Integrin interactions with extracellular matrix proteins are mediated by brief oligopeptide recognition sequences, and synthetic peptides containing such sequences can inhibit integrin binding to the matrix. The RGD peptide motif is recognized by many integrins including ␣v␤6, a specific receptor for fibronectin thought to support epithelial cell proliferation during wound healing and carcinoma progression. We report here the discovery of an unexpected non-RGD recognition motif for integrin ␣v␤6. We compared the recognition profiles of recombinant ␣v␤6 and ␣v␤3 integrins by using phage display screening employing 7-mer and 12-mer peptide libraries. As predicted, phages binding strongly to ␣v␤3 contained ubiquitous RGD sequences. However, on ␣v␤6, in addition to RGD-containing phages, one-quarter of the population from the 12-mer library contained the distinctive consensus motif DLXXL. A synthetic DLXXL peptide, RTDLDSLRTYTL, selected from the phage sequences (clone-1) was a selective inhibitor of RGD-dependent ligand binding to ␣v␤6 in isolated receptor assays (IC 50 ‫؍‬ 20 nM), and in cell adhesion assays (IC 50 ‫؍‬ 50 M). DLXXL peptides were highly specific inhibitors of ␣v␤6-fibronectin interaction as synthetic scrambled or reversed DLXXL peptides were inactive. NH 2 -and COOH-terminal modifications of the flanking amino acids suggested that the preceding two and a single trailing amino acid were also involved in interaction with ␣v␤6. The DLXXL sequence is present in several matrix components and in the ␤ chain of many integrins. Although there is as yet no precise biological role known for DLXXL, it is clearly a specific inhibitory sequence for integrin ␣v␤6 which has been unrecognized previously.Integrins are a family of heterodimeric class I transmembrane receptors involved in numerous cell-matrix and cell-cell adhesion phenomena (1). They can be grouped roughly into three classes: the ␤1 series, which are ubiquitous receptors for extracellular matrix (2); the ␤2 series, which are activatable on leukocytes and are triggered during the inflammatory response (3); and the ␣v series, which bind and mediate the cell response to provisional extracellular matrices found during wound repair and other pathological processes (4).The integrins ␣5␤1 (5), ␣IIb␤3 (6), ␣8␤1 (7), ␣v␤1 (8), ␣v␤3(9), and ␣v␤6 (10) all bind the Arg-Gly-Asp-(RGD) peptide sequence in fibronectin, where it is presented in a constrained loop (11). Soluble RGD-containing peptides can inhibit the interaction of each of these integrins with fibronectin. However, to analyze the function of individual fibronectin receptors in a particular cellular environment it is useful to have more specific inhibitors, and a variety of inhibitory antibodies, modified peptides, and non-peptidic substances has been developed. However, as yet no inhibitor has been discovered specific for ␣v␤6. ␣v␤6 is a rare integrin, induced during repair processes in epithelia (10, 12). Its only known specificities are for fibronectin (10), where it can be the dominant receptor me...

Fibronectin is ubiquitously expressed in the extracellular matrix, and experimental evidence has shown that it modulates blood vessel formation. The relative contribution of local and circulating fibronectin to blood vessel formation in vivo remains unknown despite evidence for unexpected roles of circulating fibronectin in various diseases. Using transgenic mouse models, we established that circulating fibronectin facilitates the growth of bone metastases by enhancing blood vessel formation and maturation. This effect is more relevant than that of fibronectin produced by endothelial cells and pericytes, which only exert a small additive effect on vessel maturation. Circulating fibronectin enhances its local production in tumors through a positive feedback loop and increases the amount of vascular endothelial growth factor (VEGF) retained in the matrix. Both fibronectin and VEGF then cooperate to stimulate blood vessel formation. Fibronectin content in the tumor correlates with the number of blood vessels and tumor growth in the mouse models. Consistent with these results, examination of three separate arrays from patients with breast and prostate cancers revealed that a high staining intensity for fibronectin in tumors is associated with increased mortality. These results establish that circulating fibronectin modulates blood vessel formation and tumor growth by modifying the amount of and the response to VEGF. Furthermore, determination of the fibronectin content can serve as a prognostic biomarker for breast and prostate cancers and possibly other cancers.

Plasma fibronectin is a circulating protein that facilitates phagocytosis by connecting bacteria to immune cells. A fibronectin isoform, which includes a sequence of 90 AA called extra-domain B (EDB), is synthesized de novo at the messenger RNA (mRNA) level in immune cells, but the reason for its expression remains elusive. We detected an 80-fold increase in EDB-containing fibronectin in the cerebrospinal fluid of patients with bacterial meningitis that was most pronounced in staphylococcal infections. A role for this isoform in phagocytosis was further suggested by enhanced EDB fibronectin release after internalization of Staphylococcus aureus in vitro. Using transgenic mouse models, we established that immune cell production of fibronectin contributes to phagocytosis, more so than circulating plasma fibronectin, and that accentuated release of EDB-containing fibronectin by immune cells improved phagocytosis. In line with this, administration of EDB fibronectin enhanced in vitro phagocytosis to a larger extent than plasma fibronectin. This enhancement was mediated by αvβ3 integrin as shown using inhibitors or cells from β3 integrin knockout mice. Thus, we identified both a novel function for EDB fibronectin in augmenting phagocytosis over circulating plasma fibronectin, as well as the mediating receptor. Our data also establish for the first time, a direct role for β3 integrin in bacterial phagocytosis in mammals.Key messages• Fibronectin containing an extra domain called EDB is released in bacterial meningitis.• EDB-containing fibronectin enhances phagocytosis more than plasma fibronectin.• The enhancement is mediated by activation of αvβ3 integrin in the presence of EDB.Electronic supplementary materialThe online version of this article (doi:10.1007/s00109-015-1373-0) contains supplementary material, which is available to authorized users.