Prdx6 Regulates Nlrp3 Inflammasome Activation-Driven Inflammatory Response in Lens Epithelial Cells.

The continuum of antioxidant response dysregulation in aging/oxidative stress-driven Nlrp3 inflammasome activation-mediated inflammatory response is associated with age-related diseases. Peroxiredoxin (Prdx) 6 is a key antioxidant that provides cytoprotection by regulating redox homeostasis. Herein, using lens epithelial cells (LECs) derived from the targeted inactivation of Prdx6 gene and aging lenses, we present molecular evidence that Prdx6-deficiency causes oxidative-driven Nlrp3 inflammasome activation, resulting in pyroptosis in aging/redox active cells wherein Prdx6 availability offsets the inflammatory process. We observed that Prdx6-/- and aging LECs harboring accumulated reactive oxygen species (ROS) showed augmented activation of Nlrp3 and bioactive inflammatory components, like Caspase-1, IL-1ß, ASC and Gasdermin-D. Similar to lipopolysaccharide treatment, oxidative exposure led to further ROS amplification with increased activation of the Nlrp3 inflammasome pathway. Mechanistically, we found that oxidative stress enhanced Kruppel-like factor 9 (Klf9) expression in aging/Prdx6-/- mLECs, leading to a Klf9-dependent increase in Nlrp3 transcription, while the elimination of ROS by the delivery of Prdx6 or by silencing Klf9 prevented the inflammatory response. Altogether, our data identify the biological significance of Prdx6 as an intrinsic checkpoint for regulating the cellular health of aging or redox active LECs and provide opportunities to develop antioxidant-based therapeutic(s) to prevent oxidative/aging-related diseases linked to aberrant Nlrp3 inflammasome activation.

EAAT5 glutamate transporter rapidly binds glutamate with micromolar affinity in mouse rods.

Light responses of rod photoreceptor cells in the retina are encoded by changes in synaptic glutamate release that is in turn shaped by reuptake involving EAAT5 plasma membrane glutamate transporters. Heterologously expressed EAAT5 activates too slowly upon glutamate binding to support significant uptake. We tested EAAT5 activation in mouse rods in vivo by stimulating glutamate transporter anion currents (IA(glu)) with UV flash photolysis of MNI-glutamate, varying flash intensity to vary glutamate levels. Responses to uncaging rose rapidly with time constants of 2-3 ms, similar to IA(glu) events arising from spontaneous release. Spontaneous release events and IA(glu) evoked by weak flashes also declined with similar time constants of 40-50 ms. Stronger flashes evoked responses that decayed more slowly. Time constants were twofold faster at 35°C, suggesting that they reflect transporter kinetics, not diffusion. Selective EAAT1 and EAAT2 inhibitors had no significant effect, suggesting IA(glu) in rods arises solely from EAAT5. We calibrated glutamate levels attained during flash photolysis by expressing a fluorescent glutamate sensor iGluSnFr in cultured epithelial cells. We compared fluorescence at different glutamate concentrations to fluorescence evoked by photolytic uncaging of MNI-glutamate. The relationship between flash intensity and glutamate yielded EC50 values for EAAT5 amplitude, decay time, and rise time of ∼10 µM. Micromolar affinity and rapid activation of EAAT5 in rods show it can rapidly bind synaptic glutamate. However, we also found that EAAT5 currents are saturated by the synchronous release of only a few vesicles, suggesting limited capacity and a role for glial uptake at higher release rates.

Hydralazine Revives Cellular and Ocular Lens Health-Span by Ameliorating the Aging and Oxidative-Dependent Loss of the Nrf2-Activated Cellular Stress Response.

A major hallmark of aging-associated diseases is the inability to evoke cellular defense responses. Transcriptional protein Nrf2 (nuclear factor erythroid-derived 2-related factor) plays a pivotal role in the oxidative stress response, cellular homeostasis, and health span. Nrf2's activation has been identified as a therapeutic target to restore antioxidant defense in aging. Here, we demonstrated that FDA-approved drug, hydralazine (Hyd), was a reactivator of the Nrf2/ARE (antioxidant response element) pathway in various ages and types of mouse (m) or human (h) lens epithelial cells (LECs) and mice lenses in-vitro/in-vivo. This led to Hyd-driven abatement of carbonyls, reduced reactive oxygen species (ROS), and reduced 4-HNE/MDA-adducts with cytoprotection, and extended lens healthspan by delaying/preventing lens opacity against aging/oxidative stress. We elucidated that Hyd activated the protective signaling by inducing Nrf2 to traverse from the cytoplasm to the nucleus and potentiated the ARE response by direct interaction of Nrf2 and ARE sequences of the promoter. Loss-of-function study and cotreatment of Hyd and antioxidant, N-acetyl cysteine (NAC) or Peroxiredoxin (Prdx)6, specified that Nrf2/ARE-driven increase in the promoter activity was Hyd-dependent. Our study provides proof-of concept evidence and, thereby, paves the way to repurposing Hyd as a therapeutic agent to delay/prevent aging and oxidative-related disorders.

Obligatory Role of AMPK Activation and Antioxidant Defense Pathway in the Regulatory Effects of Metformin on Cellular Protection and Prevention of Lens Opacity.

Increasing levels of oxidative-stress due to deterioration of the Nrf2 (NFE2-related factor)/ARE (antioxidant response element) pathway is found to be a primary cause of aging pathobiology. Metformin having anti-aging effects can delay/halt aging-related diseases. Herein, using lens epithelial cell lines (LECs) of human (h) or mouse (m) and aging h/m primary LECs along with lenses as model systems, we demonstrated that Metformin could correct deteriorated Bmal1/Nrf2/ARE pathway by reviving AMPK-activation, and transcriptional activities of Bmal1/Nrf2, resulting in increased antioxidants enzymatic activity and expression of Phase II enzymes. This ensued reactive oxygen species (ROS) mitigation with cytoprotection and prevention of lens opacity in response to aging/oxidative stress. It was intriguing to observe that Metformin internalized lens/LECs and upregulated OCTs (Organic Cation Transporters). Mechanistically, we found that Metformin evoked AMPK activation-dependent increase of Bmal1, Nrf2, and antioxidants transcription by promoting direct E-Box and ARE binding of Bmal1 and Nrf2 to the promoters. Loss-of-function and disruption of E-Box/ARE identified that Metformin acted by increasing Bmal1/Nrf2-mediated antioxidant expression. Data showed that AMPK-activation was a requisite for Bmal1/Nrf2-antioxidants-mediated defense, as pharmacologically inactivating AMPK impeded the Metformin's effect. Collectively, the results for the first-time shed light on the hitherto incompletely uncovered crosstalk between the AMPK and Bmal1/Nrf2/antioxidants mediated by Metformin for blunting oxidative/aging-linked pathobiology.

Switching of Redox Signaling by Prdx6 Expression Decides Cellular Fate by Hormetic Phenomena Involving Nrf2 and Reactive Oxygen Species.

Changes in intracellular reactive oxygen species (ROS) levels due to remodeling of antioxidant defense can affect the status of biological homeostasis in aging/oxidative stress. Peroxiredoxin 6 (Prdx6), an antioxidant gene downstream target for the Nrf2 pathway, plays a role in regulating ROS homeostasis. Using aging human (h) lens epithelial cells (LECs) or Prdx6-deficient (Prdx6-/-) mouse (m) LECs, here we showed that dichlorofluorescein (DCF) oxidation or H2O2 were strictly controlled by Prdx6. We observed that a moderate degree of oxidative stress augmented Nrf2-mediated Prdx6 expression, while higher doses of H2O2 (≥100 µM) caused a dramatic loss of Prdx6 expression, resulting in increased DCF oxidation and H2O2 amplification and cell death. Mechanistically, at increased oxidative stress, Nrf2 upregulated transcriptional factor Klf9, and that Klf9 bound to the promoter and repressed the Prdx6 gene. Similarly, cells overexpressing Klf9 displayed Klf9-dependent Prdx6 suppression and DCF oxidation with H2O2 amplification, while ShKlf9 reversed the process. Our data revealed that H2O2 and DCF oxidation levels play a hormetical role, and the Nrf2-Klf9-Prdx6 pathway is pivotal for the phenomena under the conditions of oxidative load/aging. On the whole, the results demonstrate that oxidative hormetical response is essentially based on levels of oxidative triggering and the status of Klf9-Prdx6 pathway activation; thus, Klf9 can be considered as a therapeutic target for hormetic shifting of cellular defense to improve protective resilience to oxidative stress.

Engineered Sumoylation-Deficient Prdx6 Mutant Protein-Loaded Nanoparticles Provide Increased Cellular Defense and Prevent Lens Opacity.

Aberrant Sumoylation-mediated protein dysfunction is involved in a variety of oxidative and aging pathologies. We previously reported that Sumoylation-deficient Prdx6K(lysine)122/142R(Arginine) linked to the TAT-transduction domain gained stability and protective efficacy. In the present study, we formulated wild-type TAT-HA-Prdx6WT and Sumoylation-deficient Prdx6-loaded poly-lactic-co-glycolic acid (PLGA) nanoparticles (NPs) to further enhance stability, protective activities, and sustained delivery. We found that in vitro and subconjuctival delivery of Sumoylation-deficient Prdx6-NPs provided a greater protection of lens epithelial cells (LECs) derived from human and Prdx6-/--deficient mouse lenses against oxidative stress, and it also delayed the lens opacity in Shumiya cataract rats (SCRs) than TAT-HA-Prdx6WT-NPs. The encapsulation efficiencies of TAT-HA-Prdx6-NPs were ≈56%-62%. Dynamic light scattering (DLS) and atomic force microscopy (AFM) analyses showed that the NPs were spherical, with a size of 50-250 nm and a negative zeta potential (≈23 mV). TAT-HA-Prdx6 analog-NPs released bioactive TAT-HA-Prdx6 (6%-7%) within 24 h. Sumoylation-deficient TAT-HA-Prdx6-NPs provided 35% more protection by reducing the oxidative load of LECs exposed to H2O2 compared to TAT-HA-Prdx6WT-NPs. A subconjuctival delivery of TAT-HA-Prdx6 analog-NPs demonstrated that released TAT-HA-Prdx6K122/142R could reduce lens opacity by ≈60% in SCRs. Collectively, our results demonstrate for the first time that the subconjuctival delivery of Sumoylation-deficient Prdx6-NPs is efficiently cytoprotective and provide a proof of concept for potential use to delay cataract and oxidative-related pathobiology in general.

Clock Protein Bmal1 and Nrf2 Cooperatively Control Aging or Oxidative Response and Redox Homeostasis by Regulating Rhythmic Expression of Prdx6.

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.

Sulforaphane-Induced Klf9/Prdx6 Axis Acts as a Molecular Switch to Control Redox Signaling and Determines Fate of Cells.

Sulforaphane (SFN), an activator of transcription factor Nrf2 (NFE2-related factor), modulates antioxidant defense by Nrf2-mediated regulation of antioxidant genes like Peroxiredoxin 6 (Prdx6) and affects cellular homeostasis. We previously observed that dose levels of SFN are crucial in determining life or death of lens epithelial cells (LECs). Herein, we demonstrated that higher doses of SFN (>6 µM) activated death signaling by overstimulation of Nrf2/ARE (antioxidant response element)-mediated Kruppel-like factor (Klf9) repression of Prdx6 expression, which increased reactive oxygen species (ROS) load and cell death. Mechanistically, Klf9 bound to its repressive Klf9 binding elements (RKBE; 5-CA/GCCC-3) in the Prdx6 promoter, and repressed Prdx6 transcription. Under the condition of higher dose of SFN, excessive Nrf2 abundance caused death signaling by enforcing Klf9 activation through ARE (5-RTGAYnnnGC-3) in Klf9 promoter that suppress antioxidant genes such as Prdx6 via a Klf9-dependent fashion. Klf9-depletion showed that Klf9 independently caused ROS reduction and subsequent cell survival, demonstrating that Klf9 upregulation caused cell death. Our work revealed the molecular mechanism of dose-dependent altered activity of SFN in LECs, and demonstrated that SFN activity was linked to levels of Nrf2/Klf9/Prdx6 axis. We proposed that in the development of therapeutic interventions for aging/oxidative disorders, combinations of Klf9-ShRNA and Nrf2 inducers may prove to be a promising strategy.

Ginkgolic Acid Rescues Lens Epithelial Cells from Injury Caused by Redox Regulated-Aberrant Sumoylation Signaling by Reviving Prdx6 and Sp1 Expression and Activities.

Sumoylation is a downstream effector of aging/oxidative stress; excess oxidative stress leads to dysregulation of a specificity protein1 (Sp1) and its target genes, such as Peroxiredoxin 6 (Prdx6), resulting in cellular damage. To cope with oxidative stress, cells rely on a signaling pathway involving redox-sensitive genes. Herein, we examined the therapeutic efficacy of the small molecule Ginkgolic acid (GA), a Sumoylation antagonist, to disrupt aberrant Sumoylation signaling in human and mouse lens epithelial cells (LECs) facing oxidative stress or aberrantly expressing Sumo1 (small ubiquitin-like modifier). We found that GA globally reduced aberrant Sumoylation of proteins. In contrast, Betulinic acid (BA), a Sumoylation agonist, augmented the process. GA increased Sp1 and Prdx6 expression by disrupting the Sumoylation signaling, while BA repressed the expression of both molecules. In vitro DNA binding, transactivation, Sumoylation and expression assays revealed that GA enhanced Sp1 binding to GC-boxes in the Prdx6 promoter and upregulated its transcription. Cell viability and intracellular redox status assays showed that LECs pretreated with GA gained resistance against oxidative stress-driven aberrant Sumoylation signaling. Overall, our study revealed an unprecedented role for GA in LECs and provided new mechanistic insights into the use of GA in rescuing LECs from aging/oxidative stress-evoked dysregulation of Sp1/Prdx6 protective molecules.

Sumoylation-deficient Prdx6 repairs aberrant Sumoylation-mediated Sp1 dysregulation-dependent Prdx6 repression and cell injury in aging and oxidative stress.

Progressive deterioration of antioxidant response in aging is a major culprit in the initiation of age-related pathobiology induced by oxidative stress. We previously reported that oxidative stress leads to a marked reduction in transcription factor Sp1 and its mediated Prdx6 expression in lens epithelial cells (LECs) leading to cell death. Herein, we examined how Sp1 activity goes awry during oxidative stress/aging, and whether it is remediable. We found that Sp1 is hyper-Sumoylated at lysine (K) 16 residue in aging LECs. DNA binding and promoter assays revealed, in aging and oxidative stress, a significant reduction in Sp1 overall binding, and specifically to Prdx6 promoter. Expression/overexpression assay revealed that the observed reduction in Sp1-DNA binding activity was connected to its hyper-Sumoylation due to increased reactive oxygen species (ROS) and Sumo1 levels, and reduced levels of Senp1, Prdx6 and Sp1. Mutagenesis of Sp1 at K16R (arginine) residue restored steady-state, and improved Sp1-DNA binding activity and transactivation potential. Extrinsic expression of Sp1K16R increased cell survival and reduced ROS levels by upregulating Prdx6 expression in LECs under aging/oxidative stress, demonstrating that Sp1K16R escapes the aberrant Sumoylation processes. Intriguingly, the deleterious processes are reversible by the delivery of Sumoylation-deficient Prdx6, an antioxidant, which would be a candidate molecule to restrict aging pathobiology.

Sulforaphane reactivates cellular antioxidant defense by inducing Nrf2/ARE/Prdx6 activity during aging and oxidative stress.

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.

Prdx6 retards senescence and restores trabecular meshwork cell health by regulating reactive oxygen species.

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 TGFßs 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 TGFßs and oxidative-stress-induced cellular changes, showing increased levels of lipid peroxidation, oxidative DNA damage, and senescence markers p16, p21 and SA-ßgal 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.

Sumoylation-deficient Prdx6 gains protective function by amplifying enzymatic activity and stability and escapes oxidative stress-induced aberrant Sumoylation.

Aberrant Sumoylation of protein(s) in response to oxidative stress or during aging is known to be involved in etiopathogenesis of many diseases. Upon oxidative stress, Peroxiredoxin (Prdx) 6 is aberrantly Sumoylated by Sumo1, resulting in loss of functions and cell death. We identified lysines (K) 122 and 142 as the major Sumo1 conjugation sites in Prdx6. Intriguingly, the mutant Prdx6 K122/142 R (arginine) gained protective efficacy, increasing in abundance and promoting glutathione (GSH) peroxidase and acidic calcium-independent phospholipase A2 (aiPLA2) activities. Using lens epithelial cells derived from targeted inactivation of Prdx6-/- gene and relative enzymatic and stability assays, we discovered dramatic increases in GSH-peroxidase (30%) and aiPLA2 (37%) activities and stability in the K122/142 R mutant, suggesting Sumo1 destabilized Prdx6 integrity. Prdx6-/-LECs with EGFP-Sumo1 transduced or co-expressed with mutant TAT-HA-Prdx6K122/142 R or pGFP-Prdx6K122/142 R were highly resistant to oxidative stress, demonstrating mutant protein escaped and interrupted the Prdx6 aberrant Sumoylation-mediated cell death pathway. Mutational analysis of functional sites showed that both peroxidase and PLA2 active sites were necessary for mutant Prdx6 function, and that Prdx6 phosphorylation (at T177 residue) was essential for optimum PLA2 activity. Our work reveals the involvement of oxidative stress-induced aberrant Sumoylation in dysregulation of Prdx6 function. Mutant Prdx6 at its Sumo1 sites escapes and abates this adverse process by maintaining its integrity and gaining function. We propose that the K122/142R mutant of Prdx6 in the form of a TAT-fusion protein may be an easily applicable intervention for pathobiology of cells related to aberrant Sumoylation signaling in aging or oxidative stress.

Delivery of a protein transduction domain-mediated Prdx6 protein ameliorates oxidative stress-induced injury in human and mouse neuronal cells.

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.

Aberrant sumoylation signaling evoked by reactive oxygen species impairs protective function of Prdx6 by destabilization and repression of its transcription.

Loss of the cytoprotective protein peroxiredoxin 6 (Prdx6) in cells that are aging or under oxidative stress is known to be linked to the 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 lens epithelial cells (LECs) facing oxidative stress and aging lenses, we found a significant increase in the levels of small ubiquitin-like modifier (Sumo)1 conjugates. These cells displayed increased levels of Sumo1 and reduced the 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 specificity protein 1 (Sp1), mRNA and protein with increased levels of reactive oxygen species; 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 Sumo-specific protease (Senp)1. Experiments with Sumo1-fused Prdx6 and Prdx6 promoter-linked to chloramphenicol acetyltransferase reporter gene constructs indicated that Sumo1 dysregulated Prdx6 activity by reducing its abundance and attenuating its transcription; in contrast, the delivery of Senp1 or Prdx6 reversed the process. The data show that reactive oxygen species-evoked aberrant sumoylation signaling affects Prdx6 activity by reducing Prdx6 abundance, as well as transcription. The findings of the present study may provide a foundation for a strategy to repair deleterious oxidative signaling generated by a reduced activity of Prdx6.

Curcumin abates hypoxia-induced oxidative stress based-ER stress-mediated cell death in mouse hippocampal cells (HT22) by controlling Prdx6 and NF-κB regulation.

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% O(2) or CoCl(2) treatment) by enhancing peroxiredoxin 6 (Prdx6) expression. Cells exposed to CoCl(2) 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.

Epigenetic repression of LEDGF during UVB exposure by recruitment of SUV39H1 and HDAC1 to the Sp1-responsive elements within LEDGF promoter CpG island.

Expression level of lens epithelial derived growth factor (LEDGF) is vital for LEDGF-mediated cell survival and cytoprotection against proapoptotic stimuli. We previously demonstrated that LEDGF is transcriptionally regulated by Sp1-responsive elements within a CpG island in the LEDGF promoter. Herein, we report on the existence of epigenetic signaling involved in the repression of LEDGF transcription in lens epithelial cells (LECs) facing UVB. UVB exposure led to histone H3 dimethylation and deacetylation at its CpG island, where a histone deacetylase/histone methylase (HDAC1/SUV39H1) complex was recruited. Exposure of LECs to UVB stress altered LEDGF protein and mRNA expression as well as promoter activity, while failing to methylate the CpG island. These events were correlated with increased reactive oxygen species (ROS) and increased cell death. LEDGF promoter activity and expression remained unaltered after 5-Aza treatment, but were relieved with tricostatin A, an inhibitor of HDACs. Expression analysis disclosed that UVB radiation altered the global expression levels of acetylated histone proteins, diminished total histone acetyltransferase (HAT) activity and increased HDAC activity and HDAC1 expression. In silico analysis of LEDGF proximal promoter and ChIP analyses disclosed HDAC1/SUV39H1 complex anchored to the -170/-10 nt promoter regions at Sp1-responsive elements and also attenuated Sp1 binding, resulting in HDAC1- and SUV39H1-dependent deacetylation and dimethylation of H3 at K9. Acetylation of H3K9 was essential for LEDGF active transcription, while enrichment of H3K9me2 at Sp1-responsive elements within CpGs (-170/-10) by UVB radiation repressed LEDGF transcription. Our study may contribute to understanding diseases associated with LEDGF aberrant expression due to specific epigenetic modifications, including blinding disorders.

Lens epithelium-derived growth factor deSumoylation by Sumo-specific protease-1 regulates its transcriptional activation of small heat shock protein and the cellular response.

Lens epithelium-derived growth factor (LEDGF), a ubiquitously expressed nuclear protein, acts by interacting with DNA and protein and is involved in widely varying cellular functions. Despite its importance, the mechanism(s) that regulate naturally occurring LEDGF activity are unidentified. In the present study, we report that LEDGF is constitutively Sumoylated, and that the dynamical regulatory mechanism(s) (i.e. Sumoylation and deSumoylation) act as a molecular switch in modulating the DNA-binding and transcriptional activity of LEDGF with the functional consequences. Using bioinformatics analysis coupled with in vitro and in vivo Sumoylation assays, we found that lysine (K) 364 of LEDGF was Sumoylated, repressing its transcriptional activity. Conversely, mutation of K364 to arginine (R) or deSumoylation by small ubiquitin-like modifier (Sumo)-specific protease-1, a nuclear deSumoylase, enhanced the transactivation capacity of LEDGF and its cellular abundance. The enhancements were directly correlated with an increase in the DNA-binding activity and small heat shock protein transcription of LEDGF, whereas the process was reversed in cells overexpressing Sumo1. Interestingly, cells expressing Sumoylation-deficient pEGFP-K364R protein showed increased cellular survival compared to wild-type LEDGF protein. The findings provide insights into the regulation and regulatory functions of LEDGF in Sumoylation-dependent transcriptional control that may be essential for modifying the physiology of cells to maintain cellular homeostasis. These studies also provide new evidence of the important role of post-translational modification in controlling LEDGF function.

Transcriptional protein Sp1 regulates LEDGF transcription by directly interacting with its cis-elements in GC-rich region of TATA-less gene promoter.

LEDGF/p75 interacts with DNA/protein to regulate gene expression and function. Despite the recognized diversity of function of LEDGF/p75, knowledge of its transregulation is in its infancy. Here we report that LEDGF/p75 gene is TATA-less, contains GC-rich cis elements and is transcriptionally regulated by Sp1 involving small ubiquitin-like modifier (Sumo1). Using different cell lines, we showed that Sp1 overexpression increased the level of LEDGF/p75 protein and mRNA expression in a concentration-dependent fashion. In contrast, RNA interference depletion of intrinsic Sp1 or treatment with artemisinin, a Sp1 inhibitor, reduced expression of LEDGF/p75, suggesting Sp1-mediated regulation of LEDGF/p75. In silico analysis disclosed three evolutionarily conserved, putative Sp1 sites within LEDGF/p75 proximal promoter (-170/+1 nt). DNA-binding and transactivation assays using deletion and point mutation constructs of LEDGF/p75 promoter-CAT revealed that all Sp1 sites (-50/-43, -109/-102 and -146/-139) differentially regulate LEDGF/p75. Cotransfection studies with Sp1 in Drosophila cells that were Sp1-deficient, showed increased LEDGF/p75 transcription, while in lens epithelial cells (LECs) promoter activity was inhibited by artemisinin. These events were correlated with levels of endogenous Sp1-dependent LEDGF/p75 expression, and higher resistance to UVB-induced cell death. ChIP and transactivation assays showed that Sumoylation of Sp1 repressed its transcriptional activity as evidenced through its reduced binding to GC-box and reduced ability to activate LEDGF/p75 transcription. As whole, results revealed the importance of Sp1 in regulating expression of LEDGF/p75 gene and add to our knowledge of the factors that control LEDGF/p75 within cellular microenvironments, potentially providing a foundation for LEDGF/p75 expression-based transcription therapy.

Deficiency of Prdx6 in lens epithelial cells induces ER stress response-mediated impaired homeostasis and apoptosis.

The multifunctional cytoprotective protein peroxiredoxin 6 (Prdx6) maintains cellular homeostasis and membrane integrity by regulating expression of intracellular reactive oxygen species (ROS) and phospholipid turnover. Using cells derived from targeted inactivation of Prdx6 gene or its depletion by RNA interference or aging, we showed that Prdx6 deficiency in cells evoked unfolded protein response (UPR), evidenced by increased expression or activation of proapoptotic factors, CHOP, ATF4, PERK, IRE-α and eIF2-α and by increased caspases 3 and 12 processing. Those cells displayed enhanced and sustained expression of endoplasmic reticulum (ER) stress-related chaperon proteins, Bip/glucose-regulated protein 78, calnexin, and calreticulin. Under cellular stress induced by hypoxia (1% O(2) or CoCl(2) treatment) or tunicamycin, Prdx6-deficient cells exhibited aberrant activation of ER stress-responsive genes/protein with higher expression of ROS, and died with apoptosis. Wild-type cells exposed to tunicamycin or hypoxia remained relatively insensitive with lower expression of ROS and ER-responsive genes than did Prdx6-deficient cells, but upregulation of ER stress responsive proteins or chaperones mimicked the UPR response of Prdx6-deficient or aging cells. Expression of Prdx6 blocked ER stress-induced deleterious signaling by optimizing physiologically aberrant expression of ER stress responsive genes/proteins in Prdx6-deficient cells or cells facing stressors, and rescued the cells from apoptosis. These findings demonstrate that impaired homeostasis and progression of pathogenesis in Prdx6-deficient lens epithelial cells or in aging cells should be blocked by a supply of Prdx6. The results provide a new molecular basis for understanding the etiology of several age-associated degenerative disorders, and potentially for developing antioxidant Prdx6-based therapeutics.