Sanda Win

c-Jun N-terminal Kinase (JNK)-dependent Acute Liver Injury from Acetaminophen or Tumor Necrosis Factor (TNF) Requires Mitochondrial Sab Protein Expression in Mice

Sustained JNK activation plays a critical role in hepatotoxicity by acetaminophen or GalN/TNF-α. 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.

Silencing glycogen synthase kinase-3β 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-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.

JNK interaction with Sab mediates ER stress induced inhibition of mitochondrial respiration and cell death

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.

Receptor interacting protein kinase 1 mediates murine acetaminophen toxicity independent of the necrosome and not through necroptosis

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.(Hepatology 2015;62:1847–1857)

Role of cAMP-responsive Element-binding Protein (CREB)-regulated Transcription Coactivator 3 (CRTC3) in the Initiation of Mitochondrial Biogenesis and Stress Response in Liver Cells

Peroxisome proliferator-activated receptor α, coactivator 1α (PGC-1α) is the master regulator of mitochondrial biogenesis. PGC-1α expression is under the control of the transcription factor, cAMP-responsive element-binding protein (CREB). In searching for candidate transcription factors that mediate mitochondrial stress-initiated mitochondria-to-nucleus signaling in the regulation of mitochondrial biogenesis, we assessed the effect of silencing CREB-regulated transcription co-activators (CRTC). CRTC isoforms are co-activators of CREB-regulated transcription by a CREB phosphorylation-independent pathway. Using cultured HepG2 cells and primary mouse hepatocytes, we determined that mitochondrial stress imposed by the complex I inhibitor rotenone elicited mitochondrial biogenesis, which was dependent on an induction of PGC-1α, which was inhibited by silencing PGC-1α. PGC-1α induction in response to rotenone was inhibited by silencing the expression of CRTC3, which blocked downstream mitochondria biogenesis. In contrast, silencing CRTC2 did not affect the induction of this pathway in response to rotenone. Thus, CRTC3 plays a selective role in mitochondrial biogenesis in response to rotenone.

c‐Jun N‐terminal kinase mediates mouse liver injury through a novel Sab (SH3BP5)‐dependent pathway leading to inactivation of intramitochondrial Src

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. 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. (Hepatology 2016;63:1987‐2003)

Sab (Sh3bp5) dependence of JNK mediated inhibition of mitochondrial respiration in palmitic acid induced hepatocyte lipotoxicity

BACKGROUND & AIMS 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. METHODS Primary mouse hepatocytes (PMH) from adeno-shlacZ or adeno-shSab treated mice and HuH7 cells were used. RESULTS In PMH, PA dose-dependently up to 1mM stimulated oxygen consumption rate (OCR) due to mitochondrial β-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-1α 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. CONCLUSIONS 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.

Immune responses against allogeneic and syngeneic tumors in aged C57BL/6 mice

Aged C57BL/6 (B6) mice could reject allogeneic BALB/c RL male 1 tumor as efficiently as young B6 mice. However, in vitro analysis showed impaired generation of cytotoxic T cell response in aged B6 mice against allogeneic tumor. The reaction could be augmented by the addition of recombinant interleukin‐2 (rIL‐2). Enzyme‐linked immunospots (ELISPOT) produced by CD8+ T cells purified from spleen cells showed no reduction in aged mice. The findings suggested that the number of CD8+ T cells capable of reacting against allogeneic H‐2 antigens was similar in young and aged B6 mice. Low cytotoxic T lymphocyte (CTL) responsiveness in aged B6 mice appeared to have resulted from low responsiveness of CD4+ T cells producing IL‐2. Although CTL generation was apparently impaired, strong multiple antigenicity of allogeneic tumor evoked a rejection response in aged B6 mice. On the other hand, no rejection response was observed against syngeneic EL4 tumor in aged B6 mice even after depletion of CD4CD25+ immunoregulatory cells. Depletion of CD4+CD25+ cells caused rejection of EL4 tumor in young B6 mice. The findings suggested that aged B6 mice were incapable of inducing effector cells against weak tumor antigens. Only marginal CTL response and small number of ELISPOTs were generated in young but not aged B6 mice against EL4. Addition of rIL‐2 to the culture augmented EL4 killing and ELISPOTs in spleen cells from young and aged B6 mice.

Hepatic FcRn regulates albumin homeostasis and susceptibility to liver injury

Significance Neonatal crystallizable fragment receptor (FcRn) regulates immunity and homeostasis of the two most abundant circulating proteins, IgG and albumin. FcRn is expressed in hepatocytes, but hepatic FcRn function is unknown. We show that hepatic FcRn regulates albumin biodistribution. Absence of FcRn in the liver leads to hypoalbuminemia by preventing efficient albumin delivery into the circulation, causing albumin retention within hepatocytes and increasing biliary albumin excretion. Blockade of albumin–FcRn interactions protects liver from damage induced by acetaminophen, a hepatotoxin. This protection results from hepatocyte accumulation of albumin, which scavenges superoxide radicals, and from the redirection of albumin-bound acetaminophen into the bile. Therefore, FcRn-mediated homeostatic distribution of albumin into the bloodstream renders hepatocytes susceptible to acute hepatotoxin exposure, and inhibition of FcRn in the hepatocyte is protective. The neonatal crystallizable fragment receptor (FcRn) is responsible for maintaining the long half-life and high levels of the two most abundant circulating proteins, albumin and IgG. In the latter case, the protective mechanism derives from FcRn binding to IgG in the weakly acidic environment contained within endosomes of hematopoietic and parenchymal cells, whereupon IgG is diverted from degradation in lysosomes and is recycled. The cellular location and mechanism by which FcRn protects albumin are partially understood. Here we demonstrate that mice with global or liver-specific FcRn deletion exhibit hypoalbuminemia, albumin loss into the bile, and increased albumin levels in the hepatocyte. In vitro models with polarized cells illustrate that FcRn mediates basal recycling and bidirectional transcytosis of albumin and uniquely determines the physiologic release of newly synthesized albumin into the basal milieu. These properties allow hepatic FcRn to mediate albumin delivery and maintenance in the circulation, but they also enhance sensitivity to the albumin-bound hepatotoxin, acetaminophen (APAP). As such, global or liver-specific deletion of FcRn results in resistance to APAP-induced liver injury through increased albumin loss into the bile and increased intracellular albumin scavenging of reactive oxygen species. Further, protection from injury is achieved by pharmacologic blockade of FcRn–albumin interactions with monoclonal antibodies or peptide mimetics, which cause hypoalbuminemia, biliary loss of albumin, and increased intracellular accumulation of albumin in the hepatocyte. Together, these studies demonstrate that the main function of hepatic FcRn is to direct albumin into the circulation, thereby also increasing hepatocyte sensitivity to toxicity.

In Vitro Assays of Mitochondrial Function/Dysfunction

In drug‐induced hepatotoxicity, type 2 diabetes, and alcoholic and nonalcoholic fatty liver diseases, liver mitochondria have been recognized to be dysfunctional. Mitochondrial function can be assessed in isolated mitochondria and in intact cells by using O2‐dependent quenching of porphyrin‐based phosphors or by using amperometric O2 sensors to measure O2 consumption.