Cadmium hijacks the high zinc response by binding and activating the HIZR-1 nuclear receptor

Significance

Zinc is essential for animal life, and zinc levels are carefully regulated by homeostatic mechanisms. In the model organism Caenorhabditis elegans, the HIZR-1 nuclear receptor directly binds zinc and mediates high zinc homeostasis by regulating transcription. Cadmium is structurally similar to zinc but is a prevalent environmental toxin. Here, we explore how animals respond to these similar metals. The results suggest that cadmium hijacks the high zinc homeostasis response by directly binding and activating HIZR-1. This is an example of cadmium functioning as an activating ligand for a zinc-regulated protein in contrast to the prevailing model that cadmium binding causes protein dysfunction. These results elucidate the interplay between these two metals and reveal an unexpected mechanism of cadmium transcriptional control.

Abstract

Cadmium is an environmental pollutant and significant health hazard that is similar to the physiological metal zinc. In Caenorhabditis elegans, high zinc homeostasis is regulated by the high zinc activated nuclear receptor (HIZR-1) transcription factor. To define relationships between the responses to high zinc and cadmium, we analyzed transcription. Many genes were activated by both high zinc and cadmium, and hizr-1 was necessary for activation of a subset of these genes; in addition, many genes activated by cadmium did not require hizr-1, indicating there are at least two mechanisms of cadmium-regulated transcription. Cadmium directly bound HIZR-1, promoted nuclear accumulation of HIZR-1 in intestinal cells, and activated HIZR-1–mediated transcription via the high zinc activation (HZA) enhancer. Thus, cadmium binding promotes HIZR-1 activity, indicating that cadmium acts as a zinc mimetic to hijack the high zinc response. To elucidate the relationships between high zinc and cadmium detoxification, we analyzed genes that function in three pathways: the pcs-1/phytochelatin pathway strongly promoted cadmium resistance but not high zinc resistance, the hizr-1/HZA pathway strongly promoted high zinc resistance but not cadmium resistance, and the mek-1/sek-1/kinase signaling pathway promoted resistance to high zinc and cadmium. These studies identify resistance pathways that are specific for high zinc and cadmium, as well as a shared pathway.

Cadmium is a metal that lacks physiological roles in animals but is a well-established environmental toxicant. Human exposures to cadmium arise predominantly from mining, refining and electroplating industries, dietary contamination of food and water, and inhalation of polluted air, including tobacco smoke. The toxicity depends on the concentration, duration, and route of cadmium exposure. In humans, cadmium adversely affects the lung when inhaled and the liver when consumed, and it accumulates in the kidney in both cases (1–4).* Because of its potent toxicity and the frequency of human exposure, cadmium is highly ranked on the Substance Priority List (5). While exposure prevention is a key public health goal, it is also important to elucidate the mechanisms of cadmium toxicity to develop strategies to treat the deleterious effects of cadmium exposure.

Two main theories have been proposed to explain the toxicity of cadmium (6): 1) Cadmium causes chemical reactions that produce reactive oxygen species, leading to oxidative stress and cellular damage, and 2) cadmium binds macromolecules, leading to a loss of function. Since half of all enzymes are predicted to bind a metal ion (7), cadmium may substitute for physiological metal ions such as zinc, manganese, or copper in the metal-binding sites of metalloproteins, causing protein dysfunction (8, 9). Cadmium can bind hormone, kinase, and calcium receptors on the cell surface (10–12). Cadmium has been proposed to disrupt zinc homeostasis, bone homeostasis, oxidative stress, and reactive oxygen species signaling; dysregulate hormone signaling; and interfere with iron, copper, and manganese function and homeostasis (9, 13, 14).

A critical goal in the field is to understand how animals sense and respond to cadmium since this may suggest new therapeutic strategies to enhance cellular defense mechanisms. In principle, cellular cadmium resistance might involve limiting the damage caused by cadmium by preventing entry, promoting excretion, or chelation to form a nontoxic chemical complex. However, routes of cadmium entry and excretion have not been well defined. Metallothionein proteins bind cadmium and are proposed to detoxify by a chelation mechanism, and small molecules such as phytochelatins also bind and detoxify (15). A second line of defense might be repairing cellular damage caused by cadmium. Damage repair might involve pathways specialized for cadmium damage or more general stress response pathways triggered by multiple insults such as other metals, heat, or oxidative stress. It is well established that cadmium exposure causes transcriptional changes, including genes involved in stress resistance such as heat shock proteins (16–19).

Zinc is an essential metal that is chemically similar to cadmium since both elements are divalent d-block elements belonging to group 12 of the periodic table. Zinc is estimated to bind about 10% of the human proteome to provide structural support, drive enzymatic catalysis, or regulate protein function (20). High zinc concentrations can be toxic, but in humans, the acute toxic concentration of zinc is so high that fatalities arise exclusively from occupational overdose known as metal fume fever (21). Because high concentrations of zinc are toxic, animals have evolved homeostatic mechanisms to maintain the concentration of zinc in an appropriate range. Caenorhabditis elegans has been a useful model to define mechanisms of high zinc homeostasis. High dietary zinc activates transcription of specific genes in C. elegans (22). Roh et al. (23) identified an evolutionarily conserved high zinc activation (HZA) element in the promoter regions of multiple genes that are induced by high dietary zinc and cadmium (16). The HZA enhancer is necessary for transcriptional activation of multiple genes in response to high dietary zinc and sufficient to mediate the activation of a basal promoter in response to dietary zinc and cadmium. Warnhoff et al. (24) discovered the high zinc activated nuclear receptor (HIZR-1), a nuclear receptor transcription factor that functions as the high zinc sensor and the master regulator of high zinc homeostasis. The ligand-binding domain of HIZR-1 binds zinc with high affinity, indicating that zinc is the ligand for this receptor. Zinc binding to HIZR-1 triggers nuclear accumulation in intestinal cells, the site of the high zinc homeostasis response. HIZR-1 directly binds the HZA enhancer and mediates activation of target genes, including genes encoding the cation diffusion facilitator (CDF) transporters CDF-2 and TTM-1B, which store and excrete excess zinc, respectively.

Although cadmium has been proposed to disrupt zinc homeostasis in metazoans, the mechanisms involved are not well defined. To define the role of HIZR-1 and the high zinc homeostasis response during cadmium exposure in C. elegans, we used multiple methods to compile a list of 145 cadmium-regulated genes. HIZR-1 was necessary for activation of a subset of these genes, indicating that cadmium-regulated changes in transcript levels include both hizr-1–dependent and hizr-1–independent genes. To elucidate the mechanism of this transcriptional response, we showed that cadmium directly binds the HIZR-1 ligand-binding domain similar to zinc. Cadmium promotes nuclear accumulation of HIZR-1 in intestinal cells and activation of HIZR-1–dependent transcription via the HZA enhancer. To determine the function of the HIZR-1–mediated transcriptional response, we analyzed loss-of-function mutants in specific target genes and hizr-1(lf) animals that lack the activation of many target genes. Loss of hizr-1 did not cause cadmium hypersensitivity, suggesting that cadmium hijacks the high zinc homeostasis response, although it has little effect on cadmium resistance. Genetic studies showed that the phytochelatin pathway confers resistance to cadmium but not high zinc, whereas the mek-1/sek-1 signaling pathway promotes resistance to both high zinc and cadmium toxicity. These studies show that hizr-1 specifically promotes high zinc resistance, the phytochelatin pathway specifically promotes cadmium resistance, and the mek-1/sek-1 signaling pathway promotes resistance to both. Furthermore, the dramatic transcriptional response to cadmium exposure that is mediated by HIZR-1 shows that cadmium can function as a zinc mimetic to activate the high zinc homeostasis pathway.

Results

Multiple Strategies Were Used to Identify Cadmium-Regulated Transcripts in Wild-Type C. elegans.

Cadmium exposure influences the transcript levels of many genes in C. elegans (16, 25). To investigate the mechanism and function of cadmium-regulated transcription, we used multiple search strategies to assemble a list of candidate genes. We reasoned that using a wide range of search strategies would produce a more diverse and comprehensive list. The first strategy involved a literature review to identify genes for which loss-of-function mutations cause hypersensitivity to cadmium toxicity (25). These genes play functional roles in cadmium resistance and might be regulated by cadmium. This list included 14 candidate genes (Fig. 1A). The second strategy involved bioinformatic identification of genes that contain a predicted HZA enhancer since this enhancer is implicated in high zinc and cadmium-regulated transcription (23). Roh et al. (23) identified a list of 136 candidate genes containing both HZA and GATA elements in the promoter region (Fig. 1A). The third strategy involved direct comparisons of transcript levels between control and cadmium-exposed animals. The effects of cadmium exposure may depend on the concentration of cadmium, the duration of exposure, and the developmental stage of the animals, so these are important variables in these experiments. A literature review by Dietrich et al. (25) identified a list of 37 candidate genes that were reported by multiple investigators who used a variety of cadmium concentrations, exposure durations, and developmental stages. In addition, Cui et al. (16) used microarray technology to assemble a list of 281 candidate genes that display higher transcript levels in mixed-stage wild-type (WT) animals exposed to 100 µM cadmium in S-medium for 4 or 24 h. To perform our own search, we used the method of RNA-sequencing (RNA-seq) to compare transcriptional profiles of mixed-stage WT animals exposed to 0 or 100 µM cadmium for 16 h, resulting in a list of 87 candidate genes. These approaches are conceptually similar in directly comparing transcript levels but differed in cadmium dose and duration, developmental stage of animals, and method to detect transcript levels; they identified 347 unique candidate genes since there were some overlaps between these three lists (Fig. 1A). Together, these search strategies resulted in a total of 447 candidate genes: 4 were uniquely identified by mutant hypersensitivity, 96 were uniquely identified by a predicted HZA enhancer, 301 were uniquely identified by transcript analysis, and 46 were identified by multiple strategies (Fig. 1A).

Fig. 1.

Cadmium exposure regulated transcription of many genes, and hizr-1 was necessary for regulation of a subset of these genes. (A) We assembled a list of 447 genes that were candidates to be regulated by cadmium using three strategies: Mutant hypersensitivity to cadmium reported in the literature (25), a predicted HZA enhancer reported in the literature (23), and direct analysis of gene expression in cadmium exposed C. elegans reported in the literature (16, 25) or described here. The Venn diagram shows the number of candidate genes identified by each strategy (denominator) and the number of genes validated as cadmium-regulated by qPCR (numerator). (B–S) WT and hizr-1(am286) mutant animals at the L4 stage were exposed to 0 or 100 µM cadmium for 4 h. Transcript levels were determined by qPCR. Comparing WT animals in the presence and absence of cadmium, 127 genes were activated by cadmium, and 18 genes were repressed by cadmium. Pie charts for activated (B) and repressed (C) genes illustrate categories of hizr-1 involvement; comparing WT and hizr-1(lf) revealed that hizr-1 was not necessary for the regulation or baseline expression of 83 genes (white), necessary for regulation but not baseline expression of 25 genes (dark blue), necessary for regulation and baseline expression of 13 genes (medium blue), and necessary for baseline expression but not regulation of 24 genes (light blue/red). (D–S) Bars indicate average mRNA level in arbitrary units (A.U.) for three biological replicates, and error bars indicate S.D.; values were normalized by setting the value for WT with 0 µM cadmium to an average of 1.0 for each gene. Colors correspond to the pie charts. Numbers in or above the bars are the ratio +Cd/−Cd. For each gene, we performed five comparisons: four comparisons are shown by horizontal lines that indicate two values were significantly different (WT −Cd to WT +Cd, WT +Cd to hizr-1 +Cd, hizr-1 −Cd to hizr-1 +Cd, and WT −Cd to hizr-1 −Cd) (P < 0.05). The absence of a horizontal line indicates no significant difference. One comparison is shown with an asterisk (*) that indicates the ratio +Cd/−Cd of hizr-1(lf) is significantly different from the ratio +Cd/−Cd of WT (P < 0.05). The absence of an asterisk indicates no significant difference.

To analyze these 447 candidate genes, we performed qPCR analysis of each gene using a unique pair of forward and reverse amplification primers (SI Appendix, Table S1). To maximize the cadmium-specific transcriptional response and limit the nonspecific stress response that results from cadmium toxicity, we chose a high-dose/short-duration exposure. WT animals were synchronized at the L4 stage and cultured with 0 or 100 µM CdCl₂ for 4 h and RNA was analyzed by qPCR reactions. ama-1, which encodes the large subunit of RNA polymerase II, was used as the control gene. Based on the gene expression values, we categorized cadmium-regulated genes in two major groups: 1) “Cadmium-activated” genes had an average 2^−∆∆Ct value of greater than 2 (double the transcript level of the gene without cadmium), and the change was statistically significant based on three biological replicates (P value less than 0.05); 2) “cadmium-repressed” genes had an average 2^−∆∆Ct value of less than 0.5 (half the transcript level of the gene without cadmium), and the change was statistically significant based on three biological replicates. Based on these criteria, we identified 127 cadmium-activated genes and 18 cadmium-repressed genes in WT animals (Fig. 1 B and C and SI Appendix, Tables S2–S4). None were uniquely identified by a hypersensitive mutant phenotype, 4 were uniquely identified by a predicted HZA element, 111 were uniquely identified by transcriptional studies, and 28 were identified by multiple strategies (Fig. 1A).

The magnitude of cadmium activation varied greatly (SI Appendix, Fig. S1). Three genes were activated more than 200-fold: mtl-1 (301-fold), cdr-1 (250-fold), and T08G5.1 (227-fold); two genes were activated ∼100-fold: F09C6.3 (110-fold), pals-28 (101-fold); and seven genes were activated ∼50 fold: gst-38 (63-fold), numr-1/-2 (55-fold), clec-74 (54-fold), fbxa-163 (48-fold), hsp-16.48 (46-fold), and pals-29 (35-fold). An additional 52 genes displayed greater than 5-fold higher transcript levels in response to cadmium, including previously analyzed genes such as mtl-2 (25-fold), which encodes a metallothionein protein, and ttm-1b (6-fold) and cdf-2 (5-fold), which encode zinc transporters (SI Appendix, Tables S2 and S3). The remaining 62 genes displayed twofold to fivefold higher transcript levels in response to cadmium (SI Appendix, Tables S2 and S3). For the 18 genes that displayed cadmium repression, the largest magnitude was a 10-fold decrease in transcript levels (SI Appendix, Table S4). Although reduced transcript levels might reflect nonspecific degradation due to cadmium toxicity, this seems unlikely since only ∼4% of candidate genes (18/447) displayed this effect. Genes that did not meet our criteria might still be regulated by cadmium in different experimental conditions, such as a different dose, duration of exposure, or developmental stage. Furthermore, because the criterion of at least a twofold increase or decrease is arbitrary, genes that displayed less than a twofold change may be true targets of cadmium regulation. We identified 55 genes that displayed a statistically significant increase that was less than twofold in WT animals exposed to cadmium (SI Appendix, Table S5) and 51 genes that displayed a statistically significant decrease that was greater than 0.5-fold in WT animals exposed to cadmium (SI Appendix, Table S6).

hizr-1 Is Necessary for a Subset of Transcripts to Be Regulated by Cadmium Exposure.

The transcriptional response to high dietary zinc is mediated by the HIZR-1 transcription factor binding to the HZA enhancer (23, 24). To determine the role of hizr-1 in cadmium-regulated transcription, we analyzed the 127 cadmium-activated genes and 18 cadmium-repressed genes in hizr-1(am286) mutant animals. The hizr-1(am286) nonsense mutation is a strong loss-of-function (lf) or null resulting in a Q86Stop change (24).

To determine if hizr-1 influences the expression level in the absence of cadmium, we compared transcript levels in WT and hizr-1(lf) animals cultured in standard medium. Because our culture conditions are zinc replete, some HIZR-1 protein is zinc bound and active in the absence of cadmium. We considered hizr-1 as necessary for expression level in the absence of cadmium if the average 2^−∆∆Ct value of three biological replicates was greater than 2 (double the transcript level of the gene without cadmium) or less than 0.5 (half the transcript level of the gene without cadmium) and the change was statistically significant. From the 145 cadmium-regulated genes, we identified 31 activated and 6 repressed genes (26%) where hizr-1 is necessary to control the expression level in the absence of cadmium and 108 (74%) where hizr-1 is not necessary in the absence of cadmium (Fig. 1 B and C). For example, in standard medium with no cadmium, hizr-1 repressed expression of F45D11.2/3/4 and ilys-3 (Fig. 1 N and P) and activated expression of cyp-14A4, sqst-3, and Y48E1B.8 (Fig. 1 O, Q, and S) since the expression level changes significantly in hizr-1(lf) mutants.

To determine if hizr-1 influences the expression level in the presence of cadmium, we compared transcript levels in WT and hizr-1(lf) animals in the presence of cadmium. Furthermore, we compared the ratio of expression level in the absence and presence of cadmium in the WT and hizr-1(lf) mutant to account for the possibility that hizr-1 influences expression in both conditions. For 38/127 cadmium-activated genes (30%), the 2^−∆∆Ct level of WT worms cultured on cadmium was significantly higher than the 2^−∆∆Ct level of hizr-1(lf) mutant worms cultured on cadmium (Fig. 1B and SI Appendix, Table S2). Thus, for these genes, hizr-1 is necessary for the full extent of cadmium activation. Nineteen of these genes were induced greater than fivefold in WT, including cdr-1, mtl-1, mtl-2, ttm-1b, and cdf-2 (SI Appendix, Table S2). Fourteen genes were induced twofold to fivefold in WT animals, including pgp-1 (twofold) (SI Appendix, Table S2). For some genes, the dramatic activation by cadmium was almost completely abrogated in the hizr-1(lf) mutant animals: comparing fold activation in WT and hizr-1(lf), mtl-1 changed from 300-fold to 3.0-fold, cdr-1 changed from 250-fold to 1.4-fold, mtl-2 changed from 24-fold to 0.5-fold, and cdf-2 changed from 5.5-fold to 1.1-fold (Fig. 1 D–G). For some genes, cadmium-activated transcription was reduced but still significant in the hizr-1(lf) mutants exposed to cadmium: ttm-1b changed from 6.2-fold in WT to 3.5-fold in hizr-1(lf) mutants (Fig. 1H). For the numr-1/-2 genes, the level of transcripts in the presence of cadmium was significantly reduced in hizr-1(lf) mutants; however, the hizr-1 fold change was not significantly different from the WT fold change because hizr-1 influences expression in the absence of cadmium (Fig. 1I). For 13 genes, hizr-1 is necessary for cadmium activation, and hizr-1 also influences the level of expression in the absence of cadmium (Fig. 1 B, D, F, N, and O).

For 89/127 cadmium-activated genes (70%), transcript levels in the hizr-1(lf) mutant were similar to or higher than WT (Fig. 1 J–M and SI Appendix, Tables S2 and S3). This group includes seven heat shock proteins. For 18 genes, the expression level in the absence of cadmium was significantly different in hizr-1 mutants compared to WT, indicating that hizr-1 regulates transcript levels in standard culture conditions (Fig. 1 P and Q).

For the 18 cadmium-repressed genes, hizr-1 is not necessary for transcript levels in 12 cases (67%) and affects transcript levels in the absence of cadmium in 6 cases (33%) (Fig. 1C and SI Appendix, Table S4). For example, cadmium exposure repressed fat-7 transcript levels about 10-fold in both WT and hizr-1(lf) animals (Fig. 1R). Cadmium exposure repressed Y48E1B.8 transcript levels about 10-fold in WT; hizr-1(lf) animals displayed lower transcript levels in the absence of cadmium but still displayed repression in the presence of cadmium (Fig. 1S).

Based on the qPCR expression values, we categorized cadmium-regulated genes into four groups. 1) hizr-1 is necessary for baseline expression levels in the absence of cadmium (18 activated/6 repressed), 2) hizr-1 is necessary for activation or repression in the presence of cadmium (25 activated/0 repressed), 3) hizr-1 is necessary for baseline expression levels in the absence of cadmium and activation or repression in the presence of cadmium (13 activated/0 repressed), and 4) hizr-1 is not necessary for baseline expression levels in the absence of cadmium or activation or repression in the presence of cadmium (71 activated/12 repressed) (Fig. 1 B and C and SI Appendix, Tables S2–S4).

hizr-1 Mediates Cadmium-Activated Transcription through the HZA Enhancer.

To analyze the mechanism of hizr-1 in cadmium-activated transcription, we investigated the hypothesis that HIZR-1 acts via the HZA enhancer. We analyzed the mtl-1 promoter that contains an experimentally confirmed HZA element (23). Transgenic young adult animals with an extrachromosomal array containing the mtl-1 promoter expressing GFP were cultured overnight with or without cadmium and analyzed for GFP expression by fluorescence microscopy. Cadmium exposure caused strong GFP fluorescence in intestinal cells (Fig. 2A). To determine the contribution of the HZA enhancer, we analyzed the mtl-1 promoter with a scrambled HZA sequence; these transgenic animals did not display GFP fluorescence in response to cadmium exposure (Fig. 2A). To determine if the HZA enhancer is sufficient to mediate transcriptional activation in response to cadmium, we analyzed transgenic animals with an extrachromosomal array containing a basal pes-10 promoter with three HZA elements expressing nuclear localized GFP. In response to cadmium exposure, these animals displayed strong GFP fluorescence in intestinal cells, indicating that the HZA enhancer is sufficient for the cadmium response (Fig. 2B). Thus, the HZA enhancer was necessary and sufficient for transcriptional activation in response to cadmium exposure.

Fig. 2.

The HZA enhancer and hizr-1 mediated cadmium-activated transcription. (A) Diagrams (not to scale) of the mtl-1 promoter region extending 489 bp upstream of the start codon (black outline) fused to the GFP coding region (green box). The endogenous HZA element (yellow box) was mutated by scrambling the nucleotide sequence (scr/black box) (23). Fluorescence microscopy images show hizr-1(+) transgenic animals that contain extrachromosomal arrays with these promoters that were cultured for 16 h with 0 or 1 µM cadmium starting at the L4/adult stage. Images were captured using identical settings and exposure times; bright field images are shown in insets. A white arrowhead indicates strong GFP expression in the intestine. (Scale bars, 100 µm.) (B) A diagram (not to scale) of the pes-10 minimal promoter containing three tandem copies of a 62-bp fragment of the mtl-1 promoter that includes the HZA element (yellow box) fused to the GFP coding region with a nuclear localization signal (N-GFP) (23). Transgenic animals that contain an extrachromosomal array with this promoter were hizr-1(+) or hizr-1(am286) mutants and were cultured and imaged as done previously. White arrowheads indicate nuclear localized GFP expression in intestinal cells. (Scale bars, 100 µm.)

To determine if hizr-1 mediates transcriptional activation in response to cadmium via the HZA enhancer, we analyzed hizr-1(lf) animals. Upon cadmium exposure, hizr-1(lf) transgenic animals with an extrachromosomal array containing a basal pes-10 promoter with three HZA elements did not display fluorescence (Fig. 2B). Thus, hizr-1 is necessary for this response. These results indicate that cadmium acts through HIZR-1 and the HZA enhancer to activate transcription in vivo.

Cadmium Promotes Nuclear Accumulation of HIZR-1 in Animals.

Nuclear receptor transcription factors are typically regulated by translocating to the nucleus in response to ligand binding (26–28). The HIZR-1 nuclear receptor accumulates in the nucleus of intestinal cells when animals are exposed to high dietary zinc (24). To determine if cadmium exposure also causes nuclear accumulation of HIZR-1, we analyzed transgenic animals with an extrachromosomal array expressing HIZR-1::GFP fusion protein. To quantify the HIZR-1 response, we counted the number of GFP-positive intestinal nuclei in each exposed animal. In standard culture medium, HIZR-1::GFP protein did not accumulate in intestinal nuclei. Incubation with 30, 50, 100, or 300 µM supplemental zinc for 4 to 6 h caused on average 24 of the intestinal cells to display nuclear accumulation of HIZR-1::GFP (Fig. 3 A and B). By contrast, incubation with the same concentrations of supplemental manganese, another physiological metal, did not cause appreciable nuclear accumulation of HIZR-1, indicating the effect is metal specific (Fig. 3 A and B). Incubation with the same concentrations of cadmium caused on average 10 intestinal nuclei to display accumulation of HIZR-1::GFP, a significant increase compared to standard medium or treatment with manganese but significantly lower than the effect of zinc in all concentrations of metal tested (Fig. 3 A and B and SI Appendix, Fig. S2). Thus, both zinc and cadmium caused nuclear accumulation of HIZR-1 (Fig. 3C).

Fig. 3.

Cadmium promoted nuclear accumulation of HIZR-1 in animals and directly bound the HIZR-1 ligand-binding domain (LBD) in purified extracts. (A) Diagram (not to scale) of the hizr-1 promoter region extending 444 bp upstream of the start codon (black outline), the endogenous HZA element (yellow box), and the full-length HIZR-1 coding region (red box) fused in frame to the GFP coding region (green box) (24). Fluorescence microscopy images show transgenic hizr-1(am286) animals that contain an extrachromosomal array with this promoter cultured for 6 h on noble agar minimal media (NAMM) with no supplemental metal or 100 µM supplemental zinc, cadmium, or manganese starting at the L4/adult stage. Images were captured using identical settings and exposure times; bright field images are shown in insets. White arrowhead indicates nuclear-localized GFP expression in intestinal cells. Small punctae in fluorescence images appear to be autofluorescence derived from gut granules, lysosome-related organelles that accumulate fluorescent material. (Scale bar, 100 µm.) (B) Values are the average number of GFP-positive alimentary nuclei per animal ± SEM (n = 8 to 18 animals). The values for manganese were not significantly different from the 0 µM control at these four concentrations (P > 0.10). Zinc displayed significantly higher values than cadmium, and zinc and cadmium were both significantly higher than manganese (*P < 0.05). (C) Model showing cadmium (orange circle) directly binding the HIZR-1 LBD, stimulating translocation from the cytoplasm to the nucleus. (D) The LBD of HIZR-1 (residues 101 to 412) that was fused to GST was expressed in bacteria, partially purified by affinity chromatography, and incubated with radioactive Zn-65 and no additional metal (0) or increasing concentrations of nonradioactive zinc, cadmium, or manganese. Values in arbitrary units (A.U.) indicate the average amount of Zn-65 bound to protein quantified by filter binding and scintillation counting ± SD (n = 4 to 6); the value with no additional metal was set equal to 1.0. Nonradioactive zinc and cadmium displayed significantly lower values than nonradioactive manganese, and zinc was slightly but significantly lower than cadmium (*P < 0.05) (two-tailed Student’s t test). The values for manganese were not significantly different from the 0 µM control at these three concentrations (P = 0.8 to 0.08).

Cadmium Binds the HIZR-1 Ligand-Binding Domain in Purified Extracts.

Nuclear receptors are composed of two functionally independent domains: the ligand-binding domain and the DNA-binding domain. The HIZR-1 ligand-binding domain directly binds zinc, providing biochemical support for the model that HIZR-1 functions as the high zinc sensor (24). To determine if HIZR-1 also binds cadmium, we analyzed the ability of cadmium to compete with zinc for binding to the HIZR-1 ligand-binding domain. The ligand-binding domain of HIZR-1 (residues 101 to 412) that fused to glutathione S-transferase [GST::HIZR-1(101-412)] was expressed in bacteria and partially purified using affinity chromatography. GST::HIZR-1(101-412) protein was incubated with radioactive Zn-65 and washed and bound Zn-65 was measured. Incubation with 3, 30, and 300 µM nonradioactive zinc caused a significant reduction of bound Zn-65, whereas incubation with nonradioactive manganese did not effectively displace radioactive Zn-65 (Fig. 3D). Thus, the HIZR-1 ligand-binding domain displays metal-specific binding. Nonradioactive cadmium competed effectively for binding with Zn-65—the effect was similar to nonradioactive zinc, but statistical tests indicate cadmium binds slightly less effectively than zinc (P value < 0.005) (Fig. 3D and SI Appendix, Fig. S3). Thus, the HIZR-1 ligand-binding domain can directly bind cadmium similar to zinc (Fig. 3C).

There Is Significant Overlap between Genes Activated by Cadmium and High Zinc.

If cadmium directly binds and activates HIZR-1, then we predict there will be an overlap between the genes activated by high zinc and cadmium. While several genes are established to be highly activated by both high zinc and cadmium, such as mtl-1, a systematic comparison has not been reported. To perform this analysis, we used the method of RNA-seq to compare transcriptional profiles of mixed-stage WT and hizr-1(lf) mutant animals exposed to no supplemental metal, 100 µM cadmium, or 200 µM supplemental zinc for 16 h. Compared to WT animals cultured in standard medium, 844 and 135 genes were significantly activated greater than twofold by high zinc or cadmium exposure, respectively (Fig. 4A). Eighty-two genes were significantly activated by both high zinc and cadmium, whereas 53 genes were significantly activated by cadmium but not high zinc exposure (Fig. 4A and SI Appendix, Tables S7 and S8). Thus, most but not all cadmium-activated genes are also activated by high zinc, consistent with the model that there is a shared mechanism of regulation.

Fig. 4.

Many genes are regulated similarly by cadmium, high zinc, and hizr-1. (A) RNA-seq data were used to identify genes significantly activated more than twofold by cadmium or high zinc. The Venn diagram shows overlap between these lists. (B–F) WT, hizr-1(am286), and mtl-1(tm1770) mtl-2(gk125) animals at the L4 stage were cultured for 4 h with standard medium (white), 100 µM cadmium (black), or 100 µM supplemental zinc (gray). Transcript levels were determined by qPCR. Bars indicate average mRNA level in arbitrary units (A.U.) for two or three biological replicates, and error bars indicate SD; values were normalized by setting the value for WT in standard medium to an average of 1.0 for each gene. All genes were significantly activated by cadmium and high zinc in WT. Three additional comparisons were performed as indicated by horizontal lines (WT +Cd to hizr-1 +Cd, hizr-1 +Cd to mtl-1 mtl-2 +Cd, and WT +Cd to mtl-1 mtl-2 +Cd). *P < 0.05; **P < 0.01; ns, no significant difference, P > 0.05. (G) RNA-seq data were used to identify genes that were activated by cadmium and high zinc in WT and displayed at least twofold reduced activation in hizr-1(lf) mutants. Ten such genes are shown of 18 that met these criteria. Fold change represents transcript level in metal supplemented medium compared to control medium. P value compares fold change to a value of 1.0. *P < 0.05; ***P < 0.001; ****P < 0.0001; *****P < 0.00001; ns, no significant difference, P > 0.05. HZA enhancers are predicted by bioinformatics [P (23)] or confirmed by mutagenesis [C (23, 41)]. A “?” indicates genes with unknown HZA enhancer status.

To investigate the role of hizr-1 in regulation of the 82 overlapping genes, we analyzed activation of these genes in hizr-1(lf) mutant animals. Using the criteria that the transcript level in hizr-1(lf) mutant animals was less than half the transcript level in WT animals, we identified 18 genes (Fig. 4G and SI Appendix, Table S7). These genes are significantly activated by both high zinc and cadmium, and hizr-1 is necessary for full activation with both metals.

To investigate the role of the HZA enhancer, we compared these lists to the list of genes with predicted HZA enhancers identified by Roh et al. (23). Of the 82 genes activated by both high zinc and cadmium, 18 contain a predicted HZA enhancer, including the 7 genes with the highest fold activation by cadmium (Fig. 4G and SI Appendix, Table S7). By contrast, of the 53 genes activated by cadmium but not high zinc exposure, only two contain a predicted HZA enhancer (SI Appendix, Table S8). The list of predicted HZA elements is not definitive since it is likely to be missing true positives and contain false positives (predicted enhancers that are not functional). Nevertheless, there appear to be a substantial number of genes that are activated by both high zinc and cadmium, contain a predicted HZA enhancer, and require hizr-1 for full activation by both metals.

Metallothionein Function Was Not Necessary for hizr-1 to Mediate Transcriptional Activation in Response to Cadmium.

Based on our results, we hypothesize that cadmium directly binds and activates HIZR-1 in animals. An alternative model is that cadmium displaces zinc from proteins such as metallothionein, resulting in an increase in the level of labile zinc, and zinc directly binds and activates HIZR-1 in animals. Indeed, Zhang et al. proposed such a model for the mechanism of cadmium activation of MTF in a cell-free transcription assay using vertebrate cells (29). While direct measurement of the metal that is bound to HIZR-1 in animals could distinguish these alternative models, that is not technically feasible. To address this alternative model, we analyzed double mutant animals that lack mtl-1 and mtl-2. L4 stage WT, hizr-1(lf) mutants, and mtl-1(lf) mtl-2(lf) double mutants were cultured with no supplemental metal, 100 µM cadmium, or 100 µM supplemental zinc for 4 h, and RNA levels were analyzed by qPCR. As expected, mtl-1 and mtl-2 transcripts were not detectable in mtl-1(lf) mtl-2(lf) double mutant animals (Fig. 4 B and C). cdf-2 and cdr-1 were significantly activated by high zinc and cadmium, and hizr-1 is necessary for this activation. By contrast, mtl-1 mtl-2 is not necessary for activation by either metal, and cdr-1 levels were actually significantly higher in mtl-1 mtl-2 mutants exposed to cadmium compared to WT (Fig. 4 D and E). pals-28 was significantly activated by high zinc and cadmium, and hizr-1 is partially necessary for activation by cadmium. By contrast, mtl-1 mtl-2 is not necessary for activation by cadmium (Fig. 4F). These results do not support the model that metallothionein plays an important role in HIZR-1–mediated transcriptional activation in response to cadmium by providing a source of labile zinc.

Most Cadmium-Activated Genes Do Not Play a Major Functional Role in Cadmium Resistance.

If a gene is transcriptionally activated by cadmium exposure, then that gene is a candidate to play a functional role in cadmium detoxification. To test this possibility, we analyzed loss-of-function mutants for hypersensitivity to cadmium toxicity. Growth rate is a sensitive measure of physiology that is reduced by cadmium exposure and can be readily quantified (25). To measure growth rate, we synchronized animals at the L1 stage, cultured them with 2.5 µM cadmium for 3 d, and measured the length of animals. To provide a basis for comparison, we first analyzed mutations in genes reported to cause hypersensitivity to cadmium toxicity. Phytochelatin synthase is an enzyme that catalyzes the oligomerization of glutathione monomers into a multimer of glutathione, a nongenetic tripeptide called a phytochelatin. Phytochelatins are important for detoxification of many chemicals (30, 31). The C. elegans genome sequence led to the discovery of pcs-1, which encodes a homolog of phytochelatin synthase (32). pcs-1(lf) mutants are extremely hypersensitive to cadmium exposure (33), and we observed a significant reduction in growth rate for cadmium-exposed pcs-1(lf) mutants (Fig. 5A). hmt-1 encodes a functional homolog of the human ABC6 transporter (34), and hmt-1(lf) mutants displayed extreme hypersensitivity to cadmium exposure (Fig. 5A). mek-1, sek-1, and mlk-1 encode kinases involved in the stress response to multiple toxins (35). The mlk-1 single mutant and the mek-1 sek-1 double mutant strains displayed moderate hypersensitivity to cadmium exposure (Fig. 5A). Thus, all of these genes play important functional roles in cadmium detoxification, consistent with previous reports. Notably, none of these genes met our criteria for transcriptional activation by cadmium. mek-1, mlk-1, and hmt-1 displayed no significant difference in transcript levels in WT animals exposed to cadmium, and pcs-1 displayed a 1.8-fold increase that was statistically significant but below our criteria of twofold regulation (Fig. 5B). Thus, these genes play important functional roles, but their transcript levels are not strongly regulated by cadmium exposure.

Fig. 5.

Mutations in many cadmium-activated genes did not cause hypersensitivity to cadmium exposure. (A) WT and mutant strains were synchronized at the L1 stage and cultured on noble agar minimal media (NAMM) dishes with 2.5 µM cadmium for 3 d, and the length of animals was measured. Bars indicate average worm length in arbitrary units (A.U.), and lines indicate SD (n = 7 to 16 animals per strain); values were normalized by setting the value for WT to an average of 1.0. WT is black, positive control genes reported to cause hypersensitivity to cadmium are gray, and experimental genes are blue or white corresponding to Fig. 1B. The horizontal black line indicates the length of L1 stage worms at the start of the experiment. An asterisk (*) indicates a significant difference compared to WT (P < 0.05) (Kruskal-Wallis ANOVA). Alleles are described in the SI Appendix, Table S10A. (B) WT and hizr-1(am286) mutant animals at the L4 stage were exposed to 0 or 100 µM cadmium for 4 h. Transcript levels were determined by qPCR. Bars indicate average mRNA level in arbitrary units (A.U.) for three biological replicates, and error bars indicate SD; values were normalized by setting the value for WT with 0 µM cadmium to an average of 1.0 for each gene. Comparisons are shown as described in Fig. 1 D–S.

To analyze genes that are transcriptionally activated by cadmium exposure, we examined deletion mutations that are likely to cause a strong loss of function. Deletion mutations for many C. elegans genes are publicly available, and we obtained mutations of 13 genes that are activated by cadmium and required hizr-1 for full activation (bcmo-2, swt-6, cdf-2, ttm-1, pgp-1, gst-33, sqst-3, ilys-3, hsp-16.41, math-34, C29F7.1, and the double mutant mtl-1 mtl-2). For 11 mutant strains, the growth rate in the presence of cadmium was not significantly different from WT (Fig. 5A). For one mutant strain (C29F7.1), the growth rate was significantly less than WT; however, the defect was relatively mild since the C29F7.1 growth rate was higher than the four mutant strains used as positive controls. We obtained mutations of 11 genes that are activated by cadmium and did not require hizr-1 for full activation (rrf-2, hsp-17, PDB1.1, gst-5, pgp-9, F55H12.2, skr-5, F49H6.5, ins-7, cld-9, and W03G1.5). For 11 mutant strains, the growth rate in the presence of cadmium was not significantly different from WT (Fig. 5A). For one mutant strain (W03G1.5), the growth rate was significantly less than WT; however, the defect was relatively mild since the W03G1.5 growth rate was higher than the four mutant strains used as positive controls. Thus, of the 24 genes analyzed, only two mutations caused mild cadmium hypersensitivity, whereas 22 genes are not necessary for animals to display a normal growth rate during cadmium exposure.

The hizr-1/HZA Pathway Mediates Resistance to High Zinc but Not Cadmium, the pcs-1/Phytochelatin Pathway Mediates Resistance to Cadmium but Not High Zinc, and the mek-1/sek-1/Kinase Signaling Pathway Mediates Resistance to High Zinc and Cadmium.

hizr-1(lf) mutants are extremely hypersensitive to high zinc toxicity, as indicated by a significantly reduced growth rate when cultured with supplemental zinc (Fig. 6A) (24). To determine the role of hizr-1 in cadmium resistance, we thoroughly analyzed growth using 10 different concentrations of cadmium ranging from 1 to 10 µM. hizr-1(lf) mutants were not significantly different from WT at most concentrations. However, hizr-1(lf) mutants displayed a small but significant increase in growth rate at 5, 8, 9, and 10 µM cadmium, suggesting the activity of hizr-1 makes animals slightly sensitive to cadmium toxicity (Fig. 6B). Thus, hizr-1 is necessary for resistance to high zinc toxicity but is not necessary for resistance to cadmium toxicity.

Fig. 6.

Genetic analysis of hizr-1, pcs-1, hmt-1, mtl-1 mtl-2, and mek-1 sek-1 in high zinc and cadmium toxicity. Animals were synchronized as eggs or L1 larva and cultured on noble agar minimal media (NAMM) dishes with supplemental zinc or cadmium. After 3.5 d for eggs or 3 d for L1 larva, worms were imaged, and individual worm length was measured. With no additional metal added, these culture times were adequate for animals to grow and develop to gravid adults. (A and B) hizr-1(am286); (C and D) pcs-1(tm1748); (E and F) mtl-1(tm1170) mtl-2(gk125); (G and H) hmt-1(gk161); and (I and J) mek-1(ks54) sek-1(qd127). All double and triple mutants contained these alleles. Values are average animal length ± SD, n = 3 to 48 animals. The dotted line indicates the average length of L1 stage worms at the beginning of the assay (∼200 µm); values similar to this line indicate no growth after 3 d. In A and B, *P < 0.05.

To further explore the role of hizr-1, we tested for possible redundancy with other genes involved in cadmium resistance by generating strains containing the hizr-1(lf) mutation in combination with pcs-1(lf), hmt-1(lf), and mek-1(lf) sek-1(lf). The pcs-1(lf) mutant strain displayed a slightly reduced growth rate in standard medium compared to WT. When cultured with increasing levels of supplemental zinc, the pcs-1(lf) mutant and WT displayed similar patterns of dose-dependent decrease in growth since the pcs-1(lf) curve was parallel to WT (Fig. 6C). The pcs-1(lf); hizr-1(lf) double mutant strain was similar to the hizr-1(lf) single mutant strain in high zinc sensitivity. Thus, pcs-1 is not necessary for high zinc resistance. By contrast, the pcs-1(lf) mutant displayed extreme hypersensitivity to cadmium exposure; in the presence of 0.3 µM cadmium, the pcs-1(lf) mutant strain displayed almost complete failure to grow. Interestingly, the pcs-1(lf); hizr-1(lf) double mutant strain displayed even greater sensitivity to cadmium; in the presence of 0.03 µM cadmium, the double mutant strain displayed almost complete failure to grow (Fig. 6D). Thus, pcs-1 is necessary for resistance to cadmium toxicity but is not necessary for resistance to high zinc toxicity. In the absence of the pcs-1 gene, hizr-1 is necessary for cadmium resistance, indicating that hizr-1 may promote cadmium resistance in a manner that is redundant with pcs-1.

To explore the function of metallothioneins, we analyzed mtl-1(lf) mtl-2(lf) double mutants. When cultured with increasing levels of supplemental zinc or cadmium, mtl-1(lf) mtl-2(lf) double mutants displayed growth similar to WT (Fig. 6 E and F). Thus, these genes are not necessary for growth in the presence of zinc or cadmium in an otherwise WT background. To investigate redundancy with pcs-1, we generated a pcs-1(lf); mtl-1(lf) mtl-2(lf) triple mutant. The pcs-1(lf) mutant displayed strong hypersensitivity to cadmium exposure, and the pcs-1(lf); mtl-1(lf) mtl-2(lf) triple mutant strain displayed even greater sensitivity to cadmium; in the presence of 0.003 µM cadmium, the double mutant strain displayed almost complete growth failure (Fig. 6F). When analyzed in parallel, the pcs-1(lf); mtl-1(lf) mtl-2(lf) triple mutant was more sensitive to cadmium exposure than the pcs-1(lf); hizr-1(lf) double mutant (SI Appendix, Fig. S4). Thus, in the absence of the pcs-1 gene, mtl-1 mtl-2 is necessary for cadmium resistance, indicating that metallothioneins may promote cadmium resistance in a manner that is redundant with pcs-1.

When cultured with increasing levels of supplemental zinc, the hmt-1(lf) mutant and WT displayed similar patterns of dose-dependent decrease in growth (Fig. 6G). The hmt-1(lf); hizr-1(lf) double mutant strain was similar to the hizr-1(lf) single mutant strain in high zinc sensitivity. Thus, hmt-1 is not necessary for high zinc resistance. By contrast, the hmt-1(lf) mutant displayed extreme hypersensitivity to cadmium exposure, and the hmt-1(lf); hizr-1(lf) double mutant strain was similar to the hmt-1 single mutant (Fig. 6H). Thus, hmt-1 is necessary for resistance to cadmium toxicity but is not necessary for resistance to high zinc toxicity. In the absence of the hmt-1 gene, hizr-1 is not necessary for cadmium resistance, indicating that hizr-1 is not redundant with hmt-1.

The mek-1 (ks54) mutation was reported to cause hypersensitivity to cadmium (12, 36, 37). It was recently discovered that this strain also contains a loss-of-function mutation in the tightly linked sek-1 gene, suggesting the phenotypes displayed by this strain result from loss of function of both genes (35, 38). When cultured with increasing levels of supplemental zinc, the mek-1(lf) sek-1(lf) double mutant displayed significant hypersensitivity, similar to the hizr-1(lf) mutant (Fig. 6I). Thus, mek-1/sek-1 activity promotes resistance to high zinc toxicity. The mek-1(lf) sek-1(lf) hizr-1(lf) triple mutant strain was even more sensitive, indicating the defects caused by hizr-1(lf) and mek-1(lf) sek-1(lf) are additive, and that hizr-1 and mek-1/sek-1 function by distinct mechanisms to promote resistance to high zinc. Thus, mek-1 and sek-1 are necessary for high zinc resistance, and these genes function nonredundantly with hizr-1. The mek-1(lf) sek-1(lf) mutant displayed moderate hypersensitivity to cadmium exposure (Fig. 6J). Thus, mek-1/sek-1 activity promotes resistance to cadmium toxicity. The mek-1(lf) sek-1(lf) hizr-1(lf) triple mutant strain was similar to the mek-1(lf) sek-1(lf) double mutant (Fig. 6J). Therefore, hizr-1 is not necessary for cadmium resistance in the absence of the mek-1 and sek-1 genes. Overall, these results indicate that mek-1/sek-1 is necessary for resistance to both high zinc and cadmium toxicity.

Cadmium and Supplemental Zinc Generally Cause Additive Toxicity.

In high doses, cadmium and supplemental zinc cause toxicity, and we expect that combinations will cause additive toxicity. However, it is possible that low doses of supplemental zinc might protect against cadmium toxicity or vice versa. To investigate this possibility, we exposed L1-stage WT, hizr-1(lf), pcs-1(lf), and pcs-1(lf); hizr-1(lf) animals to mixtures of cadmium and supplemental zinc for 3 d and measured length. In most cases, increasing amounts of supplemental zinc or cadmium decreased the average length of worms, indicating additive toxicity (SI Appendix, Table S9). However, there were cases in which an increase in supplemental zinc resulted in a small but statistically significant increase in length (SI Appendix, Table S9). In addition, there were cases in which an increase in cadmium resulted in a small but statistically significant increase in length (SI Appendix, Table S9). Such cases were observed in mutant animals but not necessarily at the same concentrations. These findings might reflect experimental variability since the differences were small; alternatively, they might indicate that supplemental zinc can partially ameliorate cadmium toxicity in some cases and cadmium can partially ameliorate high zinc toxicity in some cases.

Discussion

Cadmium Hijacks the High Zinc Homeostasis Response by Binding and Activating HIZR-1.

Warnhoff et al. established that HIZR-1 functions as the high zinc receptor in C. elegans: 1) the HIZR-1 ligand-binding domain directly binds zinc; 2) HIZR-1 accumulates in the nucleus of intestinal cells in response to high dietary zinc; 3) the HIZR-1 DNA-binding domain directly binds the HZA enhancer; 4) HIZR-1 activates transcription of zinc homeostasis genes, including genes encoding the zinc transporters CDF-2 and TTM-1B (22, 24, 39, 40). Genetic studies demonstrate that HIZR-1 is necessary for resistance to high dietary zinc, indicating HIZR-1 is the master regulator of high zinc homeostasis (24, 41). Here, we show that the HIZR-1 ligand-binding domain directly binds cadmium in purified extracts; the affinity for cadmium was similar to the affinity for zinc based on competition experiments, although these data did not establish the K_d for either metal. Cadmium exposure caused HIZR-1 to accumulate in the nucleus of intestinal cells, where it activated transcription through the HZA enhancer. For many well-studied genes that are strongly activated by cadmium exposure, hizr-1 is necessary for robust induction. These observations suggest that HIZR-1 is the high zinc receptor and can function as a cadmium receptor (Fig. 7A).

Fig. 7.

The hizr-1/HZA pathway promotes high zinc detoxification, the pcs-1/phytochelatin pathway promotes cadmium detoxification, and the mek-1/sek-1 kinase pathway promotes both high zinc and cadmium detoxification. (A) Molecular model of the transcriptional response to cadmium. Dietary cadmium (Cd) enters intestinal cells, binds the ligand-binding domain of HIZR-1, and promotes nuclear accumulation, HZA enhancer binding, and transcriptional activation of hizr-1–dependent genes. Cadmium activates transcription of additional genes independent of HIZR-1 by an undefined mechanism. (B) HIZR-1 can directly bind zinc, and the Zn/HIZR-1 complex activates cdf-2 and ttm-1 to promote zinc detoxification. Cadmium can also directly bind HIZR-1, and the Cd/HIZR-1 complex also activates cdf-2 and ttm-1. pcs-1 promotes formation of phytochelatin, which can directly bind cadmium, leading to cadmium detoxification. The mek-1/sek-1 pathway can promote both high zinc and cadmium detoxification. The dotted line from HIZR-1 to mtl-1 mtl-2 indicates that this pathway may promote cadmium detoxification in a manner redundant with pcs-1.

Nuclear receptors are characterized by a modular structure: the ligand-binding domain regulates activity, and the DNA-binding domain interacts with specific enhancer elements. Nuclear receptors are found throughout the animal kingdom, even in creatures without endocrine systems, where they may play roles in environmental sensing (27). There are about 50 nuclear receptors in humans, and about half of these receptors have known activating ligands, which are typically small hydrophobic molecules such as steroid hormones or lipids (26). The description of HIZR-1 as a nuclear receptor that uses zinc as a ligand established metal ions as a new class of ligands for a nuclear receptor. Here, we extend this observation by showing that a second metal ion, cadmium, can also function as a ligand for this nuclear receptor. These results raise the possibility that one or more orphan human nuclear receptors may also use metals as ligands. Shomer et al. recently reported that HIZR-1 interacts with MDT-15, a protein that is part of the mediator complex and important for a variety of transcriptional responses to stress (41). Cadmium promoted this binding interaction, which is consistent with our model that cadmium can function as a ligand for HIZR-1. An alternative model is that cadmium exposure displaces zinc bound to metallothioneins, thereby increasing the level of labile zinc, and zinc directly binds and activates HIZR-1 (29). The results presented here do not rigorously exclude this alternative model because the metal bound to HIZR-1 in animals was not determined. However, we demonstrated that metallothionein genes are not necessary for cadmium to induce HIZR-1–dependent transcription, indicating that metallothionein is not a necessary source of labile zinc.

The toxicity of cadmium in biological systems is well established, but the mechanisms of toxicity remain poorly defined. One prominent model is that cadmium binds proteins in physiological binding sites for another metal, such as zinc, copper, or calcium, displacing the physiological metal and decreasing protein function. A variation of this model is that cadmium binds to cryptic binding sites and decreases protein function. However, there is little direct evidence for these models. In principle, cadmium binding to a protein has three potential effects: 1) decreased or abolished function, 2) no effect, or 3) promotion of normal function or aberrant activation. A recent study of the intermolecular zinc-binding site in the hook domain of RAD50 found this metal-binding domain had a substantially greater affinity for cadmium (42). The irreversible binding of cadmium to the hook domain could be interpreted as decreased or abolished function of RAD50 in DNA damage sensing and repair. Here, we show that cadmium binding to HIZR-1 caused activation, including nuclear localization in intestinal cells and activation of transcription. If HIZR-1 is a physiological cadmium receptor that evolved to sense cadmium as well as zinc, then this activation can be interpreted as promotion of normal function. Alternatively, because HIZR-1 is the physiological high zinc receptor, activation by cadmium exposure can be interpreted as aberrant activation in a situation where zinc levels are not high. According to this interpretation, cadmium functions as a zinc mimetic to hijack the high zinc homeostasis response by binding and activating HIZR-1. Thus, cadmium binding to HIZR-1 promotes normal function or aberrant activation, but it does not appear to decrease or abolish function.

Other proteins have been reported to be functional when bound to cadmium. In terrestrial systems, the enzyme carbonic anhydrase requires zinc for function. Interestingly, a carbonic anhydrase was discovered in the marine diatom Thalassiosira weissflogii that functions when it is bound to cadmium (43). This protein enabled the diatoms to grow in zinc-deficient conditions when cadmium was present. These results suggest that in marine systems where zinc may be limiting, a protein evolved to use cadmium instead of zinc. Metallothioneins are small proteins that were first discovered because of their ability to bind cadmium (44, 45), and metallothionein genes are strongly transcriptionally activated by cadmium exposure. These observations led to the model that metallothioneins bind cadmium and thereby protect against cadmium toxicity (46–48). According to this model, cadmium binding constitutes normal metallothionein function. However, metallothioneins also bind physiological metals such as zinc and copper. Thus, an alternative interpretation is that cadmium binding displaces physiological metals such as zinc, decreasing protein function. To distinguish between these models, it is important to measure the function of metallothioneins. If cadmium binding to metallothioneins plays an important role in cadmium tolerance, then mutations that eliminate metallothionein function are predicted to cause hypersensitivity to cadmium toxicity. By contrast, if cadmium binding decreases the function of metallothioneins, then mutations that eliminate metallothionein function are predicted to have little effect on cadmium toxicity. Here, we show that a double mutant strain with deletions of both C. elegans metallothionein genes was not significantly more sensitive to cadmium toxicity or high zinc toxicity compared to WT. This is consistent with previous studies in C. elegans showing metallothionein mutant strains display weak or negligible cadmium hypersensitivity (15, 46). By contrast, deletion of both C. elegans metallothionein genes enhanced the cadmium hypersensitivity displayed by a pcs-1 mutant that is defective in generating phytochelatins, which is consistent with a previous report (15). Thus, C. elegans metallothioneins may promote cadmium detoxification in a manner that is redundant with pcs-1. In mice, which do not appear to generate phytochelatin, the loss of two metallothionein genes makes the animals more sensitive to cadmium-induced hepatotoxicity (49). The analysis of HIZR-1 presented here advances the understanding of how cadmium exposure affects protein function by demonstrating a specific protein that binds and is activated by cadmium in a terrestrial animal.

The Transcriptional Response to Cadmium Includes hizr-1–Dependent and –Independent Genes.

We identified 127 genes that were significantly activated by cadmium exposure in WT animals. For about 30% of these genes, the activation by cadmium was significantly reduced or eliminated in hizr-1(lf) mutants. These transcripts included some of the most dramatically activated genes, such as mtl-1 (300-fold to 3-fold), cdr-1 (250-fold to 1-fold), T08G5.1 (220-fold to 2-fold), and mtl-2 (24-fold to 0.5-fold). To evaluate the function of these activated genes in resistance to cadmium toxicity, we analyzed a selection of strong loss-of-function or null mutations. Most of these mutant strains were not significantly different compared to WT. These results suggest that these genes do not play a major role in cadmium resistance, and their activation in response to cadmium is unlikely to significantly blunt the toxic effects of cadmium. It is possible that these genes play redundant functions or minor roles in cadmium resistance that were not measured by the growth rate phenotype.

Another approach to evaluating the functionality of these hizr-1 target genes is to analyze the hizr-1(lf) mutant strain that lacks activation of all these genes. Whereas hizr-1(lf) mutants are dramatically hypersensitive to high zinc toxicity, hizr-1(lf) mutants were not hypersensitive to cadmium toxicity and actually were slightly resistant. These results suggest that hizr-1 is not necessary for cadmium resistance and that activation of hizr-1 may even promote vulnerability to high levels of cadmium. These results are consistent with the model that cadmium hijacks the high zinc response and do not support the model that hizr-1 evolved as a cadmium receptor to promote cadmium tolerance.

Why are hizr-1(lf) mutants slightly resistant to cadmium toxicity? One possible interpretation is that cadmium activates the high zinc homeostasis response, which reduces the level of zinc, thereby causing a zinc deficiency that contributes to cadmium toxicity. This process is referred to in the field of metal biology as conditioned deficiency. Studies of high zinc toxicity in humans suggest that some of the signs, symptoms, and phenotypes actually arise from conditioned copper deficiency (50). If cadmium causes conditioned zinc deficiency, then supplemental zinc might ameliorate cadmium toxicity. Power and de Pomerai reported that cadmium-induced beta galactosidase activity was reversed by supplemental zinc (51). Furthermore, while a zinc:cadmium mixture did not affect the mortality of worms, additional zinc rescued the behavioral effects of cadmium toxicity (52). Here, we exposed animals to combinations of supplemental zinc and cadmium. In general, toxicity was additive. However, in some cases, low levels of zinc slightly increased the growth of cadmium-exposed animals. These observations suggest that cadmium exposure may cause zinc deficiency, which might contribute to cadmium toxicity. However, supplemental zinc may blunt cadmium toxicity for multiple reasons, such as competing with cadmium for entry into the organism or binding sites on proteins.

For about 70% of the 127 cadmium-activated genes, the hizr-1 gene was not necessary for activation. This group included multiple heat shock genes such as hsp-16.48 (46-fold) and hsp-16.1 (29-fold). Although our data do not identify the mechanism of activation of these genes, we speculate they are activated by stress response pathways that detect molecular or cellular damage caused by cadmium exposure. To evaluate the function of these activated genes in resistance to cadmium toxicity, we analyzed a selection of strong loss-of-function or null mutations. Most of these mutant strains were not significantly different compared to WT. These results suggest that these genes do not play a major role in cadmium resistance, and their activation in response to cadmium is unlikely to significantly blunt the toxic effects of cadmium. It is possible that these genes play redundant functions or minor roles in cadmium resistance that were not measured by the growth rate phenotype.

Parallel and Shared Pathways for Zinc and Cadmium Detoxification.

Previous studies have identified two pathways that play important roles in promoting cadmium resistance: the pcs-1/phytochelatin pathway and the mek-1/sek-1/kinase signaling pathway. Vatamaniuk et al. reported that the pcs-1 and hmt-1 genes promote cadmium resistance (33, 53–56). pcs-1 encodes a phytochelatin synthase that catalyzes the formation of phytochelatins from glutathione monomers; phytochelatins are proposed to directly bind cadmium and thereby promote detoxification. hmt-1 encodes a transporter that is proposed to export cadmium-bound phytochelatins. Loss-of-function mutations in pcs-1 or hmt-1 cause dramatic hypersensitivity to cadmium toxicity, which we confirmed here using the growth rate assay. These genetic results support the model that phytochelatins bind and detoxify cadmium. Neither pcs-1 or hmt-1 transcripts were strongly activated by cadmium exposure, suggesting this pathway is not cadmium regulated at the level of transcription; it is possible the pathway is regulated at another level, such as increased synthesis or decreased degradation of phytochelatins (15). To analyze the metal specificity of these genes, we analyzed high zinc resistance. pcs-1(lf) and hmt-1(lf) mutants were only slightly hypersensitive to high zinc toxicity. Furthermore, these mutations did not enhance the hypersensitivity to high zinc toxicity caused by the hizr-1(lf) mutations. These results suggest the phytochelatin pathway is relatively specific for cadmium resistance and does not promote zinc resistance in C. elegans. Similar experiments in Arabidopsis thaliana have shown a major role for phytochelatins in cadmium detoxification as well as a role in zinc detoxification, though the increase of phytochelatin synthesis is greater following cadmium exposure (57, 58). Differences between Arabidopsis and C. elegans could arise from evolutionary divergence in the protein structure of phytochelatin synthase or technical and biological differences in the assays used to measure sensitivity. A. thaliana phytochelatin synthase also has a noncatalytic immune function that has not yet been observed in C. elegans (30).

The mek-1/sek-1 kinase signaling pathway promotes resistance to multiple stresses, such as starvation, metal ions, and pathogens (12, 16, 35–38, 59, 60). These genes encode MAP kinase kinases that participate in a signaling pathway that ultimately influences transcription. Mizuno et al. reported that mek-1(lf)/sek-1(lf) mutants are hypersensitive to cadmium toxicity, suggesting this pathway plays a protective role (36). We confirmed that mek-1(lf)/sek-1(lf) mutants displayed strong hypersensitivity to cadmium. To analyze the specificity of this pathway, we analyzed high zinc resistance. mek-1(lf)/sek-1(lf) mutants were strongly hypersensitive to high zinc toxicity. Furthermore, these mutations enhanced the hypersensitivity to high zinc caused by the hizr-1(lf) mutations; this additive phenotype suggests the mek-1/sek-1 signaling pathway functions in parallel to the hizr-1 pathway. These results suggest the mek-1/sek-1 pathway promotes cadmium and zinc resistance, perhaps by a similar mechanism.

The hizr-1(lf) mutants display dramatic hypersensitivity to zinc toxicity, suggesting activation of hizr-1 target genes is critical for high zinc resistance. Two key targets of hizr-1 are the zinc transporters CDF-2, which sequesters zinc in lysosome-related organelles, and TTM-1B, which transports zinc across the apical membrane of intestinal cells (22, 39). The hizr-1(lf) mutation did not cause hypersensitivity to cadmium toxicity. Furthermore, the hizr-1(lf) mutation did not enhance the hypersensitivity of hmt-1(lf) or mek-1(lf)/sek-1(lf) mutants to cadmium exposure. The hizr-1(lf) mutation did enhance the hypersensitivity of the pcs-1(lf) mutant to cadmium. In addition, a double mutation of mtl-1 and mtl-2 enhanced the hypersensitivity of the pcs-1(lf) mutant to cadmium. This might indicate that hizr-1 acting via the target genes mtl-1 and mtl-2 can promote cadmium tolerance in a manner that is redundant with pcs-1. Alternatively, this may reflect nonspecific sickness of these double and triple mutant strains. Overall, these results indicate that hizr-1 is relatively specific for zinc resistance.

These studies suggest a framework for understanding organismal resistance to high zinc and cadmium (Fig. 7B). HIZR-1 specifically promotes resistance to high zinc by activating zinc transporters that sequester or excrete excess zinc, and HIZR-1 is directly regulated by zinc binding. Cadmium can also bind and activate HIZR-1, but HIZR-1 is not effective at cadmium detoxification, perhaps because these CDF family transporters cannot utilize cadmium as a substrate. The phytochelatin pathway specifically promotes resistance to cadmium; PCS-1 catalyzes phytochelatin synthesis, and cadmium directly binds phytochelatins resulting in detoxification. This pathway does not contribute to high zinc resistance. The mek-1/sek-1 signaling pathway promotes both high zinc and cadmium resistance. It may sense damage caused by these stressors and activate transcripts that lead to damage control. Further studies are necessary to determine the effectors of this pathway that relate to the damage caused by cadmium or high zinc.

Summary of the Methods

Detailed methods are described in the SI Appendix, Materials and Methods. C. elegans strains were exposed to metals using noble agar minimal medium. Mutant and transgenic strains utilized in these experiments are described in the SI Appendix, Table S10. To identify cadmium-regulated genes, we used literature searches and experimental approaches to compile a list of candidate genes. To measure transcript levels of the 449 unique candidate genes, we used the method of qPCR. To analyze expression of promoter fragments containing the HZA enhancer, we performed fluorescence microscopy using established methods (23, 24). To monitor nuclear localization of HIZR-1, we analyzed hizr-1(am286); HIZR-1(1–412 WT)::GFP transgenic animals as described previously (24). We purified GST fusion proteins using a pGEX expression system and a glutathione resin as described previously (24). We measured metal binding to HIZR-1 as described previously (24). Growth rate was analyzed as described previously starting with animals at the L1 larval stage (24). Most data were analyzed utilizing the two-tailed Student’s t test of samples with unequal variation.

Data Availability

High-throughput RNA-seq data have been deposited in Gene Expression Omnibus (GSE160704). All other study data are included in the article and/or SI Appendix.