PRDX6, a member of the peroxiredoxins (PRDXs) family, is a key player in the removal of reactive oxygen species (ROS). Using targeted inactivation of the Prdx6 gene, we present evidence that the corresponding protein offsets the deleterious effects of ROS on lens epithelial cells (LECs) and regulates gene expression by limiting its levels. PRDX6-depleted LECs displayed phenotypic alterations and elevated a-smooth muscle actin and big-h3 expression (markers for cataractogenesis), indistinguishable from transforming growth factor b (TGFb)-induced changes. Biochemical assays disclosed enhanced levels of ROS, as well as high expression and activation of TGFb1 in Prdx6 À/À LECs. A CAT assay revealed transcriptional repression of lens epithelium-derived growth factor (LEDGF), HSP27, and aB-crystallin promoter activities in these cells. A gel mobility shift assay demonstrated the attenuation of LEDGF binding to heat shock or stress response elements present in these genes. A supply of PRDX6 toPrdx6 À/À LECs reversed these changes. Based on the above data, we propose a rheostat role for PRDX6 in regulating gene expression by controlling the ROS level to maintain cellular homeostasis.

A diminished level of endogenous antioxidant in cells/tissues is associated with reduced resistance to oxidative stress. Peroxiredoxin 6 (PRDX6), a protective molecule, regulates gene expression/function by controlling reactive oxygen species (ROS) levels. Using PRDX6 protein linked to TAT, the transduction domain from human immunodeficiency virus type 1 TAT protein, we demonstrated that PRDX6 was transduced into lens epithelial cells derived from rat or mouse lenses. The protein was biologically active, negatively regulating apoptosis and delaying progression of cataractogenesis by attenuating deleterious signaling. Lens epithelial cells from cataractous lenses bore elevated levels of ROS and were susceptible to oxidative stress. These cells harbored increased levels of active transforming growth factor (TGF)-beta 1 and of alpha-smooth muscle actin and beta ig-h3, markers for cataractogenesis. Importantly, cataractous lenses showed a 10-fold reduction in PRDX6 expression, whereas TGF-beta1 mRNA and protein levels were elevated. The changes were reversed, and cataractogenesis was delayed when PRDX6 was supplied. Results suggest that delivery of PRDX6 can postpone cataractogenesis, and this should be an effective approach to delaying cataracts and other degenerative diseases that are associated with increased ROS.

Antioxidant protein 2 (AOP2), a member of the newly defined family of thiol-specific antioxidant proteins, has been shown to remove H 2 O 2 and protect proteins and DNA from oxidative stress. Here we report that LEDGF is one of the regulatory factors for the AOP2 gene. We found that LEDGF bound to the heat shock element and to stress-related elements in the AOP2 promoter. It trans-activated expression of AOP2-CAT in COS-7 cells and lens epithelial cells overexpressing LEDGF. Mutations in the heat shock element and stress-related elements of the AOP2 promoter reduced LEDGF-dependent trans-activation. Lens epithelial cells showed a higher level of AOP2 mRNA in the presence of LEDGF. Cells overexpressing LEDGF exhibited a higher level of AOP2 protein, the level of which was directly related to the increase in cellular protection. Thus, LEDGF, by activating the AOP2 gene, protected and enhanced the survival of cells under oxidative stress.

No abstract

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.

No abstract

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.

Loss of the cytoprotective protein Peroxiredoxin 6 (Prdx6) in cells that are aging or under oxidative stress is known to be linked to pathobiology of many age-related diseases. However, the mechanism by which Prdx6 activity goes awry is largely unknown. Using Prdx6-deficient (Prdx6−/−) cells as a model for aging or redox active cells, human/mouse LECs facing oxidative stress, and aging lenses, we found a significant increase in the levels of Sumo1 conjugates. These cells displayed increased levels of Sumo1 and reduced expression of Prdx6. Specifically we observed that Prdx6 is a target for aberrant Sumoylation signaling, and that Sumo1 modification reduces its cellular abundance. LECs overexpressing Sumo1 showed reduced expression and activity of Prdx6 and its transactivator Sp1, mRNA and protein with increased levels of ROS; those cells were vulnerable to oxidative stress-induced cell death. A significant reduction in Prdx6, Sp1 protein and mRNA expression was observed in redox-active Prdx6−/− cells and in aging lenses/LECs. The reduction was correlated with increased expression of Sumo1 and enrichment of the inactive form (dimeric) of Sumo1 specific protease Senp1. Experiments with Sumo1-fused Prdx6 and Prdx6 promoter-linked to CAT reporter gene constructs pointed that Sumo1 dysregulated Prdx6 activity by reducing its abundance and attenuating its transcription, in contrast delivery of Senp1 or Prdx6 reversed the process. Data underpins that ROS-evoked aberrant Sumoylation signaling affects Prdx6 activity by reducing Prdx6 abundance as well as transcription. Our finding may provide a foundation for a strategy to repair deleterious oxidative signaling generated by reduced activity of Prdx6.