Abstract
In nematodes, the insulin signaling pathway can potentially participate in the regulation of various biological processes. In this chapter, we further discussed the involvement and possible pivotal function of core insulin signaling pathway in the regulation of toxicity of environmental toxicants or stresses. Moreover, we introduced the related information on the potential targets for DAF-16 and the possible upregulators for the insulin signaling pathway in regulating the toxicity of environmental toxicants or stresses. The possible formation of a large physical interaction surrounding the DAF-16 in regulating the toxicity of environmental toxicants or stresses needs to be paid more attention in nematodes.
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Keywords
- Insulin and the related signaling pathways
- Molecular regulation
- Environmental exposure
- Caenorhabditis elegans
5.1 Introduction
The nematode Caenorhabditis elegans has been successfully used in both the toxicity assessment and the toxicological study of various toxicants or stresses [1]. In nematodes, it has been shown that exposure to environmental toxicants or stresses can lead to the toxicity on many aspects of animals as reflected by a series of toxicity assessment endpoints [2,3,4,5,6,7,8,9,10]. Meanwhile, the insulin signaling pathway has been widely proven to participate in the regulation of various biological processes in organisms [11,12,13,14,15,16,17,18]. More and more data have implied the possible or even the potential pivotal role of insulin signaling pathway in the regulation of stress response in nematodes exposed to environmental toxicants or stresses.
In C. elegans, in the core insulin signaling pathway, insulin ligands bind to DAF-2/IGF-1 receptor (InR) to activate tyrosine kinase activity, which will allow to initiate the cascade of several kinases (AGE-1/phosphatidylinositol 3-kinase (PI3K), PDK-1/3-phosphoinositide-dependent kinase 1, AKT-1/2/serine/threonine kinase Akt/PKB, and SGK-1/serine or threonine-protein kinase). AKT and the SGK-1 will further phosphorylate and inactivate the transcription factor DAF-16/FOXO, which thereby blocks the transcription of its multiple target genes to regulate various biological processes [19, 20]. We here first introduced the involvement of core insulin signaling pathway in the regulation of toxicity of environmental toxicants or stresses. We also introduced and discussed the potential targets for DAF-16 in the insulin signaling pathway in the regulation of toxicity of environmental toxicants or stresses. Moreover, we further introduced and discussed the possible upregulators for insulin signaling pathway in the regulation of toxicity of environmental toxicants or stresses. So far, the obtained data imply the possible formation of a large physical interaction surrounding the DAF-16 in the regulation of toxicity of environmental toxicants or stresses in nematodes.
5.2 Environmental Toxicants or Stresses Dysregulate the Expression of Insulin Signaling Pathway
Graphene oxide (GO), an important carbon-based engineered nanomaterials, can cause several aspects of toxicity, including adverse effects on the function of both primary (such as the intestine) and secondary (such as the neurons and the reproductive organs) targeted organs, on nematodes [21,22,23,24,25,26,27]. With the GO as an example, prolonged exposure to GO (100 mg/L) could result in a significant increase in the expression levels of daf-2, age-1, akt-1, and akt-2 and decrease in the expression levels of daf-18 and daf-16 in wild-type nematodes [28]. Additionally, a significant increase in DAF-16:GFP expression in the nuclei of GO-exposed (100 mg/L) nematodes was also observed [28], which suggests that long-term exposure to GO not only affects the transcriptional activities of genes encoding the core insulin signaling pathway but also influences the nucleus-cytoplasm translocation of DAF-16. The induction of nucleus-cytoplasm translocation of DAF-16 and/or decrease in daf-16 expression could also be observed in nematodes exposed to heavy metals (such as Mn or As) or traffic-related PM2.5 [29,30,31,32]. Therefore, exposure to certain environmental toxicants or stresses may potentially dysregulate the expression of insulin signaling pathway in nematodes (Fig. 5.1).
5.3 The Insulin Signaling Pathway Regulates the Toxicity of Environmental Toxicants or Stresses
Further with GO as an example, it has been shown that mutation of daf-16 or daf-18 could induce a susceptibility to GO toxicity in decreasing locomotion behavior and in reducing lifespan, whereas mutation of daf-2, age-1, akt-1, or akt-2 could induce a resistance to GO toxicity in decreasing locomotion behavior and in reducing lifespan (Fig. 5.2) [28]. In nematodes, mutation of daf-16 could also induce a susceptibility to the toxicity of heavy metals or traffic-related PM2.5 in inducing intestinal ROS production [31, 33,34,35,36]. Additionally, mutation of daf-2 or age-1 could also suppress the toxicity of Hg in inducing deficits in development of male-specific structures, of heavy metals (Cd or Ca) or hypoxic stress in reducing lifespan, or of traffic-related PM2.5 in inducing intestinal ROS production [31, 33,34,35,36]. These studies performed on different environmental toxicants or stresses demonstrate the important function of insulin signaling pathway in regulating the toxicity of environmental toxicants or stresses in nematodes.
5.4 Genetic Interactions of Genes in the Insulin Signaling Pathway in Regulating the Toxicity of Environmental Toxicants or Stresses
In nematodes, genetic interaction analysis demonstrated that DAF-16 acted downstream of DAF-2, AGE-1, AKT-1, or AKT-2 to regulate the GO toxicity in reducing the longevity, because mutation of daf-16 could effectively decrease the lifespan in daf-2(e1370), age-1(hx546), akt-1(ok525), or akt-2(ok393) mutant nematodes exposed to GO (Fig. 5.3) [28]. Therefore, a signaling cascade of DAF-2-AGE-1-AKT-1/2-DAF-16 in the insulin signaling pathway was identified to be involved in the control of GO toxicity. Meanwhile, it was found that mutation of daf-18 could effectively reduce the lifespan in age(hx546) mutant exposed to GO (Fig. 5.3) [28], which suggests the suppressor role of DAF-18 on the function of AGE-1 in the regulation of GO toxicity. The raised signaling cascade in the insulin signaling in regulating toxicity of environmental toxicants or stresses was further supported or confirmed by other toxicological studies performed in nematodes. It was also observed that mutation of daf-16 could suppress the resistance of daf-2 mutant nematodes to the traffic-related PM2.5 toxicity in inducing intestinal ROS production or enhancing intestinal permeability, to the combined Ca/Cd toxicity in reducing the lifespan, or to the As toxicity in inducing ROS production [31, 32, 35].
5.5 Targets of DAF-16 in Regulating the Toxicity of Environmental Toxicants or Stresses
5.5.1 SOD-3
In C. elegans, daf-16 is expressed in almost all tissues, including the intestine, the neurons, the muscle, and the pharynx. Nevertheless, expression of the daf-16 in neurons, muscle, or pharynx could not significantly affect the GO toxicity in decreasing locomotion behavior and in reducing lifespan in daf-16(mu86) mutant nematodes [28]. In contrast, intestinal expression of daf-16 could effectively augment the decreased locomotion behavior or reduced lifespan in GO-exposed daf-16(mu96) mutant nematodes [28], which demonstrating that the DAF-16 acts primarily in the intestine to regulate the toxicity of environmental toxicants or stresses. Actually, the core insulin signaling pathway can act in the intestine to regulate the GO toxicity in nematodes [28].
SOD-3, a mitochondrial iron/manganese superoxide dismutase, is expressed in the pharynx in the head, the intestine, the muscle, the vulva, and the tail. Intestinal RNAi knockdown of sod-3 could induce a susceptibility to GO toxicity in reducing lifespan [28]. Genetic interaction analysis suggested that DAF-16 acted upstream of SOD-3 to regulate the GO toxicity, because the resistance of transgenic strain of Ex(Pges-1-daf-16) overexpressing intestinal DAF-16 to GO toxicity in reducing lifespan and in inducing intestinal ROS production could be inhibited by sod-3 mutation (Fig. 5.4) [28]. This observation also implies that prolonged exposure to GO could inhibit the function of DAF-16 within the insulin signaling and, thereby, result in the further suppression of the function of SOD-3, which plays an important role in defense against oxidative stress in nematodes [28].
5.5.2 Antimicrobial Proteins
In nematodes, among the candidate targeted genes for DAF-16, some genes (lys-1, lys-7, lys-8, dod-6, F55G11.4, spp-1, spp-12, and dod-22) encoding potential antimicrobial proteins have also been identified to act as the targeted genes for daf-16 in regulating the toxicity of GO toxicity [37,38,39,40]. Among these genes encoding potential antimicrobial proteins, RNAi knockdown of lys-1, dod-6, F55G11.4, lys-8, or spp-1 could significantly suppress the resistance of nematodes overexpressing intestinal daf-16 to GO toxicity in inducing intestinal ROS production and in decreasing locomotion behavior (Fig. 5.5) [41], suggesting that LYS-1, DOD-6, F55G11.4, LYS-8, and SPP-1 act as downstream targets for intestinal DAF-16 in regulating the toxicity of environmental toxicants or stresses.
Among LYS-1, DOD-6, F55G11.4, LYS-8, and SPP-1, F55G11.4 and SPP-1 acted further downstream of SOD-3 in the regulation of GO toxicity, because RNAi knockdown of F55G11.4 or spp-1 could significantly suppress the resistance of nematodes overexpressing intestinal sod-3 to GO toxicity in inducing intestinal ROS production and in decreasing locomotion behavior [41]. The antimicrobial proteins of F55G11.4 and SPP-1 affected the expression of gas-1 encoding a subunit of mitochondrial complex I that is required for the oxidative phosphorylation in GO-exposed nematodes [41], implying the important effect of F55G11.4 and SPP-1 on GAS-1-mediated molecular basis for oxidative stress in GO-exposed nematodes.
5.5.3 MTL-1 and MTL-2
In nematodes, mtl-1 and mtl-2 encode metallothioneins and can be expressed in the intestine. Exposure to the outdoor PM2.5 could induce the significant expression of MTL-1 and MTL-2 [42]. Meanwhile, mutation of the mtl-1 or mtl-2 resulted in a susceptibility to the outdoor PM2.5 toxicity [42], because a more severe decrease in locomotion behavior and a more significant induction of intestinal ROS production were observed in mtl-1(tm1770) or mtl-2(gk125) mutants exposed to outdoor PM2.5 (10 mg/L) compared with those in wild-type nematodes [42]. After PM2.5 exposure, the head thrash and body bend in the double mutant of daf-16(mu86);mtl-1(RNAi) were similar to those in daf-16(mu86) or mtl-1(RNAi) nematodes, and the head thrash and body bend in the double mutant of daf-16(mu86);mtl-2(RNAi) were similar to those in daf-16(mu86) or mtl-2(RNAi) nematodes (Fig. 5.6) [42]. These observations suggest that MTL-1 or MTL-2 acted in the same genetic pathway with DAF-16 in regulating the toxicity of environmental toxicants or stresses. Moreover, it was observed that the outdoor PM2.5 exposed daf-2(e1370);mtl-1(RNAi) mutant exhibited a similar head thrash and body bend to those in outdoor PM2.5 exposed mtl-1(RNAi) nematodes, and the outdoor PM2.5 exposed daf-2(e1370);mtl-2(RNAi) mutant exhibited a similar head thrash and body bend to those in outdoor PM2.5 exposed mtl-2(RNAi) nematodes (Fig. 5.6) [42]. That is, MTL-1 and MTL-2 may act further downstream of the DAF-16 to regulate the toxicity of environmental toxicants or stresses in nematodes.
5.5.4 NATC-1
In nematodes, natc-1 encodes an evolutionarily conserved subunit of the N-terminal acetyltransferase C (NAT) complex, and the N-terminal acetylation is a useful modification for eukaryotic proteins. NATC-1 is expressed in many cells and tissues and localizes to the cytoplasm [43]. Loss-of-function mutation of natc-1 caused a resistance to a broad-spectrum of physiologic stressors, including multiple metals (such as Zn), heat stress, and oxidation stress [43]. DAF-16 was predicted to directly bind the natc-1 promoter, and natc-1 mRNA levels were repressed by DAF-16 activity, indicating the role of natc-1 as a physiological target of DAF-16 (Fig. 5.7) [43]. Additionally, the daf-2 mutants displayed a twofold decrease in natc-1 expression compared to wild-type nematodes [43]. Genetic interaction analysis demonstrated that natc-1(am138) could enhance the dauer formation in daf-2(e1370) mutant nematodes, and the daf-2(e1370);natc-1(am138) double mutant nematodes displayed enhanced stress resistance compared to either single mutant animal (Fig. 5.7) [43]. Moreover, the daf-16(mu86);natc-1(am138) double mutant animals displayed heat stress resistance similar to natc-1 single mutant animals, although the daf-16(mu86) mutant displayed a mild sensitivity to the heat stress (Fig. 5.7) [43]. Therefore, NATC-1 functions downstream of DAF-16 to mediate the resistance of nematodes to environmental toxicants or stresses.
5.5.5 HSF-1
Pseudomonas aeruginosa is a commonly considered bacterial pathogen for human beings [44,45,46,47]. In nematodes, both the daf-2(e1370) mutant nematodes and the nematodes carrying additional daf-16 gene copies were resistant to P. aeruginosa and showed higher levels of HSP90 than wild-type animals (Fig. 5.8) [48], suggesting that a higher activity of HSF-1 may be in part responsible for the increased resistance of nematodes to P. aeruginosa. In contrast, this enhanced resistance of daf-2(e1370) and daf-16::gfp animals to P. aeruginosa was reduced by the RNAi knockdown of hsf-1 (Fig. 5.8) [48]. Additionally, the heat-shock protection was not detected in daf-16 RNAi nematodes (Fig. 5.8) [48]. Therefore, the HSF-1-regulated proteins may be effectors for the signaling cascade of DAF-2-DAF-16 required for the pathogen resistance in nematodes.
5.5.6 Genetic Interaction Between SOD-3 and Antimicrobial Proteins in the Regulation of Toxicity of Environmental Toxicants or Stresses
In nematodes, it has been further observed that RNAi knockdown of sod-3 could not affect the resistance of nematodes overexpressing intestinal lys-1 or lys-8 to GO toxicity in inducing intestinal ROS production and in decreasing locomotion behavior, although RNAi knockdown of sod-3 could suppress the resistance of nematodes overexpressing intestinal dod-6 to GO toxicity in inducing intestinal ROS production and in decreasing locomotion behavior (Fig. 5.9) [41]. These results imply that LYS-1 and LYS-8 acted in a different genetic pathway from the DAF-16-SOD-3 signaling cascade, and DOD-6 acted upstream of SOD-3 to regulate the toxicity of environmental toxicants or stresses.
LYS-1 and LYS-8 are two members of the lysozyme family. Genetic interaction analysis demonstrated that LYS-1 and LYS-8 functioned redundantly in the regulation of GO toxicity, because the GO-exposed double mutant of lys-8(ok3504);lys-1(ok2445) exhibited the more severe induction of intestinal ROS production and decrease in locomotion behavior than that in GO-exposed lys-1(ok2445) mutant or in GO-exposed lys-8(ok3504) mutant [41]. Meanwhile, after GO (10 mg/L) exposure, mutation of lys-1 or lys-8 did not influence the expressions of clk-1, gas-1, and isp-1, which are required for the control of oxidative stress [41].
Moreover, it was found that mutation of lys-1 significantly decreased the transcriptional expression of tub-2 in GO (10 mg/L) exposed nematodes, and intestine-specific RNAi of tub-2 significantly suppressed the resistance of nematodes overexpressing intestinal LYS-1 to GO toxicity in inducing ROS production and in decreasing locomotion behavior (Fig. 5.10) [41]. tub-2 encodes an ortholog of human tubby like protein 4, and intestine-specific RNAi of tub-2 could cause a susceptibility to GO toxicity [41]. Besides this, it was also observed that loss-of-function mutation of lys-8 significantly decreased the transcriptional expression of daf-8 and increased the transcriptional expression of daf-5 in GO (10 mg/L) exposed nematodes [41]. In nematodes, daf-5 encodes a transcriptional factor, and daf-8 encodes a R-Smad protein in the TGF-b signaling pathway. Intestine-specific RNAi of daf-8 could cause a susceptibility to GO toxicity in inducing ROS production, whereas intestine-specific RNAi of daf-5 caused a resistance to GO toxicity in inducing ROS production [41]. Furthermore, intestine-specific RNAi of daf-8 could significantly inhibit the resistance of transgenic strain overexpressing intestinal lys-8 to GO toxicity in inducing ROS production and in decreasing locomotion behavior (Fig. 5.10) [41]. Therefore, these results suggest the formation of signaling cascades of DAF-16-LYS-1-TUB-2 and DAF-16-LYS-8-DAF-8-DAF-5 in regulating the toxicity of environmental toxicants or stresses in nematodes.
5.6 Upregulators of Insulin Signaling Pathway in Regulating the Toxicity of Environmental Toxicants or Stresses
So far, many upregulators of insulin signaling pathway in regulating the biological processes, especially the longevity, have been raised. However, only limited upregulators of insulin signaling pathway in regulating the toxicity of environmental toxicants or stresses have been identified.
5.6.1 SMK-1
In nematodes, smk-1 encodes a homolog to mammalian SMEK. Under the normal conditions, the smk-1(mn156) mutant nematodes have a reduced lifespan, normal locomotion behavior, and no significant induction of intestinal ROS production [49]. After prolonged exposure to GO (100 mg/L), the smk-1(mn156) mutant showed the more severe reduction in lifespan, decrease in locomotion behavior, and induction of intestinal ROS production than wild-type nematodes [49]. Similarly, the smk-1(mn156) mutant also exhibited the susceptibility to the toxicity of coal combustion-related fine particulate matter (PM2.5) in inducing intestinal ROS production and in decreasing locomotion behavior [50]. These results suggest the potential susceptibility of smk-1(mn156) mutant nematodes to the toxicity of environmental toxicants or stresses.
In nematodes, smk-1 is expressed in the intestine, pharynx, neurons, muscle, and hypodermis. The tissue-restricted expression of smk-1 in the pharynx, the neurons, the muscle, or the hypodermis did not affect the GO toxicity in reducing lifespan and in decreasing locomotion behavior in smk-1(mn156) mutant nematodes; however, expression of smk-1 in the intestine could significantly suppress the GO toxicity in reducing lifespan and in decreasing locomotion behavior in smk-1(mn156) mutant nematodes [49]. That is, SMK-1 acted in the intestine to regulate the GO toxicity. Moreover, it was observed that the transgenic strain of Is(Pges-1-smk-1) overexpressing intestinal smk-1 had a resistance to the GO toxicity in reducing lifespan and in decreasing locomotion behavior [49], which further confirmed the tissue-specific activity of SMK-1 in the intestine in the regulation of GO toxicity.
Moreover, it has been shown that the lifespan and the locomotion behavior in GO (100 mg/L) exposed double mutant of daf-16(RNAi);smk-1(mn156) were similar to those in GO (100 mg/L) exposed single mutant of smk-1(mn156) or daf-16(RNAi) nematodes [49], suggesting that SMK-1 and DAF-16 may act in the same genetic pathway to regulate the toxicity of environmental toxicants or stresses. More importantly, it was found that RNAi knockdown of daf-16 could significantly suppress the protective effects of smk-1 overexpression on both the lifespan and the locomotion behavior in GO-exposed nematodes (Fig. 5.11) [49], which demonstrates that SMK-1 acts upstream of DAF-16 to regulate the toxicity of environmental toxicants or stresses in nematodes.
In nematodes, it has been further found that the expressions of sod-3, sod-4, and ctl-3 were significantly decreased in smk-1 mutant nematodes compared to wild-type N2 after coal combustion-related PM2.5 exposure (Fig. 5.12) [50]. SOD-3 (an iron/manganese superoxide dismutase), SOD-4 (an extracellular Cu2+/Zn2+ superoxide dismutase), and CTL-3 (a catalase) are considered as the possible downstream targets of DAF-16. After exposure to coal combustion-related PM2.5, sod-3(RNAi), sod-4(RNAi), or ctl-3(RNAi) nematodes had a significantly higher induction of intestinal ROS production and decrease in locomotion compared to the wild-type N2 (Fig. 5.12) [50], suggesting the susceptibility of sod-3(RNAi), sod-4(RNAi), or ctl-3(RNAi) nematodes to the toxicity of coal combustion-related PM2.5. These results imply that the possible signaling cascade of SMK-1-DAF-16-SOD-3/SOD-4/CTL-3 may exist in regulating the toxicity of environmental toxicants or stresses in nematodes.
5.6.2 AAK-2
In nematodes, aak-2 encodes a catalytic alpha subunit of AMP-activated protein kinases (AMPKs). Multi-walled carbon nanotubes (MWCNTs) is another important carbon-based nanomaterials widely used in different fields [51,52,53,54]. Previous study has indicated that AAK-2 may function upstream of DAF-16 in insulin signaling pathway to regulate the longevity [19]. In nematodes, mutation of aak-2 induced a susceptibility to the toxicity of both MWCNTs and GO [51, 55]. Moreover, it was observed that the lifespan and the locomotion behavior at adult day-8 in MWCNTs (1 mg/L) exposed double mutant of daf-16(mu86);aak-2(om524) were similar to those in MWCNTs (1 mg/L) exposed single mutant of aak-2(om524) or daf-16(mu86) nematodes (Fig. 5.13) [51], implying that AAK-2 can further act together with DAF-16 in the same genetic pathway to form a signaling cascade of AAK-2-DAF-16 to regulate the toxicity of environmental toxicants or stresses in nematodes.
5.6.3 JNK-1
JNK-1 is a core component in the JNK signaling pathway. We have introduced the related detailed information in the Chap. 4. It was further observed that the loss-of-function mutation of jnk-1 could significantly suppress the nuclear translocation of DAF-16 caused by heat stress or ROS stress (induced by H2O2) (Fig. 5.14) [56]. The degree of nuclear translocation of DAF-16::GFP was generally and statistically significantly lower in the jnk-1 mutant than in the wild type after exposure to heat stress or ROS stress (Fig. 5.14) [56]. Moreover, loss-of-function mutation of jnk-1 could further significantly inhibit the increase in SOD-3::GFP (a direct target of DAF-16) induced by heat stress [56]. Therefore, JNK-1 may modulate the environmental toxicant- or stress-induced translocation of DAF-16 from the cytosol into the cell nucleus in nematodes.
5.6.4 HCF-1
In nematodes, hcf-1 encodes a conserved homolog of host cell factor 1. The hcf-1(pk924) mutant nematodes showed the resistant to the paraquat treatment compared to wild-type nematodes at multiple time points (Fig. 5.15) [57]. Moreover, it has been found that this paraquat resistance of the hcf-1(pk924) mutants was dependent on daf-16, as the daf-16(mgDf47);hcf-1(pk924) double mutant was sensitive to paraquat, similar to that of the daf-16(mgDf47) single mutant (Fig. 5.15) [57]. Similarly, the hcf-1(pk924) mutant nematodes were resistant to the cadmium exposure compared to wild-type nematodes at multiple time points, and the cadmium resistance of the hcf-1(pk924) mutant was also daf-16-dependent (Fig. 5.15) [57]. It has been found that the RNA levels of sod-3, mtl-1, and F21F3.3 encoding a farnesyl cysteine carboxyl methyltransferase were significantly elevated in both the hcf-1(ok559) and the hcf-1(pk924) mutants as compared to wild-type nematodes [57]. This elevated expression of sod-3, mtl-1, and F21F3.3 in the hcf-1 mutant nematodes was also dependent on daf-16, since the levels of sod-3, mtl-1, and F21F3.3 in the daf-16(mgDf47);hcf-1(ok559) double mutant remained low and was similar to that seen in the daf-16(mgDf47) single mutant nematodes [57]. Meanwhile, among the DAF-16-repressed genes, the expression level of C32H11.4 showed a greater than twofold downregulation in hcf-1 mutant nematodes compared to wild-type nematodes, and this repressed expression of C32H11.4 could also be partially dependent on daf-16 [57]. Therefore, HCF-1 may act upstream of DAF-16 and suppress the function of DAF-16 to regulate the toxicity of environmental toxicants or stresses in nematodes.
5.6.5 SIR-2.1/SIRT1
In nematodes, overexpressing sir-2.1 can confer a lifespan extension phenotype that is dependent on the DAF-16 [19]. Under the paraquat or t-BOOH exposure conditions, the sir-2.1(ok434) mutant nematodes were sensitive, and the hcf-1(pk924) mutant nematodes were resistant to the treatments (Fig. 5.16) [58]. It has been further observed that, under the paraquat or t-BOOH exposure conditions, mutation of hcf-1 could suppress the susceptibility of sir-2.1(ok434) mutant nematodes to the toxicity of paraquat or t-BOOH (Fig. 5.16) [58]. These observations imply that SIR-2.1 can act upstream of the insulin signaling pathway to regulate the toxicity of environmental toxicants or stresses by suppressing the function of HCF-1 in nematodes.
In nematodes, it was further found that the DAF-16 and the SIR-2.1 can interact even under the stress condition, and this interaction depended on the 14-3-3 proteins as the SIR-2.1 binding partners (Fig. 5.17) [59]. The 14-3-3 proteins were also required for the SIR-2.1-induced transcriptional activation of DAF-16 and the stress resistance [59]. Following the heat stress, SIR-2.1 will bind DAF-16 in a 14-3-3-dependent manner (Fig. 5.17) [59]. In contrast, the low insulin-like signaling did not promote the SIR-2.1/DAF-16 interaction, and thereby sir-2.1 and the 14-3-3 were not required for the regulation of lifespan by the insulin-like signaling pathway [59]. Therefore, very large physical interactions surrounding the DAF-16 may be formed during the regulation of toxicity of environmental toxicants or stresses in nematodes.
5.6.6 PRDX-2
In nematodes, PRDX-2 is a single cytosolic 2-Cys Prx. Loss-of-function mutation of prdx-2 increased the arsenite resistance by increasing both SKN-1 and DAF-16 activities [60]. Under the normal conditions, there was a significant increase in the nuclear localization of DAF-16::GFP in prdx-2 mutant nematodes (Fig. 5.18) [60]. Meanwhile, the expressions of several DAF-16-activated genes (mtl-1, sod-3, gst-7), as well as the expression of sod-3p::gfp, were also increased in prdx-2 mutant nematodes (Fig. 5.18) [60]. More importantly, it was observed that mutation of daf-16 or skn-1 could suppress the resistance of prdx-2(RNAi) nematodes to the toxicity of arsenite in reducing the lifespan (Fig. 5.18) [60], which suggests that PRDX-2 acts upstream of both the DAF-16 and the SKN-1 to regulate the toxicity of environmental toxicants or stresses in nematodes.
5.7 Genetic Interaction Between SKN-1 and DAF-16 or DAF-2 in Regaling the Toxicity of Environmental Toxicants or Stresses
In nematodes, both the FOXO transcriptional factor DAF-16 and the FOXO transcriptional factor SKN-1/Nrf can act downstream of the insulin receptor DAF-2 in the insulin signaling pathway to regulate various biological processes, such as the stress response [28, 61]. Using intestinal ROS production as the toxicity assessment endpoint, it has been shown that the GO toxicity in inducing intestinal ROS production in daf-16(mu86);skn-1(RNAi) was more severe than that in daf-16(mu86) or in skn-1(RNAi) (Fig. 5.19) [22], suggesting that the SKN-1 and the DAF-16 may act in parallel signaling pathways to regulate the toxicity of environmental toxicants or stresses in nematodes.
Besides this, the genetic interaction between DAF-2 in insulin signaling pathway and SKN-1 in p38 MAPK signaling pathway in regulating GO toxicity was also examined. Prolonged exposure to GO (100 mg/L) could cause the similar toxicity on lifespan in double mutant of daf-2(e1370);skn-1(RNAi) to that in skn-1(RNAi) nematodes (Fig. 5.20) [61], suggesting that RNAi knockdown of skn-1 may potentially suppress the resistance of daf-2 mutant to the GO toxicity. Therefore, both the core signaling cascade of p38 MAPK signaling pathway and the insulin receptor DAF-2 in the insulin signaling pathway can act upstream of SKN-1 to regulate the toxicity of environmental toxicants or stresses in nematodes.
5.8 Perspectives
So far, a large amount of data has highlighted the possible pivotal function or role of the core insulin signaling pathway in the regulation of environmental toxicants or stresses in nematodes. Nevertheless, the detailed insulin signaling pathway involved in the control of toxicity from different environmental toxicants or stresses may be different. At least for the kinase cascade in the insulin signaling pathway, different environmental toxicants or stresses may affect different components. More importantly, among the large amount of targeted genes (predicted) for daf-16, only several genes have been proven to act as the downstream targeted genes for daf-16 in regulating the toxicity of environmental toxicants or stresses. That is, it is still unclear whether the rest of predicted genes can also act as the targeted genes for daf-16 in the regulation of toxicity of environmental toxicants or stresses.
As introduced above, the obtained data so far may imply the formation of a large physical interaction surrounding the DAF-16 in regulating the toxicity of environmental toxicants or stresses. The identification of exact scaffold molecules in this large complex may provide an important basis for further screen of related components and the thorough elucidation of the underlying mechanism for insulin signaling pathway in regulating the toxicity of environmental toxicants or stresses in nematodes.
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Wang, D. (2019). Functions of Insulin and the Related Signaling Pathways in the Regulation of Toxicity of Environmental Toxicants or Stresses. In: Molecular Toxicology in Caenorhabditis elegans. Springer, Singapore. https://doi.org/10.1007/978-981-13-3633-1_5
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