Cell migration in wound healing and disease is critically dependent on integration with the extracellular matrix, but the receptors that couple matrix topography to migratory behavior remain obscure. Using nano-engineered fibronectin surfaces and cell-derived matrices, we identify syndecan-4 as a key signaling receptor determining directional migration. In wild-type fibroblasts, syndecan-4 mediates the matrix-induced protein kinase Cα (PKCα)–dependent activation of Rac1 and localizes Rac1 activity and membrane protrusion to the leading edge of the cell, resulting in persistent migration. In contrast, syndecan-4–null fibroblasts migrate randomly as a result of high delocalized Rac1 activity, whereas cells expressing a syndecan-4 cytodomain mutant deficient in PKCα regulation fail to localize active Rac1 to points of matrix engagement and consequently fail to recognize and respond to topographical changes in the matrix.

The fibronectin (FN)-binding integrins α4β1 and α5β1 confer different cell adhesive properties, particularly with respect to focal adhesion formation and migration. After analyses of α4+/α5+ A375-SM melanoma cell adhesion to fragments of FN that interact selectively with α4β1 and α5β1, we now report two differences in the signals transduced by each receptor that underpin their specific adhesive properties. First, α5β1 and α4β1 have a differential requirement for cell surface proteoglycan engagement for focal adhesion formation and migration; α5β1 requires a proteoglycan coreceptor (syndecan-4), and α4β1 does not. Second, adhesion via α5β1 caused an eightfold increase in protein kinase Cα (PKCα) activation, but only basal PKCα activity was observed after adhesion via α4β1. Pharmacological inhibition of PKCα and transient expression of dominant-negative PKCα, but not dominant-negative PKCδ or PKCζ constructs, suppressed focal adhesion formation and cell migration mediated by α5β1, but had no effect on α4β1. These findings demonstrate that different integrins can signal to induce focal adhesion formation and migration by different mechanisms, and they identify PKCα signaling as central to the functional differences between α4β1 and α5β1.

Abstract-We describe extracellular interactions between fibronectin (Fn) and vascular endothelial growth factor (VEGF) that influence integrin-growth factor receptor crosstalk and cellular responses. In previous work, we found that VEGF bound specifically to fibronectin (Fn) but not vitronectin or collagens.

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α4 integrins are used by leukocytes and neural crest derivatives for adhesion and migration during embryogenesis, immune responses and tumour invasion. The pro-migratory activity of α4 integrin is mediated in part through the direct binding of the cytoplasmic domain to paxillin. Here, using intermolecular FRET and biochemical analyses, we report a novel interaction of the α4 integrin cytoplasmic domain with 14-3-3ζ. This interaction depends on serine phosphorylation of α4 integrin at a site (S978) distinct from that which regulates paxillin binding (S988). Using a combination of metabolic labelling and targeted mass spectrometry by multiple reaction monitoring we demonstrate the low stoichiometry phosphorylation of S978. The interaction between α4 integrin and 14-3-3ζ is enhanced by the direct association between 14-3-3ζ and paxillin, resulting in the formation of a ternary complex that stabilises the recruitment of each component. Although pair-wise interaction between α4 integrin and paxillin is sufficient for normal Rac1 regulation, the integrity of the ternary complex is essential for focused Cdc42 activity at the lamellipodial leading edge and directed cell movement. Taken together, these data identify a key signalling nexus mediating α4 integrin-dependent migration.

Our data demonstrates that E + Q plays a protective role against the imbalance elicited by CsA between the production of free radicals and antioxidant defence systems, and suggests that a combination of these two antioxidants may find clinical application where cellular damage is a consequence of ROS.

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Although integrins are known to mediate connections between extracellular adhesion molecules and the intracellular actin cytoskeleton, the mechanisms that are responsible for coupling ligand binding to intracellular signaling, for generating diversity in signaling, and for determining the efficacy of integrin signaling in response to ligand engagement are largely unknown. By characterizing the class of anti-integrin monoclonal antibodies (mAbs) that stimulate integrin activation and ligand binding, we have identified integrin-ligand-mAb complexes that exhibit differential signaling properties. Specifically, addition of 12G10 mAb to cells adhering via integrin ␣4␤1 was found to trigger disruption of the actin cytoskeleton and prevent cell attachment and spreading, whereas mAb addition to cells adhering via ␣5␤1 stimulated all of these processes. In contrast, soluble ligand binding to either ␣4␤1 or ␣5␤1 was augmented or unaffected by 12G10. The regions of the integrin responsible for differential signaling were then mapped using chimeras. Surprisingly, a chimeric ␣5 integrin containing the ␤-propeller domain from the ligand binding pocket of ␣4 exhibited the same signaling properties as the full-length ␣4 integrin, whereas exchanging or removing cytoplasmic domains had no effect. Thus the mAb 12G10 demonstrates dual functionality, inhibiting cell adhesion and spreading while augmenting soluble ligand binding, via a mechanism that is determined by the extracellular ␤-propeller domain of the associating ␣-subunit. These findings therefore demonstrate a direct and variable agonistic link between the ligand binding pocket of integrins and the cell interior that is independent of the ␣ cytoplasmic domains. We propose that either ligand-specific transmembrane conformational changes or ligand-specific differences in the kinetics of transmembrane domain separation underlie integrin agonism.Integrin cell adhesion receptors are uniquely positioned at a nexus regulating the signaling flux between the outside of the cell and the cell interior. Integrins dynamically link the deformable meshwork of the extracellular matrix and the contractile actin microfilament system and thereby enable cells to direct membrane protrusions and apply contractile force to adhesive extracellular sites (1). These adhesion-dependent signals are mediated by the clustering of integrins and the congregation of signaling adaptors and enzymes into specialized morphological structures, including focal complexes, focal adhesions, and fibrillar adhesions (2). In this way, the integrincytoskeletal junction is thought to impose temporal and spatial control on adhesion-related signaling events (3).Integrins are non-covalently-linked ␣␤ heterodimers. In mammals, 18 ␣-and 8 ␤-subunits combine to form 24 different receptors, with ligand-binding specificity being determined by the particular ␣␤ combination. Both subunits have a conserved, modular domain structure, except that 9 ␣-subunits contain an additional extracellular domain that is homologous to the A dom...