Direct binding to integrins and loss of disulfide linkage in interleukin-1β (IL-1β) are involved in the agonistic action of IL-1β

Abstract

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.

Keywords: IL-1β; NF-κB (NF-KB); agonistic action; cell signaling; disulfide linkage; integrin; interleukin-1 (IL-1); mutagenesis.

Figure 1.

Specific binding of IL-1β to integrin αvβ3 in an activation-dependent manner is shown. We studied if IL-1β binds to integrin αvβ3. a, IL-1β binds to soluble integrin αvβ3 in the presence of 1 mm Mn²⁺. Wells of 96-well microtiter plate were coated with IL-1β and remaining protein-binding sites were blocked with BSA. Soluble αvβ3 in Hepes-Tyrode's buffer +1 mm Mn²⁺ was added to wells and incubated. Bound αvβ3 was determined using anti-β3 mAb. The data are shown as means ± S.E. of triplicate experiments. b, IL-1β binds to αvβ3 in surface plasmon resonance study. SPR study of the interaction between αvβ3 and WT IL-1β. Recombinant soluble αvβ3 was immobilized to a sensor chip and IL-1β is in a solution phase. Mg²⁺ (1 mm) was included in the binding buffer. c, FITC–IL-1β does not bind to CHO cells (IL1R-negative), but binds to MCF7 cells (IL1R-positive). CHO cells or MCF7 cells were incubated with FITC-labeled IL-1β (1 μg/ml) in PBS/1 mm EDTA for 1 h at 4 °C, and bound FITC was measured in flow cytometry. d and e, binding of IL-1β to cell-surface αvβ3. Parent CHO cells (d) and β3-CHO cells (e). Integrins are activated in the presence of Mg²⁺ (Hepes-Tyrode's buffer +1 mm Mg²⁺, TH-Mg²⁺), but not in DMEM, which contains >1 mm Ca²⁺. Wells of 96-well microtiter plate were coated with IL-1β (10 μg/ml), and incubated with parent IL1R-negative CHO cells or CHO cells that express recombinant αvβ3 (β3-CHO cells). Bound cells were quantified using endogenous phosphatase activity. The data are shown as means ± S.E. of triplicate experiments.

Figure 2.

The E105K and E128K mutations in the IL1R-binding sites of IL-1β markedly enhance integrin binding. a and c, β3-CHO cells (a) and CHO cells (c) (both IL1R-negative) adhered much better to E105K and E128K than to WT IL-1β or IL1RN in DMEM. Adhesion assays were performed as described in Fig. 1. The data are shown as means ± S.E. of triplicate experiments. b, surface plasmon resonance study of the interaction between αvβ3 and E128K IL-1β. Recombinant soluble αvβ3 was immobilized to a sensor chip and IL-1β is in a solution phase. Mg²⁺ (1 mm) was included in the binding buffer. The results suggest that E128K has much higher affinity to αvβ3 than to WT IL-1β. d, CHO cells (integrin α5β1-positive) adhere to E128K, but the B2 variant of CHO cells (α5β1-negative) did not in DMEM. Adhesion assays were performed as described in Fig. 1. e, adhesion of CHO and β3-CHO cells with or without transfection of IL1R to E128K in the presence of IL1RN. Adhesion assays were performed as described in Fig. 1.

Figure 3.

Identification of amino acid residues critical for integrin binding in IL-1β by mutagenesis is shown. We used the E128K mutant of IL-1β for mapping integrin-binding site in IL-1β. a, alignment of IL-1β (PDB ID 9ILB) and IL1RN (PDB ID 1IRA). The positions shown are of Lys residues (blue), E105 and E128 involved in IL1R binding (red), and disulfide linkage (yellow). The alignment shows that the several Lys residues (Lys-55, Lys-63, Lys-64, Lys-74, and Lys-88) are present in IL-1β but are changed to other neutral amino acids in IL1RN. A disulfide linkage is present in IL1RN, but not present in IL-1β because of mutation (the C117F mutation). b, the Lys residues (Lys-55, Lys-63, Lys-64, Lys-74, and Lys-88) are exposed to the surface in the IL1R/IL-1β/IL1RAcP complex (PDB ID 4DEQ). The arrow indicates the predicted integrin binding site in IL-1β. Lys residues exposed to the surface of IL-1β that are not in the IL1R-binding sites were mutated to Glu. c and d, the ability of β3-CHO (αvβ3+, α5β1+) or CHO cells (α5β1+) to the IL-1β mutant in adhesion assays in DMEM. The data are shown as means ± S.E. of triplicate experiments. *, the binding to integrins is significantly low compared with E128K (p < 0.05, n = 3). The results suggest that several Lys residues in IL-1β are critical for integrin binding. Note that IL1RN did not bind to integrins under the conditions used.

Figure 4.

The integrin-binding–defective Lys mutations reduce agonistic activity of IL-1β, and reintroduction of the missing disulfide linkage effectively suppresses remaining agonistic activity of the Lys mutants. We studied whether integrin-binding–defective mutations affect agonistic activity of IL-1β. a, dose response of Il-1β mutants to induce NF-κB activation. We treated MCF7 cells that stably express NF-κB reporter gene with Il-1β for 4 h and measured the luciferase activity in cell lysates. Luciferase activity was normalized using that induced by 10 ng WT IL-1β as 100. b and c, summary of NF-κB activation by IL-1β mutants at 10 ng/ml (b) and at 1 ng/ml (c). The data are shown as means ± S.E. of triplicate experiments. d, binding of IL-1β mutants to IL1R. Wells of 96-well microtiter plate were coated with IL-1β (His-tagged, WT and mutants of IL-1β and IL1RN, 20 or 40 μg/ml) and blocked with BSA. Wells were incubated with soluble IL1R (10 μg/ml), and bound IL1R was quantified using anti-IL1R antibody. The data are shown as means ± S.E. of triplicate experiments. The columns represent 0, 20, and 40 μg/ml IL-1β, in this order. e, CD spectra of WT or mutant IL-1β.