JAK/STAT1 signaling promotes HMGB1 hyperacetylation and nuclear translocation

Abstract

Extracellular high-mobility group box (HMGB)1 mediates inflammation during sterile and infectious injury and contributes importantly to disease pathogenesis. The first critical step in the release of HMGB1 from activated immune cells is mobilization from the nucleus to the cytoplasm, a process dependent upon hyperacetylation within two HMGB1 nuclear localization sequence (NLS) sites. The inflammasomes mediate the release of cytoplasmic HMGB1 in activated immune cells, but the mechanism of HMGB1 translocation from nucleus to cytoplasm was previously unknown. Here, we show that pharmacological inhibition of JAK/STAT1 inhibits LPS-induced HMGB1 nuclear translocation. Conversely, activation of JAK/STAT1 by type 1 interferon (IFN) stimulation induces HMGB1 translocation from nucleus to cytoplasm. Mass spectrometric analysis unequivocally revealed that pharmacological inhibition of the JAK/STAT1 pathway or genetic deletion of STAT1 abrogated LPS- or type 1 IFN-induced HMGB1 acetylation within the NLS sites. Together, these results identify a critical role of the JAK/STAT1 pathway in mediating HMGB1 cytoplasmic accumulation for subsequent release, suggesting that the JAK/STAT1 pathway is a potential drug target for inhibiting HMGB1 release.

Keywords: cytokine; damage-associated molecular pattern; innate immunity; pathogen-associated molecular pattern; therapy.

Fig. 1.

Pharmacological inhibition of the JAK/STAT pathway blocks LPS-induced HMGB1 nuclear translocation and release. (A–D) Mouse macrophage-like RAW 267.4 cells were stimulated with LPS in the absence or the present of pyridone 6 for 16h. The localization of cellular HMGB1 was measured by fluorescent immunostaining analysis. (Scale bars, 20 µm.) Shown in A are representative images of HMGB1 immunostaining, and shown in B are means ± SEM of two independent experiments. The levels of HMGB1 in the culture medium were measured by Western blot analysis (C). Cytotoxicity was determined by LDH assay (D). (E) Mouse macrophages were stimulated with LPS in the absence or the present of pyridone 6 for 6 h. The levels of TNF in the culture medium were assessed by ELISA. Data shown are means ± SEM of two independent experiments. ^#P < 0.05.

Fig. 2.

Activation of the JAK/STAT pathway by type 1 IFN induces HMGB1 release. (A and B) RAW 267.4 cells were stimulated with IFN-α and IFN-β at various concentrations (A) for the indicated time (B). The levels of HMGB1 in the culture medium were measured by Western blot analysis. (C and D) RAW 267.4 cells were stimulated with IFN-β (100 U/mL) in the absence or the present of pyridone 6 (C) or 2-AP (D) at various concentrations for 16 h. The levels of HMGB1 in the culture medium were measured by Western blot analysis.

Fig. 3.

JAK/STAT1 is required for LPS- or IFN-β–induced HMGB1 acetylation within NLS sites. (A) Mouse peritoneal macrophages were stimulated with indicated stimuli in the absence or the presence of pyridone 6 for 6 h. (B) Mouse peritoneal macrophages isolated from WT or STAT1 KO mice were stimulated with indicated stimuli for 6 h. The acetylation of intracellular HMGB1 protein within the NLS sites was assessed using LC-MS/MS. Shown are representative MS traces.

Fig. 4.

JAK/STAT1 signaling is dispensable for LPS- or IFN-induced HMGB1 oxidation. Mouse peritoneal macrophages isolated from WT or STAT1 KO mice were stimulated with indicated stimuli for 6 h. The redox status of intracellular HMGB1 protein was assessed by LC-MS/MS. Shown in the graphs are representative MS traces.