- Nuclear Factor, Erythroid Derived 2, Like 2 (NFE2L2 or Nrf2) is the primary transcription factor in cellular defense against oxidative and xenobiotic stresses in higher eukaryotes. This basic leucine zipper transcription factor regulates over 200 antioxidant, detoxification, and lipid metabolizing genes by binding to the Antioxidant Response Element (ARE; a cis-acting element) as a transcription enhancer. Our group, and others, have identified that the nuclear steady state levels of this highly inducible transcription factor declines with age which coincides with decreased expression of many ARE regulated genes. To elucidate the mechanism(s) involved in lowered Nrf2 levels we used hepatocytes isolated from young and old rats as a relevant cellular model that maintains the aging phenotype with respect to Nrf2 levels. Our results show that steady state Nrf2 levels decline by ~40% with age (N=4, p<0.05), which led us to investigate the multifaceted regulation of Nrf2 homeostasis. Specifically, analysis of Nrf2 mRNA levels, and the rate of Nrf2 protein turnover and translation were investigated. Surprisingly, Keap1-mediated degradation of Nrf2, its primary negative regulator, did not account for the age-related Nrf2 decline, nor did mRNA levels change with age. Rather, Nrf2 protein synthesis declines 5.3-fold with age (N=3, p<0.05). Furthermore, we identify that rno-miR-146a, a key inflammation regulating miRNA, both inhibits Nrf2 translation and increases significantly with age. Taken together, our data suggest that the age-related loss of Nrf2 stems from the loss of its translation and may be mediated through miR regulation.
Our lab previously demonstrated in rats, that the age-related loss of Nrf2 consequentially attenuates synthesis and steady state levels of liver glutathione (GSH) synthesis and manifests as a diminished level of GSH. This antioxidant is the most abundant non-protein thiol in most cells and is pivotal for the detoxification of many xenobiotics. The rate-limiting enzyme in GSH synthesis, γ-glutamyl-cysteine ligase (Gcl), is composed of a catalytic (Gclc), and a modulatory (Gclm) subunit. In mice and humans, it is established that Nrf2, through the ARE, regulates Gclc expression. However, this had not been established in rats. From a bioinformatics analysis of the 5’ upstream sequence of the rat Gclc gene, we identified 3 putative AREs (ARE1, ARE3, and ARE4), and one cis-element containing the core but not the flanking nucleotides of the ARE (ARE2). Luciferase reporter plasmids containing each individual ARE were transfected into H4IIE rat hepatoma cells to test for ARE activity. Only one, designated “ARE4”, produced appreciable luciferase activity and was dependent on Nrf2 expression, suggesting that this ARE is a “true” enhancer element. Chromatin immunoprecipitation (ChIP) and qPCR identified that Nrf2 binds ARE4 but not ARE1-3. Further ChIP analysis identified that known partner transcription factors of Nrf2 (small maf, c-Jun and c-Fos) were also bound to ARE4. Taken together, our data demonstrates that Nrf2 regulates, in part, the expression of the rat Gclc gene through ARE4.
Having identified the Gclc-AREs in the rat we sought to describe the molecular consequence of the age-related decrement of Nrf2 in hepatocytes isolated from young and old rats. ChIPs and reporter assays (ARE1-4) were used to investigate known ARE-binding transcription factors and the ARE mediated enhancement of Gclc transcription. We identified a decline in Nrf2 occupancy of ARE4 (40%) and a loss of transcriptional activity (70%; P < 0.05), consistent with the loss in Gclc levels. In addition, hepatic chromatin from old rat samples demonstrated ARE4 enrichment of the transcriptional repressor Bach1, and a loss of the histone acetyltransferase CREB-Binding Protein (CBP). These results demonstrate that the active Gclc transcriptional complex is remodeled into a repressive motif in the liver of old rats. Furthermore, ChIPs identified that Nrf2 binds ARE2 in liver lysate isolated from old rats but not young. In agreement with this finding, results from luciferase reporter assays suggest that ARE2 facilitates an Nrf2-dependent enhancement of Gclc expression in the old. Thus, a promoter switching mechanism may occur with age.
In summary, we have identified key mechanisms of how the age-related decrement in Nrf2 protein levels is controlled and also gathered preliminary evidence that a pro-inflammatory state may contribute the loss of Nrf2 synthesis via increased miR levels. Finally, we have characterized the consequences of the loss of Nrf2 to GSH synthesis as an example of age-related impairment of cellular stress response.