Integrin and Growth Factor Receptor Alliance in Angiogenesis

A sequence of events in vascular and stromal cells maintained in a highly coordinated manner regulates angiogenesis and tissue remodeling. These processes are mediated by the ability of cells to respond to environmental cues and activate surface integrins. Physiological and pathological processes in vascular biology are dependent on the specificity of important signaling mechanisms that are activated through the association between growth factors, their receptors, integrins, and their specific extracellular matrix ligands. A large body of evidence from in vitro and in vivo models demonstrates the importance of coordination of signals from the extracellular environment that activates specific tyrosine kinase receptors and integrins in order to regulate angiogenic processes in vivo. In addition to complex formation between growth factor receptors and integrins, growth factors and cytokines also directly interact with integrins, depending upon their concentration levels in the environment, and differentially regulate integrin-related processes. Recent studies from a number of laboratories including ours have provided important novel insights into the involvement of many signaling events that improve our existing knowledge on the cross-talk between growth factor receptors and integrins in the regulation of angiogenesis. In this review, our focus will be on updating the recent developments in the field of integrin-growth factor receptor associations and their implications in the vascular processes.     

ITAM-coupled receptors inhibit IFNAR signaling and alter macrophage responses to TLR4 and Listeria monocytogenes

ITAM-coupled receptors play an essential role in regulating macrophage activation and function by cross-regulating signaling from heterologous receptors. We investigated mechanisms by which ITAM-associated receptors inhibit type I IFN (IFN-α/β) signaling in primary human macrophages and tested the effects of simultaneous ligation of ITAM-associated receptors and TLR4 on TLR4-induced Jak-STAT signaling that is mediated by autocrine IFN-β. Preligation of ITAM-coupled β2 integrins and FcγRs inhibited proximal signaling by the type I IFN receptor IFNAR. Cross-inhibition of IFNAR signaling by β2 integrins resulted in decreased Jak1 activation and was mediated by partial downregulation of the IFNAR1 subunit and MAPK-dependent induction of USP18, which blocks the association of Jak1 with IFNAR2. Simultaneous engagement of ITAM-coupled β2 integrins or Dectin-1 with TLR4 did not affect TLR4-induced direct activation of inflammatory target genes such as TNF or IL6 but abrogated subsequent induction of IFN response genes that is mediated by autocrine IFN-β signaling. Type I IFNs promote macrophage death postinfection by Listeria monocytogenes. Consequently, attenuation of IFN responses by β2 integrins protected primary human macrophages from L. monocytogenes-induced apoptosis. These results provide a mechanism for cross-inhibition of type I IFN signaling by ITAM-coupled β2 integrins and demonstrate that ITAM signaling qualitatively modulates macrophage responses to pathogen-associated molecular patterns and pathogens by selectively suppressing IFN responses.     

Signal transduction by integrin receptors for extracellular matrix: cooperative processing of extracellular information.

Adhesion receptors allow cells to interact with a dynamic and information-rich environment of extracellular matrix molecules. The integrin family of adhesion receptors transduces signals from the extracellular matrix that regulate growth, gene expression and differentiation, as well as cell shape, motility and cytoskeletal architecture. Recent data support the hypothesis that integrins transduce signals cooperatively with other classes of adhesion receptors or with growth factor receptors. Furthermore, the ability of integrins to interact with the cytoskeleton appears to be fundamental to their mechanism for signal transduction.     

Beta1 integrins regulate myoblast fusion and sarcomere assembly

The mechanisms that regulate the formation of multinucleated muscle fibers from mononucleated myoblasts are not well understood. We show here that extracellular matrix (ECM) receptors of the beta1 integrin family regulate myoblast fusion. beta1-deficient myoblasts adhere to each other, but plasma membrane breakdown is defective. The integrin-associated tetraspanin CD9 that regulates cell fusion is no longer expressed at the cell surface of beta1-deficient myoblasts, suggesting that beta1 integrins regulate the formation of a protein complex important for fusion. Subsequent to fusion, beta1 integrins are required for the assembly of sarcomeres. Other ECM receptors such as the dystrophin glycoprotein complex are still expressed but cannot compensate for the loss of beta1 integrins, providing evidence that different ECM receptors have nonredundant functions in skeletal muscle fibers.     

Activity-dependent regulation of synaptic AMPA receptor composition and abundance by beta3 integrins

At synapses, cell adhesion molecules (CAMs) provide the molecular framework for coordinating signaling events across the synaptic cleft. Among synaptic CAMs, the integrins, receptors for extracellular matrix proteins and counterreceptors on adjacent cells, are implicated in synapse maturation and plasticity and memory formation. However, little is known about the molecular mechanisms of integrin action at central synapses. Here, we report that postsynaptic beta3 integrins control synaptic strength by regulating AMPA receptors (AMPARs) in a subunit-specific manner. Pharmacological perturbation targeting beta3 integrins promotes endocytosis of GluR2-containing AMPARs via Rap1 signaling, and expression of beta3 integrins produces robust changes in the abundance and composition of synaptic AMPARs without affecting dendritic spine structure. Importantly, homeostatic synaptic scaling induced by activity deprivation elevates surface expression of beta3 integrins, and in turn, beta3 integrins are required for synaptic scaling. Our findings demonstrate a key role for integrins in the feedback regulation of excitatory synaptic strength.     

Integrins as a distinct subtype of dependence receptors

In the absence of their cognate ligand, dependence receptors trigger programmed cell death. This function is the defining feature of dependence receptors, which include members of several different protein families. The integrins are a family of heterodimeric receptors for extracellular matrix (ECM) proteins, mediating cell anchorage and migration. Integrins share characteristics with dependence receptors, and integrin binding to substrate ECM ligands is essential for cell survival. Although integrins do not conform in all characteristics to the established definitions of dependence receptors, alterations in the expression of integrins and their ligands during physiological and pathological events, such as wound healing, angiogenesis and tumorigenesis, do regulate cell fate in a ligand-dependent manner. This biosensory function of integrins fits well with our current concept of dependence receptor action, and thus integrins may rightly be considered to comprise a distinct subclass of dependence receptor.     

Adaptor protein-2 exhibits alpha 1 beta 1 or alpha 6 beta 1 integrin-dependent redistribution in rhabdomyosarcoma cells

Downregulation of several signaling pathways, such as those stimulated by growth factor receptors, occurs by internalization of signaling receptors through clathrin-coated pits. The first step in internalization or endocytosis is interaction with AP-2, which results in coated pit formation by assembly of clathrin to AP-2. Changes in endocytosis are reflected in the distribution of AP-2 molecules at the cell surface. Integrins are receptors which mediate attachment to the extracellular matrix and also stimulate numerous intracellular signaling pathways; however, it is not known how signaling through integrins is terminated or downregulated. Endocytosis through clathrin-coated pits offers an attractive mechanism for this. This work explores the relationship between AP-2 and beta(1) integrins. RD cells grown for 24 h on collagen or laminin exhibit a redistribution of AP-2 to the cell periphery relative to those grown on fibronectin or polylysine. The total AP-2 protein levels in the cells are unaffected. Blocking alpha(1)beta(1) integrin ligand binding on collagen prevents this redistribution fully. On laminin where alpha(1)beta(1) and alpha(6)beta(1) integrins are engaged, both receptors must be simultaneously blocked to prevent AP-2 redistribution, confirming that the redistribution depends on the specific engagement of the receptors. Immunofluorescence reveals that the majority of alpha(1)beta(1) integrins colocalize with alpha(6)beta(1) integrins in linear structures identified as focal adhesions. A separate fraction of alpha(1)beta(1) integrins colocalize with AP-2 in coated pits. Interestingly, alpha(6)beta(1) integrins are not located in coated pits, demonstrating that integrin colocalization with AP-2 is not necessary to induce redistribution of AP-2.     

Signal transduction through integrins: A central role for focal adhesion kinase?

The integrins are receptors for proteins of the extracellular matrix, both providing a physical link to the cytoskeleton and transducing signals from the extracellular matrix. Activation of integrins leads to tyrosine and serine phosphorylation of a number of proteins, elevation of cytosolic calcium levels, cytoplasmic alkalinization, changes in phospholipid metabolism and, ultimately, changes in gene expression. The recently discovered focal adhesion kinase localizes to focal contacts, which are sites of integrin clustering, and focal adhesion kinase can physically associate with integrins in vitro. As integrins lack intrinsic catalytic activity, focal adhesion kinase is a candidate for a signaling molecule that is recruited by integrins in order to trigger the generation of intracellular second messengers. Thus, focal adhesion kinase may play a central role in signal transduction through integrins.     

Arg-Gly-Asp-containing peptides expose novel collagen receptors on fibroblasts: implications for wound healing.

Integrins are a family of cell-surface receptors intimately involved in the interactions of cells with their extracellular matrix. These receptors comprise an alpha and beta subunit in noncovalent association and many have been shown to recognize and bind an arginine-glycine-aspartate (RGD) sequence contained within their specific extracellular matrix ligand. Fibroblasts express integrin receptors belonging to two major subfamilies. Some of the members within the subfamily defined by beta 1 (VLA) are receptors for collagen but, perhaps surprisingly, the other major subfamily of integrins on fibroblasts--that defined by the alpha chain of the vitronectin receptor, alpha v--all appear to bind primarily vitronectin and/or fibronectin. In the present study we show that RGD-containing peptides expose cryptic binding sites on the alpha v-associated integrins enabling them to function as collagen receptors. The addition of RGD-containing peptides to fibroblasts cultured on type I collagen induced dramatic cell elongation and, when the cells were contained within collagen matrices, the peptides induced marked contraction of the gels. These processes were inhibited by Fab fragments of a monoclonal antibody against an alpha v integrin. Also, alpha v-associated integrins from cell lysates bound to collagen I affinity columns in the presence, but not in the absence, of RGD-containing peptides. These data suggest a novel regulatory control for integrin function. In addition, because the cryptic collagen receptors were shown to be implicated in the contraction of collagen gels, the generation of such binding forces suggests that this may be the major biological role for these integrins in processes such as wound healing.     

Human parechovirus 1 utilizes integrins alphavbeta3 and alphavbeta1 as receptors

Human parechovirus 1 (HPEV1) displays an arginine-glycine-aspartic acid (RGD) motif in the VP1 capsid protein, suggesting integrins as candidate receptors for HPEV1. A panel of monoclonal antibodies (MAbs) specific for integrins alphavbeta3, alphavbeta1, and alphavbeta5, which have the ability to recognize the RGD motif, and also a MAb specific for integrin alpha2beta1, an integrin that does not recognize the RGD motif, were tested on A549 cells. Our results showed that integrin alphav-specific MAb reduced infectivity by 85%. To specify which alphav integrins the virus utilizes, we tested MAbs specific to integrins alphavbeta3 and alphavbeta1 which reduced infectivity significantly, while a MAb specific for integrin alphavbeta5, as well as the MAb specific for alpha2beta1, showed no reduction. When a combination of MAbs specific for integrins alphavbeta3 and alphavbeta1 were used, virus infectivity was almost completely inhibited; this shows that integrins alphavbeta3 and alphavbeta1 are utilized by the virus. We therefore proceeded to test whether alphav integrins' natural ligands fibronectin and vitronectin had an effect on HPEV1 infectivity. We found that vitronectin reduced significantly HPEV1 infectivity, whereas a combination of vitronectin and fibronectin abolished infection. To verify the use of integrins alphavbeta3 and alphavbeta1 as HPEV1 receptors, CHO cells transfected and expressing either integrin alphavbeta3 or integrin alphavbeta1 were used. It was shown that the virus could successfully infect these cells. However, in immunoprecipitation experiments using HPEV1 virions and allowing the virus to bind to solubilized A549 cell extract, we isolated and confirmed by Western blotting the alphavbeta3 heterodimer. In conclusion, we found that HPEV1 utilises both integrin alphavbeta3 and alphavbeta1 as receptors; however, in cells that express both integrins, HPEV1 may preferentially bind integrin alphavbeta3.     

Plasminogen activator inhibitors regulate cell adhesion through a uPAR‐dependent mechanism

Binding of type‐1 plasminogen activator inhibitor (PAI‐1) to cell surface urokinase (uPA) promotes inactivation and internalization of adhesion receptors (e.g., urokinase receptor (uPAR), integrins) and leads to cell detachment from a variety of extracellular matrices. In this report, we begin to examine the mechanism of this process. We show that neither specific antibodies to uPA, nor active site inhibitors of uPA, can detach the cells. Thus, cell detachment is not simply the result of the binding of macromolecules to uPA and/or of the inactivation of uPA. We further demonstrate that another uPA inhibitor, protease nexin‐1 (PN‐1), also stimulates cell detachment in a uPA/uPAR‐dependent manner. The binding of both inhibitors to uPA leads to the specific inactivation of the matrix‐engaged integrins and the subsequent detachment of these integrins from the underlying extracellular matrix (ECM). This inhibitor‐mediated inactivation of integrins requires direct interaction between uPAR and those integrins since cells attached to the ECM through integrins incapable of binding uPAR do not respond to the presence of either PAI‐1 of PN‐1. Although both inhibitors initiate the clearance of uPAR, only PAI‐1 triggers the internalization of integrins. However, cell detachment by PAI‐1 or PN‐1 does not depend on the endocytosis of these integrins since cell detachment was also observed when clearance of these integrins was blocked. Thus, PAI‐1 and PN‐1 induce cell detachment through two slightly different mechanisms that affect integrin metabolism. These differences may be important for distinct cellular processes that require controlled changes in the subcellular localization of these receptors. J. Cell. Physiol. 220: 655–663, 2009. © 2009 Wiley‐Liss, Inc.     

Integrins during evolution: Evolutionary trees and model organisms

The integrins form a large family of cell adhesion receptors. All multicellular animals express integrins, indicating that the family evolved relatively early in the history of metazoans, and homologous sequences of the component domains of integrin α and β subunits are seen in prokaryotes. Some integrins, however, seem to be much younger. For example, the αI domain containing integrins, including collagen receptors and leukocyte integrins, have been found in chordates only. Here, we will discuss what conclusions can be drawn about integrin function by studying the evolutionary conservation of integrins. We will also look at how studying integrins in organisms such as the fruit fly and mouse has helped our understanding of integrin evolution-function relationships. As an illustration of this, we will summarize the current understanding of integrin involvement in skeletal muscle formation.     

Beta 1 integrins signal lipid second messengers required during cell adhesion.

Clustering of integrin receptors during cell adhesion stimulates signal transduction across the cell membrane. Second messengers are generated, activating cytosolic proteins and causing cytoskeletal assembly and rearrangement. HeLa cell adhesion to a collagen substrate has been shown to initiate an arachidonic acid-mediated signaling pathway, leading to the activation of protein kinase C (PKC) and cell spreading. To determine the role of integrin receptors in triggering this signaling pathway, monoclonal antibodies to beta 1 integrins were used to either cluster integrins on the cell surface or to provide an integrin-dependent substrate for cell adhesion. Using this approach, we have defined a pathway required for cell spreading that can be initiated by the ligation of integrins and leads to the activation of PKC. Specifically, our results indicate that clustering beta 1 integrins results in the activation of phospholipase A2 leading to the production of arachidonic acid and the activation of PKC.     

Crosstalk between hepatocyte growth factor and integrin signaling pathways

Summary Most types of normal cells require integrin-mediated attachment to extracellular matrix to be able to respond to growth factor stimulation for proliferation and survival. Therefore, a consensus that integrins are close collaborators with growth factors in signal transduction has gradually emerged. Some integrins and growth factor receptors appear to be normally in relatively close proximity, which can be induced to form complexes upon cell adhesion or growth factor stimulation. Moreover, since integrins and growth factor receptors share many common elements in their signaling pathways, it is clear tzhat there are many opportunities for integrin signals to modulate growth factor signals and vice versa. Increasing evidence indicates that integrins can crosstalk with receptor tyrosine kinases in a cell- and integrin-type-dependent manner through a variety of specific mechanisms. This review is intended specifically for summarizing recent progress uncovering how the hepatocyte growth factor receptor c-Met coordinates with integrins to transmit signals.     

Adenovirus entry into host cells: a role for alpha(v) integrins

The mechanism(s) by which nonenveloped viruses enter host cells is poorly understood. The recent identification of cell-surface alpha(v) integrins as receptors for adenovirus internalization has shed much light on this process. In addition, analysis of alpha(v) integrins as internalization receptors for adenovirus has provided further insights into the biology of integrins.     

SNX17 protects integrins from degradation by sorting between lysosomal and recycling pathways

The FERM-like domain-containing sorting nexins of the SNX17/SNX27/SNX31 family have been proposed to mediate retrieval of transmembrane proteins from the lysosomal pathway. In this paper, we describe a stable isotope labeling with amino acids in culture-based quantitative proteomic approach that allows an unbiased, global identification of transmembrane cargoes that are rescued from lysosomal degradation by SNX17. This screen revealed that several integrins required SNX17 for their stability, as depletion of SNX17 led to a loss of β1 and β5 integrins and associated a subunits from HeLa cells as a result of increased lysosomal degradation. SNX17 bound to the membrane distal NPXY motif in β integrin cytoplasmic tails, thereby preventing lysosomal degradation of β integrins and their associated a subunits. Furthermore, SNX17-dependent retrieval of integrins did not depend on the retromer complex. Consistent with an effect on integrin recycling, depletion of SNX17 also caused alterations in cell migration. Our data provide mechanistic insight into the retrieval of internalized integrins from the lysosomal degradation pathway, a prerequisite for subsequent recycling of these matrix receptors.     

Beta1 integrins are distributed in adhesion structures with fibronectin and caveolin and in coated pits.

Integrins are found in adhesion structures, which link the extracellular matrix to cytoskeletal proteins. Here, we attempt to further define the distribution of beta1 integrins in the context of their association with matrix proteins and other cell surface molecules relevant to the endocytic process. We find that beta1 integrins colocalize with fibronectin in fibrillar adhesion structures. A fraction of caveolin is also organized along these adhesion structures. The extracellular matrix protein laminin is not concentrated in these structures. The alpha4beta1 integrin exhibits a distinct distribution from other beta1 integrins after cells have adhered for 1 h to extracellular matrix proteins but is localized in adhesion structures after 24 h of adhesion. There are differences between the fibronectin receptors: alpha5beta1 integrins colocalize with adaptor protein-2 in coated pits, while alpha4beta1 integrins do not. This parallels our earlier observation that of the two laminin receptors, alpha1beta1 and alpha6beta1, only alpha1beta1 integrins colocalize with adaptor protein-2 in coated pits. Calcium chelation or inhibition of mitogen-activated protein kinase kinase, protein kinase C, or src did not affect localization of alpha1beta1 and alpha5beta1 integrins in coated pits. Likewise, the integrity of coated-pit structures or adhesion structures is not required for integrin and adaptor protein-2 colocalization. This suggests a robust and possibly constitutive interaction between these integrins and coated pits.     

P marks the spot: site-specific integrin phosphorylation regulates molecular interactions

Integrins are heterodimeric adhesion receptors at the cell membrane that function as two-way signaling devices. The short intracellular tails of integrins are devoid of catalytic activity, but are nevertheless important for adhesion and signaling, presumably, through interactions with cytoplasmic molecules. Recently, the structure of the intracellular tails has been investigated using NMR, giving important new insight into how integrins might be regulated, but many questions remain unanswered. Signaling by many cell-surface receptors involves protein phosphorylation; over the past few years, phosphorylation of the integrin tails at specific sites has started to emerge as a dynamic mechanism that regulates molecular interactions between integrins and cytoplasmic molecules. This phosphorylation might give rise to signaling specificity and fine-tuning of the integrin-mediated responses.     

Integrins can collaborate with growth factors for phosphorylation of receptor tyrosine kinases and MAP kinase activation: roles of integrin aggregation and occupancy of receptors

Integrins mediate cell adhesion, migration, and a variety of signal transduction events. These integrin actions can overlap or even synergize with those of growth factors. We examined for mechanisms of collaboration or synergy between integrins and growth factors involving MAP kinases, which regulate many cellular functions. In cooperation with integrins, the growth factors EGF, PDGF-BB, and basic FGF each produced a marked, transient activation of the ERK (extracellular signal-regulated kinase) class of MAP kinase, but only if the integrins were both aggregated and occupied by ligand. Transmembrane accumulation of total tyrosine-phosphorylated proteins, as well as nonsynergistic MAP kinase activation, could be induced by simple integrin aggregation, whereas enhanced transient accumulation of the EGF-receptor substrate eps8 required integrin aggregation and occupancy, as well as EGF treatment. Each type of growth factor receptor was itself induced to aggregate transiently by integrin ligand-coated beads in a process requiring both aggregation and occupancy of integrin receptors, but not the presence of growth factor ligand. Synergism was also observed between integrins and growth factors for triggering tyrosine phosphorylation of EGF, PDGF, and FGF receptors. This collaborative response also required both integrin aggregation and occupancy. These studies identify mechanisms in the signal transduction response to integrins and growth factors that require various combinations of integrin aggregation and ligands for integrin or growth factor receptors, providing opportunities for collaboration between these major regulatory systems.