Peroxiredoxin-mediated disulfide bond formation is required for nucleocytoplasmic translocation and secretion of HMGB1 in response to inflammatory stimuli

Abstract

The nuclear protein HMGB1 (high mobility group box 1) is secreted by monocytes-macrophages in response to inflammatory stimuli and serves as a danger-associated molecular pattern. Acetylation and phosphorylation of HMGB1 are implicated in the regulation of its nucleocytoplasmic translocation for secretion, although inflammatory stimuli are known to induce H₂O₂ production. Here we show that H₂O₂-induced oxidation of HMGB1, which results in the formation of an intramolecular disulfide bond between Cys²³ and Cys⁴⁵, is necessary and sufficient for its nucleocytoplasmic translocation and secretion. The oxidation is catalyzed by peroxiredoxin I (PrxI) and PrxII, which are first oxidized by H₂O₂ and then transfer their disulfide oxidation state to HMGB1. The disulfide form of HMGB1 showed higher affinity for nuclear exportin CRM1 compared with the reduced form. Lipopolysaccharide (LPS)-induced HMGB1 secretion was greatly attenuated in macrophages derived from PrxI or PrxII knockout mice, as was the LPS-induced increase in serum HMGB1 levels.

Keywords: H(2)O(2); HMGB1; Oxidation; Peroxiredoxin; Secretion.

Graphical abstract

Fig. 1

HMGB1 oxidation mediated by PrxI and PrxII in the nucleus. (A) Mouse BMDMs were treated with 100 ng/mL LPS for various times, after which the nuclear fraction of cell lysates was isolated and subjected to nonreducing SDS-PAGE and immunoblotting. The intensities of bands corresponding to oxidized (Ox) forms of HMGB1 and to oxidized (dimeric) forms of PrxI and PrxII were determined. Means ± SEM (n = 4). Re, reduced form. (B) Mechanistic scheme for the formation of disulfide HMGB1 catalyzed by PrxI or PrxII. I-VI indicate the mechanistic process of reactions for HMGB1 disulfide formation by Prxs. (C, D) IP of Myc–tagged human HMGB1(WT), HMGB1(C23S) or HMGB1(C45S) as well as with those for Myc/His₆-tagged human PrxI or PrxII after 50 μM H₂O₂ for 30 min stimulation in HEK293T cells. (E) WCLs prepared in (C) and (D) were also separated into cytosolic and nuclear fractions before IP. Sp1 and GAPDH were used as nucleus and cytoplasmic marker, respectively. (F) HEK293T cells were transfected with Myc-HMGB1(C23S) or Myc-HMGB1(C45S) and Myc/His₆-tagged PrxI or PrxII, and stimulated with 50 μM H₂O₂ at 30 min. WCLs were subjected to 8% nonreducing SDS-PAGE. Black, blue, and red arrows show (Prx-Myc/His₆)_2, Myc-HMGB1-Prx-Myc/His₆, Myc-HMGB1-endogenous Prx complexes, respectively. *, (Prx-Myc/His₆)₂-Myc-HMGB1; **, Prx-Myc/His₆-protein X complexes. Endo.: endogenous. (G, H) A mixture (total of 1 ml) of HMGB1 (0.5 μg/ml) with either PrxI (0.4 μg/ml) or PrxII (0.4 μg/ml) proteins was treated with various concentrations of DTT and 50 μM H₂O₂. The mixtures were immunoprecipitated with anti-HMGB1 Ab and immunoblotted with anti-PrxI/II Abs. Amounts of HMGB1-PrxI/II disulfide intermediate were analysed with reducing SDS-PAGE. IgG, irrelevant Ab. (I, J) PLA assay for detection of the interaction between endogenous HMGB1 and PrxII. PLA signal (red fluorescence) were detected with antibodies HMGB1 and PrxII after1 μg/mL LPS for 1 h in MEFs or 50 μM H₂O₂ for 30 min in HEK293T cells. Scale bars, 10 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Ablation of PrxII impedes formation of disulfide HMGB1. (A, B) Immortalized WT or PrxII KO MEFs were incubated for 30 min in the absence or presence of PMA (50 or 250 nM) or LPS (0.25 or 1 μg/mL), after which WCLs were subjected to nonreducing SDS-PAGE and immunoblotting. #: nonspecific bands, M, monomer; D, dimer. (C, D) Immortalized PrxII KO MEFs that had been transfected with expression vectors for PrxII (C172S) or PrxII (WT) were incubated for 30 min in the absence or presence of GOX (0.5 or 5 mU/mL) or H₂O₂ (50 or 250 μM), after which WCLs were subjected to nonreducing SDS-PAGE. Means ± SEM (n = 5 for A and B, n = 3 for C and D). *P < 0.05, **P < 0.01 versus PrxII KO (A and B) or empty vector (Ev, C and D), t-test. (E) Determination of disulfide formation of HMGB1 Cys²³–Cys⁴⁵ and free thiol of Cys¹⁰⁶ by LC-MS/MS analysis. WCLs of GOX exposed Myc-HMGB1 in HEK293T cells were incubated with NEM and then treated with DTT and NEM for disulfide HMGB1 analysis.

Fig. 3

Oxidation of HMGB1 is required for nucleocytoplasmic translocation and secretion. (A, B) HEK293-hTLR4/MD2/CD14 cells expressing Myc/EGFP-tagged WT, C23S, or C45S forms of HMGB1 were incubated with 1 μg/mL LPS for 2 h, and examined for EGFP fluorescence by confocal microscopy (A). Scale bars, 10 μm. The percentage of cytoplasmic EGFP was determined (B). n > 200. (C) MEF cells were stimulated with LPS (50 or 250 ng/mL) for 24 h, after which culture supernatants were harvested, and subjected to nonreducing SDS-PAGE with/without DTT. (D-F) MEF or HEK293T cells were incubated in the presence of 250 nM PMA, 10 ng/mL TSA, 50 ng/mL TNFα, 2 mM NAC or 10 μM DPI for 24 h, after which culture supernatants were subjected to IB analysis. Means ± SEM (n = 3). *P < 0.05, **P < 0.01, t-test. NS, not significant. Dotted line is cut line of same membrane. (G-J) Oxidation effect on phosphorylation or acetylation-defective HMGB1. HMGB1 secretions of the phosphorylation-defective mutant HMGB1(NLS1/2A) and the acetylation-defective mutant HMGB1(NLS1/2R) were tested after PMA, TSA, or GOX treatments. HMGB1 knockout MEF cells (I, J) were transfected with each plasmid for 36 h and stimulated for 24 h. Culture supernatants were harvested and IB analyses were performed using an anti-Myc Ab. Means ± SEM (n = 3). *P < 0.05, **P < 0.01, t-test.

Fig. 4

Binding of disulfide HMGB1 to CRM1. (A) IP of Myc–tagged HMGB1(WT), HMGB1(C23S) or HMGB1(C45S) with CRM1 after 50 μM H₂O₂ or 0.5 mU/mL GOX for 2 h stimulation in HEK293T. (B) HEK293T cells expressing Myc–tagged WT, C23S, or C45S forms of HMGB1 were incubated in the absence or presence of 50 μM H₂O₂ or GOX (5 mU/mL) for 2 h and then subjected to a PLA assay with antibodies to Myc and to CRM1. Scale bars, 10 μm.

Fig. 5

Deficiency of PrxI or PrxII attenuates HMGB1 secretion in BMDMs and vice versa. (A-D) BMDMs derived from WT, PrxI-KO (PrxI^–/–), or PrxII-KO (PrxII^–/–) mice were incubated in the absence or presence of 100 ng/mL LPS, 250 nM PMA, 50 ng/mL TSA, or 50 ng/mL TNFα for 24 h, after which culture supernatants were subjected to IB analysis. Means ± SEM (n = 3). (E, F) PrxII KO MEF cells were transfected with PrxII plasmid and incubated with the indicated concentrations of LPS (ng/mL) (E) or GOX (μU/ml) (F) for 24 h, after which culture supernatants were subjected to IB analysis. WT MEF cells were used for positive control. Means ± SEM (n = 3). *P < 0.05, **P < 0.01 versus corresponding value for PrxII KO cells, t-test.

Fig. 6

Serum HMGB1 levels in PrxI- or PrxII-KO mice injected with LPS. (A, B) WT, PrxI^–/–, or PrxII^–/– mice were injected intraperitoneally with LPS (5 mg/kg), and blood samples were collected at 1 and 16 h, and measured concentration of TNFα (A) and HMGB1 (B), respectively. Means ± SD (n = 11 per group). **P < 0.01 versus wild type, t-test. NS, not significant.

Fig. 7

A model of PrxI/II-mediated HMGB1 oxidation and secretion. Upon receiving inflammatory stimuli such as LPS, TNFα, PMA, and TSA, cytosolic H₂O₂ can be generated through mitochondria and NADPH oxidase. Nucleic H₂O₂ can be increased by diffusion of cytosolic H₂O₂ or independently by nuclear Nox IV. Increasing of nuclear H₂O₂ leads to nuclear PrxI/II oxidation step by step, oxidized to sulfenic acid (C_P-SOH) and then reacts with the C_R-SH residue to disulfide-linked dimer. Sulfenic or disulfide Prx can initially attack either Cys²³-SH or Cys⁴⁵-SH of HMGB1, resulting in the formation of intramolecular disulfide-bond (disulfide HMGB1). Disulfide HMGB1 formation in the nucleus by PrxI/II induces nucleocytoplasmic translocation in CRM1 dependent manner and then extracellular secretion.