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.


Introduction
In order to survive against oxidative stress produced by internal and external environmental stresses, cells have developed an antioxidant defense system. However, the intensity of oxidative stress can alter antioxidant response-driven survival signaling to the death pathway [1][2][3][4]. Mild or moderate stress can evoke normal physiological functions of cells due to adoptive increased resistance of Nrf2-mediated antioxidant response [1,4], while excessive stress evokes adverse signaling and that may, in turn, lead to age-related disorders, including neurodegenerative disorder, cancer, diabetes, acute respiratory distress syndrome, hypertension, obesity, ocular disorders such as cataractogenesis, glaucoma, etc. [5][6][7][8][9][10]. This biological mechanism is known as the hormetic response (hormesis) to oxidative stress and has been reported in a variety of organisms in responses to drugs or prooxidative electrophilic compounds [11][12][13][14][15][16]. It has been shown that physiological levels of reactive oxygen species (ROS), specifically H 2 O 2 play a pivotal role in regulating normal biological activities of cells [4,13], and those homeostatic activities depend upon the status of the Nrf2-antioxidants pathway within the cellular microenvironment. Recently, the hormetic effects of ROS and their influences on cell survival/death as well as aging-related diseases have been shown [17][18][19]. These argued that, if not all, some of the pathway and identified that Klf9's activation plays a pivotal role. Because aberrant stimulation of Klf9 leads to ROS amplification-dependent cell death, we propose that Klf9 should be a therapeutic target to treat or prevent oxidative stress-associated diseases.

Isolation and Generation of Primary hLECs and SRA-hLECs
Two types of human LECs were used: (1) primary hLECs isolated from different ages of deceased persons and (2) a cell line (SRA01/04) immortalized with SV40. To avoid any confusion, the remaining text will designate the different ages of primary hLECs as primary hLECs and immortalized SRA-hLECs cell lines as hLECs [12].
Primary hLECs were separated and collected from normal eye lenses of deceased subjects or healthy donors of variable ages (16,33,54,66, and 75 y) procured from the Lions Eye Bank, Nebraska Medical Center, Omaha, NE, and National Disease Research Interchange (NDRI), Inc., Philadelphia, PA, USA. In accordance with regulation HHS45CFR 46.102(f), studies involving materials from deceased individuals are not considered human subject research as defined at 45CFR46.102(f) 10(2) and do not need IRB oversight. Briefly, the lens capsule(s) was separated and processed/trimmed before explanting in 35 mm culture dishes coated with collagen IV containing complete DMEM supplemented with 15-20% fetal bovine serum (FBS), with a brief modification [12,21,[59][60][61]. The culture explants were trypsinized and re-cultured for hLECs generation. Cell cultures attaining confluency were trypsinized and used for experiments [8,62,63]. Western analysis was carried out using an antibody specific to αA-crystallin to validate the presence of αA-crystallin, a specific marker for LEC identity (data not shown).

Generation and Validation of LECs Isolated from Lenses of Prdx6 +/+ and Prdx6 −/− Mice
All animal experiments followed the recommendations set forth in the "Statement for the Use of Animals in Ophthalmic and Visual Research" by the Association for Research in Vision and Ophthalmology, and studies conducted were approved by The University of Nebraska Medical Center (UNMC). LECs isolated from lenses of Prdx6-targeted mutants (Prdx6 −/− ) and wild type (Prdx6 +/+ ) mice were cultured and maintained in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS), as described earlier [66]. Prdx6 −/− 129/Sv mice were generated at Harvard Medical School (Boston, MA, USA) under the supervision of Dr. David R. Beier. All animals were maintained under specific pathogen-free conditions in an animal facility.

Protein Expression Analysis
The total cellular extracts of LECs were prepared in ice-cold radioimmune precipitation buffer (RIPA buffer), and Western analysis was carried out as described previously [7,[70][71][72].

Quantitation of Intracellular ROS Level by H2-DCF-DA and CellROX ® Deep Red Reagent
LECs were cultured in 96-well plates (5000/well) in either the presence or absence of an oxidant. At higher oxidant concentrations, the levels of intracellular ROS were measured after 2-4 h exposure to H 2 O 2 to establish the connection between levels of ROS and cell death. Note: lethal concentrations will be highly toxic and kill the cells if the cells are incubated for a longer period, and the condition will not provide the correct value of ROS and its connection to cell death, as well as the status of the antioxidant pathway, including the molecular mechanism involved. Thus, in this work, we assessed the biological molecules in cells facing higher concentrations of H 2 O 2 as reported previously [11,13]. Nonetheless, the time and duration of the LECs treatment with H 2 O 2 have been indicated in the Figure legends. ROS were quantified using a fluorescent dye, dichlorodihydrofluorescein diacetate (H2-DCF-DA), a nonpolar compound that is converted into a polar derivative (dichlorofluorescein) by cellular esterase after incorporation into cells [10,24,65,72]. On the day of the experiment, the medium was replaced with Hank's solution containing 10 µM H2-DCF-DA dye. 30 min later, intracellular fluorescence was detected at excitation (Ex) at 485 nm/emission (Em) at 530 nm by a Spectra Max Gemini EM (Mol. Devices, Sunnyvale, CA, USA).
ROS levels were assayed following the company's protocol (CellROX ® Deep Red Oxidative Stress Reagent, Catalog No. C10422) and as described in our published protocol [6]. For the Klf9 overexpression assay, hLECs were transfected with pEGFP-Vector or pGFP-Klf9 plasmid. Then, 48 h later, transfectants were exposed to H 2 O 2 as indicated in figure legends. For the Klf9 knock down assay, LV Sh-control and LV Sh-Klf9 infected hLECs (5 × 10 3 ) cells were seeded in a 96-well plate. Then, 24 h later, these cells were treated with increasing concentrations of H 2 O 2 . 2-3 h of H 2 O 2 exposure, CellROX deep red reagent was added with a final concentration of 5 µM and were incubated at 37 • C for 30 min. A medium containing CellROX deep red reagent was removed and fixed with 3.7% formaldehyde. Then, 15 min later, the fluorescence signal was recorded at Ex 640 nm/Em 665 nm with Spectra Max Gemini EM (Mol. Devices, Sunnyvale, CA, USA).

Measurement of H 2 O 2 Generation in Lens Epithelial Cells
LECs were cultured in 96-well plates. At a predefined time as indicated in figure legends (mostly within 2 h or onwards depending upon oxidant concentrations of oxidant exposure). H 2 O 2 levels in the cells were measured using an Amplex™ Red Hydrogen Peroxide/Peroxidase Assay Kit (Catalog number A22188, Invitrogen) according to the manufacturer's protocol and published report [73]. In brief, cells were washed with Krebs-Ringer Solution (Catalog number J67591.K2, Thermo Fisher Scientific, Waltham, MA, USA). Then 100 µL reaction mixture (50 µM Amplex Red reagent and 0.1 U/mL HRP in Krebs-Ringer phosphate) was added. The generation of H 2 O 2 was measured at different time intervals. For the negative control, 20 µL of Krebs-Ringer phosphate was added to a 96-well containing cells before the addition of a prewarmed 100 µL reaction mixture. To prepare the H 2 O 2 standard curve, 50 µL of different concentrations of H 2 O 2 (0-10 µM) prepared in a 1× reaction buffer was added to the 96-well plate, and the reaction was started by adding 50 µL of Amplex Red reagent/HRP working solution (100 µM Amplex Red reagent and 0.2 U/mL HRP in 1X reaction Buffer) and incubated at room temperature in the dark. Reading was recorded at absorbance,~560 nm.

Cell Survival Assay (MTS Assay)
A colorimetric MTS assay, The CellTiter 96 ® AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA), was conducted to determine cell viability as described previously [21,65,74]. The assay was carried out by the addition of MTS reagent to 96-well containing cells. The A 490 nm (O.D.) value was recorded after 2 h with a plate reader, Spectra Max Gemini EM (Mol. Devices, Sunnyvale, CA, USA). Results were normalized with the absorbance of the untreated control(s).

Transcription Factor Nrf2 Activation Assay
Nrf2 activation assay was carried out as described in the company's protocol (TransAM Nrf2 Transcription Factor Assay Kit, Cat No. 50296, Active motif, Carlsland, CA, USA) and as described in our published protocol [11,67]. Briefly, 10 µg of nuclear extract (up to 10 µL diluted with complete lysis buffer) prepared from H 2 O 2 exposed LECs was added to the strips well following the addition of 40 µL binding buffer containing 20 pmol of the wild-type or mutated consensus oligonucleotide to each well. The plate was incubated for 1 h at room temperature (RT). Then, 100 µL primary antibody (1:1000 dilution) was added after 3 washes and then incubated at RT for 1 h. 100 µL of diluted anti-rabbit HRP conjugated antibody (1:1000 dilution) was added and incubated for 1 h at RT. Then, 100 µL of developing solution was added to wells after washing and incubated for 2 to 10 min in the dark. Finally, 100 µL of stop solution was added, and Optical Density (O.D.) was measured at 450 nm.

Chromatin Immunoprecipitation (ChIP) Assay for DNA-Protein Binding In Vivo
The ChIP assay was conducted with the ChIP-IT ® Express (Cat. No. 53008; Active Motif, Carlsbad, CA, USA) according to the manufacturer's instruction(s) as well as following our published protocol [11,12,65,67]. Antibodies used were control IgG, an antibody specific to Klf9 (Catalog No. 701888, Thermo Scientific) and Nrf2 (Catalog No. Ab180845, Abcam). RT-PCR amplification was performed with 5 µL of DNA sample. The program applied for amplification was 3 min for 94 • C (denaturation), 20 s at 95 • C, 30 s at 59 • C, and 30 s at 72 • C for 36 cycles in 25 µL reaction volume (RT-PCR). The data obtained with RT-PCR was resolved onto 1.5% agarose gel and the band was visualized under UV, and images were captured. The data obtained from qPCR is presented as a histogram. Primers used are noted below. The 5 -region of mouse Klf9 gene, spanning from −5856 to +71 bp was isolated from mouse genomic DNA using an Advantage ® Genomic PCR Kit (Cat. No. 639103 and 639104, Clontech Laboratories, Inc., Mountain View, CA, USA, 94043). A construct of −5856 to +71 bp was engineered by ligating the DNA fragment into CAT reporter plasmid, basic pCAT vector (Promega) by utilizing the MluI and XhoI sites. The plasmid was amplified and verified by sequencing as previously described [11]. The primers used for isolating the genomic DNA fragment were as follows: Forward primer with MluI site: 5 -AAAAACGCGTGGTCATCGTAGGAAAGATGTGG-3 ; and Reverse primer containing XhoI site: 5 -AAAACTCGAGCCTACGAGACACTTCTTCCC-3 .

Preparation of Human Prdx6gene Promoter-Fused to Chloramphenicol Acetyltransferase (CAT) Reporter Plasmid Vector
The 5 -promoter fragment, spanning from −1559 to +30 bp was isolated from human genomic DNA by using an Advantage ® Genomic PCR Kit (Cat. No. 639103 and 639104, Clontech Laboratories, Inc., Mountain View, CA, USA, 94043). The PCR product was cleaned and verified by sequencing as previously described [11]. A fragment containing −1559 to +30 bp with SacI and XhoI was ligated in to basic pCAT vector (Promega) using the SacI and XhoI sites. Primers containing above restriction sites used were as follows: Forward primer; 5 -GACAGAGTTGAGCTCCACACAG-3 ; and Reverse primer; 5 -CACGTCCTCGAGAAGCAGAC-3 .

Site-Directed Mutagenesis (SDM)
SDM was performed with QuikChange TM lightning site-directed mutagenesis kit (Agilent Technologies; Catalog No. 210518), as described in the company's protocol. Briefly, amino acid exchanges at the Klf9 sites (RKBE 1 and RKBE 3) mutants (RKBE 1; GC to TT and RKBE 3; AC to TT) were produced by point mutations in the human promoter of Prdx6linked to CAT plasmid as described earlier [11]. To prepare Nrf2/ARE (ARE 3 and ARE 4) sites mutants, base A was changed to C, and base T to G at ARE sequences in the mouse Klf9 promoter-linked to CAT reporter plasmid by using site-directed mutagenesis. The following complementary primers were utilized (changed nucleotides are in red boldface type and underlined).

Statistical Analysis
For all data obtained from experiments, statistical analyses were carried out by Student's t-test and by one-way ANOVA. Data were represented as mean ± S.D. of the indicated number of experiments. A significant difference between the control and treatment groups was defined as p value of <0.05 and 0.001 for two or more independent experiments.

ROS/H 2 O 2 -Dependent Biological Response and Cellular Fate Was Linked to Cellular Level of Prdx6
In accordance with several reports on the vital influence of ROS, specifically H 2 O 2, on cell signaling, from cell survival to cell death, we wanted to understand how the hormetic effect of endogenous ROS are regulated and how the antioxidant pathway plays its role in this context during oxidative stress. Towards this, we first examined levels of Prdx6 mRNA and protein (data not shown) and its correlation with ROS abundance in hLECs-derived from lenses of subjects of different ages. To examine the oxidative status and its effect on primary hLECs and Prdx6-deficient mLECs, we monitored the total intracellular ROS (DCF oxidation levels) level of H 2 O 2 in both LECs. Expression assays and quantitation of intracellular DCF oxidation levels using H2-DCF-DA [5,6] and H 2 O 2 production using Amplex Red reagent/HRP solution showed an aging-dependent increase in DCF oxidation and H 2 O 2 levels ( Figure 1A,B, 54 y onwards) [5,6,24,72], and that the increasing levels of DCF oxidation and H 2 O 2 were connected to a significant reduction in the level of Prdx6 mRNA and protein with advancing age ( Figure 1A-C). Perhaps unsurprisingly, the data demonstrated that total ROS-mediated DCF oxidation level and the level of H 2 O 2 had similar patterns of abundance in the LECs. To investigate if there is a direct inverse connection between ROS/cellROx oxidation/H 2 O 2 levels and Prdx6 expression, we knocked down the Prdx6 gene using shRNA lentiviral specific to Prdx6 in SRA-hLECs (hLECs) and examined the ROS level as well as H 2 O 2 generation in the cells. We found that, indeed, a significant amplification of CellROX oxidation and H 2 O 2 levels in Prdx6-depleted hLECs as indicated in Figure 1D,E. Figure 1F,G showed successful Prdx6 gene knock down in hLECs. These data pointed to the fact that the biological response of ROS/H 2 O 2 is tightly regulated through antioxidants, such as Prdx6, and further argued that naturally occurring Prdx6 level is essential to fine-tune ROS levels in favor of cell homeostasis. In addition, results indicated that aging is associated with redox changes due to age-dependent deterioration of antioxidant, Prdx6, thereby leading to ROS accumulation. These observations indicate that hormetical effects of ROS can be controlled by intracellular Prdx6 abundance and provided a base for this study to understand the molecular mechanism(s) involved in the ROS-mediated hormetic response. Here, we would care to mention that we utilized the SRA-hLECs (hLECs), a cell line, to knock down Prdx6 as using primary hLECs in experimentation was cumbersome due to their limited availability. deterioration of antioxidant, Prdx6, thereby leading to ROS accumulation. These observations indicate that hormetical effects of ROS can be controlled by intracellular Prdx6 abundance and provided a base for this study to understand the molecular mechanism(s) involved in the ROS-mediated hormetic response. Here, we would care to mention that we utilized the SRA-hLECs (hLECs), a cell line, to knock down Prdx6 as using primary hLECs in experimentation was cumbersome due to their limited availability. from three independent experiments (* p < 0.001). (B) H2O2 levels in primary hLECs were measured using Amplex Red reagent and Horseradish Peroxidase (HRP) solution. The data represent the mean ± S.D. from three independent experiments (* p < 0.001). (C) Total RNA was extracted from LECs isolated from lenses of human individuals of variable age groups and transcribed cDNA was submitted to real time PCR analysis with primers specific to Prdx6. The data represent the mean ± S.D. from three independent experiments (* p < 0.001). (D-G) Prdx6-depleted hLECs displayed increased ROS-induced DCF toxicity levels and production of H2O2 in cells. hLECs were Furthermore, based upon previously published studies documenting that lower or moderate levels of ROS-induced stress can be beneficial in promoting cellular homeostasis by acting as survival signaling molecules, while excessive oxidative stress further augments ROS-induced oxidative stress with loss of antioxidant response [1,3,[11][12][13]15,16,67,76], and coupled with the results of Figure 1 showing suppression of antioxidant protein Prdx6, we intended to examine if there were any connection between cellular abundance of Prdx6 and varying degree of oxidative stress, including modulation in biological responses. To investigate the changes occurring in biological responses to levels of oxidative stress, we induced different levels of oxidative stress in mouse lens epithelial cells (mLECs) by exposing them to different concentrations of H 2 O 2 ( Figure 1H-J). We found that mild/moderate stress resulted in increased Prdx6 expression-dependent regulation of ROS levels in favor of cell health, such as better growth and survival, while increasing doses of H 2 O 2 (≥100 µM, beyond the threshold levels) led to the failure of ROS homeostasis and cell death with the loss of Prdx6 ( Figure 1H-J), suggesting that ROS-mediated hormetic effect was regulated via the cellular abundance of Prdx6. Furthermore, results also revealed an inverse relationship between the ROS and Prdx6 expression ( Figure 1H-J). However, we observed increased viability of mLECs exposed to lower concentrations of H 2 O 2 ( Figure 1I). We reasoned that the observed increase in cell viability could be due to increased cell growth, as it is known that H 2 O 2 can enhance cell proliferation [77]. However, to support the above results, we next utilized Prdx6-deficient (Prdx6 −/− ) mLECs, a redox-active cell. Prdx6 −/− mLECs are highly susceptible to cell death [10,21]. Perhaps unsurprisingly, we observed a significant increase in ROS levels as well as the presence of higher levels of H 2 O 2 generation in Prdx6 −/− mLECs ( Figure 1K,L), suggesting that Prdx6 plays a pivotal role to regulate ROS homeostasis. Taken together, our data demonstrated that changes in the intracellular ROS during aging or by H 2 O 2 -induced oxidative stress were tightly controlled by an antioxidant defense gene, Prdx6. To unveil the molecular mechanism involved in the hormetic effect of ROS and the contribution of Prdx6 to fine-tune ROS homeostasis, we examined the status of Nrf2 in cells facing different degrees of oxidative stress.

Oxidative Stress-Dependent Nrf2-Klf9 Expression Controlled Prdx6 Abundance-Mediated ROS Homeostasis and Cell Fate
Knowledge regarding the redox signaling changes and the molecular event(s) in context to ROS-mediated beneficial and/or deleterious signaling(s) (i.e., the hormetic effect of ROS) is extremely limited. However, by reviewing previously published studies [13,51], we found that Klf9 can negatively regulate the antioxidant pathway during excessive stress or cells treated with excessive doses of electrophilic compounds. Thus, we examined expression levels of Nrf2, Klf9, and antioxidant protein Prdx6 and determined their correlation with ROS levels and cell viability in hLECs exposed to mild to high concentrations of H 2 O 2 as noted in Materials and Methods. Protein blot analysis of total cellular extract prepared from hLECs treated with increasing concentrations of H 2 O 2 is indicated in Figure 2. The visualization of an immunoblotted membrane with an antibody specific to Nrf2 or Klf9 or Prdx6 revealed a significant increase in Nrf2 levels, and paradoxically the increased expression of Nrf2 was associated with the aberrant expression of Klf9 (not a favorable target for Nrf2 pathway) and a dramatic attenuation of Prdx6 expression in cells treated with ≥100 µM of H 2 O 2 concentrations. Next, we asked whether the increase in Klf9 protein and reduction in Prdx6 protein occurred at their transcript level. To examine this, we performed RT-qPCR. qPCR analysis ( Figure 2B) revealed that hLECs exposed to increasing concentrations of H 2 O 2 (≥100 µM) displayed increased expression of Klf9 mRNA with a significant decrease in Prdx6 mRNA expression. Conversely, we found that cells exposed to lower concentrations of H 2 O 2 (<100 µM) had increased Prdx6 expression with only basal expression of Klf9, suggesting that a lower degree of oxidative stress could not induce aberrant Klf9 expression (Figure 2A,B; blue bar).

Oxidative Stress-Dependent Nrf2-Klf9 Expression Controlled Prdx6 Abundance-Mediated ROS Homeostasis and Cell Fate
Knowledge regarding the redox signaling changes and the molecular event(s) in context to ROS-mediated beneficial and/or deleterious signaling(s) (i.e., the hormetic effect of ROS) is extremely limited. However, by reviewing previously published studies [13,51], we found that Klf9 can negatively regulate the antioxidant pathway during excessive stress or cells treated with excessive doses of electrophilic compounds. Thus, we examined expression levels of Nrf2, Klf9, and antioxidant protein Prdx6 and determined their correlation with ROS levels and cell viability in hLECs exposed to mild to high concentrations of H2O2 as noted in Materials and Methods. Protein blot analysis of total cellular extract prepared from hLECs treated with increasing concentrations of H2O2 is indicated in Figure  2. The visualization of an immunoblotted membrane with an antibody specific to Nrf2 or Klf9 or Prdx6 revealed a significant increase in Nrf2 levels, and paradoxically the increased expression of Nrf2 was associated with the aberrant expression of Klf9 (not a favorable target for Nrf2 pathway) and a dramatic attenuation of Prdx6 expression in cells treated with ≥100 µM of H2O2 concentrations. Next, we asked whether the increase in Klf9 protein and reduction in Prdx6 protein occurred at their transcript level. To examine this, we performed RT-qPCR. qPCR analysis ( Figure 2B) revealed that hLECs exposed to increasing concentrations of H2O2 (≥100 µM) displayed increased expression of Klf9 mRNA with a significant decrease in Prdx6 mRNA expression. Conversely, we found that cells exposed to lower concentrations of H2O2 (<100 µM) had increased Prdx6 expression with only basal expression of Klf9, suggesting that a lower degree of oxidative stress could not induce aberrant Klf9 expression (   We next determined the effect of aberrant Klf9 upregulation and its associated depletion of Prdx6 expression on ROS amplification and cellular injuries during different levels of oxidative stress. ROS ( Figure 2C) and cell viability ( Figure 2D) measurements of hLECs revealed that ≤50 µM of H 2 O 2 did have distinguishable beneficial effects and provided better cell survival and growth ( Figure 2C,D; blue bar). In comparison, cells exposed to ≥100 µM of H 2 O 2 showed a dramatic augmentation of ROS levels with a subsequent reduction in cell viability ( Figure 2C,D; red bars). Together, these observations suggested that physiological redox changes can be governed by the Nrf2-Klf9-Prdx6 pathway and that the pathway could be responsible for ROS-mediated modulation of cell signaling, cell survival, or cell death during oxidant-induced stresses. Because Nrf2 and Klf9 are transcriptional proteins and appeared as the major player during oxidative stress, we wanted to identify how and in what way(s) these factors could play a role in the regulation of Prdx6 and ROS during increased oxidative stress. Thus, to unveil the molecular mechanism(s) involved in the onset of adverse signaling and the roles of Nrf2-Klf9 in the process during excessive oxidative stress, we chose higher doses of the oxidative stress inducer, H 2 O 2, to exposed hLECs for our onward experimentation.

Cellular Prdx6 Suppression and ROS Accumulation, and Cell Death Was Connected to Expression and Nuclear Accumulation of Nrf2 and Klf9
From the above experiments, it was apparent that excessively increased expression of Nrf2 and Klf9 is related to decreased expression of Prdx6 with increased ROS levels and cell death, but it was not clear whether these transcriptional proteins were present in the nucleus to exert their activity. To determine intracellular localization(s) of nuclear Nrf2 and Klf9 and cytoplasmic Prdx6 protein in hLECs in response to higher doses of H 2 O 2 exposure, we analyzed the nuclear and cytosolic fractions of H 2 O 2 -treated hLECs for 3 h (acute stress) using expression assay ( Figure 3A). We observed that Nrf2 and Klf9 migrated at 110 kDa and 32 kDa when immunoblotted with antibodies specific to Nrf2 and Klf9, respectively. Nrf2 and Klf9 in the nuclear fraction of hLECs were dramatically accumulated with increasing concentrations of H 2 O 2 ; however, the highest accumulation could be detected with 300 µM of H 2 O 2 . We observed that the increased nuclear accumulation of Nrf2 and Klf9 was directly related to a significant depletion of cytosolic Prdx6 expression (Figures 2 and 3), pointing to the possibility that Prdx6 suppression was Klf9-dependent in hLECs facing excessive oxidative pressure. To determine the fate and status of Klf9, we treated cells with higher doses of H 2 O 2 (150 and 300 µM) and examined the Klf9 and Prdx6 mRNA expression by RT-qPCR ( Figure 3B). However, unexpectedly, we noticed a dramatic inverse correlation between increased Klf9 expression and Nrf2's target gene Prdx6 mRNA in hLECs facing higher doses of H 2 O 2 . To determine if this inverse correlation was due to dysregulation of Nrf2 activity or due to increased abundance of Klf9, we assessed the Nrf2's transactivation capacity by using TransAM Nrf2 transcription factor assay (Active motif) using equal amounts of a nuclear extract isolated from H 2 O 2 exposed or unexposed hLECs. Figure 3C showed a progressive increase in Nrf2-DNA binding activity in response to increasing concentrations of H 2 O 2 , demonstrating that Nrf2 activity is not dysregulated. This finding indicated that there might be involvement of other factor(s), such as Klf9 repressing Prdx6 mRNA as Nrf2 is in an activated form ( Figure 3C). We wanted to confirm whether levels of ROS and cell death were increased, as observed in Figure 2, or some other molecular event(s) occurred at extreme oxidative stress (300 µM of H 2 O 2 ). However, results revealed that excessive concentrations of H 2 O 2 -induced oxidative stress caused a dramatic increase in intracellular ROS when observed at 3 h of H 2 O 2 exposure ( Figure 3D), leading to increased ROS-dependent increased cell death ( Figure 3E). Notably, this injurious process was well correlated with aberrant upregulation of Nrf2 and Klf9 regulation. These results (Figures 2 and 3) served as a base to further explore the role of Nrf2 and Klf9 at the molecular level in the regulation of ROS-mediated hormetical effects. However, we acknowledge that we have used unphysiological concentrations of H 2 O 2 to treat cells in vitro to explore the plausible mechanism that may occur in the regulation of ROS hormetic responses in vivo since cells cultured in vitro develop adoptive as well as reductive responses and become relatively insensitive and therefore, they require higher concentration(s) of oxidants or other biomolecules to respond [9][10][11]13,72]. However, the in vitro work conducted in the present study should uncover the hormetic effects of ROS. However, since Prdx6 was repressed in response to aberrant Klf9 expression during excessive oxidative stress, next we examined the role of Klf9 on Prdx6 regulation-dependent ROS levels in hLECs over-or under-expressing Klf9. regulation of ROS hormetic responses in vivo since cells cultured in vitro develop adoptive as well as reductive responses and become relatively insensitive and therefore, they require higher concentration(s) of oxidants or other biomolecules to respond [9][10][11]13,72]. However, the in vitro work conducted in the present study should uncover the hormetic effects of ROS. However, since Prdx6 was repressed in response to aberrant Klf9 expression during excessive oxidative stress, next we examined the role of Klf9 on Prdx6 regulation-dependent ROS levels in hLECs over-or under-expressing Klf9. Samples containing equal amount of protein were immunoblotted using antibody specific to Nrf2 or Klf9 or LaminB1 or Prdx6 or β-actin as shown, and an increased nuclear accumulation of Nrf2 and Klf9 (Aa) and reduced in Prdx6 expression (Ab) were observed in response to the increased concentrations of H2O2 treatment. Right adjacent panel reveals densitometry analysis of each protein bands. (B) hLECs were exposed to different concentrations of H2O2. 3 h later total RNA was isolated. Real-time qPCR analysis of Klf9 (Ba) and Prdx6 (Bb) with their corresponding specific primers. Data are the mean ± S.D. value of three independent experiments. * p < 0.001 versus control. (C) H2O2 treated hLECs showed a significant increase in Nrf2 activity. hLECs were treated with different concentrations of H2O2 for 3 h, and nuclear extract were analyzed for Nrf2-ARE (antioxidant responsive element) binding by ELISA (enzyme-linked immunosorbent assay). Nuclear extracts containing equal amount of protein were processed and assayed for Nrf2 activity with a commercially available kit (Active motif) as described in Materials and Methods. The data represent the mean ± S.D. from three independent experiments. p values were determined; H2O2 treated versus untreated control. * p < 0.001. Note: to avoid effect of cell death against excessive Samples containing equal amount of protein were immunoblotted using antibody specific to Nrf2 or Klf9 or LaminB1 or Prdx6 or β-actin as shown, and an increased nuclear accumulation of Nrf2 To investigate whether an increase in cellular abundance of Klf9 is, indeed, a cause for Prdx6 suppression as observed during excessive oxidative stress, we extrinsically expressed hLECs with Klf9 plasmid (Figure 4); either with pGFP-Klf9 or pGFP-empty vector plasmid. An equal amount of plasmid DNA was transfected to avoid any effect of DNA concentration on cells. Photomicrographs showed the transfection efficiency ( Figure 4A) and no detectable changes in phenotype(s) between the overexpressing Klf9 and/or EGFP-vector-transfected hLECs. A careful analysis of results obtained from western blot ( Figure 4B) and qPCR ( Figure 4C) of protein extract and total RNA isolated from the transfectants revealed that cells overexpressing Klf9 bore reduced levels of Prdx6 protein and mRNA. To determine the correlation between endogenously generated H 2 O 2 in cells and the overexpression of Klf9, first, we examined the H 2 O 2 levels in Klf9 overexpressed hLECs. Quantification of H 2 O 2 generation in cells by Amplex Red reagent/HRP assay showed significantly elevated levels of H 2 O 2 production ( Figure 4D) in Klf9 overexpressed cells, indicating that Klf9 overexpression enhanced the ROS. Next, we determined the total levels of ROS and cell death of transfectants of the same batch in response to oxidative stress, as indicated in Figure 4A-C. Quantification of ROS ( Figure 4E, red bars vs. black bars) by CellROX Deep Red dye and cell viability ( Figure 4F, red bars vs. black bars) by MTS assay revealed that Klf9 overexpression caused a significant increase in ROS-induced DCF oxidation levels and cell death, demonstrating Klf9's involvement in the regulation of ROS via antioxidants, such as Prdx6 in response to an oxidative stressor. Next, we asked the question of if cells expressing Klf9 beyond the threshold levels are responsive and further showed the stimulation of endogenous Klf9 or expression of Klf9 could be inhibited by other cellular pathways in favor of cell survival in response to oxidative stress. To examine this, hLECs overexpressing Klf9 were treated with 150 µM of H 2 O 2 as indicated in Figure 4G,H. Western analysis and qPCR of total protein and RNA isolated from the transfectants showed further increase in Klf9 and a significant decrease in Prdx6 expression, suggesting there was no dominant active survival pathway, and Klf9 -mediated adverse pathway was dominant. The data demonstrated that the induction of Klf9 and/or its extrinsic overexpression resulted in Klf9-dependent ROS amplification and cell death, which was directly related to the reduced expression of antioxidant genes such as Prdx6. Since other antioxidants failed to protect cells, we argue that here the role of the Nrf2-Klf9-Prdx6 axis is a major determinant of the hormetical effect of ROS and cell fate. hLECs were overexpressed with pEGFP-Vector or pGFP-Klf9 plasmid. 48 h later, complete media were replaced with 0.2% BSA, and cells were exposed to different concentration (0 and 150 µM) H2O2. After 3 h of H2O2 exposure, total protein (G) and RNA (H) were isolated and expression levels were examined by western blot and qPCR, respectively. Data represent the mean ± S.D. of three independent experiments. * p < 0.001; H2O2 treated versus control and pGFP-Klf9 vs. pEGFP-Vector transfected hLECs.

Klf9 Depletion Caused Reduction in ROS Level and Abated Cell Death by Increasing Prdx6 Expression
The set of outcomes of previous experiments in this study demonstrated that overexpression of Klf9 or induction and cellular accumulation of Klf9 in response to increased ROS-driven oxidative stress was injurious to cells, and it was due to Klf9-dependent repression of antioxidant genes, such as Prdx6. Next, we were interested to know whether

Klf9 Depletion Caused Reduction in ROS Level and Abated Cell Death by Increasing Prdx6 Expression
The set of outcomes of previous experiments in this study demonstrated that overexpression of Klf9 or induction and cellular accumulation of Klf9 in response to increased ROS-driven oxidative stress was injurious to cells, and it was due to Klf9-dependent re-pression of antioxidant genes, such as Prdx6. Next, we were interested to know whether Klf9 knock down caused the reduction in ROS levels with increased Prdx6 expression and hLECs viability from oxidative stress as reported for other cell types [13]. To examine this, we depleted Klf9 by stably infecting hLECs using lentiviral (LV) Sh-Control or LV Sh-Klf9. Klf9-depleted hLECs did not show any detectable morphological changes and were indistinguishable from LV Sh-Control ( Figure 5A). Protein and mRNA data using immunoblot and qPCR demonstrated a remarkable increase in Prdx6 protein ( Figure 5B) and mRNA ( Figure 5C) expression, indicating that Klf9 was directly involved in the regulation of Prdx6. Next, we examined if Klf9-depleted hLECs could confer resistance against an increased concentration of H 2 O 2 , as shown in Figure 5D,E. Quantitation of ROS by CellROX Deep Red reagent and cell viability by using MTS assay demonstrated that, indeed, Klf9-deficient hLECs conferred resistance against oxidative stress. Furthermore, we also intended to know the status of Klf9 and Prdx6 in Klf9-depleted cells in response to oxidative stress. We did this experimentation to confirm whether Klf9 inhibitors, such as ShRNA-specific to Klf9, could be a useful strategy to block its aberrant expression-mediated ROS amplification and to restore antioxidant genes suppression, such as Prdx6. We observed that Klf9-depleted cells had an increased ability to survive against H 2 O 2, as shown in Figure 5D,E, and that this was linked to levels of Prdx6 protein ( Figure 5F) and mRNA ( Figure 5G). Thus, we anticipate that the Nrf2-Klf9 axis plays a central role in controlling ROS-mediated hormetic response by altering antioxidant genes expression, such as Prdx6.
Cells 2022, 11, x FOR PEER REVIEW 17 o Klf9 knock down caused the reduction in ROS levels with increased Prdx6 expression a hLECs viability from oxidative stress as reported for other cell types [13]. To examine th we depleted Klf9 by stably infecting hLECs using lentiviral (LV) Sh-Control or LV Sh-K Klf9-depleted hLECs did not show any detectable morphological changes and were ind tinguishable from LV Sh-Control ( Figure 5A). Protein and mRNA data using immunob and qPCR demonstrated a remarkable increase in Prdx6 protein ( Figure 5B) and mRN ( Figure 5C) expression, indicating that Klf9 was directly involved in the regulation Prdx6. Next, we examined if Klf9-depleted hLECs could confer resistance against an creased concentration of H2O2, as shown in Figure 5D,E. Quantitation of ROS by CellR Deep Red reagent and cell viability by using MTS assay demonstrated that, indeed, K deficient hLECs conferred resistance against oxidative stress. Furthermore, we also tended to know the status of Klf9 and Prdx6 in Klf9-depleted cells in response to oxidat stress. We did this experimentation to confirm whether Klf9 inhibitors, such as ShRN specific to Klf9, could be a useful strategy to block its aberrant expression-mediated R amplification and to restore antioxidant genes suppression, such as Prdx6. We observ that Klf9-depleted cells had an increased ability to survive against H2O2, as shown in F ures 5D,E, and that this was linked to levels of Prdx6 protein ( Figure 5F) and mRNA (F ure 5G). Thus, we anticipate that the Nrf2-Klf9 axis plays a central role in controlling RO mediated hormetic response by altering antioxidant genes expression, such as Prdx6.

Klf9 Gene Bore Nrf2 Binding Sequences, ARE, and Nrf2/ARE Binding Increased with Increase in Oxidative Stress
Based on the results obtained from the experiments showing changes at the transcript level (Figures 1-5) in response to oxidative stress, we posited the involvement of transcriptional machinery and conducted experiments to unveil the mechanism of Nrf2 and Klf9-dependent regulation of antioxidant gene, Prdx6, and ROS levels during oxidative stress. Recent studies have shown that the Klf9 promoter bears four putative Nrf2/ARE (antioxidant response elements) binding sites [11,13]. However, sites one and two of Nrf2/ARE were found to be unresponsive (data not shown), while sites three and four of Nrf2/ARE were responsive. It is worth noting that Nrf2 is not a bona fide regulator of Klf9; paradoxically, Klf9 can be transcriptionally regulated by Nrf2 in response to higher doses of electrophilic compounds [11,13]. Thus, in this study, we wanted to determine whether Klf9 could be a target gene of Nrf2 in response to higher oxidative stress. To this end, we first examined if oxidative-stress induced Nrf2 binding to ARE present in the Klf9 promoter as observed in previous studies. To this end, hLECs were treated with different concentrations of H 2 O 2 , and a ChIP-RT-PCR assay was performed to examine the occupancy of Nrf2 on ARE sites present in the Klf9 gene promoter by using antibodies specific to Nrf2 and control IgG as described earlier [11,65,67]. The immunoprecipitated chromatin products were processed and subjected to RT-PCR with specific primers designed for Nrf2/ARE binding sites three and four in Klf9 promoter regions, as indicated in Figure 6A. As shown in Figure 6B, we observed an increased enrichment of Nrf2 on both sites (AREs 3 and 4), and the enrichment of Nrf2 at ARE sequences was dependent on concentrations of H 2 O 2 . The data analyses demonstrated that higher doses of H 2 O 2 aberrantly and unphysiologically regulated the Nrf2 expression and its accumulation into the nucleus and that in turn led to Nrf2/ARE interaction of the Klf9 gene ( Figure 6B). There was no binding with IgG control, demonstrating the specificity of Nrf2 binding at ARE sequences. Taken together, the data demonstrated that the upregulation of Klf9 might be linked to the direct binding of Nrf2 to ARE present in the Klf9 gene promoter during increased oxidative stress.

Nrf2 Upregulated Klf9 Transcription through ARE Binding and the Transcriptional Activity was Oxidative Load-Dependent
Next, we looked at if the observed physical binding of Nrf2 to its responsiv ments, ARE in the regulatory regions of the Klf9 promoter was functional and coul vate Klf9 transcription. Toward this, Figure 6 results of in vivo DNA binding exper led the way to design the CAT reporter plasmids of Klf9 promoter: (i) pCAT-Klf9 type (WT) and (ii) pCAT-Klf9 mutant(s) at all sites (mutant). To determine H2O2-in changes in Klf9 transcription, we transfected hLECs with pCAT-Klf9-WT or its m plasmid, along with EGFP-empty vector plasmid, and then these transfectants treated with an increasing concentration of H2O2 to induce oxidative stress as indica Figure 7. We observed that the Klf9 promoter activity was drastically reduced in th tant Klf9 promoter where ARE sites were disrupted ( Figure 7B; black bars) compa the WT Klf9 promoter. These results documented that Klf9 is transcriptionally reg by a Nrf2/ARE mechanism, and the increase in the transcriptional activity was depe on oxidative levels. These results indicated that the levels of ROS could be controll Klf9 regulation of the antioxidant gene, Prdx6, and we postulated that Prdx6 coul target gene for Klf9. ChIP assay was carried out with Nrf2 and control IgG antibodies. Pulled DNA fragments were subjected to RT-qPCR analysis. DNA fragments present in the immunoprecipitation were amplified with primers that specifically recognized a fragment of Klf9 promoter containing ARE site three and ARE site four as indicated. 10% chromatin was used as input. As a negative control, ChIP with control IgG was used. # and ## denotes primer pairs used for ChIP-RT-PCR.

Nrf2 Upregulated Klf9 Transcription through ARE Binding and the Transcriptional Activity Was Oxidative Load-Dependent
Next, we looked at if the observed physical binding of Nrf2 to its responsive elements, ARE in the regulatory regions of the Klf9 promoter was functional and could activate Klf9 transcription. Toward this, Figure 6 results of in vivo DNA binding experiment led the way to design the CAT reporter plasmids of Klf9 promoter: (i) pCAT-Klf9 wild-type (WT) and (ii) pCAT-Klf9 mutant(s) at all sites (mutant). To determine H 2 O 2 -induced changes in Klf9 transcription, we transfected hLECs with pCAT-Klf9-WT or its mutant plasmid, along with EGFP-empty vector plasmid, and then these transfectants were treated with an increasing concentration of H 2 O 2 to induce oxidative stress as indicated in Figure 7. We observed that the Klf9 promoter activity was drastically reduced in the mutant Klf9 promoter where ARE sites were disrupted ( Figure 7B; black bars) compared to the WT Klf9 promoter. These results documented that Klf9 is transcriptionally regulated by a Nrf2/ARE mechanism, and the increase in the transcriptional activity was dependent on oxidative levels. These results indicated that the levels of ROS could be controlled via Klf9 regulation of the antioxidant gene, Prdx6, and we postulated that Prdx6 could be a target gene for Klf9.

In Vivo DNA Binding Assay Disclosed That Oxidative Stress Level Stimulated Klf9 Bind to its New Target Gene, Prdx6 in Dose-Dependent Fashion
In this study, we found an inverse correlation between expression levels of Prdx6 a Klf9 (Figures 1-4). Furthermore, recently we have reported that aberrant expression Klf9 in response to lethal doses of SFN represses the antioxidant gene Prdx6 [11]. Ho ever, it was not clear whether excessive stress-driven Klf9-dependent ROS amplificat was linked to Klf9-mediated transcriptional repression of Prdx6. To achieve this e hLECs were treated with different concentrations of H2O2, as indicated in Figure 8, a performed a ChIP assay to examine Klf9 binding to its RKBE (repressive Klf9 bind element) present in the Prdx6 gene promoter. As shown in Figure 8, Klf9 occupied Prdx6 gene promoter at RKBEs (5′-C A/G CCC-3′), and notably, the occupancy of Klf9 w increased with an increase in oxidative stress driven by the H2O2 in a concentrationpendent manner. Chromatin immunoprecipitation of H2O2-treated hLECs with an an body specific to Klf9 and control IgG antibody showed that Klf9 bound specifically to repressive site. In the experiment, 10% chromatin was used as input (upper panel), a no amplicon was detected with IgG control (lower panel), demonstrating the specific of Klf9 antibody binding to Klf9 at RKBE sites (middle panel) present in Prdx6 gene p moter. Collectively, data revealed that Klf9 specifically interacted with RKBE sites pres in the Prdx6 promoter.

In Vivo DNA Binding Assay Disclosed That Oxidative Stress Level Stimulated Klf9 Binding to Its New Target Gene, Prdx6 in Dose-Dependent Fashion
In this study, we found an inverse correlation between expression levels of Prdx6 and Klf9 (Figures 1-4). Furthermore, recently we have reported that aberrant expression of Klf9 in response to lethal doses of SFN represses the antioxidant gene Prdx6 [11]. However, it was not clear whether excessive stress-driven Klf9-dependent ROS amplification was linked to Klf9-mediated transcriptional repression of Prdx6. To achieve this end, hLECs were treated with different concentrations of H 2 O 2, as indicated in Figure 8, and performed a ChIP assay to examine Klf9 binding to its RKBE (repressive Klf9 binding element) present in the Prdx6 gene promoter. As shown in Figure 8, Klf9 occupied the Prdx6 gene promoter at RKBEs (5 -C A/G CCC-3 ), and notably, the occupancy of Klf9 was increased with an increase in oxidative stress driven by the H 2 O 2 in a concentration-dependent manner. Chromatin immunoprecipitation of H 2 O 2 -treated hLECs with an antibody specific to Klf9 and control IgG antibody showed that Klf9 bound specifically to its repressive site. In the experiment, 10% chromatin was used as input (upper panel), and no amplicon was detected with IgG control (lower panel), demonstrating the specificity of Klf9 antibody binding to Klf9 at RKBE sites (middle panel) present in Prdx6 gene promoter. Collectively, data revealed that Klf9 specifically interacted with RKBE sites present in the Prdx6 promoter. Pulled DNA fragments with Klf9 antibody were subjected to PCR analysis for RKBE site(s) using primers that recognized a fragment of Prdx6 promoter containing RKBE as indicated. Primers used for amplification of specific region containing Klf9 sites; @ within Klf9 site, @@ within Klf9 site 2, @@@ within Klf9 site 3, @@@@ within Klf9 site 4 and @@@@@ within Klf9 site 5. Control IgG served as negative control.

Loss and Gain Experiments Disclosed That the Prdx6 Transcription was Dependent on Cellular Availability of Klf9 during Oxidative Stress
As shown in Figure 8, Klf9 occupancy was increased to its sites present in the Prdx6 gene promoter in response to increased oxidative stress. Next, we sought to determine the functionality of H2O2-induced increased Klf9 binding on Prdx6 transcription by using a transactivation assay, as shown in Figure 9. We engineered Prdx6 promoter linked to CAT reporter plasmid containing Klf9 sites ( Figure 9A), and plasmid mutated at RKBE1 and RKBE3 (RKBE1-mut + RKBE3-mut) sites created by a Site-Directed Mutagenesis (SDM) kit ( Figure 9A). hLECs expressing pCAT-Prdx6-WT promoter plasmid along with pEGFPempty vector or pGFP-Klf9 expression plasmids were exposed to different concentrations of H2O2 as indicated in Figure 9B. The results revealed that Prdx6 transcription was notably reduced in hLECs overexpressing Klf9 in comparison to the EGFP-empty vector. Perhaps not surprisingly, Prdx6 promoter activity was further reduced in response to H2O2induced stress in a dose-dependent manner in hLECs overexpressing Klf9 ( Figure 9B), demonstrating that increased expression of Klf9 was a cause for repression of Prdx6 transcription. Next, we sought to determine whether Klf9 knock down experiment restored the Prdx6 transcriptional activity; we depleted the Klf9 gene by infecting hLECs with ShRNA specific to Klf9 (LV Sh-Klf9). hLECs infected with lentiviral ShRNA Control (LV Sh-Control) or LV Sh-Klf9 were transfected with pCAT-hPrdx6-WT promoter plasmid as shown in Figure 9C and then were exposed to different concentrations of H2O2. Assay of CAT activity showed increased Prdx6 promoter activity in Klf9-depleted hLECs in comparison to control. Intriguingly, Klf9-deficient hLECs facing oxidative stress showed a significant increase in the Prdx6 promoter activity. Collectively, the results demonstrated that Prdx6 transcription is controlled by Klf9 in conditions of oxidative stress. Next, we wanted to validate whether RKBE/Klf9 is specifically involved in the regulation of Prdx6 transcription; we performed Prdx6 transactivation assay by mutating RKBEs in the Prdx6 gene promoter; to achieve this, we mutated repressive Klf9 binding sites (RKBE; C A/G CCC) one and three using site-directed mutagenesis (SDM) as shown in Figure 9A and described in Materials and Methods section and our published protocol [11]. Transfectants containing either pCAT-hPrdx6-WT or pCAT-hPrdx6-mutant (mutated at RKBE1 and Pulled DNA fragments with Klf9 antibody were subjected to PCR analysis for RKBE site(s) using primers that recognized a fragment of Prdx6 promoter containing RKBE as indicated. Primers used for amplification of specific region containing Klf9 sites; @ within Klf9 site, @@ within Klf9 site 2, @@@ within Klf9 site 3, @@@@ within Klf9 site 4 and @@@@@ within Klf9 site 5. Control IgG served as negative control.

Loss and Gain Experiments Disclosed That the Prdx6 Transcription Was Dependent on Cellular Availability of Klf9 during Oxidative Stress
As shown in Figure 8, Klf9 occupancy was increased to its sites present in the Prdx6 gene promoter in response to increased oxidative stress. Next, we sought to determine the functionality of H 2 O 2 -induced increased Klf9 binding on Prdx6 transcription by using a transactivation assay, as shown in Figure 9. We engineered Prdx6 promoter linked to CAT reporter plasmid containing Klf9 sites ( Figure 9A), and plasmid mutated at RKBE1 and RKBE3 (RKBE1-mut + RKBE3-mut) sites created by a Site-Directed Mutagenesis (SDM) kit ( Figure 9A). hLECs expressing pCAT-Prdx6-WT promoter plasmid along with pEGFPempty vector or pGFP-Klf9 expression plasmids were exposed to different concentrations of H 2 O 2 as indicated in Figure 9B. The results revealed that Prdx6 transcription was notably reduced in hLECs overexpressing Klf9 in comparison to the EGFP-empty vector. Perhaps not surprisingly, Prdx6 promoter activity was further reduced in response to H 2 O 2 -induced stress in a dose-dependent manner in hLECs overexpressing Klf9 ( Figure 9B), demonstrating that increased expression of Klf9 was a cause for repression of Prdx6 transcription. Next, we sought to determine whether Klf9 knock down experiment restored the Prdx6 transcriptional activity; we depleted the Klf9 gene by infecting hLECs with ShRNA specific to Klf9 (LV Sh-Klf9). hLECs infected with lentiviral ShRNA Control (LV Sh-Control) or LV Sh-Klf9 were transfected with pCAT-hPrdx6-WT promoter plasmid as shown in Figure 9C and then were exposed to different concentrations of H 2 O 2 . Assay of CAT activity showed increased Prdx6 promoter activity in Klf9-depleted hLECs in comparison to control. Intriguingly, Klf9-deficient hLECs facing oxidative stress showed a significant increase in the Prdx6 promoter activity. Collectively, the results demonstrated that Prdx6 transcription is controlled by Klf9 in conditions of oxidative stress. Next, we wanted to validate whether RKBE/Klf9 is specifically involved in the regulation of Prdx6 transcription; we performed Prdx6 transactivation assay by mutating RKBEs in the Prdx6 gene promoter; to achieve this, we mutated repressive Klf9 binding sites (RKBE; C A/G CCC) one and three using site-directed mutagenesis (SDM) as shown in Figure 9A and described in Section 2 and our published protocol [11]. Transfectants containing either pCAT-hPrdx6-WT or pCAT-hPrdx6-mutant (mutated at RKBE1 and RKBE3; RKBE1-mutant + RKBE3-mutant) were exposed to different concentrations of H 2 O 2 . We found a significant increase in Prdx6 promoter activity in the pCAT-hPrdx6-mutant promoter constructs mutated at RKBE site(s) compared to pCAT-hPrdx6-WT containing repressive Klf9 binding sites. Altogether our data demonstrated that RKBE sites present in the Prdx6 gene regulate Prdx6 transcription in the condition of the cellular microenvironment. RKBE3; RKBE1-mutant + RKBE3-mutant) were exposed to different concentrations of H2O2. We found a significant increase in Prdx6 promoter activity in the pCAT-hPrdx6mutant promoter constructs mutated at RKBE site(s) compared to pCAT-hPrdx6-WT containing repressive Klf9 binding sites. Altogether our data demonstrated that RKBE sites present in the Prdx6 gene regulate Prdx6 transcription in the condition of the cellular microenvironment. WT along with pEGFP-Vector or pGFP-Klf9 plasmid constructs as shown. 48 h later transfectants were exposed to H2O2 exposure for 3 h as shown, and CAT ELISA was performed to measure the Prdx6 promoter activity. Data are the mean ± S.D. of three independent experiments. * p < 0.001; GFP-Klf9 vs. EGFP-Vector transfected cells and H2O2 treated versus untreated hLECs. (C) Transcription assay displayed increased Prdx6 promoter activity in Klf9-depleted hLECs. LV Sh-Control and/or LV Sh-Klf9 infected hLECs were transfected with pCAT-hPrdx6 (-1559/+30) plasmid construct, 48 h later these cells were exposed to H2O2 as indicated. Prdx6 promoter activity were determined after 3 h of H2O2 exposure. Data are the mean ± S.D. of three independent experiments. # p < 0.05; * p < 0.001; LV Sh-Klf9 vs. LV Sh-Control and H2O2 treated vs. untreated cells. (D) hLECs were transfected with human Prdx6 gene promoter (-1559/+30) linked to CAT wild type (WT) plasmid construct or its mutant at RKBE1 plus RKBE3. 48 h later transfectants were exposed to H2O2 for 3 h as shown, and CAT ELISA was performed to measure Prdx6 promoter activity. Data are the mean ± S.D. of three independent experiments. * p < 0.001 mutant vs. wild-type and H2O2 treated vs untreated cells.

A Transcription Inhibitor, Actinomycin D Treatment Demonstrated That Klf9 Induction by Oxidative Stress Did Not Occur Post-Transcriptionally
To determine whether Klf9 regulation occurred at the transcriptional or post-transcriptional level, hLECs were incubated with Actinomycin D (5 µg/mL) for 30 min, and then these cells were exposed to H2O2 as indicated in Figure 10. Expression assays with total protein extract and RNA using an antibody specific to Klf9 demonstrated that Actinomycin D inhibited the H2O2-induced abundance of Klf9 protein and mRNA expression. WT along with pEGFP-Vector or pGFP-Klf9 plasmid constructs as shown. 48 h later transfectants were exposed to H 2 O 2 exposure for 3 h as shown, and CAT ELISA was performed to measure the Prdx6 promoter activity. Data are the mean ± S.D. of three independent experiments. * p < 0.001; GFP-Klf9 vs. EGFP-Vector transfected cells and H 2 O 2 treated versus untreated hLECs. (C) Transcription assay displayed increased Prdx6 promoter activity in Klf9-depleted hLECs. LV Sh-Control and/or LV Sh-Klf9 infected hLECs were transfected with pCAT-hPrdx6 (−1559/+30) plasmid construct, 48 h later these cells were exposed to H 2 O 2 as indicated. Prdx6 promoter activity were determined after 3 h of H 2 O 2 exposure. Data are the mean ± S.D. of three independent experiments. # p < 0.05; * p < 0.001; LV Sh-Klf9 vs. LV Sh-Control and H 2 O 2 treated vs. untreated cells. (D) hLECs were transfected with human Prdx6 gene promoter (−1559/+30) linked to CAT wild type (WT) plasmid construct or its mutant at RKBE1 plus RKBE3. 48 h later transfectants were exposed to H 2 O 2 for 3 h as shown, and CAT ELISA was performed to measure Prdx6 promoter activity. Data are the mean ± S.D. of three independent experiments. * p < 0.001 mutant vs. wild-type and H 2 O 2 treated vs. untreated cells.

A Transcription Inhibitor, Actinomycin D Treatment Demonstrated That Klf9 Induction by Oxidative Stress Did Not Occur Post-Transcriptionally
To determine whether Klf9 regulation occurred at the transcriptional or posttranscriptional level, hLECs were incubated with Actinomycin D (5 µg/mL) for 30 min, and then these cells were exposed to H 2 O 2 as indicated in Figure 10. Expression assays with total protein extract and RNA using an antibody specific to Klf9 demonstrated that Actinomycin D inhibited the H 2 O 2 -induced abundance of Klf9 protein and mRNA expression. The results indicated that activation of Klf9 by H 2 O 2 does not occur post-transcriptionally, but it was regulated at the transcriptional level. The results indicated that activation of Klf9 by H2O2 does not occur post-transcriptionally, but it was regulated at the transcriptional level.

Under-and Over-Expression Experimentation Disclosed That Klf9 Expression was Transcriptionally Regulated by Nrf2
Next, we asked whether depletion or overexpression of the Nrf2 gene affected Klf9 transcription or expression; hLECs were transiently transfected with ShRNA-Control or ShRNA specific to Nrf2 and generated a stable cell line of hLECs using antibiotic selection pressure. Transfectants were exposed to H2O2, and Nrf2 and Klf9 protein expressions in the nucleus were analyzed by immunoblotting, as shown in Figure 11A. We observed that Nrf2 and Nrf2-mediated nuclear accumulation of Klf9 protein in response to H2O2 treatment in Sh-Control hLECs, while Klf9 did not accumulate in the nucleus of Nrf2-depleted hLECs. Data demonstrated that the cellular abundance of Nrf2 was a requisite for Klf9's aberrant induction and expression. Next, we examined if Nrf2 knock down inhibited Klf9 transcription, Sh-Control and Sh-Nrf2 hLECs were transfected with pCAT-Klf9 WT promoter construct and then exposed to H2O2 as shown in Figure 11B, and Klf9 promoter activity was determined. The results demonstrated that Klf9 transcription was significantly augmented in Sh-Control hLECs treated with H2O2, while there was no significant activation of Klf9 transcription in Sh-Nrf2 hLECs as shown in Figure 11B.

Under-and Over-Expression Experimentation Disclosed That Klf9 Expression Was Transcriptionally Regulated by Nrf2
Next, we asked whether depletion or overexpression of the Nrf2 gene affected Klf9 transcription or expression; hLECs were transiently transfected with ShRNA-Control or ShRNA specific to Nrf2 and generated a stable cell line of hLECs using antibiotic selection pressure. Transfectants were exposed to H 2 O 2, and Nrf2 and Klf9 protein expressions in the nucleus were analyzed by immunoblotting, as shown in Figure 11A. We observed that Nrf2 and Nrf2-mediated nuclear accumulation of Klf9 protein in response to H 2 O 2 treatment in Sh-Control hLECs, while Klf9 did not accumulate in the nucleus of Nrf2depleted hLECs. Data demonstrated that the cellular abundance of Nrf2 was a requisite for Klf9's aberrant induction and expression. Next, we examined if Nrf2 knock down inhibited Klf9 transcription, Sh-Control and Sh-Nrf2 hLECs were transfected with pCAT-Klf9 WT promoter construct and then exposed to H 2 O 2 as shown in Figure 11B, and Klf9 promoter activity was determined. The results demonstrated that Klf9 transcription was significantly augmented in Sh-Control hLECs treated with H 2 O 2 , while there was no significant activation of Klf9 transcription in Sh-Nrf2 hLECs as shown in Figure 11B.
Furthermore, we next sought to determine the Klf9 status in Nrf2 overexpressed hLECs facing oxidative stress. To this end, hLECs overexpressed with pEGFP-vector or pEGFP-Nrf2 were exposed to H 2 O 2 . Figure 11C displayed transfection efficiency, and Figure 11D showed the levels of Nrf2 in the same transfectants through immunoblotting using an antibody specific to Nrf2. hLECs were transfected with pCAT-Klf9-WT-promoter construct along with pEGFP-Vector or pEGFP-Nrf2 plasmid and exposed to oxidative stress as indicated in Figure 11E. The data revealed that Klf9 transcriptional activity was significantly increased in hLECs overexpressing Nrf2 and that the Klf9 transcriptional activity was further increased in response to oxidative stress. On the whole, data demonstrated that Klf9 is regulated by Nrf2, and Klf9 cellular abundance is directly connected to the increased cellular availability of Nrf2. We surmise that these phenomena that occurred during oxidative stress play a pivotal role in Nrf2-Klf9-dependent regulation of Prdx6 and Prdx6 regulation of ROS levels and biological response(s). Furthermore, we next sought to determine the Klf9 status in Nrf2 overexpressed hLECs facing oxidative stress. To this end, hLECs overexpressed with pEGFP-vector or pEGFP-Nrf2 were exposed to H2O2. Figure 11C displayed transfection efficiency, and Figure 11D showed the levels of Nrf2 in the same transfectants through immunoblotting using an antibody specific to Nrf2. hLECs were transfected with pCAT-Klf9-WT-promoter construct along with pEGFP-Vector or pEGFP-Nrf2 plasmid and exposed to oxidative stress as indicated in Figure 11E. The data revealed that Klf9 transcriptional activity was significantly increased in hLECs overexpressing Nrf2 and that the Klf9 transcriptional activity was further increased in response to oxidative stress. On the whole, data demonstrated that Klf9 is regulated by Nrf2, and Klf9 cellular abundance is directly connected to the increased cellular availability of Nrf2. We surmise that these phenomena that occurred during oxidative stress play a pivotal role in Nrf2-Klf9-dependent regulation of Prdx6 and Prdx6 regulation of ROS levels and biological response(s).

Discussion
The hormetic response of oxidative stress or electrophilic compounds may be at- Figure 11. Loss and gain experiments of Nrf2 disclosed that altered Klf9 expression and its promoter activity was directly linked to cellular levels of Nrf2. (A,B) Nrf2-deficient hLECs fails to activate the Klf9 expression and its transcription. (A) Nrf2-depleted hLECs were exposed to H 2 O 2 as indicated in Figure 3A for 3 h. Nuclear extract were prepared, and equal amount of protein was immunoblotted using an antibody specific to Nrf2 or Klf9. (B) Nrf2-deficeint hLECs were transfected with WT Klf9 promoter (−5856/+71) linked to CAT vector. The transfectants were exposed to H 2 O 2 for 3 h as shown, and Klf9 promoter activity was measured and shown. Data are the mean ± S.D.

Discussion
The hormetic response of oxidative stress or electrophilic compounds may be attributed to levels of intracellular ROS, and ROS-driven beneficial or deleterious signaling [78][79][80]. Thus, maintenance of ROS homeostasis is imperative for beneficial biological response in favor of cells health. ROS include H 2 O 2, and H 2 O 2 plays a key role as an intracellular messenger to control cellular physiology in a concentration-dependent fashion [43,[81][82][83]. In this study, we intended to uncover the molecular mechanism involved in the regulation and changes of ROS and H 2 O 2 homeostasis and the molecular events which are responsible for the hormetic effects of ROS. The results from this study, for the first time, showed oxidative stress-driven hormetic effect is regulated via Nrf2-Klf9 regulation of antioxidant response at least in part at the transcriptional level. Our findings suggest that (i) a lower level of oxidative stress (below the threshold levels) results in Nrf2/Prdx6mediated maintenance of ROS homeostasis and cellular survival while excessive degree (beyond the threshold level) leads to adverse signaling-induced cells injuries; (ii) mecha-nistically, the changes in intracellular ROS were related to Nrf2-mediated expression and accumulation of Klf9, and Klf9-dependent suppression of Prdx6, resulting in increased ROS levels; (iii) the data also revealed that Nrf2 is transcriptionally regulated Klf9 transcription depending on cellular redox microenvironment; and (iv) Prdx6 expression determined the levels of ROS in response to oxidative conditions via Nrf2-Klf9 activation. It is noteworthy that the intrinsic generation of ROS at a normal physiological level is the major source of oxidants due to the malfunction of antioxidants; however, exposure to extrinsic stresses is an unavoidable factor(s) that can further augment oxidant levels. Recently, adaptive stress responses (hormetic effects), the concept of oxidative stress, and antioxidant responses have been widely accepted. This demonstrates that the lower exposure of prooxidants leads to environmental priming of antioxidant responses, and the process results in better cell survival due to increased antioxidant response [84]. Mechanistically, we reasoned that the presence of mild levels of oxidant during physiological stress within the cellular microenvironment acts as signaling molecules that make cells ready to cope with increased redox challenges, and thus cells survive well under adverse conditions. A similar phenomenon has recently been observed in enhanced survival of neuronal cells; mild/moderate stress applied to cortical neurons decreases the intensity of lipofuscin granules and pathological aggregates as well as in other cell types [85,86]. Moreover, it has been documented that variation in hormetic response is genetically determined, and the hormesis phenomenon plays a pivotal role in maintaining cellular status by regulating cell physiology [87][88][89]. Moreover, many aging-related diseases such as neurodegenerative disease, cancer, cardiovascular disease, cataract, glaucoma, diabetes, etc. are directly or indirectly associated with increased levels of ROS [6,9,[90][91][92][93][94][95][96]. To maintain ROS homeostasis, cells are equipped with antioxidant defense systems, containing antioxidant proteins, such as superoxide dismutase (SODs), catalase (CAT), peroxiredoxins (Prdxs), glutathione (GSH), glutathione peroxidase (GPx) [11,12,67,97], however how the antioxidant system goes awry and what molecular events occur, leading to ROS-driven increased cells or tissues damage in response to oxidative stress and with advancing age are still not well explored. In this study, we found that a progressive decline in cellular abundance of Prdx6 expression was directly connected to increased expression of transcriptional proteins Klf9 and Nrf2 with elevated ROS accumulation and subsequent cell death in the aging cells or cells facing oxidative stress ( Figure 1). Interestingly, as observed in Figures 1-6, the changes in Prdx6 expression were directly connected to the augmented cellular abundance of Nrf2 and Klf9 and the levels of ROS alterations, suggesting that Prdx6 is pivotal to regulate ROS-driven redox signaling. Based on these results, we think that hormetic effects of ROS may be regulated by cellular Prdx6 levels, thereby Prdx6 determines the fate of the cells in cellular microenvironment conditions. Nevertheless, we acknowledge that Prdx6 regulation of ROS levels may not only be linked to modulation in the transcription of Prdx6 but also its posttranslational modifications, specifically, oxidative modification, can play a role in cellular signaling [98,99]. ROS can do so by acting on downstream target proteins such as Prdx6 within the signal transduction pathway to alter their activities via reversible /unreversible oxidation of Cysteine residues [83,100]. However, how Prdx6 posttranslational modification contributes to the regulation of intracellular ROS and thereby in cellular signaling within the cellular microenvironment warrants further investigation.
In previous studies, we have shown that loss of Prdx6 causes an increase in intracellular ROS with increased susceptibility to oxidants-induced cell death, and we also found that other antioxidants were not effective at protecting cells [6,7,9,21,59,65,68,69,71,96,101], suggesting that the role of Prdx6 is pivotal to control ROS homeostasis. Moreover, intracellular ROS generation evoked by oxidative inducers, such as H 2 O 2 or UV radiation-induced oxidative stress, can damage the cells but also can stimulate the biological process such as cell growth, cell differentiation, and migration of the cells by inducing the Nrf2/ARE-mediated survival pathway [102]. Nonetheless, the hormetic effects of oxidative stress will depend upon levels of intracellular ROS, which are regulated via antioxidant genes such as Prdx6. In this study, we have utilized different concentrations of H 2 O 2 to uncover the molecular mechanism(s) involved in the controlling hormetic effects of ROS. Our results revealed that relatively increased level of oxidative stress, the level of transcription factor Klf9 was aberrantly expressed and accumulated in the nucleus, and this was directly correlated with increased expression of Nrf2 ( Figure 2). Interestingly, we found that paradoxically, Nrf2's increased expression-mediated Klf9 upregulation, and Klf9-dependent suppression of Prdx6 mRNA and protein and ROS accumulation (Figures 2 and 3). Our results are consistent with studies as reported by other investigators [13,47,51,56].
Furthermore, an investigation of the underlying molecular mechanism(s) involved in Klf9 activation (induction of Klf9) during oxidative stress showed that lower levels of oxidative stress induce the activation of Nrf2 and Nrf2-Prdx6-mediated cellular protection (Figures 2 and 3). In contrast, at higher levels of oxidative stress, Nrf2 is overly accumulated in the nucleus and activates the Klf9 gene transcription, which represses the antioxidant gene expression, such as Prdx6, with a further increase in ROS production ( Figure 3). Furthermore, in support of the above observations, our studies also showed that overexpression of Klf9 suppresses Prdx6 expression with increased ROS levels, and these cells were highly susceptible to death in response to oxidative stress. While depletion of the Klf9 gene in hLECs enhanced the Prdx6 gene expression and the transfectants engendered resistance with reduced levels of ROS. These results demonstrate that Nrf2-Klf9 regulates the hormetic effects of ROS via Prdx6 gene transcription (Figures 4 and 5). Moreover, oxidative stress beyond the threshold level induces the Klf9 gene expression. Cells expressing an increased level of Klf9 are found to be over-sensitive to oxidative stressinduced death [11,13]. Klf9-overexpressed NIH3T3 and WI38 cells have been reported to be sensitive to ROS amplification-mediated cell death facing toxic doses of electrophilic compound(s), and the adverse process was related to reduced expression of some antioxidant proteins [13]. Our results of this study revealed that Klf9-depleted cells engender resistance against cell death are in agreement with previously reported works [13], wherein the investigators have shown that Klf9-depletion provides resistance against oxidants such as paraquat-induced oxidative stress in NIH3T3 and WI38 cells. Furthermore, it has been shown that Klf9-deficient mice confer resistance to bleomycin-induced oxidative stress and pulmonary fibrosis. Recently, in studies with other model systems, it has been reported that activated Klf9 increases endoplasmic reticulum (ER)-stress via facilitating calcium release from ER. Conversely, Klf9-depletion decreases tunicamycin-induced ER-stress in mouse liver [103]. In addition, Klf9-depletion reduces the protease inhibitors bortezomib (BTZ) and carfilzomib (CFZ)-induced ROS levels and leads to~50% less cell death by enhancing the thioredoxin reductase 2 (Txnrd2) expression in MM1.S and RPMI-8226 cells [104]. These data suggest that the expression level of Klf9 is involved in the regulation of ROS and can determine ROS-dependent biological responses via antioxidant genes regulation, as observed in this study. Recent evidence reveals that in mammalian systems, H 2 O 2 and other forms of ROS act as secondary messengers in regulating many types of biological functions and physiological activities [105]. Superoxide radical and hydroxyl radical, key members of the ROS class, including less reactive H 2 O 2 , are found to be involved in many cellular processes [106]. Furthermore, the increase in intracellular steady-state levels of ROS due to deficiency of cellular antioxidant(s) can halt prosurvival signaling pathways, suggesting regulation of antioxidant genes and their physiological abundance are required to reverse ROS-mediated cell-death signaling. We recently showed that toxic concentration of SFN can cause aberrant expression and accumulation of Nrf2 in the nucleus, resulting in the repression of antioxidant genes via Klf9 activation [11]. Protease inhibitors BTZ and CFZ induced oxidative stress and enhanced the Klf9 gene expression, which represses the Txnrd2 expression [104]. ROS have hormetic effects on melanin synthesis and melanosome transfer; at high doses, ROS impair melanosome transfer in vitiligo, while low doses promote beneficial proliferation [107]. Additionally, hormetic regulation on angiogenesis as well as on prostaglandin production by ROS has been reported [108,109].
Mechanistically, our studies showed that the regulation of ROS hormesis involves activation of the Nrf2-Klf9-Prdx6 axis; our results of DNA binding coupled with transcriptional assays disclosed oxidative stress-dependent increased activation of Nrf2 ( Figure 3C), and its direct binding to ARE present in Klf9 gene promoter and increased transcription (Figures 7-9). Similarly, NIH3T3 cells, when exposed to higher concentrations of SFN, showed enhanced Nrf2's functional binding to ARE sequences present in the Klf9 promoter leading to Klf9 stimulation-dependent cytotoxicity with suppression of antioxidant gene [13]. This explains the hormetic effects of electrophilic compounds that occur through changes in ROS levels, as found in this study. Furthermore, it has been reported that XBP1 transcriptionally activates Klf9 under high endoplasmic stress (ER) conditions; however, the study did not show whether increased ROS are involved in these phenomena [103]. Nevertheless, our results revealed that Nrf2-induced increased activation of Klf9 was a cause for the repression of Prdx6 transcription. It occurred by Klf9 binding to its repressive binding elements (RKBE) present in some of the antioxidant gene promoters such as Prdx6 [11,13]. On the whole, our findings demonstrate that cellular expression of the Nrf2-Klf9-Prdx6 pathway is regulated at the transcriptional level, wherein Nrf2-Klf9-mediated Prdx6 expression and their cellular abundance play a central role in the regulation of ROS homeostasis, thereby determining ROS-mediated hormetic effects, beneficial or adverse signaling and biological responses. We acknowledge that oxidative stress-induced cellular signaling is complex. This can alter other cellular processes, such as translational as well as posttranslational modification(s) of genes/proteins such as antioxidant genes. These changes in molecules may also affect cellular physiology by changing ROS homeostasis during oxidative stress [24,58,110]. We also recognize that other mechanism (s) or processes could be involved in regulating ROS, such as posttranslational modification of proteins, which needs further investigation; the investigation of such signaling, specifically related to Prdx6 and hormetic effects of ROS are further research line of our laboratory. Nevertheless, our studies suggested that the Nrf2-Klf9-Prdx6 axis can regulate the hormetic effect of ROS at least in part via transcriptional repression of Prdx6 genes in conditions of oxidative stress.

Conclusions
In conclusion, our data demonstrated, for the first time, the plausible molecular mechanism of ROS regulation and ROS-mediated hormetic effects during oxidative stress and aging ( Figure 12). We found that the hormetic consequence of ROS was regulated at least in part by transcription suppression of Prdx6 and was connected to Nrf2-Klf9's cellular status. However, predicting Nrf2's beneficial or harmful effect is challenging since it is dose-dependent on inductors. Thus, it is highly imperative to take into consideration and to target the factor(s) responsible for harmful signaling during Nrf2 activation. Because our results revealed that Klf9 upregulation due to aberrant Nrf2 activation causes ROS amplification and subsequent cell death (Nrf2-Prdx6-mediated survival pathway turns to death pathway) in response to excessive oxidative stress, and the fact that these events are at the transcriptional level, we propose that the application of Klf9 ShRNA and/or Klf9 inhibitor(s) would be a possible strategy to blunt or treat age-associated disorders related to increased ROS-induced injurious signaling. However, future studies are warranted to develop Klf9 antagonists and determine the consequences that the hormetic effects of ROS may have on other physiological functions. and/or Klf9 inhibitor(s) would be a possible strategy to blunt or treat age-associated disorders related to increased ROS-induced injurious signaling. However, future studies are warranted to develop Klf9 antagonists and determine the consequences that the hormetic effects of ROS may have on other physiological functions. Figure 12. Diagrammatic Illustration of the molecular mechanism governing the bimodal effect of ROS responsible for cellular response in response to oxidative stress. Mild to moderate oxidative stress regulates the Nrf2 at physiological level while excessive oxidative stress aberrantly accumulates the Nrf2 into nucleus. Physiological regulations of Nrf2 activates its target antioxidant genes transcription and expression by binding to antioxidant response element (ARE) sites present in the regulatory region of antioxidant genes and maintain the cellular ROS homeostasis which leads to beneficial cell signaling. At higher levels of oxidative stress, aberrant accumulations of Nrf2 in nucleus, paradoxically activated Klf9 transcription by binding to its ARE sites present in Klf9 gene promoter, and this aberrant Klf9 expression repressed some of the antioxidant genes, such as Prdx6. Finally, these phenomena result in Klf9 abundance-dependent increased ROS amplification, and subsequent cell injuries/death at least in part by transcriptional suppression of Prdx6. Taken together, our data demonstrate that Nrf2-Klf9-Prdx6-axis can play a role in determining ROS-mediated signaling pathway and biological response(s) in conditions of oxidative stress. Funding: This study was supported by the National Eye Institute, NIH EY024589 (to D.P.S.).

Institutional Review Board Statement:
The human ocular samples used in this study was derived from deceased individuals. Thus according to per the regulations at 45 CFR 46. 102(f)(1)(2) this study is not considered to be human subject as they are not living at the time the samples obtained, and does not require IRB (Institutional Review Board) oversight. The all animal experiments followed the recommendation set forth in the "Statement for the use of Animals in Ophthalmic and Vision Figure 12. Diagrammatic Illustration of the molecular mechanism governing the bimodal effect of ROS responsible for cellular response in response to oxidative stress. Mild to moderate oxidative stress regulates the Nrf2 at physiological level while excessive oxidative stress aberrantly accumulates the Nrf2 into nucleus. Physiological regulations of Nrf2 activates its target antioxidant genes transcription and expression by binding to antioxidant response element (ARE) sites present in the regulatory region of antioxidant genes and maintain the cellular ROS homeostasis which leads to beneficial cell signaling. At higher levels of oxidative stress, aberrant accumulations of Nrf2 in nucleus, paradoxically activated Klf9 transcription by binding to its ARE sites present in Klf9 gene promoter, and this aberrant Klf9 expression repressed some of the antioxidant genes, such as Prdx6. Finally, these phenomena result in Klf9 abundance-dependent increased ROS amplification, and subsequent cell injuries/death at least in part by transcriptional suppression of Prdx6. Taken together, our data demonstrate that Nrf2-Klf9-Prdx6-axis can play a role in determining ROS-mediated signaling pathway and biological response(s) in conditions of oxidative stress.