Protein kinase C (PKC) participates in acetaminophen hepatotoxicity through c-jun-N-terminal kinase (JNK)-dependent and -independent signaling pathways.

UNLABELLED: This study examines the role of protein kinase C (PKC) and AMP-activated kinase (AMPK) in acetaminophen (APAP) hepatotoxicity. Treatment of primary mouse hepatocytes with broad-spectrum PKC inhibitors (Ro-31-8245, Go6983), protected against APAP cytotoxicity despite sustained c-jun-N-terminal kinase (JNK) activation. Broad-spectrum PKC inhibitor treatment enhanced p-AMPK levels and AMPK regulated survival-energy pathways including autophagy. AMPK inhibition by compound C or activation using an AMPK activator oppositely modulated APAP cytotoxicity, suggesting that p-AMPK and AMPK regulated energy survival pathways, particularly autophagy, play a critical role in APAP cytotoxicity. Ro-31-8245 treatment in mice up-regulated p-AMPK levels, increased autophagy (i.e., increased LC3-II formation, p62 degradation), and protected against APAP-induced liver injury, even in the presence of sustained JNK activation and translocation to mitochondria. In contrast, treatment of hepatocytes with a classical PKC inhibitor (Go6976) protected against APAP by inhibiting JNK activation. Knockdown of PKC-α using antisense (ASO) in mice also protected against APAP-induced liver injury by inhibiting JNK activation. APAP treatment resulted in PKC-α translocation to mitochondria and phosphorylation of mitochondrial PKC substrates. JNK 1 and 2 silencing in vivo decreased APAP-induced PKC-α translocation to mitochondria, suggesting PKC-α and JNK interplay in a feed-forward mechanism to mediate APAP-induced liver injury. CONCLUSION: PKC-α and other PKC(s) regulate death (JNK) and survival (AMPK) proteins, to modulate APAP-induced liver injury.

Dynamic adaptation of liver mitochondria to chronic alcohol feeding in mice: biogenesis, remodeling, and functional alterations.

Liver mitochondria undergo dynamic alterations following chronic alcohol feeding to mice. Intragastric alcohol feeding to mice resulted in 1) increased state III respiration (109% compared with control) in isolated liver mitochondria, probably due to increased levels of complexes I, IV, and V being incorporated into the respiratory chain; 2) increased mitochondrial NAD(+) and NADH levels (∼2-fold), with no change in the redox status; 3) alteration in mitochondrial morphology, with increased numbers of elongated mitochondria; and 4) enhanced mitochondrial biogenesis in the liver, which corresponded with an up-regulation of PGC-1α (peroxisome proliferator-activated receptor γ coactivator-1α). Oral alcohol feeding to mice, which is associated with less liver injury and steatosis, slightly enhanced respiration in isolated liver mitochondria (30.8% compared with control), lower than the striking increase caused by intragastric alcohol feeding. Mitochondrial respiration increased with both oral and intragastric alcohol feeding despite extensive N-acetylation of mitochondrial proteins. The alcohol-induced mitochondrial alterations are probably an adaptive response to enhance alcohol metabolism in the liver. Isolated liver mitochondria from alcohol-treated mice had a greater rate of acetaldehyde metabolism and respiration when treated with acetaldehyde than control. Aldehyde dehydrogenase-2 levels were unaltered in response to alcohol, suggesting that the greater acetaldehyde metabolism by isolated mitochondria from alcohol-treated mice was due to increased mitochondrial respiration that regenerated NAD(+), the rate-limiting substrate in alcohol/acetaldehyde metabolism. Overall, our work suggests that mitochondrial plasticity in the liver may be an important adaptive response to the metabolic stress caused by alcohol intake and could potentially play a role in many other vital functions performed by the liver.

Hepatic stellate cells maintain liver homeostasis through paracrine neurotrophin-3 signaling that induces hepatocyte proliferation.

Organ size is maintained by the controlled proliferation of distinct cell populations. In the mouse liver, hepatocytes in the midlobular zone that are positive for cyclin D1 (CCND1) repopulate the parenchyma at a constant rate to preserve liver mass. Here, we investigated how hepatocyte proliferation is supported by hepatic stellate cells (HSCs), pericytes that are in close proximity to hepatocytes. We used T cells to ablate nearly all HSCs in the murine liver, enabling the unbiased characterization of HSC functions. In the normal liver, complete loss of HSCs persisted for up to 10 weeks and caused a gradual reduction in liver mass and in the number of CCND1+ hepatocytes. We identified neurotrophin-3 (Ntf-3) as an HSC-produced factor that induced the proliferation of midlobular hepatocytes through the activation of tropomyosin receptor kinase B (TrkB). Treating HSC-depleted mice with Ntf-3 restored CCND1+ hepatocytes in the midlobular region and increased liver mass. These findings establish that HSCs form the mitogenic niche for midlobular hepatocytes and identify Ntf-3 as a hepatocyte growth factor.

Silencing glycogen synthase kinase-3beta inhibits acetaminophen hepatotoxicity and attenuates JNK activation and loss of glutamate cysteine ligase and myeloid cell leukemia sequence 1.

Previously we demonstrated that c-Jun N-terminal kinase (JNK) plays a central role in acetaminophen (APAP)-induced liver injury. In the current work, we examined other possible signaling pathways that may also contribute to APAP hepatotoxicity. APAP treatment to mice caused glycogen synthase kinase-3beta (GSK-3beta) activation and translocation to mitochondria during the initial phase of APAP-induced liver injury ( approximately 1 h). The silencing of GSK-3beta, but not Akt-2 (protein kinase B) or glycogen synthase kinase-3alpha (GSK-3alpha), using antisense significantly protected mice from APAP-induced liver injury. The silencing of GSK-3beta affected several key pathways important in conferring protection against APAP-induced liver injury. APAP treatment was observed to promote the loss of glutamate cysteine ligase (GCL, rate-limiting enzyme in GSH synthesis) in liver. The silencing of GSK-3beta decreased the loss of hepatic GCL, and promoted greater GSH recovery in liver following APAP treatment. Silencing JNK1 and -2 also prevented the loss of GCL. APAP treatment also resulted in GSK-3beta translocation to mitochondria and the degradation of myeloid cell leukemia sequence 1 (Mcl-1) in mitochondrial membranes in liver. The silencing of GSK-3beta reduced Mcl-1 degradation caused by APAP treatment. The silencing of GSK-3beta also resulted in an inhibition of the early phase (0-2 h), and blunted the late phase (after 4 h) of JNK activation and translocation to mitochondria in liver following APAP treatment. Taken together our results suggest that activation of GSK-3beta is a key mediator of the initial phase of APAP-induced liver injury through modulating GCL and Mcl-1 degradation, as well as JNK activation in liver.

Signal transduction pathways involved in drug-induced liver injury.

Hepatocyte death following drug intake is the critical event in the clinical manifestation of drug-induced liver injury (DILI). Traditionally, hepatocyte death caused by drugs had been attributed to overwhelming oxidative stress and mitochondria dysfunction caused by reactive metabolites formed during drug metabolism. However, recent studies have also shown that signal transduction pathways activated/inhibited during oxidative stress play a key role in DILI. In acetaminophen (APAP)-induced liver injury, hepatocyte death requires the sustained activation of c-Jun kinase (JNK), a kinase important in mediating apoptotic and necrotic death. Inhibition of JNK using chemical inhibitors or knocking down JNK can prevent hepatocyte death even in the presence of extensive glutathione (GSH) depletion, covalent binding, and oxidative stress. Once activated, JNK translocates to mitochondria, to induce mitochondria permeability transition and trigger hepatocyte death. Mitochondria are central targets where prodeath kinases such as JNK, prosurvival death proteins such as bcl-xl, and oxidative damage converge to determine hepatocyte survival. The importance of mitochondria in DILI is also observed in the Mn-SOD heterozygous (+/-) model, where mice with less mitochondrial Mn-SOD are sensitized to liver injury caused by certain drugs. An extensive body of research is accumulating suggesting a central role of mitochondria in DILI. Drugs can also cause redox changes that inhibit important prosurvival pathways such as NF-kappaB. The inhibition of NF-kappaB by subtoxic doses of APAP sensitizes hepatocyte to the cytotoxic actions of tumor necrosis factor (TNF). Many drugs will induce liver injury if simultaneously treated with LPS, which promotes inflammation and cytokine release. Drugs may be sensitizing hepatocytes to the cytotoxic effects of cytokines such as TNF, or vice versa. Overall many signaling pathways are important in regulating DILI, and represent potential therapeutic targets to reduce liver injury caused by drugs.

Detection of Novel Fusion Transcript VTI1A-CFAP46 in Hepatocellular Carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is now the second-highest cause of cancer death worldwide. Recent studies have discovered a wide range of somatic mutations in HCC. These mutations involve various vital signaling pathways such as: Wnt/ß-Catenin, p53, telome-rase reverse transcriptase (TERT), chromatin remodeling, RAS/MAPK signaling, and oxidative stress. However, fusion transcripts have not been broadly explored in HCC. METHODS: To identify novel fusion transcripts in HCC, in the first phase of our study, we performed targeted RNA sequencing (in HCC and paired non-HCC tissues) on 6 patients with a diagnosis of HCC undergoing liver transplantation. RESULTS: As a result of these studies, we discovered the novel fusion transcript, VTI1A-CFAP46. In the second phase of our study, we measured the expression of wild-type VTI1A in 21 HCC specimens, which showed that 10 of 21 exhibited upregulation of wild-type VTI1A in their tumors. VTI1A (Vesicle Transport via Interaction with t-SNARE homolog 1A) is a member of the Soluble N-ethylmaleimide-Sensitive Factor (NSF) attachment protein receptor (SNARE) gene family, which is essential for membrane trafficking and function in endocytosis, autophagy, and Golgi transport. Notably, it is known that autophagy is involved in HCC. CONCLUSIONS: The link between novel fusion transcript VTI1A-CFAP46 and autophagy as a potential therapeutic target in HCC patients deserves further investigation. Moreover, this study shows that fusion transcripts are worthy of additional exploration in HCC.

Autophagy is a gatekeeper of hepatic differentiation and carcinogenesis by controlling the degradation of Yap.

Activation of the Hippo pathway effector Yap underlies many liver cancers, however no germline or somatic mutations have been identified. Autophagy maintains essential metabolic functions of the liver, and autophagy-deficient murine models develop benign adenomas and hepatomegaly, which have been attributed to activation of the p62/Sqstm1-Nrf2 axis. Here, we show that Yap is an autophagy substrate and mediator of tissue remodeling and hepatocarcinogenesis independent of the p62/Sqstm1-Nrf2 axis. Hepatocyte-specific deletion of Atg7 promotes liver size, fibrosis, progenitor cell expansion, and hepatocarcinogenesis, which is rescued by concurrent deletion of Yap. Our results shed new light on mechanisms of Yap degradation and the sequence of events that follow disruption of autophagy, which is impaired in chronic liver disease.

Lysosomal Cholesterol Accumulation Sensitizes To Acetaminophen Hepatotoxicity by Impairing Mitophagy.

The role of lysosomes in acetaminophen (APAP) hepatotoxicity is poorly understood. Here, we investigated the impact of genetic and drug-induced lysosomal cholesterol (LC) accumulation in APAP hepatotoxicity. Acid sphingomyelinase (ASMase)(-/-) mice exhibit LC accumulation and higher mortality after APAP overdose compared to ASMase(+/+) littermates. ASMase(-/-) hepatocytes display lower threshold for APAP-induced cell death and defective fusion of mitochondria-containing autophagosomes with lysosomes, which decreased mitochondrial quality control. LC accumulation in ASMase(+/+) hepatocytes caused by U18666A reproduces the susceptibility of ASMase(-/-) hepatocytes to APAP and the impairment in the formation of mitochondria-containing autolysosomes. LC extraction by 25-hydroxycholesterol increased APAP-mediated mitophagy and protected ASMase(-/-) mice and hepatocytes against APAP hepatotoxicity, effects that were reversed by chloroquine to disrupt autophagy. The regulation of LC by U18666A or 25-hydroxycholesterol did not affect total cellular sphingomyelin content or its lysosomal distribution. Of relevance, amitriptyline-induced ASMase inhibition in human hepatocytes caused LC accumulation, impaired mitophagy and increased susceptibility to APAP. Similar results were observed upon glucocerebrosidase inhibition by conduritol ß-epoxide, a cellular model of Gaucher disease. These findings indicate that LC accumulation determines susceptibility to APAP hepatotoxicity by modulating mitophagy, and imply that genetic or drug-mediated ASMase disruption sensitizes to APAP-induced liver injury.

Determination of GSH, GSSG, and GSNO using HPLC with electrochemical detection.

GSNO is an important intermediate in nitric oxide metabolism and mediates many ()NO-mediated signaling pathways through the post-translational modification of redox-sensitive proteins. The detection of GSNO in biological samples has been hampered by a lack of sensitive and simple assays. In this work, we describe the utilization of HPLC with electrochemical detection for the identification and quantification of GSNO in biological samples. GSNO requires a high potential (>700 mV) for its electrochemical detection, similar to that of GSSG. A simple isocratic HPLC system can be used to separate and simultaneously detect GSH, GSSG, and GSNO electrochemically. This HPLC system can be utilized to measure the redox profile of biological samples and applied for the measurement of GSNO reductase activity in cells. Proper sample preparation is essential in GSNO measurements, because artifactual formation of GSNO occurs in acidic conditions due to the reaction between GSH and nitrite. Treatment of samples with ammonium sulfamate or N-ethylmaleimide (NEM) can prevent the artifactual formation of GSNO and accurately detect GSNO in biological samples. Overall, the HPLC with electrochemical detection is a powerful tool to measure redox status in cells and tissues.

Redox regulation of tumor necrosis factor signaling.

Tumor necrosis factor-alpha (TNF) is a key cytokine that has been shown to play important physiologic (e.g., inflammation) and pathophysiologic (e.g., various liver pathologies) roles. In liver and other tissues, TNF treatment results in the simultaneous activation of an apoptotic pathway (i.e., TRADD, RIP, JNK) and a survival pathway mediated by NF-kappaB transcription of survival genes (i.e., GADD45beta, Mn-SOD, cFLIP). The cellular response (e.g., proliferation versus apoptosis) to TNF is determined by the balance between the apoptotic signaling pathway and the NF-kappaB survival pathway stimulated by TNF. Reactive oxygen species (ROS) are important modulators of signaling pathways and can regulate both apoptotic signaling and NF-kappaB transcription triggered by TNF. ROS are important in mediating the sustained activation of JNK, to help mediate apoptosis after TNF treatment. In some cells, ROS are second messengers that mediate apoptosis after TNF stimulation. Conversely, ROS can cause redox modifications that inhibit NF-kappaB activation, which can lead to cell death triggered by TNF. Consequently, the redox status of cells can determine the biologic response that TNF will induce in cells. In many liver pathologies, ROS generated extrinsically (e.g., inflammation) or intrinsically (i.e., drugs, toxins) may act in concert with TNF to promote hepatocyte death and liver injury through redox inhibition of NF-kappaB.

Regulation of H(2)O(2)-induced necrosis by PKC and AMP-activated kinase signaling in primary cultured hepatocytes.

Recent studies have suggested that, in certain cases, necrosis, like apoptosis, may be programmed, involving the activation and inhibition of many signaling pathways. In this study, we examined whether necrosis induced by H(2)O(2) is regulated by signaling pathways in primary hepatocytes. A detailed time course revealed that H(2)O(2) treated to hepatocytes is consumed within minutes, but hepatocytes undergo necrosis several hours later. Thus, H(2)O(2) treatment induces a "lag phase" where signaling changes occur, including PKC activation, Akt (PKB) downregulation, activation of JNK, and downregulation of AMP-activated kinase (AMPK). Investigation of various inhibitors demonstrated that PKC inhibitors were effective in reducing necrosis caused by H(2)O(2) (~80%). PKC inhibitor treatment decreased PKC activity but, surprisingly, also upregulated Akt and AMPK, suggesting that various PKC isoforms negatively regulate Akt and AMPK. Akt did not appear to play a significant role in H(2)O(2)-induced necrosis, since PKC inhibitor treatment protected hepatocytes from H(2)O(2) even when Akt was inhibited. On the other hand, compound C, a selective AMPK inhibitor, abrogated the protective effect of PKC inhibitors against necrosis induced by H(2)O(2). Furthermore, AMPK activators protected against H(2)O(2)-induced necrosis, suggesting that much of the protective effect of PKC inhibition was mediated through the upregulation of AMPK. Work with PKC inhibitors suggested that atypical PKC downregulates AMPK in response to H(2)O(2). Knockdown of PKC-alpha using antisense oligonucleotides also slightly protected (~22%) against H(2)O(2). Taken together, our data demonstrate that the modulation of signaling pathways involving PKC and AMPK can alter H(2)O(2)-induced necrosis, suggesting that a signaling "program" is important in mediating H(2)O(2)-induced necrosis in primary hepatocytes.