Endothelial cell migration on fibronectin is regulated by syntaxin 6-mediated alpha5beta1 integrin recycling

The α5β1 integrin heterodimer regulates many processes that contribute to embryonic development and angiogenesis, in both physiological and pathological contexts. As one of the major adhesion complexes on endothelial cells, it plays a vital role in adhesion and migration along the extracellular matrix. We recently showed that angiogenesis is modulated by syntaxin 6, a Golgi- and endosome-localized t-SNARE, and that it does so by regulating the post-Golgi trafficking of VEGFR2. Here we show that syntaxin 6 is also required for α5β1 integrin-mediated adhesion of endothelial cells to, and migration along, fibronectin. We demonstrate that syntaxin 6 and α5β1 integrin colocalize in EEA1-containing early endosomes, and that functional inhibition of syntaxin 6 leads to misrouting of β1 integrin to the degradation pathway (late endosomes and lysosomes) rather transport along recycling pathway from early endosomes; an increase in the pool of ubiquitinylated α5 integrin and its lysosome-dependent degradation; reduced cell spreading on fibronectin; decreased Rac1 activation; and altered Rac1 localization. Collectively, our data show that functional syntaxin 6 is required for the regulation of α5β1-mediated endothelial cell movement on fibronectin. These syntaxin 6-regulated membrane trafficking events control outside-in signaling via haptotactic and chemotactic mechanisms.     

The integrin alpha9beta1 mediates adhesion to activated endothelial cells and transendothelial neutrophil migration through interaction with vascular cell adhesion molecule-1

The integrin alpha9beta1 has been shown to be widely expressed on smooth muscle and epithelial cells, and to mediate adhesion to the extracellular matrix proteins osteopontin and tenascin-C. We have found that the peptide sequence this integrin recognizes in tenascin-C is highly homologous to the sequence recognized by the closely related integrin alpha4beta1, in the inducible endothelial ligand, vascular cell adhesion mole-cule-1 (VCAM-1). We therefore sought to determine whether alpha9beta1 also recognizes VCAM-1, and whether any such interaction would be biologically significant. In this report, we demonstrate that alpha9beta1 mediates stable cell adhesion to recombinant VCAM-1 and to VCAM-1 induced on human umbilical vein endothelial cells by tumor necrosis factor-alpha. Furthermore, we show that alpha9beta1 is highly and selectively expressed on neutrophils and is critical for neutrophil migration on VCAM-1 and tenascin-C. Finally, alpha9beta1 and alpha4 integrins contribute to neutrophil chemotaxis across activated endothelial monolayers. These observations suggest a possible role for alpha9beta1/VCAM-1 interactions in extravasation of neutrophils at sites of acute inflammation.     

Maspin, the molecular bridge between the plasminogen activator system and beta1 integrin that facilitates cell adhesion

Maspin is a non-inhibitory serine protease inhibitor (serpin) that influences many cellular functions including adhesion, migration, and invasion. The underlying molecular mechanisms that facilitate these actions are still being elucidated. In this study we determined the mechanism by which maspin mediates increased MCF10A cell adhesion. Utilizing competition peptides and mutation analyses, we discovered two unique regions (amino acid residues 190-202 and 260-275) involved in facilitating the increased adhesion function of maspin. In addition, we demonstrate that the urokinase-type plasminogen activator (uPA)/uPA receptor (uPAR) complex is required for the localization and adhesion function of maspin. Finally, we showed that maspin, uPAR, and β1 integrin co-immunoprecipitate, suggesting a novel maspin-uPA-uPAR-β1 integrin mega-complex that regulates mammary epithelial cell adhesion.     

Cross-talk between integrins alpha1beta1 and alpha2beta1 in renal epithelial cells

The collagen-binding integrins α1β1 and α2β1 have profoundly different functions, yet they are often co-expressed in epithelial cells. When both integrins are expressed in the same cell, it has been suggested that α1β1 negatively regulates integrin α2β1-dependent functions. In this study we utilized murine ureteric bud (UB) epithelial cells, which express no functionally detectable levels of endogenous integrins α1β1 and α2β1, to determine the mechanism whereby this regulation occurs. We demonstrate that UB cells expressing integrin α2β1, but not α1β1 adhere, migrate and proliferate on collagen I as well as form cellular cords in 3D collagen I gels. Substitution of the transmembrane domain of the integrin α2 subunit with that of α1 results in decreased cell adhesion, migration and cord formation. In contrast, substitution of the integrin α2 cytoplasmic tail with that of α1, decreases cell migration and cord formation, but increases proliferation. When integrin α1 and α2 subunits are co-expressed in UB cells, the α1 subunit negatively regulates integrin α2β1-dependent cord formation, adhesion and migration and this inhibition requires expression of both α1 and α2 tails. Thus, we provide evidence that the transmembrane and cytoplasmic domains of the α2 integrin subunit, as well as the α1 integrin subunit, regulate integrin α2β1 cell function.     

Rhinocetin, a venom-derived integrin-specific antagonist inhibits collagen-induced platelet and endothelial cell functions

Snaclecs are small non-enzymatic proteins present in viper venoms reported to modulate hemostasis of victims through effects on platelets, vascular endothelial, and smooth muscle cells. In this study, we have isolated and functionally characterized a snaclec that we named "rhinocetin" from the venom of West African gaboon viper, Bitis gabonica rhinoceros. Rhinocetin was shown to comprise α and β chains with the molecular masses of 13.5 and 13 kDa, respectively. Sequence and immunoblot analysis of rhinocetin confirmed this to be a novel snaclec. Rhinocetin inhibited collagen-stimulated activation of human platelets in a dose-dependent manner but displayed no inhibitory effects on glycoprotein VI (collagen receptor) selective agonist, CRP-XL-, ADP-, or thrombin-induced platelet activation. Rhinocetin antagonized the binding of monoclonal antibodies against the α2 subunit of integrin α2β1 to platelets and coimmunoprecipitation analysis confirmed integrin α2β1 as a target for this venom protein. Rhinocetin inhibited a range of collagen-induced platelet functions such as fibrinogen binding, calcium mobilization, granule secretion, aggregation, and thrombus formation. It also inhibited integrin α2β1-dependent functions of human endothelial cells. Together, our data suggest rhinocetin to be a modulator of integrin α2β1 function and thus may provide valuable insights into the role of this integrin in physiological and pathophysiological scenarios, including hemostasis, thrombosis, and envenomation.     

Interaction of nectin-like molecule 2 with integrin alpha6beta4 and inhibition of disassembly of integrin alpha6beta4 from hemidesmosomes

In normal epithelial cells, integrin α(6)β(4) is abundantly expressed and forms hemidesmosomes, which is a cellular structure that mediates cell-extracellular matrix binding. In many types of cancer cells, integrin α(6)β(4) is up-regulated, laminin is cleaved, and hemidesmosomes are disrupted, eventually causing an enhancement of cancer cell movement and facilitation of their invasion. We previously showed that the immunoglobulin-like cell adhesion molecule Necl-2 (Nectin-like molecule 2), known as a tumor suppressor, inhibits cancer cell movement by suppressing the ErbB3/ErbB2 signaling. We show here that Necl-2 interacts in cis with integrin α(6)β(4). The binding of Necl-2 with integrin β(4) was mediated by its extracellular region. In human colorectal adenocarcinoma Caco-2 cells, integrin α(6)β(4) was localized at hemidesmosomes. Small interfering RNA-mediated suppression of Necl-2 expression enhanced the phorbol ester-induced disruption of the integrin α(6)β(4) complex at hemidesmosomes, whereas expression of Necl-2 suppressed the disruption of this structure. These results indicate that tumor-suppressive functions of Necl-2 are mediated by the stabilization of the hemidesmosome structure in addition to the inhibition of the ErbB3/ErbB2 signaling.     

Integrin alpha9beta1 directly binds to vascular endothelial growth factor (VEGF)-A and contributes to VEGF-A-induced angiogenesis

Vascular endothelial growth factor A (VEGF-A) is a potent inducer of angiogenesis. We now show that VEGF-A-induced adhesion and migration of human endothelial cells are dependent on the integrin alpha9beta1 and that VEGF-A is a direct ligand for this integrin. Adhesion and migration of these cells on the 165 and 121 isoforms of VEGF-A depend on cooperative input from alpha9beta1 and the cognate receptor for VEGF-A, VEGF receptor 2 (VEGF-R2). Unlike alpha3beta1or alphavbeta3 integrins, alpha9beta1 was also found to bind the 121 isoform of VEGF-A. This interaction appears to be biologically significant, because alpha9beta1-blocking antibody dramatically and specifically inhibited angiogenesis induced by VEGF-A165 or -121. Together with our previous findings that alpha9beta1 directly binds to VEGF-C and -D and contributes to lymphangiogenesis, these results identify the integrin alpha9beta1 as a potential pharmacotherapeutic target for inhibition of pathogenic angiogenesis and lymphangiogenesis.     

Blocking αvβ3 integrin by a recombinant RGD disintegrin impairs VEGF signaling in endothelial cells

Vascular endothelial growth factor (VEGF) and αvβ3 integrin are key molecules that actively participate in tumor angiogenesis and metastasis. Some integrin-blocking molecules are currently under clinical trials for cancer and metastasis treatment. However, the mechanism of action of such inhibitors is not completely understood. We have previously demonstrated the anti-angiogenic and anti-metastatic properties of DisBa-01, a recombinant His-tag RGD-disintegrin from Bothrops alternatus snake venom in some experimental models. DisBa-01 blocks αvβ3 integrin binding to vitronectin and inhibits integrin-mediated downstream signaling cascades and cell migration. Here we add some new information on the mechanism of action of DisBa-01 in the tumor microenvironment. DisBa-01 supports the adhesion of fibroblasts and MDA-MB-231 breast cancer cells but it inhibits the adhesion of these cells to type I collagen under flow in high shear conditions, as a simulation of the blood stream. DisBa-01 does not affect the release of VEGF by fibroblasts or breast cancer cells but it strongly decreases the expression of VEGF mRNA and of its receptors, vascular endothelial growth factor receptors 1 and 2 (VEGFR1 and VEGFR2) in endothelial cells. DisBa-01 at nanomolar concentrations also modulates metalloprotease 2 (MMP-2) and 9 (MMP-9) activity, the latter being decreased in fibroblasts and increased in MDA-MB-231 cells. In conclusion, these results demonstrate that αvβ3 integrin inhibitors may induce distinct effects in the cells of the tumor microenvironment, resulting in blockade of angiogenesis by impairing of VEGF signaling and in inhibition of tumor cell motility.     

RGD-independent cell adhesion via a tissue transglutaminase-fibronectin matrix promotes fibronectin fibril deposition and requires syndecan-4/2 and {alpha}5{beta}1 integrin co-signaling

Fibronectin (FN) deposition mediated by fibroblasts is an important process in matrix remodeling and wound healing. By monitoring the deposition of soluble biotinylated FN, we show that the stress-induced TG-FN matrix, a matrix complex of tissue transglutaminase (TG2) with its high affinity binding partner FN, can increase both exogenous and cellular FN deposition and also restore it when cell adhesion is interrupted via the presence of RGD-containing peptides. This mechanism does not require the transamidase activity of TG2 but is activated through an RGD-independent adhesion process requiring a heterocomplex of TG2 and FN and is mediated by a syndecan-4 and β1 integrin co-signaling pathway. By using α5 null cells, β1 integrin functional blocking antibody, and a α5β1 integrin targeting peptide A5-1, we demonstrate that the α5 and β1 integrins are essential for TG-FN to compensate RGD-induced loss of cell adhesion and FN deposition. The importance of syndecan-2 in this process was shown using targeting siRNAs, which abolished the compensation effect of TG-FN on the RGD-induced loss of cell adhesion, resulting in disruption of actin skeleton formation and FN deposition. Unlike syndecan-4, syndecan-2 does not interact directly with TG2 but acts as a downstream effector in regulating actin cytoskeleton organization through the ROCK pathway. We demonstrate that PKCα is likely to be the important link between syndecan-4 and syndecan-2 signaling and that TG2 is the functional component of the TG-FN heterocomplex in mediating cell adhesion via its direct interaction with heparan sulfate chains.     

Integrin {alpha}1{beta}1 promotes caveolin-1 dephosphorylation by activating T cell protein-tyrosine phosphatase

Integrin α1β1 is a collagen receptor that down-regulates collagen and reactive oxygen species (ROS) production, and mice lacking this receptor show increased ROS levels and exacerbated glomerular sclerosis following injury. Caveolin-1 (Cav-1) is a multifunctional protein that is tyrosine-phosphorylated in response to injury and has been implicated in ROS-mediated injury. Cav-1 interacts with integrins, and integrin α1β1 binds/activates T cell protein-tyrosine phosphatase (TCPTP), which is homologous to the tyrosine phosphatase PTP1B known to dephosphorylate Cav-1. In this study, we analyzed whether phosphorylated Cav-1 (pCav-1) is a substrate of TCPTP and if integrin α1β1 is essential for promoting TCPTP-mediated Cav-1 dephosphorylation. We found that Cav-1 phosphorylation is significantly higher in cells lacking integrin α1β1 at base line and following oxidative stress. Overexpression of TCPTP leads to reduced pCav-1 levels only in cells expressing integrin α1β1. Using solid phase binding assays, we demonstrated that 1) purified Cav-1 directly interacts with TCPTP and the integrin α1 subunit, 2) pCav-1 is a substrate of TCPTP, and 3) TCPTP-mediated Cav-1 dephosphorylation is highly increased by the addition of purified integrin α1β1 or an integrin α1 cytoplasmic peptide to which TCPTP has been shown to bind. Thus, our results demonstrate that pCav-1 is a new substrate of TCPTP and that integrin α1β1 acts as a negative regulator of Cav-1 phosphorylation by activating TCPTP. This could explain the protective function of integrin α1β1 in oxidative stress-mediated damage and why integrin α1-null mice are more susceptible to fibrosis following injury.     

Anti-human activin receptor-like kinase 1 (ALK1) antibody attenuates bone morphogenetic protein 9 (BMP9)-induced ALK1 signaling and interferes with endothelial cell sprouting

Genetic and molecular studies suggest that activin receptor-like kinase 1 (ALK1), a transforming growth factor β (TGF-β) type I receptor, and endoglin, a TGF-β co-receptor, play an essential role in vascular development and pathological angiogenesis. Several agents that interfere with ALK1 and endoglin function are currently in clinical trials for antiangiogenic activity in cancer therapy. One of these agents, PF-03446962 (anti-hALK1 antibody), shows promising results in the clinic. However, its effects on endothelial cell function and mechanism of action are unclear. Here we demonstrate that anti-hALK1 antibody selectively recognizes human ALK1. The anti-hALK1 antibody interfered with bone morphogenetic protein 9 (BMP9)-induced signaling in endothelial cells. Consistent with this notion, anti-hALK1 antibody was found to compete highly efficiently with the binding of the ALK1 ligand BMP9 and TGF-β to ALK1. Moreover, it prevented BMP9-dependent recruitment of co-receptor endoglin into this angiogenesis-mediating signaling complex. In addition, we demonstrated that anti-hALK1 antibody inhibited endothelial cell sprouting but did not directly interfere with vascular endothelial growth factor (VEGF) signaling, VEGF-induced proliferation, and migration of endothelial cells. Finally, we demonstrated that BMP9 in serum is essential for endothelial sprouting and that anti-hALK1 antibody inhibits this potently. Our data suggest that both the VEGF/VEGF receptor and the BMP9/ALK1 pathways are essential for stimulating angiogenesis, and targeting both pathways simultaneously may be an attractive strategy to overcome resistance to antiangiogenesis therapy.     

Distinct ligand binding sites in integrin alpha3beta1 regulate matrix adhesion and cell-cell contact

The integrin alpha3beta1 mediates cellular adhesion to the matrix ligand laminin-5. A second integrin ligand, the urokinase receptor (uPAR), associates with alpha3beta1 via a surface loop within the alpha3 beta-propeller (residues 242-246) but outside the laminin binding region, suggesting that uPAR-integrin interactions could signal differently from matrix engagement. To explore this, alpha3-/- epithelial cells were reconstituted with wild-type (wt) alpha3 or alpha3 with Ala mutations within the uPAR-interacting loop (H245A or R244A). Wt or mutant-bearing cells showed comparable expression and adhesion to laminin-5. Cells expressing wt alpha3 and uPAR dissociated in culture, with increased Src activity, up-regulation of SLUG, and down-regulation of E-cadherin and gamma-catenin. Src kinase inhibition or expression of Src 1-251 restored the epithelial phenotype. The H245A and R244A mutants were unaffected by coexpression of uPAR. We conclude that alpha3beta1 regulates both cell-cell contact and matrix adhesion, but through distinct protein interaction sites within its beta-propeller. These studies reveal an integrin- and Src-dependent pathway for SLUG expression and mesenchymal transition.     

Vesicle-associated membrane protein 2 mediates trafficking of α5β1 integrin to the plasma membrane

Integrins are major receptors for cell adhesion to the extracellular matrix (ECM). As transmembrane proteins, the levels of integrins at the plasma membrane or the cell surface are ultimately determined by the balance between two vesicle trafficking events: endocytosis of integrins at the plasma membrane and exocytosis of the vesicles that transport integrins. Here, we report that vesicle-associated membrane protein 2 (VAMP2), a SNARE protein that mediates vesicle fusion with the plasma membrane, is involved in the trafficking of α5β1 integrin. VAMP2 was present on vesicles containing endocytosed β1 integrin. Small interfering RNA (siRNA) silencing of VAMP2 markedly reduced cell surface α5β1 and inhibited cell adhesion and chemotactic migration to fibronectin, the ECM ligand of α5β1, without altering cell surface expression of α2β1 integrin or α3β1 integrin. By contrast, silencing of VAMP8, another SNARE protein, had no effect on cell surface expression of the integrins or cell adhesion to fibronectin. In addition, VAMP2-mediated trafficking is involved in cell adhesion to collagen but not to laminin. Consistent with disruption of integrin functions in cell proliferation and survival, VAMP2 silencing diminished proliferation and triggered apoptosis. Collectively, these data indicate that VAMP2 mediates the trafficking of α5β1 integrin to the plasma membrane and VAMP2-dependent integrin trafficking is critical in cell adhesion, migration and survival.     

Vascular endothelial growth factor-induced migration of multiple myeloma cells is associated with beta 1 integrin- and phosphatidylinositol 3-kinase-dependent PKC alpha activation.

In multiple myeloma (MM), migration is necessary for the homing of tumor cells to bone marrow (BM), for expansion within the BM microenvironment, and for egress into the peripheral blood. In the present study we characterize the role of vascular endothelial growth factor (VEGF) and beta(1) integrin (CD29) in MM cell migration. We show that protein kinase C (PKC) alpha is translocated to the plasma membrane and activated by adhesion of MM cells to fibronectin and VEGF. We identify beta(1) integrin modulating VEGF-triggered MM cell migration on fibronectin. We show that transient enhancement of MM cell adhesion to fibronectin triggered by VEGF is dependent on the activity of both PKC and beta(1) integrin. Moreover, we demonstrate that PKC alpha is constitutively associated with beta(1) integrin. These data are consistent with PKC alpha-dependent exocytosis of activated beta(1) integrin to the plasma membrane, where its increased surface expression mediates binding to fibronectin; conversely, catalytically active PKC alpha-driven internalization of beta(1) integrin results in MM cell de-adhesion. We show that the regulatory subunit of phosphatidylinositol (PI) 3-kinase (p85) is constitutively associated with FMS-like tyrosine kinase-1 (Flt-1). VEGF stimulates activation of PI 3-kinase, and both MM cell adhesion and migration are PI 3-kinase-dependent. Moreover, both VEGF-induced PI 3-kinase activation and beta(1) integrin-mediated binding to fibronectin are required for the recruitment and activation of PKC alpha. Time-lapse phase contrast video microscopy (TLVM) studies confirm the importance of these signaling components in VEGF-triggered MM cell migration on fibronectin.     

Polydom/SVEP1 is a ligand for integrin α9β1

A variety of proteins, including tenascin-C and osteopontin, have been identified as ligands for integrin α9β1. However, their affinities for integrin α9β1 are apparently much lower than those of other integrins (e.g. α3β1, α5β1, and α8β1) for their specific ligands, leaving the possibility that physiological ligands for integrin α9β1 still remain unidentified. In this study, we found that polydom (also named SVEP1) mediates cell adhesion in an integrin α9β1-dependent manner and binds directly to recombinant integrin α9β1 with an affinity that far exceeds those of the known ligands. Using a series of recombinant polydom proteins with N-terminal deletions, we mapped the integrin-binding site to the 21st complement control protein domain. Alanine-scanning mutagenesis revealed that the EDDMMEVPY sequence (amino acids 2636-2644) in the 21st complement control protein domain was involved in the binding to integrin α9β1 and that Glu(2641) was the critical acidic residue for the integrin binding. The importance of this sequence was further confirmed by integrin binding inhibition assays using synthetic peptides. Immunohistochemical analyses of mouse embryonic tissues showed that polydom colocalized with integrin α9 in the stomach, intestine, and other organs. Furthermore, in situ integrin α9β1 binding assays using frozen mouse tissues showed that polydom accounts for most, but not all, of the integrin α9β1 ligands in tissues. Taken together, the present findings indicate that polydom is a hitherto unknown ligand for integrin α9β1 that functions as a physiological ligand in vivo.     

Identification and characterization of the integrin alpha2beta1 binding motif in chondroadherin mediating cell attachment

Chondroadherin is a leucine-rich repeat protein known to mediate adhesion of isolated cells via the integrin α(2)β(1) and to interact with collagen. In this work, we show that cell adhesion to chondroadherin leads to activation of MAPKs but does not result in cell spreading and division. This is in contrast to the spreading and dividing of cells grown on collagen, although the binding is mediated via the same α(2)β(1) receptor. We identified a cell binding motif, CQLRGLRRWLEAK(318) by mass spectrometry after protease digestion of chondroadherin. Cells adhering to the synthetic peptide CQLRGLRRWLEAK(318) remained round, as was observed when they bound to the intact protein. The peptide added in solution was able to inhibit cell adhesion to the intact protein in a dose-dependent manner and was also verified to bind to the α(2)β(1) integrin. A cyclic peptide, CQLRGLRRWLEAKASRPDATC(326), mimicking the structural constraints of this sequence in the intact protein, showed similar efficiency in inhibiting binding to chondroadherin. The unique peptide motif responsible for cellular binding is primarily located in the octamer sequence LRRWLEAK(318). Binding of cells to the active peptide or to chondroadherin immobilized on cell culture plates rapidly induces intracellular signaling (i.e. ERK phosphorylation). Thus, chondroadherin interaction with cells may be central for maintaining the adult chondrocyte phenotype and cartilage homeostasis. The peptides, particularly the more stable cyclic peptide, open new opportunities to modulate cell behavior in situations of tissue pathology.     

Structure-function analysis of tetraspanin CD151 reveals distinct requirements for tumor cell behaviors mediated by α3β1 versus α6β4 integrin

The basement membrane protein laminin-332 (laminin-5) mediates both stable cell adhesion and rapid cell migration and thus has the potential to either restrain or promote tumor cell metastasis. The major cellular receptors for laminin-332 are integrin α3β1, which mediates rapid tumor cell migration, and integrin α6β4, which often mediates stable cell attachment. Tetraspanin protein CD151 interacts directly with both α3β1 and α6β4 integrins and with other tetraspanins, thereby promoting α3β1 and α6β4 association with tetraspanin-enriched microdomains on the cell surface. To explore the possibility of selectively modulating tumor cell responses to laminin-332, we re-expressed a series of CD151 mutants in epidermoid carcinoma cells with near total, RNAi-mediated silencing of endogenous CD151. The interactions of CD151 with its integrin partners or its interactions with other tetraspanins were selectively disrupted by specific mutations in the CD151 large extracellular loop (EC2 domain) or in intracellular CD151 palmitoylation sites, respectively. CD151-integrin association and CD151-tetraspanin association were both important for α3β1 integrin-dependent initial adhesion and rapid migration on laminin-332. Remarkably, however, only CD151-integrin association was required for stable, α6β4 integrin-dependent cell attachment on laminin-332. In addition, we found that a QRD amino acid motif in the CD151 EC2 domain, which had been thought to be crucial for CD151-integrin interaction, is not essential for CD151-integrin association or for the ability of CD151 to promote several different integrin functions. These new data suggest potential strategies for selectively modulating migratory cell responses to laminin-332, while leaving stable cell attachment on laminin-332 intact.