, Schwann cell culture experiments showed that α1β1 integrin levels are strongly upregulated by transforming growth factor-βs and phorbol esters. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 914–928, 1997     

Rap1 controls activation of the αMβ2 integrin in a talin‐dependent manner

The small GTPase Rap1 and the cytoskeletal protein talin regulate binding of C3bi‐opsonised red blood cells (RBC) to integrin α_Mβ₂ in phagocytic cells, although the mechanism has not been investigated. Using COS‐7 cells transfected with α_Mβ₂, we show that Rap1 acts on the β₂ and not the α_M chain, and that residues 732–761 of the β₂ subunit are essential for Rap1‐induced RBC binding. Activation of α_Mβ₂ by Rap1 was dependent on W747 and F754 in the β₂ tails, which are required for talin head binding, suggesting a link between Rap1 and talin in this process. Using talin1 knock‐out cells or siRNA‐mediated talin1 knockdown in the THP‐1 monocytic cell line, we show that Rap1 acts upstream of talin but surprisingly, RIAM knockdown had little effect on integrin‐mediated RBC binding or cell spreading. Interestingly, Rap1 and talin influence each other's localisation at phagocytic cups, and co‐immunoprecipitation experiments suggest that they interact together. These results show that Rap1‐mediated activation of α_Mβ₂ in macrophages shares both common and distinct features from Rap1 activation of α_IIbβ₃ expressed in CHO cells. J. Cell. Biochem. 111: 999–1009, 2010. © 2010 Wiley‐Liss, Inc.     

Brain‐derived neurotrophic factor induces migration of endothelial cells through a TrkB–ERK–integrin αVβ3–FAK cascade

Brain‐derived neurotrophic factor (BDNF) promotes the regeneration of periodontal tissue. Since angiogenesis is important for tissue regeneration, investigating effect of BDNF on endothelial cell function may help to reveal its mechanism, whereby, BDNF promotes periodontal tissue regeneration. In this study, we examined the influence of BDNF on migration in human microvascular endothelial cells (HMVECs), focusing on the effects on extracellular signal‐regulated kinase (ERK), integrin α_Vβ₃, and focal adhesion kinase (FAK). The migration of endothelial cells was assessed with a modified Boyden chamber and a wound healing assay. The expression of integrin α_Vβ₃ and the phosphorylation of ERK and FAK were analyzed by immunoblotting and immunofluorescence microscopy. BDNF (25 ng/ml) induced cell migration. PD98059, an ERK inhibitor, K252a, a specific inhibitor for TrkB, a high affinity receptor of BDNF, and an anti‐integrin α_Vβ₃ antibody suppressed the BDNF‐induced migration. BDNF increased the levels of integrin α_Vβ₃ and phosphorylated ERK1/2 and FAK. The ERK inhibitor and TrkB inhibitor also reduced levels of integrin α_Vβ₃ and phosphorylated FAK. We propose that BDNF stimulates endothelial cell migration by a process involving TrkB/ERK/integrin α_Vβ₃/FAK, and this may help to enhance the regeneration of periodontal tissue. J. Cell. Physiol. 227: 2123–2129, 2012. © 2011 Wiley Periodicals, Inc.     

Plasma gelsolin facilitates interaction between β2 glycoprotein I and α5β1 integrin

Antiphospholipid syndrome (APS) is characterized by thrombosis and the presence of antiphospholipid antibodies (aPL) that directly recognizes plasma β₂‐glycoprotein I (β₂GPI). Tissue factor (TF), the major initiator of the extrinsic coagulation system, is induced on monocytes by aPL in vitro, explaining in part the pathophysiology in APS. We previously reported that the mitogen‐activated protein kinase (MAPK) pathway plays an important role in aPL‐induced TF expression on monocytes. In this study, we identified plasma gelsolin as a protein associated with β₂GPI by using immunoaffinity chromatography and mass spectrometric analysis. An in vivo binding assay showed that endogenous β₂GPI interacts with plasma gelsolin, which binds to integrin a₅β₁ through fibronectin. The tethering of β₂GPI to monoclonal anti‐β₂GPI autoantibody on the cell surface was enhanced in the presence of plasma gelsolin. Immunoblot analysis demonstrated that p38 MAPK protein was phosphorylated by monoclonal anti‐β₂GPI antibody treatment, and its phosphorylation was attenuated in the presence of anti‐integrin a₅β₁ antibody. Furthermore, focal adhesion kinase, a downstream molecule of the fibronectin‐integrin signalling pathway, was phosphorylated by anti‐β₂GPI antibody treatment. These results indicate that molecules including gelsolin and integrin are involved in the anti‐β₂GPI antibody‐induced MAPK pathway on monocytes and that integrin is a possible therapeutic target to modify a prothrombotic state in patients with APS.     

Role of the α1β1 integrin complex in collagen gel contraction in vitro by fibroblasts

Matrix remodeling, critical to embryonic morphogenesis and wound healing, is dependent on the expression of matrix components, their receptors, and matrix proteases. The collagen gel assay has provided an effective model for the examination of the functional role(s) of each of these groups of molecules in matrix remodeling. Previous investigations have indicated that collagen gel contraction involves the β1 integrin family of matrix receptors and is stimulated by several growth factors, including TGF-β, PDGF, and angiotensin II. In particular, collagen gel remodeling by human cells involves the α2β1 and, to a lesser extent the α1β1 integrin complexes. The present studies were undertaken to determine the role of the α1 integrin chain, a collagen/laminin receptor, in collagen gel contration by rodent and avian fibroblasts. A high degree of correlation was found between the expression of the α1β1 integrin complex and the relative ability of cells to contract collagen gels. Further studies using antibodies and antisense oligonucleotides against the α1 integrin indicated a significant role for this integrin chain in contraction of collagen gels by rat cardiac fibroblasts. In addition, antibodies to the α1 integrin chain inhibited migration of these fibroblasts on a collagen substratum, suggesting that at least one role of this integrin is in migration of cells in collagen gels. These results indicate that the α1β integrin complex plays a significant role in cellular interactions with interstital collagen that are involved in matrix remodeling such as is seen during morphogenesis and wound healing. © 1995 Wiley-Liss, Inc.     

1,25-dihydroxyvitamin D3 selectively induces increased expression of the Na,K-ATPase β1 subunit in avian myelomonocytic cells without a concomitant change in Na,K-ATPase activity

Treatment of avian myelomonocytic cells with 1,25-dihydroxyvitamin D₃ (1,25(OH)2D3) results in an approximately two fold increase in levels of Na,K-ATPase β1 subunit mRNA and protein (both total and plasma membrane-associated). The changes in β1 subunit expression occur in the absence of a detectable increase in expression of any of the three α subunit isoforms or in Na,K-ATPase activity. The selective induction of the expression of the β subunit in avian myelomonocytic cells by 1,25(OH)2D3 reveals a previously unobserved feature of the regulation of Na,K-ATPase expression, while the targeting of β subunit polypeptides to the plasma membrane in the absence of a corresponding increase in active Na,K-ATPase suggests that, in these cells, transport of the β subunit to the plasma membrane may be independent of its binding to the α subunit. J. Cell. Physiol. 172:221–229, 1997. © 1997 Wiley-Liss, Inc.     

The matrix protein CCN1/CYR61 is required for αVβ5‐mediated cancer cell migration

CYR61 is one of the six proteins of the CCN family of proteins known to play diverse roles in angiogenesis, cellular proliferation, survival, migration and wound healing. However, the specific function of CYR61 in cancer is unclear, and the literature remains controversial. We used quantitative real‐time PCR to establish the expression profile of CYR61 and integrin α_Vβ₅ in three non–small cell lung cancer, five colorectal cancer, one breast cancer and one oesophageal squamous carcinoma cell lines. We showed that the levels of CYR61 were significantly increased in oesophageal squamous carcinoma cell line along with the enhanced levels of α_Vβ₅ integrin. Further, we investigated whether tumour cell–secreted CYR61 can facilitate cell migration by interacting with the α_Vβ₅ integrin. Using tumour cell lines with low, intermediate and high CYR61 expression and their isogenic variants as a cellular model, we determined that integrin α_Vβ₅ expressed on these tumour cells is required for cell migration. Moreover, we showed that the modulation of expression levels of CYR61 in these cancer cells affected their capacity for migration. These results represent an advance to the understanding of the role of CYR61 and α_vβ₅ integrin as proteins that cooperate to mediate cancer cell migration. Copyright © 2012 John Wiley & Sons, Ltd.     

Norcantharidin,a clinical used chemotherapeutic agent,acts as a powerful inhibitor by interfering with fibrinogen–integrin αIIbβ3 binding in human platelets

During platelet activation, fibrinogen binds to its specific platelet receptor, integrin α_IIbβ₃, thus completing the final common pathway for platelet aggregation. Norcantharidin (NCTD) is a promising anticancer agent in China from medicinal insect blister beetle. In this study, we provided the evidence to demonstrate NCTD (0.1–1.0 μM) possesses very powerful antiplatelet activity in human platelets; nevertheless, it had no effects on surface P‐selectin expression and only slight inhibition on ATP‐release reaction in activated platelets. Moreover, NCTD markedly hindered integrin α_IIbβ₃ activation by interfering with the binding of FITC‐labelled PAC‐1. It also markedly reduced the number of adherent platelets and the single platelet spreading area on immobilized fibrinogen as well as clot retraction. Additionally, NCTD attenuated phosphorylation of proteins such as integrin β₃, Src and FAK in platelets spreading on immobilized fibrinogen. These results indicate that NCTD restricts integrin α_IIbβ₃‐mediated outside‐in signalling in human platelets. Besides, NCTD substantially prolonged the closure time in human whole blood and increased the occlusion time of thrombotic platelet plug formation and prolonged the bleeding time in mice. In conclusion, NCTD has dual activities, it can be a chemotherapeutic agent for cancer treatment, and the other side it possesses powerful antiplatelet activity for treating thromboembolic disorders.