There is a strong link between integrins and interleukin-1β (IL-1β), but the specifics of the role of integrins in IL-1β signaling are unclear. We describe that IL-1β specifically bound to integrins αvβ3 and α5β1. The E128K mutation in the IL1R-binding site enhanced integrin binding. We studied whether direct integrin binding is involved in IL-1β signaling. We compared sequences of IL-1β and IL-1 receptor antagonist (IL1RN), which is an IL-1β homologue but has no agonistic activity. Several surface-exposed Lys residues are present in IL-1β, but not in IL1RN. A disulfide linkage is present in IL1RN, but is not in IL-1β because of natural C117F mutation. Substitution of the Lys residues to Glu markedly reduced integrin binding of E128K IL-1β, suggesting that the Lys residues mediate integrin binding. The Lys mutations reduced, but did not completely abrogate, agonistic action of IL-1β. We studied whether the disulfide linkage plays a role in agonistic action of IL-1β. Reintroduction of the disulfide linkage by the F117C mutation did not affect agonistic activity of WT IL-1β, but effectively reduced the remaining agonistic activity of the Lys mutants. Also, deletion of the disulfide linkage in IL1RN by the C116F mutation did not make it agonistic. We propose that the direct binding to IL-1β to integrins is primarily important for agonistic IL-1β signaling, and that the disulfide linkage indirectly affects signaling by blocking conformational changes induced by weak integrin binding to the Lys mutants. The integrin-IL-1β interaction is a potential target for drug discovery.

Background

Crosstalk between integrins and FGF receptors has been implicated in FGF signaling, but the specifics of the crosstalk are unclear. We recently discovered that 1) FGF1 directly binds to integrin alphavbeta3, 2) the integrin-binding site and FGF receptor (FGFR) binding site are distinct, and 3) the integrin-binding-defective FGF1 mutant (R50E) is defective in inducing FGF signaling although R50E still binds to FGFR and heparin and induces transient ERK1/2 activation.

Principal findings

We tested if excess R50E affect DNA synthesis and cell survival induced by WT FGF1 in BaF3 mouse pro-B cells expressing human FGFR1. R50E suppressed DNA synthesis and cell proliferation induced by WT FGF1. We tested if WT FGF1 and R50E generate integrin-FGF1-FGFR ternary complex. WT FGF1 induced ternary complex formation (integrin-FGF-FGFR1) and recruitment of SHP-2 to the complex in NIH 3T3 cells and human umbilical endothelial cells, but R50E was defective in these functions. It has been reported that sustained ERK1/2 activation is integrin-dependent and crucial to cell cycle entry upon FGF stimulation. We thus determined the time-course of ERK1/2 activation induced by WT FGF1 and R50E. We found that WT FGF1 induced sustained activation of ERK1/2, but R50E was defective in this function.

Conclusions/significance

Our results suggest that 1) R50E is a dominant-negative mutant, 2) Ternary complex formation is involved in FGF signaling, 3) The defect of R50E to bind to integrin may be directly related to the antagonistic action of R50E. Taken together, these results suggest that R50E has potential as a therapeutic in cancer.

Fibroblast growth factor-1 (FGF1) and FGF2 play a critical role in angiogenesis, a formation of new blood vessels from existing blood vessels. Integrins are critically involved in FGF signaling through crosstalk. We previously reported that FGF1 directly binds to integrin αvβ3 and induces FGF receptor-1 (FGFR1)-FGF1-integrin αvβ3 ternary complex. We previously generated an integrin binding defective FGF1 mutant (Arg-50 to Glu, R50E). R50E is defective in inducing ternary complex formation, cell proliferation, and cell migration, and suppresses FGF signaling induced by WT FGF1 (a dominant-negative effect) in vitro. These findings suggest that FGFR and αvβ3 crosstalk through direct integrin binding to FGF, and that R50E acts as an antagonist to FGFR. We studied if R50E suppresses tumorigenesis and angiogenesis. Here we describe that R50E suppressed tumor growth in vivo while WT FGF1 enhanced it using cancer cells that stably express WT FGF1 or R50E. Since R50E did not affect proliferation of cancer cells in vitro, we hypothesized that R50E suppressed tumorigenesis indirectly through suppressing angiogenesis. We thus studied the effect of R50E on angiogenesis in several angiogenesis models. We found that excess R50E suppressed FGF1-induced migration and tube formation of endothelial cells, FGF1-induced angiogenesis in matrigel plug assays, and the outgrowth of cells in aorta ring assays. Excess R50E suppressed FGF1-induced angiogenesis in chick embryo chorioallantoic membrane (CAM) assays. Interestingly, excess R50E suppressed FGF2-induced angiogenesis in CAM assays as well, suggesting that R50E may uniquely suppress signaling from other members of the FGF family. Taken together, our results suggest that R50E suppresses angiogenesis induced by FGF1 or FGF2, and thereby indirectly suppresses tumorigenesis, in addition to its possible direct effect on tumor cell proliferation in vivo. We propose that R50E has potential as an anti-cancer and anti-angiogenesis therapeutic agent ("FGF1 decoy").