Sab (Sh3bp5) dependence of JNK mediated inhibition of mitochondrial respiration in palmitic acid induced hepatocyte lipotoxicity.
ABSTRACT: Sustained c-Jun N-terminal kinase (JNK) activation by saturated fatty acids plays a role in lipotoxicity and the pathogenesis of non-alcoholic steatohepatitis (NASH). We have reported that the interaction of JNK with mitochondrial Sab leads to inhibition of respiration, increased reactive oxygen species (ROS), cell death and hepatotoxicity. We tested whether this pathway underlies palmitic acid (PA)-induced lipotoxicity in hepatocytes.Primary mouse hepatocytes (PMH) from adeno-shlacZ or adeno-shSab treated mice and HuH7 cells were used.In PMH, PA dose-dependently up to 1mM stimulated oxygen consumption rate (OCR) due to mitochondrial ?-oxidation. At ?1.5mM, PA gradually reduced OCR, followed by cell death. Inhibition of JNK, caspases or treatment with antioxidant butylated hydroxyanisole (BHA) protected PMH against cell death. Sab knockdown or a membrane permeable Sab blocking peptide prevented PA-induced mitochondrial impairment, but inhibited only the late phase of both JNK activation (beyond 4h) and cell death. In PMH, PA increased p-PERK and its downstream target CHOP, but failed to activate the IRE-1? arm of the UPR. However, Sab silencing did not affect PA-induced PERK activation. Conversely, specific inhibition of PERK prevented JNK activation and cell death, indicating a major role upstream of JNK activation.The effect of p-JNK on mitochondria plays a key role in PA-mediated lipotoxicity. The interplay of p-JNK with mitochondrial Sab leads to impaired respiration, ROS production, sustained JNK activation, and apoptosis.
Project description:Palmitic acid (PA) induces hepatocyte apoptosis and fuels de novo ceramide synthesis in the endoplasmic reticulum (ER). Myristic acid (MA), a free fatty acid highly abundant in copra/palmist oils, is a predictor of nonalcoholic steatohepatitis (NASH) and stimulates ceramide synthesis. Here we investigated the synergism between MA and PA in ceramide synthesis, ER stress, lipotoxicity and NASH. Unlike PA, MA is not lipotoxic but potentiated PA-mediated lipoapoptosis, ER stress, caspase-3 activation and cytochrome c release in primary mouse hepatocytes (PMH). Moreover, MA kinetically sustained PA-induced total ceramide content by stimulating dehydroceramide desaturase and switched the ceramide profile from decreased to increased ceramide 14:0/ceramide16:0, without changing medium and long-chain ceramide species. PMH were more sensitive to equimolar ceramide14:0/ceramide16:0 exposure, which mimics the outcome of PA plus MA treatment on ceramide homeostasis, than to either ceramide alone. Treatment with myriocin to inhibit ceramide synthesis and tauroursodeoxycholic acid to prevent ER stress ameliorated PA plus MA induced apoptosis, similar to the protection afforded by the antioxidant BHA, the pan-caspase inhibitor z-VAD-Fmk and JNK inhibition. Moreover, ruthenium red protected PMH against PA and MA-induced cell death. Recapitulating in vitro findings, mice fed a diet enriched in PA plus MA exhibited lipodystrophy, hepatosplenomegaly, increased liver ceramide content and cholesterol levels, ER stress, liver damage, inflammation and fibrosis compared to mice fed diets enriched in PA or MA alone. The deleterious effects of PA plus MA-enriched diet were largely prevented by in vivo myriocin treatment. These findings indicate a causal link between ceramide synthesis and ER stress in lipotoxicity, and imply that the consumption of diets enriched in MA and PA can cause NASH associated with lipodystrophy.
Project description: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 + 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.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. (Hepatology 2016;63:1987-2003).
Project description:The c-jun N-terminal kinases (JNKs) are responsive to stress stimuli leading to activation of proapoptotic proteins and transcription. Additionally, JNK mitochondrial localization has been reported. To selectively target mitochondrial JNK signaling, we exploited JNK interaction with its mitochondrial scaffold, Sab, using small interfering RNAs (siRNAs) and a cell-permeable peptide corresponding to the KIM1 domain of Sab. Gene silencing and peptide interference of this interaction disrupted JNK translocation to the mitochondria and reduced phosphorylation of Bcl-2 without significant impact on c-Jun phosphorylation or AP-1 transcription. In contrast, the JNK inhibitory peptide (TI-JIP1) prevented these three functions. Tat-Sab(KIM1) selectivity was also demonstrated in anisomycin-stressed HeLa cells where Tat-Sab(KIM1) prevented Bcl-2 phosphorylation, cell death, loss of mitochondrial membrane potential, and superoxide generation but not c-Jun phosphorylation. Conversely, TI-JIP1 prevented all aforementioned stress-induced events. This probe introduces a means to evaluate JNK-mediated events on the mitochondria without intervening in nuclear functions of JNK.
Project description:c-Jun-N-terminal kinase (JNK) activity plays a critical role in modulating cell death, which depends on the level and duration of JNK activation. The kinase cascade from MAPkinase kinase kinase (MAP3K) to MAPkinase kinase (MAP2K) to MAPKinase (MAPK) can be regulated by a number of direct and indirect post-transcriptional modifications, including acetylation, ubiquitination, phosphorylation, and their reversals. Recently, a JNK-mitochondrial SH3-domain binding protein 5 (SH3BP5/SAB)-ROS activation loop has been elucidated, which is required to sustain JNK activity. Importantly, the level of SAB expression in the outer membrane of mitochondria is a major determinant of the set-point for sustained JNK activation. SAB is a docking protein and substrate for JNK, leading to an intramitochondrial signal transduction pathway, which impairs electron transport and promotes reactive oxygen species (ROS) release to sustain the MAPK cascade.
Project description: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.
Project description:Our aim was to better understand the mechanism and importance of sustained c-Jun N-terminal kinase (JNK) activation in endoplasmic reticulum (ER) stress and effects of ER stress on mitochondria by determining the role of mitochondrial JNK binding protein, Sab. Tunicamycin or brefeldin A induced a rapid and marked decline in basal mitochondrial respiration and reserve-capacity followed by delayed mitochondrial-mediated apoptosis. Knockdown of mitochondrial Sab prevented ER stress-induced sustained JNK activation, impaired respiration, and apoptosis, but did not alter the magnitude or time course of activation of ER stress pathways. P-JNK plus adenosine 5'-triphosphate (ATP) added to isolated liver mitochondria promoted superoxide production, which was amplified by addition of calcium and inhibited by a blocking peptide corresponding to the JNK binding site on Sab (KIM1). This peptide also blocked tunicamycin-induced inhibition of cellular respiration. In conclusion, ER stress triggers an interaction of JNK with mitochondrial Sab, which leads to impaired respiration and increased mitochondrial reactive oxygen species, sustaining JNK activation culminating in apoptosis.
Project description:Sustained JNK activation plays a critical role in hepatotoxicity by acetaminophen or GalN/TNF-?. To address the importance of JNK translocation to mitochondria that accompanies sustained activation in these models, we assessed the importance of the expression of a potential initial target of JNK in the outer membrane of mitochondria, namely Sab (SH3 domain-binding protein that preferentially associates with Btk), also known as Sh3bp5 (SH3 domain-binding protein 5). Silencing the expression of Sab in the liver using adenoviral shRNA inhibited sustained JNK activation and mitochondrial targeting of JNK and the upstream MKK4 (MAPK kinase 4), accompanied by striking protection against liver injury in vivo and in cultured hepatocytes in both toxicity models. We conclude that mitochondrial Sab may serve as a platform for the MAPK pathway enzymes and that the interaction of stress-activated JNK with Sab is required for sustained JNK activation and toxicity.
Project description:trans-Fatty acids (TFAs) are unsaturated fatty acids that contain one or more carbon-carbon double bonds in trans configuration. Epidemiological evidence has linked TFA consumption with various disorders, including cardiovascular diseases. However, the underlying pathological mechanisms are largely unknown. Here, we show a novel toxic mechanism of TFAs triggered by DNA damage. We found that elaidic acid (EA) and linoelaidic acid, major TFAs produced during industrial food manufacturing (so-called as industrial TFAs), but not their corresponding cis isomers, facilitated apoptosis induced by doxorubicin. Consistently, EA enhanced UV-induced embryonic lethality in C. elegans worms. The pro-apoptotic action of EA was blocked by knocking down Sab, a c-Jun N-terminal kinase (JNK)-interacting protein localizing at mitochondrial outer membrane, which mediates mutual amplification of mitochondrial reactive oxygen species (ROS) generation and JNK activation. EA enhanced doxorubicin-induced mitochondrial ROS generation and JNK activation, both of which were suppressed by Sab knockdown and pharmacological inhibition of either mitochondrial ROS generation, JNK, or Src-homology 2 domain-containing protein tyrosine phosphatase 1 (SHP1) as a Sab-associated protein. These results demonstrate that in response to DNA damage, TFAs drive the mitochondrial JNK-Sab-ROS positive feedback loop and ultimately apoptosis, which may provide insight into the common pathogenetic mechanisms of diverse TFA-related disorders.
Project description:3'-Hydroxyacetanilide orN-acetyl-meta-aminophenol (AMAP) is generally regarded as a non-hepatotoxic analog of acetaminophen (APAP). Previous studies demonstrated the absence of toxicity after AMAP in mice, hamsters, primary mouse hepatocytes and several cell lines. In contrast, experiments with liver slices suggested that it may be toxic to human hepatocytes; however, the mechanism of toxicity is unclear. To explore this,we treated primary human hepatocytes (PHH) with AMAP or APAP for up to 48 h and measured several parameters to assess metabolism and injury. Although less toxic than APAP, AMAP dose-dependently triggered cell death in PHH as indicated by alanine aminotransferase (ALT) release and propidium iodide (PI) staining. Similar to APAP, AMAP also significantly depleted glutathione (GSH) in PHH and caused mitochondrial damage as indicated by glutamate dehydrogenase (GDH) release and the JC-1 assay. However, unlike APAP, AMAP treatment did not cause relevant c-jun-N-terminal kinase (JNK) activation in the cytosol or phospho-JNK translocation to mitochondria. To compare, AMAP toxicity was assessed in primary mouse hepatocytes (PMH). No cytotoxicity was observed as indicated by the lack of lactate dehydrogenase release and no PI staining. Furthermore, there was no GSH depletion or mitochondrial dysfunction after AMAP treatment in PMH. Immunoblotting for arylated proteins suggested that AMAP treatment caused extensive mitochondrial protein adduct formation in PHH but not in PMH. In conclusion, AMAP is hepatotoxic in PHH and the mechanism involves the formation of mitochondrial protein adducts and mitochondrial dysfunction.
Project description:BACKGROUND & AIMS:Inclusion of liver grafts from cardiac death donors (CDD) would increase the availability of donor livers but is hampered by a higher risk of primary non-function. Here, we seek to determine mechanisms that contribute to primary non-function of liver grafts from CDD with the goal to develop strategies for improved function and outcome, focusing on c-Jun-N-terminal kinase (JNK) activation and mitochondrial depolarization, two known mediators of graft failure. METHODS:Livers explanted from wild-type, inducible nitric oxide synthase knockout (iNOS(-/-)), JNK1(-/-) or JNK2(-/-) mice after 45-min aorta clamping were implanted into wild-type recipients. Mitochondrial depolarization was detected by intravital confocal microscopy in living recipients. RESULTS:After transplantation of wild-type CDD livers, graft iNOS expression and 3-nitrotyrosine adducts increased, but hepatic endothelial NOS expression was unchanged. Graft injury and dysfunction were substantially higher in CDD grafts than in non-CDD grafts. iNOS deficiency and inhibition attenuated injury and improved function and survival of CDD grafts. JNK1/2 and apoptosis signal-regulating kinase-1 activation increased markedly in wild-type CDD grafts, which was blunted by iNOS deficiency. JNK inhibition and JNK2 deficiency, but not JNK1 deficiency, decreased injury and improved function and survival of CDD grafts. Mitochondrial depolarization and binding of phospho-JNK2 to Sab, a mitochondrial protein linked to the mitochondrial permeability transition, were higher in CDD than in non-CDD grafts. iNOS deficiency, JNK inhibition and JNK2 deficiency all decreased mitochondrial depolarization and blunted ATP depletion in CDD grafts. JNK inhibition and deficiency did not decrease 3-nitrotyrosine adducts in CDD grafts. CONCLUSION:The iNOS-JNK2-Sab pathway promotes CDD graft failure via increased mitochondrial depolarization, and is an attractive target to improve liver function and survival in CDD liver transplantation recipients.