Role of JNK translocation to mitochondria leading to inhibition of mitochondria bioenergetics in acetaminophen-induced liver injury

Abstract

Previously, we demonstrated JNK plays a central role in acetaminophen (APAP)-induced liver injury (Gunawan, B. K., Liu, Z. X., Han, D., Hanawa, N., Gaarde, W. A., and Kaplowitz, N. (2006) Gastroenterology 131, 165-178). In this study, we examine the mechanism involved in activating JNK and explore the downstream targets of JNK important in promoting APAP-induced liver injury in vivo. JNK inhibitor (SP600125) was observed to significantly protect against APAP-induced liver injury. Increased mitochondria-derived reactive oxygen species were implicated in APAP-induced JNK activation based on the following: 1) mitochondrial GSH depletion (maximal at 2 h) caused increased H2O2 release from mitochondria, which preceded JNK activation (maximal at 4 h); 2) treatment of isolated hepatocytes with H2O2 or inhibitors (e.g. antimycin) that cause increased H2O2 release from mitochondria-activated JNK. An important downstream target of JNK following activation was mitochondria based on the following: 1) JNK translocated to mitochondria following activation; 2) JNK inhibitor treatment partially protected against a decline in mitochondria respiration caused by APAP treatment; and 3) addition of purified active JNK to mitochondria isolated from mice treated with APAP plus JNK inhibitor (mitochondria with severe GSH depletion, covalent binding) directly inhibited respiration. Cyclosporin A blocked the inhibitory effect of JNK on mitochondria respiration, suggesting JNK was directly inducing mitochondrial permeability transition in isolated mitochondria from mice treated with APAP plus JNK inhibitor. Addition of JNK to mitochondria isolated from control mice did not affect respiration. Our results suggests that APAP-induced liver injury involves JNK activation, due to increased reactive oxygen species generated by GSH-depleted mitochondria, and translocation of activated JNK to mitochondria where JNK induces mitochondrial permeability transition and inhibits mitochondria bioenergetics.

FIGURE 1.

Protective effects of JNK inhibitor (SP600125) on APAP-induced toxicity in vivo in C57BL/6 and TNF-R1 knock-out mice. Protective effects of JNK inhibitor against APAP dissolved in warm PBS in C57BL/6 mice. ▴, acetaminophen; ▪, APAP plus JNK inhibitor. APAP (600 mg/kg) was dissolved in warm PBS prior to injection of mice. In all experiments mice were pretreated with JNK inhibitor (10 mg/kg in DMSO (8.3%) and PBS) or equivalent amounts of DMSO in PBS as vehicle control, 1 h prior to APAP treatment. Serum ALT levels were measured 24 h after APAP treatment. n = 4–12 mice per group. Results are mean ± S.D. *, p < 0.05 versus APAP treatment alone.

FIGURE 2.

Time course of GSH depletion, JNK activation, and liver injury following APAP treatment in vivo. A, total liver homogenate GSH; B, mitochondrial GSH; C, JNK activation; D, JNK inhibitor prevents JNK activation (4 h following APAP treatment); E, early time course of serum ALT reflecting liver injury. C57BL/6 mice were treated with APAP (600 mg/kg) dissolved in warm PBS and pretreated with either JNK inhibitor (10 mg/kg in DMSO (8.3%) and PBS) or equivalent amounts of DMSO in PBS 1 h prior APAP treatment. At indicated times liver were taken, ALT measurements made in serum, GSH measured using HPLC with electrochemical detection, and Western blot analysis was performed using antisera against phospho-JNK, JNK, and actin as loading control. ♦, control; ▴, APAP; ▪, APAP plus JNK inhibitor. Results are mean ± S.D. *, p < 0.05 versus APAP treatment alone. n = 3–5 mice.

FIGURE 3.

Effect of AMAP on liver GSH levels and JNK activation. A, total liver homogenate GSH; B, mitochondrial GSH; C, JNK activation. C57BL/6 mice were treated either APAP (400 mg/kg) or AMAP (600 mg/kg) dissolved in warm PBS. 2 h following APAP or AMAP treatment, liver mitochondria was isolated. Total liver homogenate and mitochondria GSH levels were measured using HPLC with electrochemical detection, and Western blot analysis was performed using antisera against phospho-JNK, JNK, and actin as loading control. n = 4 mice.

FIGURE 4.

Activation of JNK by mitochondrial ROS. A, APAP treatment induces increased H₂O₂ generation from mitochondria. C57BL/6 mice were treated with either APAP (400 mg/kg) or AMAP (600 mg/kg) dissolved in warm PBS. 1 h following APAP treatment, livers were taken and mitochondria was isolated using discontinuous Percoll gradient centrifugation, and H₂O₂ release from mitochondria was measured by monitoring fluorescence of p-hydroxyphenyl acetate oxidation in the presence of horseradish peroxidase. H₂O₂ measurements were made with mitochondria treated with complex I substrates (glutamate/malate 7.5 mm). B, H₂O₂ and inhibitors that increase mitochondrial H₂O₂ generation induce JNK activation in isolated primary cultured hepatocytes. Primary cultured hepatocytes were treated with various doses of H₂O₂ (300 and 400 μm for 30 min) or rotenone (2.5 and 5 μm for 60 min) or antimycin (5 and 10 μm for 60 min). Western blot analysis was performed using antisera against phospho-JNK, JNK, and actin as loading control.

FIGURE 5.

JNK inhibitor protects against a decline in mitochondria bioenergetics caused by APAP in vivo. A, state III respiration (glutamate/malate plus ADP) in isolated liver mitochondria. B, state IV respiration (glutamate/malate). C, RCR (state III/state IV). D, ATP levels in liver homogenate. C57BL/6 mice were treated with APAP (600 mg/kg) dissolved in warm PBS and pretreated with either JNK inhibitor (10 mg/kg in DMSO (8.3%) and PBS) or equivalent amounts of DMSO in PBS 1 h prior APAP treatment. At the indicated times livers were taken, mitochondria was isolated using differential centrifugation, and mitochondrial oxygen consumption was measured using a Clarke type electrode. ATP measurements were made using HPLC with UV detection as described under “Experimental Procedures.” ♦, control; ▴, APAP; ▪, APAP plus JNK inhibitor. Results are mean ± S.D. *, p < 0.05 versus APAP treatment alone. n = 3 mice per time point per group.

FIGURE 6.

Effect of APAP and JNK inhibitor on bcl2 family members and apoptotic factors in cytoplasm and mitochondria in vivo. A, cytoplasm; B, mitochondria. Bax, Bad, Bak, Bid, tBid, Bcl_xL, Bim_EL, and cytochrome c were measured by Western blot analysis at 4 h after APAP (600 mg/kg) treatment pretreated with either JNK inhibitor or DMSO-PBS vehicle. A positive control for tBid was made by treating primary culture hepatocytes with actinomycin D (10 μm) plus TNF (20 ng) for 6 h (data not shown). Densitometry was performed using Scion. AU = arbitrary units. Results are mean ± S.D. *, p < 0.05 versus control.

FIGURE 7.

Time course of JNK translocation to mitochondria following APAP treatment in vivo: modulation by JNK inhibitor. A, time course of JNK translocation to mitochondria following APAP treatment. B, JNK inhibitor prevents JNK translocation to mitochondria (4 h following APAP treatment). C57BL/6 mice were treated APAP (600 mg/kg) dissolved in warm PBS and pretreated with either JNK inhibitor (10 mg/kg in DMSO (8.3%) and PBS) or equivalent amounts of DMSO in PBS 1 h prior APAP treatment. At indicated times livers were taken, and mitochondria were separated from cytoplasm by differential centrifugation. Western blot analysis was performed using antisera against phospho-JNK, JNK, and cytochrome oxidase as loading control.

FIGURE 8.

Effect of knocking down JNK1 and/or -2 on JNK translocation to mitochondria, mitochondria bioenergetics, and liver injury. A, effect of JNK1 and -2 ASO on JNK translocation to mitochondria induced by APAP treatment. B, effect of silencing JNK1 and -2 on mitochondria bioenergetics following APAP treatment C, effect of silencing JNK1, JNK2, or JNK1 and -2 on APAP-induced liver injury. One set of C56BL/6 mice were treated with control or JNK1 or JNK2 antisense (ASO) every other day for 2 weeks (7 doses). The other set of C56BL/6 mice were treated with control (double ASO dose) or JNK1 plus JNK2 antisense (ASO) every other day for 2 weeks (7 doses). Mice were subsequently treated with APAP (300 mg/kg) dissolved in warm PBS. For mitochondria measurements, livers were taken 2 h following APAP treatment, and mitochondria were separated from cytoplasm by differential centrifugation. Western blot analysis was performed using antisera against JNK with cytochrome oxidase and actin serving as loading controls, and mitochondria bioenergetics was measured as described under “Experimental Procedures.” In other experiments, serum ALT levels were measured 24 h after APAP treatment. n = 4–8 mice. Results are mean ± S.D. *, p < 0.05 versus APAP treatment alone.

FIGURE 9.

Addition of purified active JNK1 or JNK2 inhibits mitochondria bioenergetics and induces MPT in redox altered mitochondria. Isolated mitochondria from control and JNK inhibitor plus APAP treated (redox altered due to GSH depletion and covalent binding) mice were incubated with purified active JNK1 or JNK2. A, state III respiration; B, RCR; C, Western blot of JNK1 and JNK2 associated with mitochondria following incubation. ♦, control mitochondria; ▴, mitochondria from APAP-treated mice; ▪, mitochondria from APAP plus JNK inhibitor-treated mice; □, mitochondria from APAP plus JNK inhibitor-treated mice treated with purified active JNK and cyclosporin A. Mitochondria were incubated with purified active JNK1 or JNK2 (0.35 μg/mg of mitochondria) or with buffer alone (control) for 10 min in 230 mm mannitol, 70 mm sucrose, 30 mm Tris-HCl, 5 mm KH₂PO₄, 1 mm EDTA, 10 mm MgCl₂, 600 μm ATP, pH 7.4. In some samples isolated mitochondria were pretreated with cyclosporin A (CsA, 2 μm) 5 min prior to JNK treatment. The mitochondria were centrifuged, washed, and centrifuged one more time. Mitochondria respiration was measured with a Clark type electrode using complex I substrates (glutamate/malate, 7.5 mm) and ADP. *, p < 0.05 versus untreated mitochondria; #, p < 0.05 versus APAP plus JNK inhibitor plus active JNK-treated mitochondria.

FIGURE 10.

JNK inhibitor given after APAP treatment still partially protects against JNK-induced mitochondria dysfunction. A, protective effects of JNK inhibitor treatment following APAP administration in C57BL/6 mice. ▴, APAP; ▪, APAP plus JNK inhibitor. Mice received either JNK inhibitor or vehicle (DMSO-PBS) at time points indicated following APAP treatments. Serum ALT levels were measured 24 h after APAP. B, effect of delaying JNK inhibitor (2 h following APAP) on JNK activation and translocation to mitochondria (at 4 h following APAP). C, effect of delaying JNK inhibitor (2 h following APAP) on mitochondria state III respiration (at 4 h following APAP treatment). Mitochondria respiration was measured using an oxygen electrode in the presence of complex I substrate (glutamate/malate) plus ADP. Results are mean ± S.D. *, p < 0.05 versus APAP treatment alone. n = 4 mice.

FIGURE 11.

Proposed model of the JNK-mitochondria signaling loop important in mediating liver injury. APAP is metabolized to NAPQI by CYP2E1, which depletes mitochondria GSH and covalently binds to protein in mitochondria. Mitochondria GSH depletion and possible covalent binding cause a partial decrease in mitochondria respiration. Mitochondrial GSH levels are inadequate to detoxify H₂O₂ formed in mitochondria, causing an increase release of H₂O₂ from mitochondria. The H₂O₂ released from mitochondria activates JNK in cytoplasm and triggers its translocation to mitochondria. Activated JNK also promotes bax translocation to mitochondria (not shown) which may contribute to mitochondrial dysfunction in a redundant fashion. JNK associated with mitochondria induces MPT and promotes cytochrome c release, which inhibits mitochondria bioenergetics. The mechanism of MPT could be a direct effect of JNK on a pore constituent or possibly through an intermediary target, which then induces pore opening.