Sustained c-Jun N-terminal kinase (JNK) activation has been implicated in many models of cell death and tissue injury. Phosphorylated JNK (p-JNK) interacts with the mitochondrial outer membrane SH3 homology associated BTK binding protein (Sab, or SH3BP5). Using knockdown or liver-specific deletion of Sab, we aimed to elucidate the consequences of this interaction on mitochondrial function in isolated mitochondria and liver injury models in vivo. Respiration in isolated mitochondria was directly inhibited by p-JNK 1 adenosine triphosphate. Knockdown or liver-specific knockout of Sab abrogated this effect and markedly inhibited sustained JNK activation and liver injury from acetaminophen or tumor necrosis factor/galactosamine. We then elucidated an intramitochondrial pathway in which interaction of JNK and Sab on the outside of the mitochondria released protein tyrosine phosphatase, nonreceptor type 6 (SHP1, or PTPN6) from Sab in the inside of the mitochondrial outer membrane, leading to its activation and transfer to the inner membrane, where it dephosphorylates P-Y419Src (active), which required a platform protein, docking protein 4 (DOK4), on the inner membrane. Knockdown of mitochondrial DOK4 or SHP1 inhibited the inactivation of mitochondrial p-Src and the effect of p-JNK on mitochondria. Conclusions: The binding to and phosphorylation of Sab by p-JNK on the outer mitochondrial membrane leads to SHP1-dependent and DOK4-dependent inactivation of p-Src on the inner membrane; inactivation of mitochondrial Src inhibits electron transport and increases reactive oxygen species release, which sustains JNK activation and promotes cell death and organ injury.
The c‐Jun‐N‐terminal‐kinase (JNK) family is highly conserved across species such as Drosophila, C. elegans, zebrafish and mammals, and plays a central role in hepatic physiologic and pathophysiologic responses. These responses range from cell death to cell proliferation and carcinogenesis, as well as metabolism and survival, depending on the specific context and duration of activation of the JNK signaling pathway. Recently, several investigators identified the key molecules in the JNK activation loop which include apoptosis signal‐regulating kinase (ASK1) and SH3‐domain binding protein 5 (Sab) and their involvement in acute or chronic liver disease models. Thus, regulating JNK activation through modulating the JNK activation loop may represent an important new strategy in the prevention and treatment of acute and chronic liver diseases. In this review, we will discuss the molecular pathophysiology of the JNK activation loop and its role in the pathogenesis of liver diseases. (Hepatology 2018;67:2013‐2024).
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 3. SAB expression in male mice determines the susceptibility to APAP-induced liver injury in a JNK-dependent fashion. WT C57BL/6N male mice received Ad-lacZ or Ad-SAB. (A) Fourteen days later, SAB expression was analyzed by immunoblotting, and (B) APAP (150 mg/kg) was given i.p., and H&E staining was performed and serum ALT levels were determined after 24 hours. Original magnification, ×10. Scale bars: 100 μm. n = 3/group. ALT: *P < 0.05 versus Ad-lacZ-injected Sab iΔHep mice, by unpaired, 2-tailed Student's t test. Data are presented as the mean ± SD. (C) Sab fl/fl or Sab iΔHep mice received 0.05 5 × 10 9 to approximately 5 × 10 9 IU Ad-lacZ or Ad-SAB. Fourteen days later, SAB expression was determined by immunoblotting. Immunoblot is representative of 3 separate experiments. n = 3/group. *P < 0.05 versus Sab iΔHep + Ad-lacZ, by 1-way ANOVA with Bonferroni's correction. Data are presented as the mean ± SEM. (D) Mice received APAP (300 mg/kg, i.p.), and 24 hours later, liver sections were stained with H&E, and the necrotic area (percentage) was measured. Serum ALT (± SEM) (U/L) was measured. Scale bars: 100 μm. n = 3 mice/group. ALT: *P < 0.05, versus Ad-lacZ-injected Sab fl/fl mice; **P < 0.05, versus Ad-lacZ-injected Sab iΔHep mice, by unpaired, 2-tailed Student's t test. Data are presented as the mean ± SD. (E and F) Jnk1/2 fl/fl mice received AAV8-TBG-GFP (n = 3), AAV8-TBG-Cre (n = 5), or AAV8-TBG-Cre plus Ad-SAB (n = 3). Fourteen days later, JNK and SAB expression levels were determined by immunoblotting, or mice were treated with APAP to analyze liver injury and serum ALT levels. Scale bars: 100 μm. ALT: *P < 0.05, versus AAV8-TBG-GFP, by unpaired, 2-tailed Student's t test. Data are presented as the mean ± SD.
Background and Aims The hepatic mitogen‐activated protein kinase (MAPK) cascade leading to c‐Jun N‐terminal kinase (JNK) activation has been implicated in the pathogenesis of nonalcoholic fatty liver (NAFL)/NASH. In acute hepatotoxicity, we previously identified a pivotal role for mitochondrial SH3BP5 (SAB; SH3 homology associated BTK binding protein) as a target of JNK, which sustains its activation through promotion of reactive oxygen species production. Therefore, we assessed the role of hepatic SAB in experimental NASH and metabolic syndrome. Approach and Results In mice fed high‐fat, high‐calorie, high‐fructose (HFHC) diet, SAB expression progressively increased through a sustained JNK/activating transcription factor 2 (ATF2) activation loop. Inducible deletion of hepatic SAB markedly decreased sustained JNK activation and improved systemic energy expenditure at 8 weeks followed by decreased body fat at 16 weeks of HFHC diet. After 30 weeks, mice treated with control–antisense oligonucleotide (control‐ASO) developed steatohepatitis and fibrosis, which was prevented by Sab‐ASO treatment. Phosphorylated JNK (p‐JNK) and phosphorylated ATF2 (p‐ATF2) were markedly attenuated by Sab‐ASO treatment. After 52 weeks of HFHC feeding, control N‐acetylgalactosamine antisense oligonucleotide (GalNAc‐Ctl‐ASO) treated mice fed the HFHC diet exhibited progression of steatohepatitis and fibrosis, but GalNAc‐Sab‐ASO treatment from weeks 40 to 52 reversed these findings while decreasing hepatic SAB, p‐ATF2, and p‐JNK to chow‐fed levels. Conclusions Hepatic SAB expression increases in HFHC diet–fed mice. Deletion or knockdown of SAB inhibited sustained JNK activation and steatohepatitis, fibrosis, and systemic metabolic effects, suggesting that induction of hepatocyte Sab is an important driver of the interplay between the liver and the systemic metabolic consequences of overfeeding. In established NASH, hepatocyte‐targeted GalNAc‐Sab‐ASO treatment reversed steatohepatitis and fibrosis.
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