Sirtuin 3 (Sirt3), a major mitochondrial NAD+-dependent deacetylase, targets various mitochondrial proteins for lysine deacetylation and regulates important cellular functions such as energy metabolism, aging, and stress response. In this study, we identified the human 8-oxoguanine-DNA glycosylase 1 (OGG1), a DNA repair enzyme that excises 7,8-dihydro-8-oxoguanine (8-oxoG) from damaged genome, as a new target protein for Sirt3. We found that Sirt3 physically associated with OGG1 and deacetylated this DNA glycosylase and that deacetylation by Sirt3 prevented the degradation of the OGG1 protein and controlled its incision activity. We further showed that regulation of the acetylation and turnover of OGG1 by Sirt3 played a critical role in repairing mitochondrial DNA (mtDNA) damage, protecting mitochondrial integrity, and preventing apoptotic cell death under oxidative stress. We observed that following ionizing radiation, human tumor cells with silencing of Sirt3 expression exhibited deteriorated oxidative damage of mtDNA, as measured by the accumulation of 8-oxoG and 4977 common deletion, and showed more severe mitochondrial dysfunction and underwent greater apoptosis in comparison with the cells without silencing of Sirt3 expression. The results reported here not only reveal a new function and mechanism for Sirt3 in defending the mitochondrial genome against oxidative damage and protecting from the genotoxic stress-induced apoptotic cell death but also provide evidence supporting a new mtDNA repair pathway.

Repression of the NF-κB pathway has been extensively researched due to its pivotal role in inflammation. We investigated the potential of the aryl hydrocarbon receptor (AHR) to suppress NF-κB regulated gene expression, especially acute phase genes, such as serum amyloid A (Saa). Using AHR mutants, it was determined that nuclear translocation and heterodimerization with ARNT are essential, but DNA-binding is not involved in AHR-mediated Saa repression. A number of AHR ligands were capable of repressing saa3 expression. AHR activation leads to a decrease in RELA and C/EBP/β recruitment to and histone acetylation at Saa3 gene promoter. A battery of acute-phase genes (e.g. C-reactive protein and haptoglobin) induced by cytokine exposure was repressed by AHR activation in mouse hepatocytes. Dietary exposure to an AHR ligand represses cytokine induced acute-phase response in liver. Use of a human liver-derived cell line revealed similar repression of Saa mRNA levels and secreted protein. Repression of AHR expression also enhanced Saa induction in response to cytokines, suggesting that AHR is capable of constitutively repressing Saa gene expression. These results establish a role for AHR in inflammatory signaling within the liver, presenting a new therapeutic opportunity, and signify AHR’s ability to function in a DNA-independent manner.

The aryl hydrocarbon receptor (AHR) is a ligand activated transcription factor. Activation of AHR mediates the expression of target genes (e.g. CYP1A1), by binding to dioxin response element (DRE) sequences in their promoter region. To understand the multiple mechanisms of AHR-mediated gene regulation, a microarray analysis on liver isolated from ligand-treated transgenic mice expressing a wild-type Ahr or a DRE-binding mutant Ahr (A78D) on an ahr-null background was performed. Results revealed that AHR DRE-binding is not required for suppression of genes involved in cholesterol synthesis. Quantitative RT- PCR performed on both mouse liver and primary human hepatocyte RNA demonstrated a coordinate repression of genes involved in cholesterol biosynthesis, namely HMGCR, FDFT1, SQLE and LSS following receptor activation. An additional transgenic mouse line was established expressing a liver-specific Ahr-A78D on a CreAlb/Ahrflox/flox background. These mice displayed a similar repression of cholesterol biosynthetic genes compared to Ahrflox/flox mice, further indicating that the observed modulation is AHR-specific and occurs in a DRE-independent manner. Elevated hepatic transcriptional levels of the genes of interest were noted in congenic C57BL/6J-Ahd allele mice, when compared to the WT C57BL/6J mice, which carry the Ahb allele. Down-regulation of ARNT levels using siRNA in a human cell line revealed no effect on the expression of cholesterol biosynthetic genes. Finally, cholesterol secretion was shown to be significantly decreased in human cells following AHR activation. Conclusion These data firmly establish an endogenous role for AHR as a regulator of the cholesterol biosynthesis pathway independent of its DRE-binding ability and suggest that AHR may be a previously unrecognized therapeutic target.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor responsible for mediating the cellular response to the toxic compound 2,3,7,8,-tetrachlorodibenzo-p-dioxin. An essential role for the AhR in cellular biology has been established previously, but no high-affinity endogenous ligand has yet been identified. We have confirmed the presence of a putative endogenous ligand(s) in CV-1 cells through transient transfection with various cytochrome P450 isoforms. Expression of cytochromes P450 1A1, 1A2, or 1B1 reduced AhR-mediated luciferase reporter activity, whereas cytochrome P450 2E1 exhibited no significant effect. Studies with 2,4,3Ј,5Ј-tetramethoxystilbene, a potent and specific inhibitor of cytochrome P450 1B1, was able to partially block cytochrome P450 1B1-mediated reduction in reporter gene activity. These results provide evidence of the existence of a possible feedback mechanism in which AhR-regulated cytochromes P450 from the CYP1A and CYP1B families are able to metabolically alter putative endogenous ligand(s). Several experiments were performed to provide initial characterization of these putative endogenous ligands, including electrophoretic mobility shift assay analyses, which demonstrated that these ligands directly activate the AhR. Soluble extracts from various C57BL/6J and Ahr-null mouse tissues were also analyzed for the presence of AhR activators. Studies revealed that Ahr-null mouse lung tissue had a 4-fold increase in AhR-mediated reporter activity in cells. Quantitative polymerase chain reaction analysis revealed that lung tissue exhibits relatively high constitutive CYP1A1 mRNA levels. These results suggest that there is an autoregulatory feedback loop between the AhR and cytochrome P450 1A1 in mouse lung.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is known to cause a large number of adverse effects, mediated largely by its binding to the aryl-hydrocarbon receptor (AhR) and subsequent modulation of gene expression. It is thought that AhR mediates these effects through the untimely and disproportionate expression of specific genes. However, the exact mechanism, or the genes involved, through which TCDD leads to these effects is still unknown. This study reports the discovery of a novel target gene, epiregulin, which is regulated by TCDD-activated AhR. Epiregulin is a growth regulator which belongs to the epidermal growth factor (EGF) family. Using real time quantitative PCR (qPCR), it was established that TCDD upregulates epiregulin gene expression. The promoter region of epiregulin has a dioxin responsive element (DRE) 56 nucleotides upstream of the transcription start site, along with three potential Sp1 binding sites. Chromatin immunoprecipitation (ChIP) assays with an anti-AhR antibody showed promoter occupancy upon TCDD treatment. Luciferase reporter assays using a vector harboring the first 125 base pairs of the epiregulin rat promoter revealed an increase in signal on TCDD treatment, which was lost upon mutation of the DRE. Epiregulin and TCDD treatment mediated a dose-dependent increase in primary mouse keratinocyte growth. These results demonstrate that AhR directly increases epiregulin expression, which could play an important role in TCDD mediated tumor promotion observed in rodent models.