Although necrosis in the acetaminophen (APAP) model is known to be regulated by c‐Jun NH2‐terminal kinase (JNK) through interaction with mitochondria, the role of necroptosis through receptor‐interacting proteins 1 and 3 (RIPK1 and RIPK3) has also been suggested. Our aim was to determine the relationship between these two mechanisms of cell death. To verify the participation of RIPK1, we used antisense knockdown and confirmed protection comparable to the RIPK1 inhibitor, necrostatin, in vivo and in vitro . However, we found no evidence that RIPK3 is expressed in primary mouse hepatocytes under basal conditions or after APAP and RIPK3 −/− mice were not protected. RIPK3 was exclusively expressed in nonparenchymal cells. RIPK1 knockdown protected RIPK3 −/− mice to the same extent as wild‐type mice, underscoring the independent role of RIPK1. We confirmed that necroptosis is not involved in APAP toxicity by using mixed lineage kinase domain‐like protein (MLKL) knockout mice, which were not protected from APAP. Next, we addressed whether there is interplay between RIPK1 and JNK. RIPK1 knockdown decreased the level of JNK activation and translocation to mitochondria and abrogated subsequent translocation of dynamin‐related protein 1 (Drp1). Interestingly, APAP induced translocation of RIPK1 to mitochondria, which was unaffected by knockdown of the mitochondrial JNK docking protein, Sh3 homology 3 binding protein 5 (Sab). Conclusion : RIPK1 participates in APAP‐induced necrosis upstream of JNK activation whereas RIPK3 and MLKL are dispensable, indicating that necroptosis does not contribute to APAP‐induced necrosis and RIPK1 has a unique, independent role.(H epatology 2015;62:1847–1857)

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-alpha using antisense (ASO) in mice also protected against APAP-induced liver injury by inhibiting JNK activation. APAP treatment resulted in PKC-alpha translocation to mitochondria and phosphorylation of mitochondrial PKC substrates. JNK 1 and 2 silencing in vivo decreased APAP-induced PKC-alpha translocation to mitochondria, suggesting PKC-alpha and JNK interplay in a feed-forward mechanism to mediate APAP-induced liver injury. Conclusion: PKC-alpha and other PKC(s) regulate death (JNK) and survival (AMPK) proteins, to modulate APAP-induced liver injury. (Hepatology 2014;59:1543-1554)

The Th2 cytokines IL-4 and IL-1 3 mediate allergic pulmonary inflammation and airways hyperreactivity (AHR) in asthma models through signaling dependent upon the IL-4 receptor-a chain (IL-4R alpha). IL-13 has been further implicated in the overproduction of mucus by the airway epithelium and in lung remodeling that commonly accompanies chronic inflammation. IL-4R alpha-deficient mice are resistant to allergen-induced asthma, highlighting the therapeutic promise of selective molecular inhibitors of IL-4R alpha. We designed a chemically modified IL-4R alpha antisense oligonucleotide (IL-4R alpha ASO) that specifically inhibits IL-4R alpha protein expression in lung eosinophils, macrophages, dendritic cells, and airway epithelium after inhalation in allergen-challenged mice. Inhalation of IL-4R alpha ASO attenuated allergen-induced AHR, suppressed airway eosinophilia and neutrophilia, and inhibited production of airway Th2 cytokines and chemokines in previously allergen-primed and -challenged mice. Histologic analysis of lungs from these animals demonstrated reduced goblet cell metaplasia and mucus staining that correlated with inhibition of Muc5AC gene expression in lung tissue. Therapeutic administration of inhaled IL-4R alpha ASO in chronically allergen-challenged mice produced a spectrum of anti-inflammatory activity similar to that of systemically administered Dexamethasone with the added benefit of reduced airway neutrophilia. These data support the potential utility of a dual IL-4 and IL-13 oligonucleotide inhibitor in allergy/asthma, and suggest that local inhibition of IL-4R alpha in the lung is sufficient to suppress allergen-induced pulmonary inflammation and AHR.

Antisense oligonucleotides (ASO) containing 2'-O-methoxyethyl ribose (2'-MOE) modifications have been shown to possess both excellent pharmacokinetic properties and robust pharmacological activity in several animal models of human disease. 2'-MOE ASOs are generally well tolerated, displaying minimal to mild proinflammatory effect at doses far exceeding therapeutic doses. Although the vast majority of 2'-MOE ASOs are safe and well tolerated, a small subset of ASOs inducing acute inflammation in mice has been identified. The mechanism for these findings is not clear at this point, but the effects are clearly sequence-specific. One of those ASOs, ISIS 147420, causes a severe inflammatory response atypical of this class of oligonucleotides characterized by induction in interferon-β (IFN-β) at 48 h followed by acute transaminitis and extensive hepatocyte apoptosis and necrosis at 72 h. A large number of interferon-stimulated genes were significantly up-regulated in liver as early as 24 h. We speculated that a specific sequence motif might cause ISIS 147420 to be mistaken for viral RNA or DNA, thus triggering an acute innate immune response. ISIS 147420 toxicity was independent of Toll-like receptors, because there was no decrease in IFN-β in Toll/interleukin-1 receptor-domain-containing adapter-inducing IFN-β or Myd88-deficient mice. The involvement of cytosolic retinoic acid-inducible gene (RIG)-I-like pattern recognition receptors was also investigated. Pretreatment of mice with melanoma differentiation-associated gene 5 (MDA5) and IFN-β promoter stimulator-1 ASOs, but not RIG-I or laboratory of genetics and physiology 2 (LGP2) ASOs, prevented the increase in IFN-β and alanine aminotransferase induced by ISIS 147420. These results revealed a novel mechanism of oligonucleotide-mediated toxicity requiring both MDA5 and IPS-1 and resulting in the activation of the innate immune response.

BACKGROUND & AIMS:After liver injury, bone marrow-derived liver sinusoidal endothelial cell progenitor cells (BM SPCs) repopulate the sinusoid as liver sinusoidal endothelial cells (LSECs). After partial hepatectomy, BM SPCs provide hepatocyte growth factor, promote hepatocyte proliferation, and are necessary for normal liver regeneration. We examined how hepatic vascular endothelial growth factor (VEGF) regulates recruitment of BM SPCs and their effects on liver injury.METHODS:Rats were given injections of dimethylnitrosamine to induce liver injury, which was assessed by histology and transaminase assays. Recruitment of SPCs was analyzed by examining BM SPC proliferation, mobilization to the circulation, engraftment in liver, and development of fenestration (differentiation).RESULTS:Dimethylnitrosamine caused extensive denudation of LSECs at 24 hours, followed by centrilobular hemorrhagic necrosis at 48 hours. Proliferation of BM SPCs, the number of SPCs in the bone marrow, and mobilization of BM SPCs to the circulation increased 2- to 4-fold by 24 hours after injection of dimethylnitrosamine; within 5 days, 40% of all LSECs came from engrafted BM SPCs. Allogeneic resident SPCs, infused 24 hours after injection of dimethylnitrosamine, repopulated the sinusoid as LSECs and reduced liver injury. Expression of hepatic VEGF messenger RNA and protein increased 5-fold by 24 hours after dimethylnitrosamine injection. Knockdown of hepatic VEGF with antisense oligonucleotides completely prevented dimethylnitrosamine-induced proliferation of BM SPCs and their mobilization to the circulation, reduced their engraftment by 46%, completely prevented formation of fenestration after engraftment as LSECs, and exacerbated dimethylnitrosamine injury.CONCLUSIONS:BM SPC recruitment is a repair response to dimethylnitrosamine liver injury in rats. Hepatic VEGF regulates recruitment of BM SPCs to liver and reduces this form of liver injury.

BACKGROUND & AIMS:Capillarization, characterized by loss of differentiation of liver sinusoidal endothelial cells (LSECs), precedes the onset of hepatic fibrosis. We investigated whether restoration of LSEC differentiation would normalize crosstalk with activated hepatic stellate cells (HSC) and thereby promote quiescence of HSC and regression of fibrosis. METHODS:Rat LSECs were cultured with inhibitors and/or agonists and examined by scanning electron microscopy for fenestrae in sieve plates. Cirrhosis was induced in rats using thioacetamide, followed by administration of BAY 60-2770, an activator of soluble guanylate cyclase (sGC). Fibrosis was assessed by Sirius red staining; expression of α-smooth muscle actin was measured by immunoblot analysis. RESULTS:Maintenance of LSEC differentiation requires vascular endothelial growth factor-A stimulation of nitric oxide-dependent signaling (via sGC and cyclic guanosine monophosphate) and nitric oxide-independent signaling. In rats with thioacetamide-induced cirrhosis, BAY 60-2770 accelerated the complete reversal of capillarization (restored differentiation of LSECs) without directly affecting activation of HSCs or fibrosis. Restoration of differentiation to LSECs led to quiescence of HSCs and regression of fibrosis in the absence of further exposure to BAY 60-2770. Activation of sGC with BAY 60-2770 prevented progression of cirrhosis, despite continued administration of thioacetamide. CONCLUSIONS:The state of LSEC differentiation plays a pivotal role in HSC activation and the fibrotic process.

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-3β (GSK-3β) activation and translocation to mitochondria during the initial phase of APAP-induced liver injury (∼1 h). The silencing of GSK-3β, but not Akt-2 (protein kinase B) or glycogen synthase kinase-3α (GSK-3α), using antisense significantly protected mice from APAP-induced liver injury. The silencing of GSK-3β 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-3β 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-3β translocation to mitochondria and the degradation of myeloid cell leukemia sequence 1 (Mcl-1) in mitochondrial membranes in liver. The silencing of GSK-3β reduced Mcl-1 degradation caused by APAP treatment. The silencing of GSK-3β 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-3β 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.

Activation of c-Jun N-terminal kinase (JNK) has been implicated as a mechanism in the development of steatohepatitis. This finding, together with the reported role of JNK signaling in the development of obesity and insulin resistance, two components of the metabolic syndrome and predisposing factors for fatty liver disease, suggests that JNK may be a central mediator of the metabolic syndrome and an important therapeutic target in steatohepatitis. To define the isoform-specific functions of JNK in steatohepatitis associated with obesity and insulin resistance, the effects of JNK1 or JNK2 ablation were determined in developing and established steatohepatitis induced by a high-fat diet (HFD). HFD-fed jnk1 null mice failed to develop excessive weight gain, insulin resistance, or steatohepatitis. In contrast, jnk2(-/-) mice fed a HFD were obese and insulin-resistant, similar to wild-type mice, and had increased liver injury. In mice with established steatohepatitis, an antisense oligonucleotide knockdown of jnk1 decreased the amount of steatohepatitis in concert with a normalization of insulin sensitivity. Knockdown of jnk2 improved insulin sensitivity but had no effect on hepatic steatosis and markedly increased liver injury. A jnk2 knockdown increased hepatic expression of the proapoptotic Bcl-2 family members Bim and Bax and the increase in liver injury resulted in part from a Bim-dependent activation of the mitochondrial death pathway. Conclusion: JNK1 and JNK2 both mediate insulin resistance in HFD-fed mice, but the JNK isoforms have distinct effects on steatohepatitis, with JNK1 promoting steatosis and hepatitis and JNK2 inhibiting hepatocyte cell death by blocking the mitochondrial death pathway. (HEPATOLOGY 2009;49:87-96.)

Previously, we demonstrated JNK plays a central role in acetaminophen (APAP)-induced liver injury (Gunawan, B. K., Liu, Z. X., Han, D., Hanawa, N., Gaarde, W. A., and Kaplowitz, N. (2006) Gastroenterology 131, 165-178). In this study, we examine the mechanism involved in activating JNK and explore the downstream targets of JNK important in promoting APAP-induced liver injury in vivo. JNK inhibitor (SP600125) was observed to significantly protect against APAP-induced liver injury. Increased mitochondria-derived reactive oxygen species were implicated in APAP-induced JNK activation based on the following: 1) mitochondrial GSH depletion (maximal at 2 h) caused increased H2O2 release from mitochondria, which preceded JNK activation (maximal at 4 h); 2) treatment of isolated hepatocytes with H2O2 or inhibitors (e.g. antimycin) that cause increased H2O2 release from mitochondria-activated JNK. An important downstream target of JNK following activation was mitochondria based on the following: 1) JNK translocated to mitochondria following activation; 2) JNK inhibitor treatment partially protected against a decline in mitochondria respiration caused by APAP treatment; and 3) addition of purified active JNK to mitochondria isolated from mice treated with APAP plus JNK inhibitor (mitochondria with severe GSH depletion, covalent binding) directly inhibited respiration. Cyclosporin A blocked the inhibitory effect of JNK on mitochondria respiration, suggesting JNK was directly inducing mitochondrial permeability transition in isolated mitochondria from mice treated with APAP plus JNK inhibitor. Addition of JNK to mitochondria isolated from control mice did not affect respiration. Our results suggests that APAP-induced liver injury involves JNK activation, due to increased reactive oxygen species generated by GSH-depleted mitochondria, and translocation of activated JNK to mitochondria where JNK induces mitochondrial permeability transition and inhibits mitochondria bioenergetics.

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.