Aspirin's Active Metabolite Salicylic Acid Targets High Mobility Group Box 1 to Modulate Inflammatory Responses

Abstract

Salicylic acid (SA) and its derivatives have been used for millennia to reduce pain, fever and inflammation. In addition, prophylactic use of acetylsalicylic acid, commonly known as aspirin, reduces the risk of heart attack, stroke and certain cancers. Because aspirin is rapidly de-acetylated by esterases in human plasma, much of aspirin's bioactivity can be attributed to its primary metabolite, SA. Here we demonstrate that human high mobility group box 1 (HMGB1) is a novel SA-binding protein. SA-binding sites on HMGB1 were identified in the HMG-box domains by nuclear magnetic resonance (NMR) spectroscopic studies and confirmed by mutational analysis. Extracellular HMGB1 is a damage-associated molecular pattern molecule (DAMP), with multiple redox states. SA suppresses both the chemoattractant activity of fully reduced HMGB1 and the increased expression of proinflammatory cytokine genes and cyclooxygenase 2 (COX-2) induced by disulfide HMGB1. Natural and synthetic SA derivatives with greater potency for inhibition of HMGB1 were identified, providing proof-of-concept that new molecules with high efficacy against sterile inflammation are attainable. An HMGB1 protein mutated in one of the SA-binding sites identified by NMR chemical shift perturbation studies retained chemoattractant activity, but lost binding of and inhibition by SA and its derivatives, thereby firmly establishing that SA binding to HMGB1 directly suppresses its proinflammatory activities. Identification of HMGB1 as a pharmacological target of SA/aspirin provides new insights into the mechanisms of action of one of the world's longest and most used natural and synthetic drugs. It may also provide an explanation for the protective effects of low-dose aspirin usage.

Figure 1

Identification of HMGB1 as an SA-binding protein and its SA-binding sites. (A) Affinity purification of SABPs from human HeLa cells. Total protein extracts prepared from human HeLa cells were chromatographed on an SA-immobilized PharmaLink column. After washing with increasing concentrations of the biologically inactive SA analog, 4-hydroxy benzoic acid (4-HBA), the retained proteins were eluted with 5 mmol/L SA, fractionated by gel electrophoresis and resolved by silver staining. Subsequent MS analyses identified band a as HMGB1 (GI: 4504425). Note that the band denoted by “b” was identified as soybean trypsin inhibitor, added to inhibit protein degradation. MWM, molecular weight markers. (B) Photoaffinity labeling of HMGB1 using 4AzSA. HMGB1 was incubated with or without 0.1 mmol/L 4AzSA in the absence or presence of the indicated concentrations of SA for 1 h, and then treated with UV light (50 mJ). Proteins labeled with 4AzSA were detected by immunoblot analysis with α-SA antibodies. Proteins stained with Ponceau S served as a loading control. (C) HMGB1 has strong affinity for 3AESA with an apparent K_d of 1.48 nmol/L. Sensorgrams of concentration-dependent HMGB1 interacting with 3AESA immobilized on the SPR sensor chip. (D and E) Identification of SA binding sites on HMGB1. (D) ¹⁵N-¹H-HSQC spectra for HMGB1-ΔC were generated in the presence (blue) or absence (red) of 10 mmol/L SA; expanded regions of the spectra were then superimposed. Residues that show significant chemical shift perturbations (CSPs) due to SA binding are labeled. (E) SA-induced CSPs mapped onto the 3D structure of human HMGB1-ΔC (PDB id: 2YRQ, residues 6–164). The colors in the space-filling model correspond to the amplitude of the observed CSPs [cyan: Δδ_(N–H) < 12 ppb; blue: 12 < Δδ_(N–H) < 25 ppb; red: Δδ_(N–H) > 25 ppb, as indicated by the scale]. Residues for which the backbone ¹⁵N-¹H resonance assignments are not available due to rapid exchange of surface amide protons and/or proline residues are shown in white.

Figure 2

Ac3AESA and amorfrutin B1 bind in the SA-binding sites of HMGB1. (A) Chemical structures of SA and its synthetic and natural derivatives. Conserved hydroxyl (-OH) and carboxyl (-COOH) groups of salicylates are shown in blue. (B and C) 2D ¹⁵N-¹H HSQC spectra for full-length (FL) HMGB1. Spectra were recorded in the absence (black) or presence of 3 mmol/L ac3AESA (blue) (B) or 2 mmol/L amorfrutin B1 (green) (C). Expanded regions of the spectra were then superimposed. Residues that show significant CSPs due to ligand binding are labeled. (D–F) ¹⁵N-¹H Δδ_(N–H) CSPs due to SA (D), ac3AESA (E), and amorfrutin B1 (F) binding are mapped onto the 3D structure of human HMGB1 (PDB id: 2YRQ, residues 6–164), which is oriented to highlight the similarities and differences in the SA-binding sites of Box A and Box B. The colors correspond to the amplitude of the observed CSPs [cyan: Δδ_(N–H) < 12 ppb; blue: 12 < Δδ_(N–H) < 25 ppb; red: Δδ_(N–H) > 25 ppb]; residues that both exhibit significant Δδ_(N–H) CSPs and were mutated to Ala are shown in green.

Figure 3

Arg24 and Lys28 are required for binding SA. (A and B) Comparison of 3D structure of WT versus R24A/K28A mutant FL HMGB1 by ¹⁵N-¹H HSQC 2D NMR spectroscopy. (A) R24A/K28A has a 3D structure similar to the WT HMGB1. This is demonstrated by superimposed ¹⁵N-¹H HSQC spectra of WT HMGB1 (blue) and of R24A/K28A (red). Most NH sites exhibit little or no CSP due to this double mutation. (B) Amide NHs that show significant CSPs in R24A/K28A (red) compared with the WT (blue) are all localized in or near the SA-binding sites in both Box A and Box B of FL HMGB1 and include many of the NH sites that exhibit CSP due to SA binding, which are labeled in this expanded region of the superimposed spectra. (C–F) Mutant HMGB1 does not bind SA. (C) ¹⁵N-¹H HSQC spectra for FL WT HMGB1 (~0.1 mmol/L) were generated in the presence (blue) or absence (red) of 15 mmol/L SA. (D) Residues that show significant CSPs due to SA binding are labeled in expanded regions of the superimposed spectra. (E) ¹⁵N-¹H HSQC spectra for R24A/K28A (~0.1 mmol/L) were generated in the presence (green) or absence (red) of 15 mmol/L SA. (F) Residues that show significant CSPs due to SA binding of WT, but not of R24A/K28A, are labeled in the expanded regions of the superimposed spectra.

Figure 4

SA specifically inhibits fully reduced HMGB1’s chemoattractant activity. (A–C) Dose-dependent inhibition of HMGB1’s chemoattractant activity by SA (A), ac3AESA (B) and amorfrutin B1 (amo B1) (C). Migration of mouse 3T3 fibroblasts in the presence of 1 nmol/L fully reduced HMGB1, but not of 0.1 nmol/L fMLP, was blocked by indicated concentrations of SA, ac3AESA and amo B1. (D) Arg24 and Lys28 are required for binding SA and its derivatives and for their inhibition of HMGB1’s chemoattractant activity by SA, ac3AESA and amo B1. The data represent the mean ± SD (n = 3). Means that are statistically different (Tukey HSD test, P < 0.05) are indicated by different letters over the bar. Means that are not statistically different are indicated by the same letter.

Figure 5

Effects of salicylates on disulfide HMGB1’s cytokine-inducing activities in human macrophages. (A) Dose-dependent (left panels) and time-course (right panels) expression of PTGS2 gene (coding for COX-2) and inflammatory cytokine genes (IL6 and TNF) by following exposure to disulfide HMGB1. For the dose-effect experiment, macrophages were activated for 3 h with the indicated concentrations of HMGB1. For the time-course experiment, 10 μg/mL HMGB1 (~300 nmol/L) or 10 ng/mL LPS were used. (B) Inhibition of the cytokine-inducing activity of HMGB1, but not of LPS, by SA and ac3AESA. Human macrophages were activated for 3 h with 1 μg/mL HMGB1 (~30 nmol/L) (left panels) or 10 ng/mL LPS (right panels) in the absence or presence of 100 μmol/L SA or 1 μmol/L ac3AESA. The data are the mean ± SD (n = 3). Means that are statistically different (Tukey HSD test, P < 0.05) are indicated by different letters over the bar. Means that are not statistically different are indicated by the same letter.

Figure 6

Schematic representation of SA’s mechanisms of suppression of HMGB1’s proinflammatory activities. Extracellular HMGB1 is recognized by two different receptors, CXCR4 and TLR4 (16). The fully reduced HMGB1 induces CXCR4-mediated cell migration. SA inhibits cell migration induced by HMGB1. The disulfide HMGB1 induces TLR4-mediated activation of the NF-κB signaling pathway (16,17), resulting in transcriptional activation of genes coding for COX-2 (PTGS2) and proinflammatory cytokines (IL6 and TNF ). SA suppresses disulfide HMGB1-induced PTGS2, IL6 and TNF expression.