Hepatic apoptosis postburn is mediated by c-Jun N-terminal kinase 2

Abstract

The trauma of a severe burn injury induces a hypermetabolic response that increases morbidity and mortality. Previously, our group showed that insulin resistance after burn injury is associated with endoplasmic reticulum (ER) stress. Evidence suggests that c-Jun N-terminal kinase (JNK) 2 may be involved in ER stress-induced apoptosis. Here, we hypothesized that JNK2 contributes to the apoptotic response after burn injury downstream of ER stress. To test this, we compared JNK2 knockout mice (-/-) with wild-type mice after inducing a 30% total body surface area thermal injury. Animals were killed after 1, 3, and 5 days. Inflammatory cytokines in the blood were measured by multiplex analysis. Hepatic ER stress and insulin signaling were assessed by Western blotting, and insulin resistance was measured by a peritoneal glucose tolerance test. Apoptosis in the liver was quantified by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining. Liver function was quantified by aspartate aminotransferase and alanine aminotransferase activity assays. Endoplasmic reticulum stress increased after burn in both JNK2 and wild-type mice, indicating that JNK2 activation is downstream of ER stress. Knockout of JNK2 did not affect serum inflammatory cytokines; however, the increase in interleukin 6 mRNA expression was prevented in the knockouts. Serum insulin did not significantly increase in the JNK2 group. On the other hand, insulin signaling (PI3K/Akt pathway) and glucose tolerance tests did not improve in JNK2. As expected, apoptosis in the liver increased after burn injury in wild-type mice but not in JNK2. Aspartate aminotransferase/alanine aminotransferase activity revealed that liver function recovered more quickly in JNK2. This study indicates that JNK2 is a central mediator of hepatic apoptosis after a severe burn.

Figure 1

JNK2 knockout genotype confirmed.

Figure 2. Hepatic ER stress

was initiated post-burn in both wildtype and JNK2 knock out (JNK2 −/−) mice. Bip (A) and activated ATF6 (B) levels were quantified 1, 3 and 5 days post-burn relative to β-actin. Representative blots from sham and burn groups at day 1 are shown above each graph. Downstream effectors of ER stress were transcribed in wildtype mice but not JNK2 knockout mice, as indicated by mRNA expression of XBP1s, Pdia3, and Dnajb9 (C–E) measured by real time PCR of day 5 liver samples. Data presented are mean ± SEM. * p < 0.05 and *** p < 0.001 vs. Sham.

Figure 3. Serum insulin, leptin and resistin

(A) Serum insulin was measured by ELISA before and 1, 3, 5 days after burn injury. * p < 0.05 vs. Day 0. # p < 0.05 vs. Day 3. Serum leptin (B) and resistin (C) were measured by multiplex analysis before and 1, 3, 5 days post-burn. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. Day 0. # p < 0.05, ## p < 0.01, ### p < 0.001 vs. Day 1. ΨΨΨ p < 0.001 vs. Day 3. γ p < 0.05 vs. Wildtype. Mean ± SEM.

Figure 4. Inflammatory cytokines

in the serum were measured by ELISA (A). IL-6 mRNA expression in the liver was measured by qPCR relative to 18S rRNA (B). Mean ± SEM. ** p < 0.01 vs. all other means.

Figure 5. Hepatic apoptosis

was reduced in JNK2 −/− mice. Apoptosis was measured by TUNEL staining (A,B). Red (propidium iodide) staining indicates all nuclei and green staining indicates TUNEL positive cells (highlighted by white arrows). ** p < 0.01 vs. Sham. * p < 0.05 vs. Wildtype at day 5. Pro-survival (anti-apoptotic signaling in the liver was measured by Western blotting (C,D). Representative blots from day 1 post-burn are shown above each graph. Mean ± SEM. ** p < 0.01 and *** p < 0.001 vs. Sham. # p < 0.05 vs. Wildtype.

Figure 6. Liver injury and function

A) AST activity indicated that liver function improved more quickly in the JNK2 −/− mice. B) The peak in ALT activity was lower in JNK2 −/− mice (although not significant). C) Albumin mRNA levels were significantly lower in JNK2 −/− mice at 5 days post-burn. Mean ± SEM. * p < 0.05 vs. Day 0. # p < 0.05, ## p < 0.01 vs. Day 1. γ p < 0.05 vs. JNK2 −/−.