Protective role of HO-1 and carbon monoxide in ethanol-induced hepatocyte cell death and liver injury in mice.

BACKGROUND & AIMS: Alcoholic liver disease is associated with inflammation and cell death. Heme oxygenase-1 (HO-1) is a stress-inducible enzyme with anti-apoptotic and anti-inflammatory properties. Here we tested the hypothesis that induction of HO-1 or treatment with a carbon monoxide releasing molecule (CORM) during chronic ethanol exposure protects and/or reverses ethanol-induced liver injury. METHODS: Female C57BL/6J mice were allowed free access to a complete liquid diet containing ethanol or to pair-fed control diets for 25days. Mice were treated with cobalt protoporphyrin (CoPP) to induce HO-1 expression during ethanol feeding or once liver injury had been established. Mice were also treated with CORM-A1, a CO-releasing molecule (CORM), after ethanol-induced liver injury was established. The impact of HO-1 induction on ethanol-induced cell death was investigated in primary cultures of hepatocytes. RESULTS: Induction of HO-1 during or after ethanol feeding, as well as treatment with CORM-A1, ameliorated ethanol-induced increases in AST and expression of mRNAs for inflammatory cytokines. Treatment with CoPP or CORM-A1 also reduced hepatocyte cell death, indicated by decreased accumulation of CK18 cleavage products and reduced RIP3 expression in hepatocytes. Exposure of primary hepatocyte cultures to ethanol increased their sensitivity to TNFα-induced cell death; this response was attenuated by necrostatin-1, an inhibitor of necroptosis, but not by caspase inhibitors. Induction of HO-1 with CoPP or CORM-3 treatment normalized the sensitivity of hepatocytes to TNFα-induced cell death after ethanol exposure. CONCLUSIONS: Therapeutic strategies to increase HO-1 and/or modulate CO availability ameliorated chronic ethanol-induced liver injury in mice, at least in part by decreasing hepatocellular death.

Antioxidant-induced INrf2 (Keap1) tyrosine 85 phosphorylation controls the nuclear export and degradation of the INrf2-Cul3-Rbx1 complex to allow normal Nrf2 activation and repression.

INrf2 (Keap1) serves as a negative regulator of the cytoprotective transcription factor Nrf2. At basal levels, INrf2 functions as a substrate adaptor to sequester Nrf2 into the Cul3-Rbx1 E3 ligase complex for ubiquitylation and proteasomal degradation. In response to antioxidants, Nrf2 is released from the INrf2-Cul3-Rbx1 complex and translocates into the nucleus, where it activates ARE-mediated cytoprotective gene expression. The present studies demonstrate that INrf2, Cul3 and Rbx1 export out of the nucleus and are degraded during the early or pre-induction response to antioxidants. Mutation of Tyr85 in INrf2 stymied the nuclear export of INrf2, suggesting that tyrosine phosphorylation controls the pre-induction nuclear export and degradation in response to antioxidants. The nuclear export of Cul3-Rbx1 were also blocked when INrf2Tyr85 was mutated, suggesting that INrf2-Cul3-Rbx1 undergo nuclear export as a complex. INrf2 siRNA also inhibited the nuclear export of Cul3-Rbx1, confirming that Cul3-Rbx1 requires INrf2 for nuclear export. Newly synthesized INrf2-Cul3-Rbx1 is imported back into the nucleus during the post-induction period to ubiquitylate and degrade Nrf2. Mutation of INrf2Tyr85 had no effect on activation of Nrf2 but led to nuclear accumulation of Nrf2 during the post-induction period owing to reduced export and degradation of Nrf2. Our results also showed that nuclear export and degradation followed by the new synthesis of INrf2-Cul3-Rbx1 controls the cellular abundance of the proteins during different phases of antioxidant responses. In conclusion, the early or pre-induction nuclear export of INrf2 in response to antioxidants is controlled by tyrosine phosphorylation, whereas the nuclear export of Cul3 and Rbx1 is controlled by INrf2, allowing normal activation or repression of Nrf2.

Tyrosine phosphorylation controls nuclear export of Fyn, allowing Nrf2 activation of cytoprotective gene expression.

Fyn, an Src kinase family member, acts as a negative regulator of NF-E2-related factor 2 (Nrf2). Under stressful conditions, Nrf2 translocates into the nucleus and binds to the antioxidant response element (ARE), activating defensive gene expression. Once Nrf2 completes activation, Fyn phosphorylates tyrosine 568 of Nrf2, resulting in the nuclear export and degradation of Nrf2. The present studies demonstrate that within 0.5 h of antioxidant treatment in human hepatoblastoma (HepG2) cells, Fyn exports out of the nucleus, allowing Nrf2 unimpeded movement to the ARE. Mutation of tyrosine 213 of Fyn stymied nuclear export, suggesting that tyrosine phosphorylation controls nuclear export. Mass spectrometry confirmed tyrosine 213 as the site of phosphorylation. ChIP and real-time PCR assays revealed that FynY213A mutant caused decreased binding of Nrf2 to the promoter of defensive gene NAD(P)H:quinone oxidoreductase 1 (NQO1) and decreased NQO1 expression by 5-fold (P<0.0001) compared to wild-type Fyn. In addition, a putative nuclear export signal (NES) was identified, and mutation of it also inhibited nuclear export of Fyn. Furthermore, FynY213A caused an increased susceptibility to cell death following treatment with etoposide in mouse hepatoma (Hepa-1) cells. The preinduction regulation of Nrf2 is controlled by the nuclear export of Fyn, allowing for activation of defensive gene expression.

An autoregulatory loop between Nrf2 and Cul3-Rbx1 controls their cellular abundance.

The INrf2 (Keap1)/Cul3-Rbx1 complex constantly degrades Nrf2 under normal conditions. When a cell encounters oxidative or electrophilic stress, Nrf2 dissociates from the INrf2/Cul3-Rbx1 complex and translocates into the nucleus. In the nucleus, Nrf2 activates a myriad of antioxidant and defensive genes that protect cells. Nrf2 is then exported out of the nucleus and degraded. INrf2 serves as a substrate adaptor to link Nrf2 to Cul3 and Rbx1. Cul3 and Rbx1 make up the ubiquitin ligase complex that is responsible for the ubiquitination and degradation of Nrf2. Previously we have shown a feedback autoregulatory loop between Nrf2 and INrf2 indicating that Nrf2 regulates INrf2 by controlling its transcription. Here we are extending this research by demonstrating the presence of another feedback autoregulatory loop between Cul3-Rbx1 and Nrf2. Experiments using Hepa-1 and HepG2 cells indicate that Nrf2 controls its own degradation by regulating expression and induction of Cul3-Rbx1 genes. Treatment with the antioxidant tert-Butylhydroquinone (t-BHQ) leads to induction of Cul3-Rbx1 genes. Mutagenesis and transfection experiments identified an antioxidant response element in the forward and reverse strands of the proximal Cul3 and Rbx1 promoters, respectively, that Nrf2 binds and regulates expression and antioxidant induction of the Cul3-Rbx1 genes. In addition, short interfering RNA inhibition and overexpression of Nrf2 led to a respective decrease and increase in Cul3-Rbx1 gene expression. The increase in Cul3-Rbx1 leads to ubiquitination and degradation of Nrf2. These data suggest that Nrf2 regulates Cul3-Rbx1 by controlling regulation of expression and induction of Cul3-Rbx1. The induction of Cul3-Rbx1 control Nrf2 by increasing degradation.

Antioxidant-induced phosphorylation of tyrosine 486 leads to rapid nuclear export of Bach1 that allows Nrf2 to bind to the antioxidant response element and activate defensive gene expression.

Antioxidants cause stabilization and nuclear translocation of NF-E2-related factor 2 (Nrf2), where it binds to the antioxidant response element (ARE) and induces up-regulation of defensive genes that protect cells against oxidative and electrophilic stress. Bach1, the negative regulator of Nrf2, competes with Nrf2 for binding to the ARE in the human NQO1 promoter. In this study, we demonstrate that Bach1 exits the nucleus within 1-2 h upon antioxidant treatment. Genistein, an inhibitor of tyrosine kinases, blocked nuclear export of Bach1. Site-directed mutagenesis and immunoprecipitation assays identified tyrosine 486 that was phosphorylated in response to the antioxidant and was essential for nuclear export of Bach1. Chromatin immunoprecipitation assays revealed a competitive interplay between Bach1 and Nrf2 at 1-2 and 4 h for binding to the human NQO1 ARE. Luciferase and real time PCR assays showed a significant decrease in antioxidant induction of reporter activity and mRNA levels in cells transfected with mutant Bach1 compared with wild type. This decrease was due to the absence of nuclear export of the mutant protein. Bach1 levels inside the nucleus returned to normal at 4 h after antioxidant treatment in the absence but not in the presence of protein synthesis inhibitor cycloheximide. In addition, antioxidant treatment increased the transcription of Bach1 as shown by pulse chase and real time PCR experiments. Taken together, these results indicate that increased synthesis of Bach1 restored its nuclear levels to normal at 4 h. In conclusion, antioxidant-induced tyrosine 486 phosphorylation leads to nuclear exit of Bach1, thus allowing Nrf2 access to the ARE.

Nrf2 signaling and cell survival.

Nrf2:INrf2 acts as a sensor for oxidative/electrophilic stress. INrf2 serves as an adaptor to link Nrf2 to the ubiquitin ligase Cul3-Rbx1 complex that ubiquitinate and degrade Nrf2. Under basal conditions, cytosolic INrf2/Cul3-Rbx1 is constantly degrading Nrf2. When a cell encounters stress Nrf2 dissociates from the INrf2 and translocates into the nucleus. Oxidative/electrophilic stress induced modification of INrf2Cysteine151 and/or protein kinase C (PKC)-mediated phosphorylation of Nrf2Serine40 controls Nrf2 release from INrf2 followed by stabilization and nuclear translocation of Nrf2. Nrf2 binds to the antioxidant response element (ARE) and activates a myriad of genes that protect cells against oxidative/electrophilic stress and neoplasia. A delayed response of oxidative/electrophilic stress activates GSK-3beta that phosphorylates Fyn at unknown threonine residue(s). Phosphorylated Fyn translocates to the nucleus and phosphorylates Nrf2Tyrosine568 that leads to nuclear export and degradation of Nrf2. Prothymosin-alpha mediated nuclear translocation of INrf2 also degrades nuclear Nrf2. The degradation of Nrf2 both in cytosol and nuclear compartments rapidly brings down its levels to normal resulting in suppression of Nrf2 downstream gene expression. An auto-regulatory loop between Nrf2 and INrf2 controls their cellular abundance. Nrf2 regulates INrf2 by controlling its transcription, and INrf2 controls Nrf2 by degrading it. In conclusion, switching on and off of Nrf2 combined with promoting an auto-regulatory loop between them regulates activation/deactivation of defensive genes leading to protection of cells against adverse effects of oxidative and electrophilic stress and promote cell survival.

Nrf2:INrf2 (Keap1) signaling in oxidative stress.

Nrf2:INrf2 (Keap1) are cellular sensors of chemical- and radiation-induced oxidative and electrophilic stress. Nrf2 is a nuclear transcription factor that controls the expression and coordinated induction of a battery of defensive genes encoding detoxifying enzymes and antioxidant proteins. This is a mechanism of critical importance for cellular protection and cell survival. Nrf2 is retained in the cytoplasm by an inhibitor, INrf2 which functions as an adapter for Cul3/Rbx1-mediated degradation of Nrf2. In response to oxidative/electrophilic stress, Nrf2 is switched on and then off by distinct early and delayed mechanisms. Oxidative/electrophilic modification of INrf2 cysteine 151 and/or protein kinase C phosphorylation of Nrf2 serine 40 results in the escape or release of Nrf2 from INrf2. Nrf2 is stabilized and translocates to the nucleus, forms heterodimers with unknown proteins, and binds the antioxidant response element, which leads to coordinated activation of gene expression. It takes less than 15 min from the time of exposure to switch on nuclear import of Nrf2. This is followed by activation of a delayed mechanism that controls the switching off of Nrf2 activation of gene expression. GSK3beta phosphorylates Fyn at an unknown threonine residue(s), leading to the nuclear localization of Fyn. Fyn phosphorylates Nrf2 tyrosine 568, resulting in the nuclear export of Nrf2, binding with INrf2, and degradation of Nrf2. The switching on and off of Nrf2 protects cells against free radical damage, prevents apoptosis, and promotes cell survival.