In the retina, integrins in the beta1 family have been shown to be important in many phases of neuronal development, particularly neuroblast migration and axon outgrowth. However, the functions of specific integrin heterodimers are not well defined. In this study, we investigated the functions of beta1 integrins in developing chicken retina by expression of a dominant-negative beta1A construct using a replication-competent retrovirus. Inhibition of integrins using this approach resulted in alteration of cell morphology and increased apoptosis, but did not preclude migration and axon elongation. In an attempt to identify which specific beta1 heterodimer was important, expression and function of the alpha4beta1 heterodimer were also investigated. At early developmental stages, alpha4 protein and mRNA were detected in undifferentiated neuroblasts throughout the retina. At later stages, expression was confined to retinal ganglion cells (RGCs) and amacrine cells. A small molecule antagonist of alpha4 integrins was shown to inhibit neurite outgrowth on recombinant soluble vascular cell adhesion molecule-1 (VCAM-1), a known ligand of alpha4beta1. Introduction of alpha4 antagonist in vivo gave rise to increased apoptosis and led to a thinning of the retina and reduced numbers of retinal ganglion cells (RGCs). We conclude that the integrin alpha4beta1 is important for survival of developing retinal neurons, including RGCs.

Thrombospondin (TSP)-1 has been reported to modulate T cell behavior both positively and negatively. We found that these opposing responses arise from interactions of TSP1 with two different T cell receptors. The integrin alpha4beta1 recognizes an LDVP sequence in the NH2-terminal domain of TSP1 and was required for stimulation of T cell adhesion, chemotaxis, and matrix metalloproteinase gene expression by TSP1. Recognition of TSP1 by T cells depended on the activation state of alpha4beta1 integrin, and TSP1 inhibited interaction of activated alpha4beta1 integrin on T cells with its counter receptor vascular cell adhesion molecule-1. The alpha4beta1 integrin recognition site is conserved in TSP2. A recombinant piece of TSP2 containing this sequence replicated the alpha4beta1 integrin-dependent activities of TSP1. The beta1 integrin recognition sites in TSP1, however, were neither necessary nor sufficient for inhibition of T cell proliferation and T cell antigen receptor signaling by TSP1. A second TSP1 receptor, CD47, was not required for some stimulatory responses to TSP1 but played a significant role in its T cell antigen receptor antagonist and antiproliferative activities. Modulating the relative expression or function of these two TSP receptors could therefore alter the direction or magnitude of T cell responses to TSPs.

The extent of disability caused by spinal cord injury (SCI) relates to secondary tissue destruction arising partly from an intraspinal influx of neutrophils and monocyte/macrophages after the initial injury. The integrin alpha4beta1, expressed by these leukocytes, is a key to their activation and migration into/within tissue. Therefore, blocking this integrin's functions may afford significant neuroprotection. Rats were treated intravenously with a blocking monoclonal antibody (mAb) to the alpha4 subunit of alpha4beta1 at 2 and 24 h after thoracic clip-compression SCI. Anti-alpha4beta1 treatment significantly decreased neutrophil and monocyte/macrophage influx at 3 d by 47% and 53%, respectively, and decreased neutrophil influx by 61% at 7 d after SCI. Anti-alpha4beta1 treatment also significantly reduced oxidative activity in injured cord homogenates at 3 d. For example, myeloperoxidase activity decreased by 38%, inducible nitric oxide by 44%, dichlorofluorescein (marking free radicals) by 33% and lipid peroxidation (malondialdehyde) by 42%. At 2-8 weeks after SCI, motor function improved by up to 2 points on an open-field locomotor scale. Treated rats supported weight with their hind paws instead of sweeping. At 2-4 weeks after SCI, anti-alpha4beta1 treatment decreased blood pressure responses during autonomic dysreflexia by as much as 43% and, at 2-8 weeks, decreased mechanical allodynia elicited from the trunk and hind paw by up to 54% and 40%, respectively. This improved functional recovery correlated with spared myelin-containing white matter and >10-fold more bulbospinal serotonergic axons below the injury than were in controls. The significant neurological improvement offered by this neuroprotective strategy underscores the potential for an anti-integrin treatment for SCI.

Erythropoietin (Epo) is essential for the terminal proliferation and differentiation of erythroid progenitor cells. Fibronectin is an important part of the erythroid niche, but its precise role in erythropoiesis is unknown. By culturing fetal liver erythroid progenitors, we show that fibronectin and Epo regulate erythroid proliferation in temporally distinct steps: an early Epo-dependent phase is followed by a fibronectin-dependent phase. In each phase, Epo and fibronectin promote expansion by preventing apoptosis partly through bcl-xL. We show that alpha(4), alpha(5), and beta(1) are the principal integrins expressed on erythroid progenitors; their down-regulation during erythropoiesis parallels the loss of cell adhesion to fibronectin. Culturing erythroid progenitors on recombinant fibronectin fragments revealed that only substrates that engage alpha(4)beta(1)-integrin support normal proliferation. Collectively, these data suggest a two-phase model for growth factor and extracellular matrix regulation of erythropoiesis, with an early Epo-dependent, integrin-independent phase followed by an Epo-independent, alpha(4)beta(1)-integrin-dependent phase.

Activation of integrins in response to inside-out signaling serves as a basis for leukocyte arrest on endothelium, and migration of immune cells. Integrin-dependent adhesion is controlled by the conformational state of the molecule (i.e. change in the affinity for the ligand and molecular unbending (extension)), which is regulated by seven-transmembrane Guanine nucleotide binding Protein-Coupled Receptors (GPCRs). alpha4beta1-integrin (CD49d/CD29, Very Late Antigen-4, VLA-4) is expressed on leukocytes, hematopoietic stem cells, hematopoietic cancer cells, and others. Affinity and extension of VLA-4 are both rapidly up-regulated by inside-out signaling through several Galphai-coupled GPCRs. The goal of the current report was to study the effect of Galphas-coupled GPCRs upon integrin activation.

Lymphocyte integrins mediate cell arrest on endothelium during immune surveillance after activation by chemokine-stimulated inside-out signals. Here we show that a Vav1-talin complex in T cells is a key target for chemokine-triggered inside-out signaling leading to integrin alpha4beta1 activation. Thus, Vav1 dissociation from talin was required to generate high-affinity alpha4beta1 conformations. Assembly of the Vav1-talin complex required PtdIns(4,5)P(2), which was provided by talin-bound phosphatidylinositol phosphate kinase Igamma. Chemokine-promoted Vav1 dissociation from talin followed an initial increase in talin binding to alpha4beta1. This process was dependent on ZAP-70, which binds to and phosphorylates Vav1 in the complex, leading to further alpha4beta1 activation and cell adhesion strengthening. Moreover, Vav1-talin dissociation was needed for Rac1 activation, thus indicating that alpha4beta1 and Rac1 activation can be coupled by chemokine-stimulated ZAP-70 function. Our data suggest that Vav1 might function as a repressive adaptor of talin that must dissociate from alpha4beta1-talin complexes for efficient integrin activation.

Junctional adhesion molecules (JAMs) are endothelial and epithelial adhesion molecules involved in the recruitment of circulating leukocytes to inflammatory sites. We show here that JAM-L, a protein related to the JAM family, is restricted to leukocytes and promotes their adhesion to endothelial cells. Cis dimerization of JAM-L is required to engage in heterophilic interactions with its cognate counter-receptor CAR (coxsackie and adenovirus receptor). Interestingly, JAM-L expressed on neutrophils binds CAR independently of integrin activation. However, on resting monocytes and T lymphocytes, which express the integrin VLA-4, JAM-L molecules engage in complexes with VLA-4 and mainly accumulate in their monomeric form. Integrin activation is required for the dissociation of JAM-L-VLA-4 complexes and the accumulation of functional JAM-L dimers, which indicates that the leukocyte integrin VLA-4 controls JAM-L function in cis by controlling its dimerization state. This provides a mechanism through which VLA-4 and JAM-L functions are coordinately regulated, allowing JAM-L to strengthen integrin-dependent adhesion of leukocytes to endothelial cells.

Integrins coordinate spatial signaling events essential for cell polarity and directed migration. Such signals from alpha4 integrins regulate cell migration in development and in leukocyte trafficking. Here, we report that efficient alpha4-mediated migration requires spatial control of alpha4 phosphorylation by protein kinase A, and hence localized inhibition of binding of the signaling adaptor, paxillin, to the integrin. In migrating cells, phosphorylated alpha4 accumulated along the leading edge. Blocking alpha4 phosphorylation by mutagenesis or by inhibition of protein kinase A drastically reduced alpha4-dependent migration and lamellipodial stability. alpha4 phosphorylation blocks paxillin binding in vitro; we now find that paxillin and phospho-alpha4 were in distinct clusters at the leading edge of migrating cells, whereas unphosphorylated alpha4 and paxillin colocalized along the lateral edges of those cells. Furthermore, enforced paxillin association with alpha4 inhibits migration and reduced lamellipodial stability. These results show that topographically specific integrin phosphorylation can control cell migration and polarization by spatial segregation of adaptor protein binding.

Ischemia/reperfusion injury is a major cause of the highly dysfunctional rate observed in marginal steatotic orthotopic liver transplantation. In this study, we document that the interactions between fibronectin, a key extracellular matrix protein, and its integrin receptor alpha4beta1, expressed on leukocytes, specifically up-regulated the expression and activation of metalloproteinase-9 (MMP-9, gelatinase B) in a well-established steatotic rat liver model of ex vivo ice-cold ischemia followed by isotransplantation. The presence of the active form of MMP-9 was accompanied by massive intragraft leukocyte infiltration, high levels of proinflammatory cytokines, such as interleukin-1beta and tumor necrosis factor-alpha, and impaired liver function. Interestingly, MMP-9 activity in steatotic liver grafts was, to a certain extent, independent of the expression of its natural inhibitor, the tissue inhibitor of metalloproteinases-1. Moreover, the blockade of fibronectin-alpha4beta1-integrin interactions inhibited the expression/activation of MMP-9 in steatotic orthotopic liver transplantations without significantly affecting the expression of metalloproteinase-2 (MMP-2, gelatinase A). Finally, we identified T lymphocytes and monocytes/macrophages as major sources of MMP-9 in steatotic liver grafts. Hence, these findings reveal a novel aspect of the function of fibronectin-alpha4beta1 integrin interactions that holds significance for the successful use of marginal steatotic livers in transplantation.

Bronchus-associated lymphoid tissue (BALT) participates in airway immune responses. However, little is known about the lymphocyte-endothelial adhesion cascades that recruit lymphocytes from blood into BALT. We show that high endothelial venules (HEVs) in BALT express substantial levels of VCAM-1, in marked contrast to HEVs in other secondary lymphoid tissues. BALT HEVs also express the L-selectin ligand PNAd. Anti-L-selectin, anti-PNAd, and anti-LFA-1 mAbs almost completely block the homing of B and T lymphocytes into BALT, whereas anti-alpha4 integrin and anti-VCAM-1 mAbs inhibit homing by nearly 40%. alpha4beta7 integrin and MAdCAM-1 are not involved. Importantly, we found that mAbs against alpha4 integrin and VCAM-1 significantly block the migration of total T cells (80% memory phenotype) but not naive T and B cells to BALT. These results suggest that an adhesion cascade, which includes L-selectin/PNAd, alpha4beta1 integrin/VCAM-1, and LFA-1, targets specific lymphocyte subsets to BALT. This high level of involvement of alpha4beta1 integrin/VCAM-1 is unique among secondary lymphoid tissues, and may help unify lymphocyte migration pathways and immune responses in BALT and other bronchopulmonary tissues.

The capacity of integrins to mediate adhesiveness is modulated by their cytoplasmic associations. In this study, we describe a novel mechanism by which alpha4-integrin adhesiveness is regulated by the cytoskeletal adaptor paxillin. A mutation of the alpha4 tail that disrupts paxillin binding, alpha4(Y991A), reduced talin association to the alpha4beta1 heterodimer, impaired integrin anchorage to the cytoskeleton, and suppressed alpha4beta1-dependent capture and adhesion strengthening of Jurkat T cells to VCAM-1 under shear stress. The mutant retained intrinsic avidity to soluble or bead-immobilized VCAM-1, supported normal cell spreading at short-lived contacts, had normal alpha4-microvillar distribution, and responded to inside-out signals. This is the first demonstration that cytoskeletal anchorage of an integrin enhances the mechanical stability of its adhesive bonds under strain and, thereby, promotes its ability to mediate leukocyte adhesion under physiological shear stress conditions.

The regeneration of peripheral nerve is associated with a change in the alternative splicing of the fibronectin primary gene transcript to re-express embryonic isoforms containing a binding site for alpha4beta1 integrins that promote neurite outgrowth. Here we use PC12 cells to examine the role of the interaction between paxillin and the alpha4 integrin cytoplasmic domain in neurite outgrowth.

Engagement of integrin receptors by the extracellular matrix (ECM) protein fibronectin (FN) activates intracellular signaling, cytoskeletal reorganization and cellular tension. The soluble factor lysophosphatidic acid (LPA) acts through Rho GTPase and its effector Rho kinase (ROCK) to enhance alpha5beta1 integrin-mediated cell spreading on the Arg-Gly-Asp (RGD) cell-binding domain of FN. A second cell-binding site for alpha4 integrins resides in the CS1 segment of the alternatively spliced V region of FN. We show here that LPA treatment of alpha4beta1-expressing CHOalpha4 cells on FN induced a significant decrease in spread cell area. LPA also decreased apoptosis induced by serum-deprivation in CHOalpha4 and human A375 melanoma cells in an alpha4beta1-dependent manner. Improvement in cell viability and changes in cell morphology were dependent on ROCK and on the number of substrate binding sites for alpha4beta1. LPA signaling combined with alpha4beta1-mediated adhesion appears to sustain cell viability in situations where FN matrix is limiting. Such cooperation may impact dynamic cellular events such as wound healing, fibrosis, and metastasis.

The fibronectin (FN)-binding integrins alpha4beta1 and alpha5beta1 confer different cell adhesive properties, particularly with respect to focal adhesion formation and migration. After analyses of alpha4+/alpha5+ A375-SM melanoma cell adhesion to fragments of FN that interact selectively with alpha4beta1 and alpha5beta1, we now report two differences in the signals transduced by each receptor that underpin their specific adhesive properties. First, alpha5beta1 and alpha4beta1 have a differential requirement for cell surface proteoglycan engagement for focal adhesion formation and migration; alpha5beta1 requires a proteoglycan coreceptor (syndecan-4), and alpha4beta1 does not. Second, adhesion via alpha5beta1 caused an eightfold increase in protein kinase Calpha (PKCalpha) activation, but only basal PKCalpha activity was observed after adhesion via alpha4beta1. Pharmacological inhibition of PKCalpha and transient expression of dominant-negative PKCalpha, but not dominant-negative PKCdelta or PKCzeta constructs, suppressed focal adhesion formation and cell migration mediated by alpha5beta1, but had no effect on alpha4beta1. These findings demonstrate that different integrins can signal to induce focal adhesion formation and migration by different mechanisms, and they identify PKCalpha signaling as central to the functional differences between alpha4beta1 and alpha5beta1.

Integrin-mediated adhesion and B cell antigen receptor (BCR) signaling play a critical role in B cell development and function, including antigen-specific B cell differentiation. Here we show that the BCR controls integrin alpha4beta1 (VLA-4)-mediated adhesion of B cells to vascular cell adhesion molecule-1 and fibronectin. Molecular dissection of the underlying signaling mechanism by a combined biochemical, pharmacological, and genetic approach demonstrates that this BCR-controlled integrin-mediated adhesion requires the (consecutive) activation of Lyn, Syk, phosphatidylinositol 3-kinase, Bruton's tyrosine kinase (Btk), phospholipase C (PLC)gamma2, IP3R-mediated Ca2+ release, and PKC. In contrast, activation of mitogen-activated protein kinase kinase (MEK) or extracellular signal-regulated kinase (ERK) is not required, and simultaneous activation of MEK, ERK, and PKB is not sufficient either. Furthermore, Btk is also involved in the control of integrin-mediated adhesion of preB cells. The control of integrin alpha4beta1-mediated B cell adhesion by the BCR involves cytoskeletal reorganization and integrin clustering. These results reveal a novel function for the BCR and Btk, i.e., regulation of integrin alpha4beta1 activity, thereby providing new insights into the control of B cell development and differentiation, as well as into the pathogenesis of the immunodeficiency disease X-linked agammaglobulineamia (XLA).

The crystal structure of human fibronectin (FN) type III repeats 12-14 reveals the primary heparin-binding site, a clump of positively charged residues in FN13, and a putative minor site approximately 60 A away in FN14. The IDAPS motif implicated in integrin alpha4beta1 binding is at the FN13-14 junction, rendering the critical Asp184 inaccessible to integrin. Asp184 clamps the BC loop of FN14, whose sequence (PRARI) is reminiscent of the synergy sequence (PHSRN) of FN9. Mutagenesis studies prompted by this observation reveal that both arginines of the PRARI sequence are important for alpha4beta1 binding to FN12-14. The PRARI motif may represent a new class of integrin-binding sites. The spatial organization of the binding sites suggests that heparin and integrin may bind in concert.

In cancer and angiogenesis, coagulation-independent roles of tissue factor (TF) in cell migration are incompletely understood. Immobilized anti-TF extracellular domain antibodies induce cell spreading, but this phenomenon is epitope specific and is not induced by anti-TF 5G9. Spreading on anti-TF is beta1 integrin-dependent, indicating functional interactions of the TF extracellular domain 5G9 epitope (a presumed integrin-binding site) and integrins. Recombinant TF extracellular domain supports adhesion of cells expressing alphavbeta3 or certain beta1 integrin heterodimers (alpha3beta1, alpha4beta1, alpha5beta1, alpha6beta1, alpha9beta1) and adhesion is blocked by specific anti-integrin antibodies or mutations in the integrin ligand-binding site. Although several studies have linked TF to cell migration, we here demonstrate that TF specifically regulates alpha3beta1-dependent migration on laminin 5. Expression of TF suppresses alpha3beta1-dependent migration, but only when the TF cytoplasmic domain is not phosphorylated. Suppression of migration can be reversed by 5G9, presumably by disrupting integrin interaction, or by the protease ligand VIIa, known to induce PAR-2-dependent phosphorylation of TF. In both cases, release of alpha3beta1 inhibition is prevented by mutation of critical phosphorylation sites in the TF cytoplasmic domain. Thus, TF influences integrin-mediated migration through cooperative intra- and extracellular interactions and phosphorylation regulates TF's function in cell motility.

Previous studies have shown that integrin alpha chain tails make strong positive contributions to integrin-mediated cell adhesion. We now show here that integrin alpha4 tail deletion markedly impairs static cell adhesion by a mechanism that does not involve altered binding of soluble vascular cell adhesion molecule 1 ligand. Instead, truncation of the alpha4 cytoplasmic domain caused a severe deficiency in integrin accumulation into cell surface clusters, as induced by ligand and/ or antibodies. Furthermore, alpha4 tail deletion also significantly decreased the membrane diffusivity of alpha4beta1, as determined by a single particle tracking technique. Notably, low doses of cytochalasin D partially restored the deficiency in cell adhesion seen upon alpha4 tail deletion. Together, these results suggest that alpha4 tail deletion exposes the beta1 cytoplasmic domain, leading to cytoskeletal associations that apparently restrict integrin lateral diffusion and accumulation into clusters, thus causing reduced static cell adhesion. Our demonstration of integrin adhesive activity regulated through receptor diffusion/clustering (rather than through altered ligand binding affinity) may be highly relevant towards the understanding of inside-out signaling mechanisms for beta1 integrins.

Allylamine, a selective cardiovascular toxin that induces oxidative stress, is known to alter expression of extracellular matrix and cell adhesion proteins that are central to arterial remodeling. Our goals were to determine whether AAM treatment in rats modulates integrin/matrix-dependent arteriolar function, and to what extent integrin expression correlated to these alterations. Integrins are transmembrane proteins that facilitate mechanical and molecular signaling between the extracellular matrix and cytoskeleton, and so are suitable candidates for involvement in phenotypic and functional alterations of smooth muscle in response to oxidative stress. Arg-Gly-Asp (RGD) and Leu-Asp-Val (LDV), two integrin-binding motifs found in ECM proteins such as collagens and fibronectin, are known to interact with integrins alphavbeta3 and alpha4beta1, respectively. Previously, we found that RGD containing peptides induce vasodilation through alphavbeta3, while LDV containing peptides induce vasoconstriction through alpha4beta1 of normal rat cremasteric arterioles. In allylamine-treated rats (AAM), the vasomotor response to LDV, but not RGD, was attenuated in a dose-dependent manner. To determine whether changes in integrin subunit mRNA levels correlated with these functional changes, we performed reverse transcription and Real-time PCR for alpha4 and beta3 integrin subunits on RNA isolated from single, first-order cremasteric arterioles. AAM treatment caused a dose-dependent decrease in alpha4 mRNA expression, but not beta3 mRNA expression, suggesting that the changes in vasomotor activity to LDV peptides may be attributable in part to reduced alpha4 expression upon exposure to AAM. These data are supported by similar decreases in alpha4integrin cell surface protein expression in cultured vascular smooth muscle cells treated either in vivo and in vitro with AAM.

The role of integrin-mediated signaling events in T cell function remains incompletely characterized. We report here that alpha4beta1 integrin stimulation of H9 T cells and normal human T cell blasts results in rapid and transient tyrosine phosphorylation of the adapter protein, SH2 domain-containing 76-kDa protein (SLP-76)-associated phosphoprotein of 130 kDa (SLAP-130)/FYB at levels comparable to those observed following TCR stimulation. Stimulation of T cells via the alpha4beta1 integrin enhances the association of tyrosine phosphorylated SLAP-130/FYB with the SH2 domain of the src tyrosine kinase p59fyn. Activation of normal T cells, but not H9 T cells, via alpha4beta1 leads to tyrosine phosphorylation of SLP-76 as well as SLAP-130/FYB. Overexpression of SLAP-130/FYB in normal T cells enhances T cell migration through fibronectin-coated filters in response to the chemokine stromal cell-derived factor (SDF)-1alpha. These results identify SLAP-130/FYB as a new tyrosine phosphorylated substrate in beta1 integrin signaling and suggest a novel function for SLAP-130/FYB in regulating T lymphocyte motility.