HMGB1 release induced by liver ischemia involves Toll-like receptor 4 dependent reactive oxygen species production and calcium-mediated signaling

Abstract

Ischemic tissues require mechanisms to alert the immune system of impending cell damage. The nuclear protein high-mobility group box 1 (HMGB1) can activate inflammatory pathways when released from ischemic cells. We elucidate the mechanism by which HMGB1, one of the key alarm molecules released during liver ischemia/reperfusion (I/R), is mobilized in response to hypoxia. HMGB1 release from cultured hepatocytes was found to be an active process regulated by reactive oxygen species (ROS). Optimal production of ROS and subsequent HMGB1 release by hypoxic hepatocytes required intact Toll-like receptor (TLR) 4 signaling. To elucidate the downstream signaling pathways involved in hypoxia-induced HMGB1 release from hepatocytes, we examined the role of calcium signaling in this process. HMGB1 release induced by oxidative stress was markedly reduced by inhibition of calcium/calmodulin-dependent kinases (CaMKs), a family of proteins involved in a wide range of calcium-linked signaling events. In addition, CaMK inhibition substantially decreased liver damage after I/R and resulted in accumulation of HMGB1 in the cytoplasm of hepatocytes. Collectively, these results demonstrate that hypoxia-induced HMGB1 release by hepatocytes is an active, regulated process that occurs through a mechanism promoted by TLR4-dependent ROS production and downstream CaMK-mediated signaling.

Figure 1.

Oxidative stress induces HMGB1 release from hepatocytes. (A) Western blot analysis of supernatants from hepatocytes that underwent 8, 12, or 24 h of hypoxia (1% O₂). Cells were plated in Williams' medium E or Williams' medium E containing 50 mM NAC. The blot shown is representative of three different experiments with similar results. (B) Hepatocytes were stimulated with 125, 250, or 500 μM H₂O₂ for 8 h, and supernatants were sampled and subjected to Western blot analysis for HMGB1. The blot shown is representative of three different experiments with similar results. (C) Hepatocytes were exposed to 500 μM H₂O₂ for 30 min before treatment with NAC or Trolox in a dose–response fashion. Supernatants were sampled at 8 h and subjected to Western blot analysis for HMGB1. The blot shown is representative of three different experiments with similar results. (D) Viability was determined by crystal violet assay on hepatocytes subjected to either 24 h of hypoxia (1% O₂) or 8 h of oxidative stress from H₂O₂. Results are expressed as the mean ± SEM. (E) Hepatocytes were exposed to normoxia (N) or hypoxia (H) for 8 and 24 h, and supernatants were sampled for Western blot analysis for HMGB1. The blot shown is representative of multiple experiments with similar results.

Figure 2.

HMGB1 release from hepatocytes under oxidative stress is calcium dependent. (A) Hepatocytes were treated with A23187 (calcium ionophore), bapta (calcium chelator), or KN62 (CaMK inhibitor) for 30 min to alter intracellular calcium signaling. Cells were then exposed to 500 μM H₂O₂ for 8 h or to hypoxia (1% O₂) for 24 h. Supernatants were sampled and subjected to Western blot analysis for HMGB1. The blot shown is representative of three different experiments with similar results. (B) CaMK mRNA expression levels in rat primary hepatocytes (lane 1) were detected by RT-PCR using specific primers for CaMK I, IIα, IIβ, IIγ, IIδ, and IV, respectively. Rat cerebral cortex extract (lane 2) was used as a positive control. (C) Plated cells from the experiment in A underwent assessment of cell viability by crystal violet assay after supernatant samples had been taken. Results are expressed as the mean ± SEM. The experiment shown is representative of three different experiments with similar results. (D) Hepatocytes were transfected with siRNA to each CaMK (I, IIδ, and IV) or the upstream CaMKK (CaMKK α and CaMKK β2). The specificity of each siRNA was confirmed by Western blot (CaMK I, CaMK IV, CaMKK β2, and CaMK IIδ,) or RT-PCR (CaMKK α). (E) HMGB1 release was examined in the transfected cells stimulated with or without H₂O₂. The experiment shown is representative of three different experiments with similar results.

Figure 3.

CaMK inhibition protects against liver I/R injury. (A) Mice were given 16-mg/kg intraperitoneal injections of KN93, a CaMK inhibitor, or KN92, an inactive analogue, 12 and 1 h before ischemia. Mice underwent 1 h of ischemia and 6 h of reperfusion before serum samples were taken and tested for ALT levels. Sham animals that were injected with the same solutions were subjected only to laparotomy without ischemia. Data represent means ± SE (n = 6–10 animals). *,P < 0.05 versus mice subjected to I/R and treated with KN92 control. (B) Western blot analysis for HMGB1 was also performed on serum samples from animals. Each lane represents a different animal. The blot shown is representative of two experiments with similar results. (C) Immunofluorescent stain of HMGB1 from sections of livers subjected to sham operation or 60 min of ischemia and 6 h of reperfusion. Mice were given intraperitoneal injections of KN93, a CaMK inhibitor, or KN92, an inactive analogue, 12 and 1 h before ischemia. Images are representative liver sections from six mice per group (green, HMGB1; blue, nuclei; red, F-actin). Bars: (×200) 50 μm; (×800) 10 μm.

Figure 4.

Functional TLR4 signaling required for optimal ROS production. (A) Hepatocytes from TLR4 wild-type and KO mice were plated and incubated with 10 μM DCF-DA for 30 min before treatment. Cells were subjected to normoxia or hypoxia (1% O₂) for times up to 50 min, and ROS production was measured by fluorescence spectrophotometry. Data represent means ± SE. *, P < 0.05 versus TLR4 KO hepatocytes subjected to hypoxia. (B) Hepatocytes plated in 10-cm plates were exposed to either normoxia or hypoxia for 2 h. Baseline samples, in addition to 2-h samples, were assayed for byproducts of lipid peroxidation, MDA and 4-HAE. *, P < 0.05 versus 2-h normoxic controls. (C) Hepatocytes from TLR4 wild-type and KO mice were transfected with adenoviral vector encoding either murine TLR4 (Ad-TLR4) or empty adenoviral vector control (Ad-null). Cells were subjected to 30 min of hypoxia. ROS production measured by DCF assay shown is the percent increase relative to respective normoxic controls. *, P < 0.05 versus TLR4 wild-type controls treated with Ad-null virus. (D) Hepatocytes were treated with either anti-HMGB1 neutralizing antibody or IgG control and exposed to normoxia or hypoxia (1% O₂) for 30 min. ROS production was measured by DCF assay, and results are reported as the percentage relative to normoxic controls. Results in B–D are expressed as the mean ± SEM.

Figure 5.

Functional TLR4 signaling required for HMGB1 release from hepatocytes during oxidative stress. (A) TLR4 wild-type and KO hepatocytes were plated and subjected to hypoxic (1% O₂) conditions for various times up to 24 h. The supernatants were sampled at predetermined time points and subjected to Western blot analysis for HMGB1. The blot shown is representative of three different experiments with similar results. (B) Supernatants from Fig. 4 C were sampled at 24 h and subjected to Western blot analysis for HMGB1. The blot shown is a representative blot from multiple experiments. (C) TLR4 wild-type and KO hepatocytes were treated with A23187, KN62, or bapta and subjected to normoxia or hypoxia (1% O₂). Supernatants were sampled at 24 h and subjected to Western blot analysis for HMGB1. The blot shown is representative of three different experiments with similar results. (D) TLR4 wild-type and KO hepatocytes were subjected to hypoxia (1% O₂) with or without 50-mM NAC treatment. Supernatant was sampled at 24 h and subjected to Western blot analysis for HMGB1. The blot shown is representative of three different experiments with similar results. (E) Baseline intracellular HMGB1 levels were determined in TLR4 wild-type and KO hepatocytes. β-actin was used as a loading control. (F) H₂O₂ was used to stimulate TLR4 wild-type and KO hepatocytes to release HMGB1. Cells were subjected to 250 or 500 μM H₂O₂ for 8 h, and supernatants were subjected to Western blot analysis for HMGB1. The blot shown is representative of three different experiments with similar results.

Figure 6.

Antioxidant-mediated hepatic protection during I/R is TLR4 dependent. (A) TLR4 wild-type and mutant mice were used for in vivo study of antioxidant effect. Mice were given 300-mg/kg intravenous injections of NAC or control normal saline immediately before ischemia and immediately after reperfusion. Mice underwent 1 h of ischemia and 6 h of reperfusion before serum samples were taken and tested for ALT levels. Data represent means ± SE (n = 4–6 mice per group). *, P < 0.05 versus TLR4 wild-type mice that were subjected to I/R and given control normal saline. (B) TLR4 wild-type and mutant mice were treated as described for ischemia and were given 1 h of reperfusion. Hepatic tissue was taken and used for approximating the oxidant stress state by measuring levels of MDA and 4-HAE, lipid peroxidation by-products produced from oxidant stress. Data represent means ± SE (n = 4–6 mice per group). *, P < 0.05 versus TLR4 wild-type mice that were subjected to I/R and given control normal saline. (C) Hepatic TNF and IL-6 mRNA expression were obtained in TLR4 wild-type and mutant mice after ischemia and 6 h of reperfusion. Mice were treated as described with NAC or control normal saline. Results are expressed as the relative increase of mRNA expression compared with sham animals. Data represent means ± SE (n = 4–6 mice per group). *, P < 0.05 versus TLR4 wild-type mice that were subjected to I/R and given control normal saline. (D) Serum samples from mice subjected to ischemia and 6 h of reperfusion underwent Western blot analysis for HMGB1. Each lane represents a different animal. The blot shown is representative of two experiments with similar results.