Aspirin’s Active Metabolite Salicylic Acid Targets High Mobility Group Box 1 to Modulate Inflammatory Responses
- 45 Downloads
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.
The plant-derived phenolic compound salicylic acid (SA) and its derivatives, known collectively as salicylates, have long been used to reduce pain, fever, and inflammation (1, 2, 3). Records from the third century B.C. indicate that Hippocrates prescribed willow bark and leaves, which contain salicylates, to relieve pain and fever (4). The best-known salicylate is acetylsalicylic acid, commonly known as aspirin. In addition to its antiinflammatory, antipyretic and analgesic effects (5, 6, 7), prophylactic use of aspirin reduces the risk of heart attack, stroke and certain cancers (3,8,9). Aspirin’s primary mechanism of action in mammals has been attributed to disruption of eicosanoid biosynthesis through irreversible inhibition via acetylation of cyclooxygenases (COX) 1 and 2, thereby altering the levels of prostaglandins, hormones that are involved in inflammation and pain (7). Aspirin is rapidly deacetylated to SA by esterases in human plasma, with a half-life of conversion of 13–19.5 min (10). SA’s half maximal inhibitory concentration (IC50) for COX-2 enzymatic activity in vitro is much higher (>100 mg/L, or ∼500 µmol/L) than aspirin’s (6.3 mg/L, or ∼35 µmol/L); yet SA and aspirin have largely the same pharmacological effects (7). Thus, aspirin/SA likely have additional mechanisms of action that are only partially understood.
In plants, SA is involved in many physiological processes, including immunity, where it plays a central role (3). To decipher SA’s mechanisms of action, we have identified several plant SA-binding proteins (SABPs) (3,11,12). By applying the approaches developed for identifying plant SABPs to mammalian cells, we have discovered a new target of SA in humans, the high mobility group box 1 protein, HMGB1.
HMGB1 is an abundant, chromatinassociated protein that is present in all animal cells; fungi and plants have related proteins (13). Structurally, HMGB1 is composed of two basic DNA-binding domains, designated HMG boxes A and B, and a highly acidic C-terminal tail that participates in specific intramolecular interactions (14). In the nucleus, HMGB1 binds DNA to facilitate nucleosome formation and transcription factor binding (15). HMGB1 also acts as a DAMP molecule, with chemoattractant and cytokine-inducing activities upon its release into the extracellular milieu from necrotic, damaged or severely stressed cells (16).
Extracellular HMGB1 mediates a range of biological responses in association with multiple receptors, such as the receptor for advanced glycation end products (RAGE), Toll-like receptor 2 (TLR2), TLR4 and C-X-C chemokine receptor type 4 (CXCR4) (16). HMGB1 has multiple redox states, which in part depend on a reversible intramolecular disulfide bond formed between cysteine residues 23 and 45 (17). Disulfide HMGB1 signaling through TLR4 leads to activation of nuclaar factor kappa-B (NF-κB) and transcription of proinflammatory cytokines (17,18), whereas recognition by CXCR4 of a complex formed by fully reduced HMGB1 with the C-X-C motif chemokine 12 (CXCL12) promotes the recruitment of inflammatory cells to damaged tissue (19). HMGB1’s diverse activities and receptors likely account for its multiple roles in human disease, including sepsis and arthritis (20,21), atherosclerotic plaque formation (22) and cancer (23, 24, 25). Consequently, HMGB1 has attracted considerable attention as an important drug target for various human diseases (13,16,20, 21, 22, 23, 24, 25).
We show here that SA, as well as synthetic and natural SA derivatives, bind HMGB1 in two distinct binding sites and inhibit its extracellular chemoattractant and cytokine-inducing activities. Mutations in one of the SA-binding sites, which disrupt binding of SA and its derivatives, also suppress inhibition by SA and its derivatives of HMGB1’s chemoattractant activity.
Materials and Methods
Identification of SA-Binding Protein from HeLa Cells
Approximately 3.5 × 107 HeLa cells were trypsinized and pelleted after neutralization and resuspended in 2 mL of 0.2 mol/L Tris-HCl (pH 7.4) containing 137 mmol/L NaCl, 1 mmol/L EDTA, 0.5% (v/v) Triton X-100, 1 mmol/L phenylmethanesulfonylfluoride (PMSF) and a protease inhibitor cocktail (Sigma-Aldrich). The suspension was then subjected to two freeze-thaw cycles, and cells were disrupted by ultrasound. The solution was clarified by a 10-min spin at 20,000g and dialyzed against loading buffer, 50 mmol/L KPO4 (pH 7.0) containing 50 mmol/L NaCl, a protease inhibitor cocktail (Sigma-Aldrich) and 0.1 % (v/v) Triton X-100.
The preparation of SA-immobilized resin and purification of SABPs with this resin were previously described (12). Three enriched proteins from the SA eluate were excised and subjected to electrospray ionization-tandem mass spectrometry (ESI-MS/MS) for peptide identification (Donald Danforth Plant Science Center, St Louis, MO, USA).
Cloning, Expression, and Purification of HMGB1
The rat HMGB1 expression plasmid pET30 Xa/LIC HMGB1 was kindly provided by S Lippard (26). Because there are two amino acid differences between rat and human HMGB1, we generated a human HMGB1 expression clone, pET30 Xa/LIC hHMGB1, by introducing PCR-based point mutations that converted residues 189D and 202E to 189E and 202D. Box A (residues 10–80) and Box B (residues 96–164) domains were amplified from pET30 Xa/LIC HMGB1 and cloned into KpnI and EcoRI sites of pET30 Xa/LIC. Recombinant human HMGB1, Box A and Box B proteins were expressed in Escherichia coli strain BL21 cells grown at 37°C to OD600 = 0.7. HMGB1 expression was induced by adding 0.1 mmol/L isopropylthiogalactoside (IPTG) for 16 h at room temperature. Cells were collected by centrifugation at 6,000g for 30 min, and stored at −20°C. For protein purification, cells were resuspended in lysis buffer, buffer A (20 mmol/L Tris-HCl/8.0, 0.15 mol/L NaCl, 2 mmol/L β-mercap-toethanol and 10% glycerol) containing 0.2% NP-40, 1 mg/mL lysozyme, 1 mmol/L PMSF and 10 mmol/L imidazole. After sonication and centrifugation at 20,000g for 1 h, soluble 6xHis-tagged HMGB1 was purified by affinity chromatography using nickel nitrilotriacetic acid (Ni-NTA) agarose resin (Novagen); after washing the HMGB1-bound resin with buffer A containing 20 mmol/L imidazole, HMGB1 was eluted in buffer A containing 300 mmol/L imidazole. The 6xHis tag was removed by overnight incubation at 4°C with factor Xa (for fulllength HMGB1) and thrombin (for Box A or Box B). The cleaved HMGB1 was further purified using gel filtration chromatography on a HiLoad 16/60 Superdex 200 column (GE Healthcare) equilibrated in buffer A.
To prepare fully reduced HMGB1, purified HMGB1 was incubated with 5 mmol/L dithiothreitol for 1 h at 4°C, and then desalted using a PD-10 desalting column (GE Healthcare) equilibrated in 20 mmol/L Tris-HCl/8.0 and 20 mmol/L NaCl plus 0.5 mmol/L DTT. Disulfide HMGB1 was prepared in the absence of any reducing agent. Contaminating lipopolysaccharide (LPS) were removed from all HMGB1 preparations used for biological assays as described (17). For some experiments, fully reduced HMGB1 and disulfide HMGB1 were provided by HMGBiotech (Milan, Italy).
NMR Sample Preparation
All four HMGB1 constructs (Box A, Box B, HMGB1-δC and full length HMGB1) were cloned in either pET15Tev_NESG or Avi-NESG expression vectors, which both include a Tomato Etch Virus proteas (TEV)-cleavable N-terminal 6xHis purification tag, expressed with uniform 15N or 13C,15N-enrichment, and purified using the published protocols of the Northeast Structural Genomics Consortium (NESG) (27,28). Protein constructs were purified with a 5-mL HisTrap HP Ni-NTA column (GE Healthcare), cleaved with TEV-protease, and repurified using a 5-mL HisTrap HP column. Size exclusion chromatography was performed using a HiLoad 26/600 Superdex 75 column (GE Healthcare) Q1 with a buffer containing 50 mmol/L Tris-HCl, pH 7.5, 100 mmol/L NaCl, 0.02% NaN3, and 10 mmol/L DTT. Samples for nuclear magnetic resonance (NMR) studies were exchanged into this same buffer, containing either no SA, SA, amorfrutin B1 or ac3AESA using a Zeba 96-well spin-desalting plate (Thermo Scientific).
NMR Data Collection
Assays for Cytokine Induction
Total RNAs were isolated using the Illustra RNAspin Mini kit (GE Healthcare), and complementary DNAs (cDNAs) were obtained by reverse transcription with oligo(dT) primers (Invitrogen) and SuperScript II Reverse Transcriptase (Invitrogen) following the manufacturers’ instructions. Quantitative real-time PCR was performed using a LightCycler480 (Roche Molecular Diagnostics), in duplicates, using SYBR Green I master mix. The δC method was used for quantification, and the β-actin gene was used for normalization.
The downside of polyvinylpirrolidone (PVP)-free polycarbonate filters (8 µm pores; Millipore) was coated with 50 µg/mL fibronectin (Sigma-Aldrich). Serum-free Dulbecco modified Eagle medium (DMEM) (negative control), DMEM containing 0.1 nmol/L N-formylmethionyl-leucyl-phenylalanine (fMLP) (positive control; Sigma-Aldrich), or DMEM containing 1 nmol/L fully reduced HMGB1 or disulfide HMGB1 were placed in the lower chamber, together with different concentrations of SA (Sigma-Aldrich). Mouse 3T3 fibroblasts were cultured in DMEM media supplemented with 10% fetal bovine serum (FBS) until 90% confluence, washed twice with phosphate-buffered saline (PBS) to eliminate any floating cells and harvested with trypsin. A total of 50,000 cells in 200 µL were added to the upper chamber, and cells were left to migrate for 3 h at 37°C. Nonmigrating cells were removed with a cotton swab, and migrated cells were fixed with ethanol and stained with Giemsa.
The results are expressed as a mean ± standard deviation (SD). Statistical analysis on the group differences was performed by using a Tukey honest significant difference (HSD) test with software JMP Pro, version 10.0.0 (SAS Institute Inc.).
All supplementary materials are available online at www.molmed.org .
Identification of HMGB1 as an SA-Binding Protein
SA-binding activity of fully reduced HMGB1 was then assessed using photoaffinity crosslinking (11,12). Immunoblotting of 4-azido SA (4AzSA) crosslinked products with α-SA antibody revealed a band at the expected molecular weight for HMGB1 when 4AzSA was present in the reaction, but not in the control reaction without 4AzSA (Figure 1B). Increasing concentrations of SA inhibited crosslinking of 4AzSA to fully reduced HMGB1, indicating that this interaction reflects authentic SA-binding activity. To further confirm HMGB1’s SA-binding activity, we used surface plasmon resonance (SPR), which provides sensitive and quantitative measurements of bimolecular interactions in real-time (11). An SA derivative, 3-aminoethyl SA (3AESA), was synthesized and affixed to the CM5 sensor chip via an amide bond formed between the amine group of 3AESA and the carboxyl groups on the chip. The ethylamine group was added at the 3 position of the phenyl ring because several SA derivatives with substitutions at this position could still bind plant SABPs and induce immune responses (11) in plants. Binding was detected by flowing fully reduced HMGB1 over the 3AESA-immobilized sensor chip. A dose-dependent response was obtained with nmol/L concentrations of fully reduced HMGB1, indicating strong binding (Figure 1C).
Characterization of SA-Binding Sites in HMGB1
To identify which domain(s) of HMGB1 binds SA, four constructs were generated: (i) full-length (FL): residues 1–215, (ii) HMGB1-δC: residues 1–165, (iii) Box A: residues 8–78, and (iv) Box B: residues 86–165 (Supplementary Figure S1A). SPR analysis revealed that all four constructs exhibited SA-binding activity, indicating that Box A and Box B both have an SA-binding site (Supplementary Figure S1B, C).
The amino acid (aa) residues in fully reduced HMGB1 affected by binding of SA were identified by 15N-1H heteronuclear single quantum correlation (HSQC) 2D NMR spectroscopy at pH 7.5. Under these conditions, some surface amide protons are broadened due to solvent exchange, and cannot be observed. The sequence-specific resonance assignments for all observable backbone amide 15N and 1HN resonances of HMGB1-δC were determined using standard triple-resonance NMR methods (30,31). A region of the superimposed 15N-1H-HSQC spectra obtained in the presence or absence of 10 mmol/L SA is shown in Figure 1D. Analysis of 15N-1H HSQC spectra from HMGB1-δC showed that some backbone amide 1HN and/or N resonances exhibited significant chemical shift perturbations (CSPs) upon SA binding, including those of residues Phe18, Thr22, Arg24, Glu25, His27, Lys28, Glu40, Cys45 and Ser46 in Box A and Phe103, Ser121, Gly123, Asp124 and Ala126 in Box B. Similar results were obtained using Box A or Box B alone (Supplementary Figures S1D, E), confirming that both have SA-binding sites. The Box A and Box B domains of HMGB1 are structurally similar, and the CSPs due to SA binding are localized in corresponding regions of the two HMG-box domains (Figure 1E).
Synthetic and Natural SA Derivatives Bind to HMGB1 with Higher Affinity than SA
Comparison of the NMR spectra of FL HMGB1 in the absence versus presence of 15 mmol/L SA, 3 mmol/L ac3AESA, or 2 mmol/L amorfrutin B1 identified aa residues with significant CSPs in the presence of these compounds (Figures 2B, C and Supplementary Figure S2A–C). Mapping the ac3AESA- and amorfrutin B1-induced NMR CSPs onto the 3D structure of human HMGB1 published in the Protein Data Bank (PDB id: 2YRQ, residues 6–164) confirmed that SA, ac3AESA, and amorfrutin B1 share the same binding sites in Box A and Box B domains of the FL protein (Figures 2D–F). Furthermore, saturation transfer difference (STD) analysis (35), which detects magnetization transfer from a protein to its bound ligand, showed that ac3AESA is bound much more tightly than SA by HMGB1 (Supplementary Figure S3). At a ligand concentration of 6 mmol/L, no STD signal was detected for SA binding to FL HMGB1 (Supplementary Figure S3A), whereas significant STD signal was observed for ac3AESA at concentrations of 0.6–5.8 mmol/L (Supplementary Figure S3B).
Finally, we wished to test whether the free hydroxyl group at position 2 of the salicylate moiety is important for binding to HMGB1. WaterLogsy ligand detection experiments clearly show resonance transfer to SA, but no resonance transfer to aspirin (where the 2-OH group is acetylated) nor to 4-HBA (where the hydroxyl group in is in position 4 instead of 2) (Supplementary Figure S4). Likewise, 15N-1H HSQC 2D NMR spectroscopy indicates that SA, but neither aspirin nor 4-HBA, binds HMGB1 (Supplementary Figure S5).
Identification of an HMGB1 Mutant Unable to Bind SA or Its Derivatives
SA and Its Derivatives Inhibit the Chemoattractant Activity of HMGB1
Analysis of the R24A/K28A HMGB1 mutant revealed that it retained its chemoattractant activity which could no longer be inhibited by SA or its more potent derivatives (Figure 4D). This finding is consistent with its inability to bind these compounds (Supplementary Figures S6A–D and S7A–D).
Two more Box A double mutants were constructed—H27A/R48A and K12A/K68A. K12A/K68A retained SA-inhibitable chemoattractant activity (Supplementary Figure S8), demonstrating that only specific mutations in Box A disrupt SA binding and inhibition of HMGB1’s chemoattractant activity. Mutant H27A/R48A no longer had chemoattractant activity, thus SA’s effect on this activity could not be tested (Supplementary Figure S8).
The fact that a mutation that disrupts SA binding to the Box A domain of HMGB1 in NMR studies also abrogates SA inhibition of chemoattractant activity demonstrates that a site identified by NMR studies is functional in SA regulation of HMGB1’s chemoattractant activity and strongly argues that SA directly affects HMGB1’s proinflammatory activity.
SA and ac3AESA Suppress Disulfide HMGB1’s Induction of PTGS2 and Cytokine Genes
Although SA and aspirin are widely used as nonsteroidal antiinflammatory drugs, their cellular targets and mechanisms of action are still being discovered. In this study, we identified HMGB1 as a novel SA-binding protein from HeLa cell extracts using affinity chromatography. Photoaffinity labeling, SPR, and NMR analyses confirmed SA binding to HMGB1. NMR analyses revealed that CSPs upon SA binding are localized to residues in the HMG-box domains of both Box A and Box B. The SA-binding site in Box A identified by these NMR studies was confirmed by demonstrating that conversion of Arg24 and Lys28 to Ala did not affect HMGB1 folding or chemoattractant activity, but suppressed binding by SA and its two more potent derivatives and abrogated SA inhibition of its chemoattractant activity.
Despite weak binding of SA to HMGB1 in the conditions used for NMR experiments, SA inhibited the proinflammatory activities of HMGB1 at low µmol/L concentrations. We speculate that in vivo the conformation of HMGB1 is modified by the interaction with another molecule, so that the binding of salicylates is facilitated. Whereas this “missing molecule” has yet to be identified, several lines of evidence show that SA’s effects on cells are due to direct interaction of SA with HMGB1. First, SA, but neither aspirin nor 4-HBA, binds HMGB1. Conversion of the hydroxyl of SA to an acetyl group (aspirin) or altering the position of the hydroxyl group on the phenyl ring (4-HBA) compromised HMGB1 binding. In contrast, ac3AESA and amorfrutin B1, which have additional groups on position 3, 4 or 6, were bound by HMBG1. These findings suggest that the SA core (free carboxyl and hydroxyl groups at position 1 and 2 of the phenyl ring) is critical for specific binding with HMGB1. Second, the SA derivative ac3AESA binds in HMGB1’s SA-binding sites more tightly than SA and is a stronger inhibitor of its proinflammatory activities. Third, the binding sites for glycyrrhizin, a known HMGB1 inhibitor (32), and SA overlap (Supplementary Figure S10). Finally and most importantly, a mutant protein with alterations in the SA-binding site not only failed to bind SA and its more potent derivatives, but also failed to exhibit inhibition of its chemoattractant activity by SA and these derivatives. These results further confirm the identity of the SA-binding site and the direct effect SA exerts on the immune-related activities of HMGB proteins.
Of particular note is the fact that SA targets a DAMP, which provides a new mechanism to explain the efficiency of aspirin in limiting sterile inflammation. Notably, SA did not suppress migration of cells to fMLP, which mimics N-formylated peptides characteristic of bacteria, or the induction of proinflammatory cytokines or COX-2 in macrophages exposed to LPS. Thus, SA might preferentially suppress sterile inflammation over pathogen-initiated inflammation. This is desirable because clinical management of injury requires specific attenuation of sterile inflammation, without compromising innate immunity against pathogens. Likewise, SA inhibits the cancer-promoting activities of HMGB1 in mesothelioma, whose cells are addicted to HMGB1 for growth and survival (41).
The identification of HMGB1 as another pharmacological target of SA/aspirin provides new insights into the mechanisms of action of the most widely used drug for reducing inflammation and inflammation-associated diseases. Moreover, together the identification of HMGB1’s SA-binding sites by both NMR and site-directed mutagenesis and the discovery of natural and synthetic SA derivatives with greatly enhanced potency compared with SA in suppressing HMGB1’s proinflammatory activities hold great promise for the development of improved SA-based drugs.
Several of the authors (DF Klessig, S-W Park, HW Choi, FC Schroeder, GT Montelione, K Hamilton, F Song, ME Bianchi) have applied for a patent based in part on the work described within. This does not alter our adherence to all Molecular Medicine policies on sharing data and materials.
This work was supported by grants from (i) the Arabidopsis 2010 Initiative of the National Science Foundation USA (IOS-0820405 to DF Klessig), (ii) the Protein Structure Initiative of the National Institutes of Health USA (NIH; U54-GM094597 to GT Montelione), (iii) NIH (GM040654 to RA Cerione), the Triad Foundation (to FC Schroeder), and (iv) Associazione Italiana Ricerca sul Cancro (IG-14233 to ME Bianchi).
- 4.Aboelsoud N. (2010) Herbal medicine in ancient Egypt. J. Med. Plants Res. 4:82–6.Google Scholar
- 30.Biamonti C, Rios C, Lyons B, Montelione G. (1994) Multidimensional NMR experiments and analysis techniques for determining homo- and heteronuclear scalar coupling constants in proteins and nucleic acids. Adv. Biophys. Chem. 4:51–120.Google Scholar
Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, and provide a link to the Creative Commons license. You do not have permission under this license to share adapted material derived from this article or parts of it.
The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.
To view a copy of this license, visit (https://doi.org/creativecommons.org/licenses/by-nc-nd/4.0/)