The role of MAP2 kinases and p38 kinase in acute murine liver injury models

Abstract

c-Jun N-terminal kinase (JNK) mediates hepatotoxicity through interaction of its phospho-activated form with a mitochondrial outer membrane protein, Sh3bp5 or Sab, leading to dephosphorylation of intermembrane Src and consequent impaired mitochondrial respiration and enhanced ROS release. ROS production from mitochondria activates MAP3 kinases, such as MLK3 and ASK1, which continue to activate a pathway to sustain JNK activation, and amplifies the toxic effect of acetaminophen (APAP) and TNF/galactosamine (TNF/GalN). Downstream of MAP3K, in various contexts MKK4 activates both JNK and p38 kinases and MKK7 activates only JNK. The relative role of MKK4 versus 7 in liver injury is largely unexplored, as is the potential role of p38 kinase, which might be a key mediator of toxicity in addition to JNK. Antisense oligonucleotides (ASO) to MKK4, MKK7 and p38 (versus scrambled control) were used for in vivo knockdown, and in some experiments PMH were used after in vivo knockdown. Mice were treated with APAP or TNF/GalN and injury assessed. MKK4 and MKK7 were expressed in liver and each was efficiently knocked down with two different ASOs. Massive liver injury and ALT elevation were abrogated by MKK4 but not MKK7 ASO pretreatment in both injury models. The protection was confirmed in PMH. Knockdown of MKK4 completely inhibited basal P-p38 in both cytoplasm and mitochondria. However, ALT levels and histologic injury in APAP-treated mice were not altered with p38 knockdown versus scrambled control. p38 knockdown significantly increased P-JNK levels in cytoplasm but not mitochondria after APAP treatment. In conclusion, MKK4 is the major MAP2K, which activates JNK in acute liver injury. p38, the other downstream target of MKK4, does not contribute to liver injury from APAP or TNF/galactosamine.

Figure 1

Comparison of knockdown of MKK4 versus MKK7 in acetaminophen induced necrotic liver injury model. After injection of control or MKK4 ASO or MKK7 ASO, the mice were treated with APAP (300 mg/kg) intraperitoneally. (a) Western blots of MKK4 and MKK7 knockdown in liver lysate. Hela cell lysate used as positive control. (b) Quantitative PCR of MKK4 or MKK7 in liver of control ASO versus MKK4 or MKK7 ASO-injected mice. MKK4 or MKK7 mRNA was normalized by housekeeping gene TBP (TATA box binding protein); *P<0.05; n=5 mice per each group. (c) Serum ALT 24 h after APAP. Mean±S.D., n=5 per group; *P<0.05 (t-test), Control versus MKK4 or MKK7 KD. (d) Representative hematoxylin and eosin-stained liver histology 24 h after APAP. Scale bars represent 100 μm. (e) Effect of control, MKK4 ASO or MKK7 ASO on covalent binding of NAPQI. Western blot of liver homogenate was prepared 1 h after APAP (300 mg/kg) using anti-adduct antisera. (* indicates versus control PBS). (f) Liver GSH levels. Mean±S.D., n=5 per group and time point

Figure 2

Effect of MKK4 knockdown in TNF/galactosamine apoptotic liver injury model. After injection of control, or MKK4 ASO, or MKK7 ASO, the mice were treated with TNF (12 μg/kg)/GalN (800 mg/kg) intraperitoneally. (a) Serum ALT 6 h after TNF/GalN. Bars represent mean±S.D., n=5 per group; *P<0.05 (t-test), MKK4 KD versus control or MKK7 KD. (b) Comparison of hematoxylin and eosin or TUNEL stain of liver histology 6 h after TNF/GalN. Scale bars represent 100 μm and quantitative microscopy is described in the 'Materials and Methods' section

Figure 3

Effect of MKK4 or 7 knockdown on early signaling in cytoplasm and mitochondria in APAP toxicity. (a) Immunoblot analysis of JNK and MKK4 activation in cytoplasm and translocation to mitochondria 2 h after APAP (300 mg/kg) in ASO control versus MKK4 KD and MKK7 KD mice. (b) Immunoblots of cytoplasm and mitochondrial extracts at 1 and 2 h after APAP in control versus MKK4 KD livers. GAPDH (cytoplasm) and prohibitin (PHB1) (mitochondria) were loading controls. (* indicates versus control PBS; # indicates nonspecific band; NS, not significant). Bar graphs represent densitometric analysis of results from five separate mice per group. As JNK is not present in association with normal mitochondria, we used PHB1 for normalization of P-JNK in mitochondrial fraction

Figure 4

Effect of p38 knockdown in liver injury models. After injection of control or p38 ASO, the mice were treated with APAP (300 mg/kg) or TNF (12 μg/kg)/GalN (800 mg/kg) intraperitoneally. (a) Immunoblot analysis of p38 knockdown condition. (b) P-JNK, JNK in cytoplasm and translocation to mitochondria in 1 and 2 h after APAP in ASO control versus p38 KD mice. Bars represent mean±standard deviation, n=5 per group; *P<0.05 (t-test), Control versus p38 KD. (c) Serum ALT 24 h after APAP, 6 h after TNF/GalN. N=5 per group; (NS, not significant, ASO control versus p38 ASO-treated mice). (d) Comparison of hematoxylin and eosin liver histology 24 h after APAP and 6 h after TNF/GalN; TUNEL stain of the latter also shown. Scale bars represent 100 μm. Quantitative microscopy is described in the 'Materials and Methods' section. (e) Effect of liver-specific Sab knockout on P-p38 association with mitochondria. Mice were fed with Tam diet for 7 days. One week later, Tam-Sab^f/f Control and Tam-Sab^LPC-KO KO littermates were treated with APAP (300 mg/kg i.p.) in warm PBS in pyrogen-free PBS. Mitochondria were isolated from livers at indicated time points by differential centrifugation. Western blot analysis was performed using antisera against P-p38, p38 and PHB1. The densitometric ratio of P-p38/PHB1 is shown below the blots (n=5); no significant differences were seen by AOV

Figure 5

Effect of MKK4 knockdown on APAP and TNF-α-induced toxicity in cultured hepatocytes. Immunoblots assessing (a) APAP and (b) TNF-α /ActD-induced JNK activation in PMHs. After control ASO and MKK4 ASO injections, hepatocytes were isolated. Cultured hepatocytes from these mice were incubated with PBS or APAP (5 mM) or TNF-α (20 ng/ml)/ActD (0.5 μg/ml), and whole-cell extracts were examined by western blotting for p-JNK and JNK at the indicated times. GAPDH was used as a loading control. Bars represent densitometric analysis of immunoblots from three mice per group. (c and d) PMHs from control and MKK4 KD mice were incubated with APAP 16 h or (d) TNF-α/ActD for 6 h, and hepatocytes were stained with Hoechst 3325. Dead cells were counted as described in the 'Materials and Methods' section. *P<0.05 versus control group (n=three experiments, three different primary culture from three mice)