MEKK-3 Acts Cooperatively with NSY-1 in SKN-1-Dependent Manner against Oxidative Stress and Aging in Caenorhabditis elegans

Simple Summary Oxidative stress has been linked to myriad pathologies and aging. Here, we show that MEKK-3- and NSY-1-mediated nuclear translocation of SKN-1 is critical for protection against oxidative stress and for longevity in Caenorhabditis elegans. A genetic approach revealed that the knockdown of MEKK-3 and NSY-1, components of the p38 MAPK pathway, significantly reduced the resistance to oxidative stress achieved by SKN-1 overexpression. Mechanistic analyses showed that MEKK-3 and NSY-1 participate in the accumulation of SKN-1 in intestinal nuclei, which is required for the activation of phase II detoxification genes. These results provide molecular insights into oxidative stress resistance and reveal potential targets for aging-associated diseases. Abstract Oxidative stress resulting from reactive oxygen species and other toxic metabolites is involved in human diseases, and it plays an important role in aging. In Caenorhabditis elegans, SKN-1 is required for protection against oxidative stress and aging. As p38 mitogen-activated protein kinase signaling is activated in response to oxidative stress, SKN-1 accumulates in intestinal nuclei and induces phase II detoxification genes. However, NSY-1, a well-known mitogen-activated protein kinase kinase kinase (MAPKKK) of C. elegans, acts as a partial regulator of the SKN-1-induced oxidative stress signaling pathway, suggesting that the regulator for optimal activation of SKN-1 remains unknown. Here, we report a MAPKKK, MEKK-3, as a new regulator required for full activation of SKN-1-mediated resistance against oxidative stress and aging. In RNA-interference-based screening, we found that the simultaneous knockdown of mekk-3 and nsy-1 significantly decreased the oxidative stress resistance and survival of SKN-1 transgenic worms. MEKK-3 was induced in response to oxidative stress. Mechanistic analysis revealed that double knockdown of mekk-3 and nsy-1 completely suppressed the nuclear localization of SKN-1. These results were reproduced in mutant worms in which SKN-1 is constitutively localized to intestinal nuclei. In addition, mekk-3 and nsy-1 were required for optimal induction of SKN-1 target genes such as gcs-1 and trx-1. These data indicate that MEKK-3 plays an essential role in the SKN-1-dependent signaling pathway involved in oxidative stress resistance and longevity by cooperating with NSY-1.


Introduction
Aging is a complex process involving natural degradation [1]. Several theories have focused on the mechanisms of aging, including free radicals, metabolic stress, and telomere dysfunction [2][3][4]. According to the free radical aging theory, oxidative damage to cells and tissues causes aging, and this mechanism has been implicated in the pathophysiology of numerous diseases [5,6]. It has been hypothesized that aging is caused by a combination of

RNAi
RNAi experiments were performed by feeding the worms as previously described, with minor modifications [22]. Plates for RNAi were made by adding ampicillin at a concentration of 100 g/mL and isopropyl β-D-1-thiogalactopyranoside (IPTG, Merck-Millipore, Burlington, MA, USA) at a concentration of 2 mM to NGM. RNAi clones were grown in LB medium that was treated with 100 µg/mL ampicillin and 12.5 µg/mL tetracycline at 37 • C. The following day, the cultures were diluted (1:50), grown to an OD 600 of 1, and induced with 1 mM IPTG. This culture was used for seeding plates containing tetracycline, ampicillin, and IPTG and left to dry for 1-2 days. The gene knockdown experiments were performed by transferring the worms on these plates, including RNAi bacteria.

GFP Fusion Protein Scoring System
SKN-1 accumulation in the intestinal nuclei was scored as previously described [16]. Strong nuclear SKN-1::GFP was observed throughout the entire intestine ("High"), while high levels of SKN-1::GFP were observed anteriorly or posteriorly but were barely detectable through the intestine ("Medium"). Fluorescence images were acquired using an Axiovert 200 microscope (Zeiss, Germany) and measured using the ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Oxidative Stress Resistance Assay
To investigate resistance against oxidative stress, young adult worms were transferred onto plates containing 7.5 or 20 mM tert-butyl hydrogen peroxide (t-BOOH, Merck Millipore) in NGM agar at 20 • C. Worms were considered dead when they stopped responding to repeated pick prodding.

Lifespan Assay
Animals were synchronized by laying timed eggs overnight and were allowed to develop at 20 • C. When animals reached L4 or young adulthood, they were transferred to plates with OP50, RNAi bacteria, or t-BOOH; kept at 20 • C, and periodically scored as alive or dead in response to prodding. The data did not include nematodes or worms that escaped or died of vulval bursting.

RNA Isolation and Quantitative Real-Time PCR (qRT-PCR)
Mixed-stage worms were collected from each strain and then washed three times with M9 buffer. Total RNA was isolated using TRI Reagent (Merck Millipore), according to the manufacturer's protocol. The concentration and purity of RNA were determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher, Waltham, MA, USA). cDNA was synthesized using 2.5 µg of RNA and the SuperScript III cDNA synthesis kit (Merck-Millipore), according to the manufacturer's specifications. Gene expression levels were determined by qRT-PCR using SYBR Premix Ex Taq II (Takara Biotechnology, Dalian, China) and ViiA7 System (Thermo Fisher). The amount of target mRNA was normalized to that of act-1 mRNA. The primer sequences used are listed in Table S1.

Statistics
Statistical analyses were performed using Prism 9 (GraphPad Software, Inc., La Jolla, CA, USA). Statistical significance was determined using Student's t-test for comparing two Biology 2022, 11, 1526 4 of 11 means or a one-way/two-way analysis of variance with a post hoc test (Dunnett, Tukey) for comparing multiple means. Statistical significance was set at p < 0.05. Statistical analyses for survival were conducted using Log-rank (Mantel-Cox) test. Statistical analyses for SKN-1 nuclear localization were conducted using a chi 2 test. Data are expressed as the mean ± SEM.

Concomitant Inhibition of mekk-3 and nsy-1 Significantly Suppresses the Oxidative Stress Resistance of SKN-1 Transgenic Worms
To identify a regulator of SKN-1 in the p38 MAPK pathway in C. elegans, we screened novel MAPKKKs that regulate SKN-1 under oxidative stress conditions by comparing the survival rates of SKN-1 transgenic worms treated with RNAi against the candidate genes. Since NSY-1 is a partial regulator of SKN-1 in the oxidative stress pathway [20], we screened by simultaneous inhibition of nsy-1 and MAPKKKs, including dlk-1 (the ortholog of mammalian MAP3K12), tap-1 (the ortholog of mammalian TAB1), mom-4 (the ortholog of mammalian MAP3K7), zak-1 (the ortholog of mammalian MAP3K20), irk-1 (the ortholog of mammalian KCNJ14, KCNJ16, and KCNJ18), mtk-1 (the ortholog of mammalian MAP3K4), kin-18 (the ortholog of mammalian TAOK1, TAOK2, and TAOK3), and mekk-3 (the ortholog of mammalian MAP3K3) using specific RNAi (Figure 1a). The knockdown efficiency of each RNAi was confirmed by qRT-PCR ( Figure S1). SKN-1 transgenic worms displayed an extraordinarily high survival rate even when exposed to 5-10 mM t-BOOH, an exogenous inducer of ROS, in the conventional oxidative stress assay ( Figure S2); therefore, an extraordinarily high concentration of 20 mM t-BOOH was used for the screening. We found that concomitant inhibition of mekk-3 and nsy-1 showed the most significant decrease in the RNAi screening ( Figure 1a) and completely suppressed resistance against oxidative stress compared to a single knockdown (Figure 1b). These results showed that mekk-3 RNAi suppressed the oxidative stress resistance achieved through the overexpression of SKN-1, and mekk-3 partially complements the function of nsy-1.

Statistics
Statistical analyses were performed using Prism 9 (GraphPad Software, Inc., La Jolla, CA, USA). Statistical significance was determined using Student's t-test for comparing two means or a one-way/two-way analysis of variance with a post hoc test (Dunnett, Tukey) for comparing multiple means. Statistical significance was set at p < 0.05. Statistical analyses for survival were conducted using Log-rank (Mantel-Cox) test. Statistical analyses for SKN-1 nuclear localization were conducted using a chi 2 test. Data are expressed as the mean ± SEM.

Concomitant Inhibition of mekk-3 and nsy-1 Significantly Suppresses the Oxidative Stress Resistance of SKN-1 Transgenic Worms
To identify a regulator of SKN-1 in the p38 MAPK pathway in C. elegans, we screened novel MAPKKKs that regulate SKN-1 under oxidative stress conditions by comparing the survival rates of SKN-1 transgenic worms treated with RNAi against the candidate genes. Since NSY-1 is a partial regulator of SKN-1 in the oxidative stress pathway [20], we screened by simultaneous inhibition of nsy-1 and MAPKKKs, including dlk-1 (the ortholog of mammalian MAP3K12), tap-1 (the ortholog of mammalian TAB1), mom-4 (the ortholog of mammalian MAP3K7), zak-1 (the ortholog of mammalian MAP3K20), irk-1 (the ortholog of mammalian KCNJ14, KCNJ16, and KCNJ18), mtk-1 (the ortholog of mammalian MAP3K4), kin-18 (the ortholog of mammalian TAOK1, TAOK2, and TAOK3), and mekk-3 (the ortholog of mammalian MAP3K3) using specific RNAi (Figure 1a). The knockdown efficiency of each RNAi was confirmed by qRT-PCR ( Figure S1). SKN-1 transgenic worms displayed an extraordinarily high survival rate even when exposed to 5-10 mM t-BOOH, an exogenous inducer of ROS, in the conventional oxidative stress assay ( Figure  S2); therefore, an extraordinarily high concentration of 20 mM t-BOOH was used for the screening. We found that concomitant inhibition of mekk-3 and nsy-1 showed the most significant decrease in the RNAi screening ( Figure 1a) and completely suppressed resistance against oxidative stress compared to a single knockdown ( Figure 1b). These results showed that mekk-3 RNAi suppressed the oxidative stress resistance achieved through the overexpression of SKN-1, and mekk-3 partially complements the function of nsy-1.

MEKK-3 Overexpression Displays Increased Resistance against Oxidative Stress
To determine the expression pattern of MEKK-3, we analyzed the expression of a transgene in which GFP was fused to the C-terminus of full-length MEKK-3 (MEKK-3::GFP). Expression of MEKK-3::GFP was observed in the intestine of wild-type (N2) young adult worms (Figure 2a). Notably, after treatment with 7.5 mM t-BOOH at 20 • C for 16 h, the intestinal cells showed high levels of MEKK-3::GFP (Figure 2a,b), and the mRNA expression of MEKK-3 was significantly increased (Figure 2c). In addition, we tested whether MEKK-3 overexpression increased resistance against oxidative stress. Surprisingly, MEKK-3:: GFP transgenic worms showed approximately three times higher resistance to oxidative stress than N2 (Figures 2d and S3); however, their longevity was not significantly higher than that of N2 ( Figure S3, Table S2). Collectively, these results suggest that MEKK-3 overexpression has a strong positive effect on resistance against oxidative stress; however, it is not sufficient to extend the lifespan.
Biology 2022, 11, x FOR PEER REVIEW 5 of 12 points (h). This representative experiment involved 30 worms in each group. Data are shown as the mean ± SEM.

MEKK-3 Overexpression Displays Increased Resistance against Oxidative Stress
To determine the expression pattern of MEKK-3, we analyzed the expression of a transgene in which GFP was fused to the C-terminus of full-length MEKK-3 (MEKK-3::GFP). Expression of MEKK-3::GFP was observed in the intestine of wild-type (N2) young adult worms (Figure 2a). Notably, after treatment with 7.5 mM t-BOOH at 20 °C for 16 h, the intestinal cells showed high levels of MEKK-3::GFP (Figure 2a,b), and the mRNA expression of MEKK-3 was significantly increased (Figure 2c). In addition, we tested whether MEKK-3 overexpression increased resistance against oxidative stress. Surprisingly, MEKK-3:: GFP transgenic worms showed approximately three times higher resistance to oxidative stress than N2 (Figures 2d and S3); however, their longevity was not significantly higher than that of N2 ( Figure S3, Table S2). Collectively, these results suggest that MEKK-3 overexpression has a strong positive effect on resistance against oxidative stress; however, it is not sufficient to extend the lifespan.
Next, we investigated the effects of MEKK-3 and NSY-1 on SKN-1 localization using a phosphorylation-inhibiting mutant in which serine residues at 393 of SKN-1::GFP were replaced with alanine (SKN-1 S393A::GFP). In a previous study, the SKN-1 S393A::GFP mutant was constitutively localized to the intestinal nuclei by alteration of the critical inhibitory glycogen synthase kinase-3 (GSK-3) phosphorylation site; it increased oxidative stress resistance compared to N2 SKN-1::GFP [24]. We found that the nuclear localization of SKN-1 S393A::GFP was strong in the intestinal nuclei but was significantly decreased by genetic knockdown of mekk-3 and nsy-1 via RNAi (Figure 3d,e). These results also exclude the possibility that sek-1 and p38 signaling is simply epistatic to gsk-3 with respect to SKN-1 regulation. Interestingly, the individual and synergistic effects of mekk-3 and nsy-1 knockdown were more pronounced on the lifespan of SKN-1 S393A transgenic worms than on SKN-1 transgenic worms (Figure 3f, Table S2). This is possibly because SKN-1-dependent lifespan extension in the absence of GSK-3 inhibitory phosphorylation enhances the effect of mekk-3 and nsy-1 knockdown on longevity. Collectively, these results demonstrated that MEKK-3 is required for the nuclear translocation of SKN-1 and that it plays a synergistic role with NSY-1.

MEKK-3 Is Required for Optimal Activation of SKN-1 Target Genes
SKN-1 promotes the expression of phase II detoxification genes, which encode enzymes responsible for antioxidants and glutathione synthesis [13,16]. The significant role of MEKK-3 was determined via examination of SKN-1 target genes, such as gcs-1 and trx-1, which encode the gamma-glutamine cysteine synthase heavy chain and thioredoxin, respectively, and was induced by oxidative stress ( Figure S5). To investigate whether MEKK-3 regulates SKN-1 target genes, we used transgenic worms overexpressing gcs-1 with C-terminal GFP (GCS-1::GFP) or trx-1 with C-terminal GFP (TRX-1::GFP). Under oxidative stress conditions, mekk-3 and nsy-1 knockdown significantly decreased the expression of GCS-1::GFP (Figure 4a,b) and mRNA expression of gcs-1 (Figure 4c). Similarly, the expression of TRX-1::GFP (Figure 4d,e) and mRNA expression of trx-1 (Figure 4f) were significantly decreased following mekk-3 and nsy-1 knockdown. These results indicate that MEKK-3 is a key factor in the fine regulation and optimal activation of detoxification genes induced by SKN-1. Biology 2022, 11, x FOR PEER REVIEW 7 of 12  Table S2. (d) The nuclear localization of SKN-1 S393A mutant labeled with GFP. All procedures were performed in the same manner as described in (a). SKN-1 nuclear localization of each inset was confirmed by using DAPI staining in Figure S4. (e) Quantitative analyses of the fluorescent images of SKN-1 S393A::GFP. All procedures were performed in the same manner as described in (b) (n = 30 in each group). (f) Lifespan assay was performed in SKN-1 S393A transgenic worms. All procedures were performed in the same manner as described in Figure 1b. This experiment involved 90 worms in each group. The mean and median lifespan are shown in Table S2. ** p < 0.01 compared to control. All p values were calculated by chi 2 tests.  Table S2. (d) The nuclear localization of SKN-1 S393A mutant labeled with GFP. All procedures were performed in the same manner as described in (a). SKN-1 nuclear localization of each inset was confirmed by using DAPI staining in Figure S4. (e) Quantitative analyses of the fluorescent images of SKN-1 S393A::GFP. All procedures were performed in the same manner as described in (b) (n = 30 in each group). (f) Lifespan assay was performed in SKN-1 S393A transgenic worms. All procedures were performed in the same manner as described in Figure 1b. This experiment involved 90 worms in each group. The mean and median lifespan are shown in Table S2. ** p < 0.01 compared to control. All p values were calculated by chi 2 tests. with C-terminal GFP (GCS-1::GFP) or trx-1 with C-terminal GFP (TRX-1::GFP). Under oxidative stress conditions, mekk-3 and nsy-1 knockdown significantly decreased the expression of GCS-1::GFP (Figure 4a,b) and mRNA expression of gcs-1 (Figure 4c). Similarly, the expression of TRX-1::GFP (Figure 4d,e) and mRNA expression of trx-1 (Figure 4f) were significantly decreased following mekk-3 and nsy-1 knockdown. These results indicate that MEKK-3 is a key factor in the fine regulation and optimal activation of detoxification genes induced by SKN-1.

Discussion
In the present study, we showed that MEKK-3 is critically involved in SKN-1-dependent resistance against oxidative stress and aging, particularly by regulating the translocation of SKN-1 into the nucleus and its target genes. We found that MEKK-3 overexpression acts as a sensor of oxidative stress (Figure 2a-c). Notably, MEKK-3 exhibited a synergistic effect with NSY-1 in response to oxidative stress (Figures 1, 3 and 4). Previous reports have suggested that NSY-1 is a partial regulator of SKN-1 [20], presumably through another regulatory factor that is redundant with NSY-1. The authors hypothesized that an additional pathway would be required to activate SKN-1 via interaction with SEK-1 or PMK-1. However, the results of the present study, surprisingly, showed that MEKK-3, another MAPKKK of the p38 MAPK pathway, could partially supplement the role of NSY-1, implying that the evolutionary adaptation of existing regulators with similar functions is more efficient than the development of new molecular pathways.
The dual specificity of kinases in the MAPK pathway has been reported by multiple pieces of literature [25,26]. For example, MEK-1 and MEK-2 are dual-specificity kinases that phosphorylate both ERK substrates (ERK1 and ERK2) with high selectivity [27]. Notably, single phosphorylation of either threonine or tyrosine residues leads to partial activation of ERK protein, while dual phosphorylation of both residues is needed for complete activation of ERK. In addition, MEK proteins require dual phosphorylation of two serine residues by several kinases, including Raf, TPL2, and Mos [28]. Although the universality of the principle of dual phosphorylation in kinase-mediated pathways is unclear, dual phosphorylation mediated by kinases and phosphatases can provide a more precise regulatory system favorable to the cellular defense systems and the survival of the organism.
Recently, MEKK-3 was reported to be a regulator of the nutrient-sensing pathway and an initiator of the diet-restriction (DR) response [29] as MEKK-3-deficient C. elegans phenocopies a DR-like state. The authors suggested that knocking down mekk-3 induces metabolic reprogramming, where β-oxidation is upregulated, and production of ROS is reduced, while xenobiotic detoxification genes such as pha-4, nhr-8, and ahr-1/aha-1 are upregulated. In addition, they suggested that the coupling of the metabolism shift and xenobiotic detoxification leads to extended longevity during mekk-3 knockdownmediated DR. Interestingly, the effect of mekk-3 knockdown on lifespan extension depends on temporal conditions of the onset of RNAi onset. Knocking down the mekk-3 gene at L4 or later produced no effect, while the maximum extension of lifespan was observed when mekk-3 RNAi was initiated at the L1 stage [29], suggesting that the temporal requirements of mekk-3 are different from those of the insulin/IGF-1 signaling pathway and are similar to those of mitochondrial genes that affect lifespan [30]. Therefore, the results of the present study are consistent with those of previous reports (Figure 3c,f). Further investigation of mekk-3 requirements, including the potential role of nsy-1 in this condition, may contribute to the elucidation of the mechanism against cellular stresses such as metabolic or oxidative stress.

Conclusions
We used genetic and mechanistic approaches to demonstrate that MEKK-3 plays a key physiological role in the SKN-1-mediated oxidative stress pathway. MEKK-3 and NSY-1 deficiencies lead to defects in the nuclear localization of SKN-1, which decreases resistance against oxidative stress and aging due to a reduction in the expression of SKN-1 target genes such as gcs-1 and trx-1. In addition, high levels of MEKK-3 induced by oxidative stress would further increase the resistance against oxidative stress.
Human Nrf protein-mediated ROS protection is thought to be helpful in a wide variety of diseases, including diabetes, neurodegenerative diseases, atherosclerosis, and viral infection. This gene activation may offer a generally applicable method of cancer prevention by aiding in drug detoxification, which is crucial for chemotherapy tolerance. For example, the consumption of chemoprotective antioxidants, which are mediated by Nrf2, prevents the development of chemical carcinogenesis in mice [31] and lowers the incidence of gastrointestinal and lung cancer in humans [32,33]. The functional similarities between SKN-1 and Nrf proteins would open the way for a new therapeutic by exploring these SKN-1-mediated pathways in C. elegans, which is particularly appropriate for genetic and pharmacological screening.
Increasing evidence also suggests that the p38 MAPK pathway plays a pivotal role in aging-related diseases, such as cancer, arthritis, diabetes, and neurodegenerative diseases [34,35]. Therefore, the finding that MEKK-3 deficiency resulted in reduced resistance to oxidative stress and aging has enormous implications in the search for druggable targets against aging-associated diseases, as well as in elucidating the physiological mechanisms of aging.