Upon oxidative stress and aging, Nrf2 (NFE2-related factor2) triggers antioxidant defense genes to defends against homeostatic failure. Using human(h) or rat(r) lens epithelial cells (LECs) and aging human lenses, we showed that a progressive increase in oxidative load during aging was linked to a decline in Prdx6 expression. DNA binding experiments using gel-shift and ChIP assays demonstrated a progressive reduction in Nrf2/ARE binding (−357/−349) of Prdx6 promoter. The promoter (−918) with ARE showed a marked reduction in young vs aged hLECs, which was directly correlated to decreased Nrf2/ARE binding. A Nrf2 activator, Sulforaphane (SFN), augmented Prdx6, catalase and GSTπ expression in dose-dependent fashion, and halted Nrf2 dysregulation of these antioxidants. SFN reinforced Nrf2/DNA binding and increased promoter activities by enhancing expression and facilitating Nrf2 translocalization in nucleus. Conversely, promoter mutated at ARE site did not respond to SFN, validating the SFN-mediated restoration of Nrf2/ARE signaling. Furthermore, SFN rescued cells from UVB-induced toxicity in dose-dependent fashion, which was consistent with SFN’s dose-dependent activation of Nrf2/ARE interaction. Importantly, knockdown of Prdx6 revealed that Prdx6 expression was prerequisite for SFN-mediated cytoprotection. Collectively, our results suggest that loss of Prdx6 caused by dysregulation of ARE/Nrf2 can be attenuated through a SFN, to combat diseases associated with aging.

Many disorders of aging, including blinding-diseases, are associated with deficiency of brain and muscle arnt-like protein 1 (Bmal1) and, thereby, dysregulation of antioxidant-defense pathway. However, knowledge is limited regarding the role of Bmal1 regulation of antioxidant-pathway in the eye lens/lens epithelial cells (LECs) at the molecular level. We found that, in aging human (h)LECs, a progressive decline of nuclear factor erythroid 2-related factor 2 (Nrf2)/ARE (antioxidant response element)-mediated antioxidant genes was connected to Bmal1-deficiency, leading to accumulation of reactive oxygen species (ROS) and cell-death. Bmal1-depletion disrupted Nrf2 and expression of its target antioxidant genes, like Peroxiredoxin 6 (Prdx6). DNA binding and transcription assays showed that Bmal1 controlled expression by direct binding to E-Box in Prdx6 promoter to regulate its transcription. Mutation at E-Box or ARE reduced promoter activity, while disruption of both sites diminished the activity, suggesting that both sites were required for peak Prdx6-transcription. As in aging hLECs, ROS accumulation was increased in Bmal1-deficient cells and the cells were vulnerable to death. Intriguingly, Bmal1/Nrf2/Prdx6 and PhaseII antioxidants showed rhythmic expression in mouse lenses in vivo and were reciprocally linked to ROS levels. We propose that Bmal1 is pivotal for regulating oxidative responses. Findings also reveal a circadian control of antioxidant-pathway, which is important in combating lens/LECs damage induced by aging or oxidative stress.

Oxidative stress and endoplasmic reticulum (ER) stress are emerging as crucial events in the etiopathology of many neurodegenerative diseases. While the neuroprotective contributions of the dietary compound curcumin has been recognized, the molecular mechanisms underlying curcumin's neuroprotection under oxidative and ER stresses remains elusive. Herein, we show that curcumin protects HT22 from oxidative and ER stresses evoked by the hypoxia (1% O₂ or CoCl₂ treatment) by enhancing peroxiredoxin 6 (Prdx6) expression. Cells exposed to CoCl₂ displayed reduced expression of Prdx6 with higher reactive oxygen species (ROS) expression and activation of NF-κB with IκB phosphorylation. When NF-κB activity was blocked by using SN50, an inhibitor of NF-κB, or cells treated with curcumin, the repression of Prdx6 expression was restored, suggesting the involvement of NF-κB in modulating Prdx6 expression. These cells were enriched with an accumulation of ER stress proteins, C/EBP homologous protein (CHOP), GRP/78, and calreticulin, and had activated states of caspases 12, 9, and 3. Reinforced expression of Prdx6 in HT22 cells by curcumin reestablished survival signaling by reducing propagation of ROS and blunting ER stress signaling. Intriguingly, knockdown of Prdx6 by antisense revealed that loss of Prdx6 contributed to cell death by sustaining enhanced levels of ER stress-responsive proapoptotic proteins, which was due to elevated ROS production, suggesting that Prdx6 deficiency is a cause of initiation of ROS-mediated ER stress-induced apoptosis. We propose that using curcumin to reinforce the naturally occurring Prdx6 expression and attenuate ROS-based ER stress and NF-κB-mediated aberrant signaling improves cell survival and may provide an avenue to treat and/or postpone diseases associated with ROS or ER stress.

Peroxiredoxin 6 (Prdx6) is a pleiotropic oxidative stress-response protein that defends cells against reactive oxygen species (ROS)-induced damage. Curcumin, a naturally occurring agent, has diversified beneficial roles including cytoprotection. Using human lens epithelial cells (hLECs) and Prdx6-deficient cells, we show the evidence that curcumin protects cells by upregulating Prdx6 transcription via invoking specificity protein 1 (Sp1) activity against proapoptotic stimuli. Curcumin enhanced Sp1 and Prdx6 mRNA and protein expression in a concentration-dependent manner, as evidenced by western and real-time PCR analyses, and thereby negatively regulated ROS-mediated apoptosis by blunting ROS expression and lipid peroxidation. Bioinformatic analysis and DNA–protein binding assays disclosed three active Sp1 sites (−19/27, −61/69 and −82/89) in Prdx6 promoter. Co-transfection experiments with Sp1 and Prdx6 promoter–chloramphenicol acetyltransferase (CAT) constructs showed that CAT activity was dramatically increased in LECs or Sp1-deficient cells (SL2). Curcumin treatment of LECs enhanced Sp1 binding to its sites, consistent with curcumin-dependent stimulation of Prdx6 promoter with Sp1 sites and cytoprotection. Notably, disruption of Sp1 sites by point mutagenesis abolished curcumin transactivation of Prdx6. Also, curcumin failed to activate Prdx6 expression in the presence of Sp1 inhibitors, demonstrating that curcumin-mediated increased expression of Prdx6 was dependent on Sp1 activity. Collectively, the study may provide a foundation for developing transcription-based inductive therapy to reinforce endogenous antioxidant defense by using dietary supplements.

Oxidative stress or reduced expression of naturally occurring antioxidants during aging has been identified as a major culprit in neuronal cell/tissue degeneration. Peroxiredoxin (Prdx) 6, a protective protein with GSH peroxidase and acidic calcium-independent phospholipase A2 activities, acts as a rheostat in regulating cellular physiology by clearing reactive oxygen species (ROS) and thereby optimizing gene regulation. We found that under stress, the neuronal cells displayed reduced expression of Prdx6 protein and mRNA with increased levels of ROS, and the cells subsequently underwent apoptosis. Using Prdx6 fused to TAT transduction domain, we showed evidence that Prdx6 was internalized in human brain cortical neuronal cells, HCN-2, and mouse hippocampal cells, HT22. The cells transduced with Prdx6 conferred resistance against the oxidative stress inducers paraquat, H₂O₂, and glutamate. Furthermore, Prdx6 delivery ameliorated damage to neuronal cells by optimizing ROS levels and overstimulation of NF-κB. Intriguingly, transduction of Prdx6 increased the expression of endogenous Prdx6, suggesting that protection against oxidative stress was mediated by both extrinsic and intrinsic Prdx6. The results demonstrate that Prdx6 expression is critical to protecting oxidative stress-evoked neuronal cell death. We propose that local or systemic application of Prdx6 can be an effective means of delaying/postponing neuronal degeneration.

A progressive decline in antioxidant potential and accumulation of reactive oxygen species (ROS) are major causes of pathogenesis of several diseases, including glaucoma. Trabecular meshwork (TM) dysfunction resulting in higher intraocular pressure (IOP) is a hallmark of glaucoma, but its causes are unclear. Using human (h) TM cells derived from glaucomatous and normal subjects of different ages and cells facing oxidative-stress, we showed that specific loss of moonlighting antioxidant protein Peroxiredoxin (Prdx) 6 in aging or in glaucomatous TM cells caused ROS accumulation and pathobiological changes in TM cells. Prdx6 limits the levels of ROS, thus preventing overstimulation of genes and resultant deleterious effects. We found that Prdx6 levels declined in aging and were reduced dramatically in glaucomatous and aged TM cells. Biochemical assays revealed enhanced levels of ROS, and high expression/activation of TGFβs and its responsive extracellular matrix genes α-SM, fibronectin, TGase2 and Tsp1 in aged or glaucomatous cells. Furthermore, hTM cells displayed typical features of the combined effects of TGFβs and oxidative-stress-induced cellular changes, showing increased levels of lipid peroxidation, oxidative DNA damage, and senescence markers p16, p21 and SA-βgal activity, along with reduced levels of telomerase expression and activity. Exposure to oxidative-stress (H₂O₂) or knocking down of Prdx6 (with antisense) accelerated this process. Importantly, Prdx6 delivery to sick or aged TM cells reversed the process. We propose Prdx6 as a potential therapeutic target to guard the TM from oxidative-stress and age-dependent accumulation of ROS by balancing redox-homeostasis to prevent ocular disorders, like glaucoma.