The Society for Integrative and Comparative Biology
Changes in Nuclear Receptor and Vitellogenin Gene Expression in Response to Steroids and Heavy Metal in Caenorhabditis elegans1
1 Department of Biology, Boston University, Boston, Massachusetts 02215
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To gain basic understanding of the reproductive and developmental effects of endocrine disrupting chemicals in invertebrates, we have used C. elegans as an animal model. The completion of the C. elegans genome sequence brings to bear microarray analysis as a tool for these studies. We previously showed that the C. elegans genome was responsive to vertebrate steroid hormones, and changes in gene expression of traditional biomarkers used in environmental studies were detected; i.e., vitellogenin (vtg), cytochrome P450 (cyp450), glutathione-S-transferase (gst) and heat shock proteins (hsp). The data were interpreted to suggest that exogenous lipophilic compounds can be metabolized via cytochrome P450 proteins, and that the resulting metabolites can bind to members of the Nuclear Receptor (NR) class of proteins and regulate gene expression. In the present study, using DNA microarrays, we examined the pattern of gene expression after progesterone (10–5, 10–7 M), estradiol (10–5 M), cholesterol (10–9 M) and cadmium (0.1, 1 and 10 µM) exposure, with special attention to the members of NRs. Of approximately 284 NRs in C. elegans, expression of 25 NR genes (representing 9% of the total NRs in C. elegans) was altered after exposure to steroids. Of note, each steroid activated or inhibited different subsets of NR genes, and only estradiol regulated NR genes implicated in neurogenesis. These results suggest that NRs respond to a variety of exogenous steroids, which regulate important metabolic and developmental pathways. The response of the C. elegans genome to cholesterol and cadmium was analyzed in more detail. Cholesterol is a probable precursor to signaling molecules that may interact with NRs and we focused on expression of genes related to lipid metabolism (cyp450), transport and storage (i.e., vitellogenin). Worms exposed to cadmium respond principally by activating the expression of genes encoding stress-responsive proteins, such as mtl-2 and cdr-1, and no significant changes in expression of NRs or vtg genes were observed. The possible implications of these results with regard to the evolution of steroid receptors, endocrine disruption and the role of vitellogenin as a lipid transporter are discussed.
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INTRODUCTION |
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Nuclear receptors (NRs) are a large superfamily of transcriptional regulators exclusively found in metazoans (The Arabidopsis Genome Initiative, 2000
). Nuclear receptors can be identified on the basis of their well-conserved DNA binding domain (DBD), which comprises two Cys2-Cys2 zinc-coordinating modules, and on the basis of a less conserved domain, called the ligand binding-domain (LBD), located at the C-terminal. This domain participates in ligand binding, homo- and heterodimerization, and transcriptional regulation (see review; Freedman, 1997). These proteins are involved in embryonic pattern formation, development and differentiation of multiple tissue types, sex determination, metamorphosis, fertility, regulation of cellular metabolism and homeostasis (Giguere, 1999; Miyabayashi et al., 1999; Owen and Zelent, 2000; Sluder and Maina, 2001; Enmark and Gustafsson, 2001; Maglich et al., 2001; Sullivan and Thummel, 2003; Francis et al., 2003). In addition, NRs are implicated in diseases such as cancer, diabetes or hormone resistance syndromes, and are extensively investigated for drug development. More recently, the identification in the environment of many small molecules (xenobiotics) that bind these NRs has led to the recognition of the phenomenon of endocrine disruption (ED), and it is important to understand the effects of xenobiotic exposure on receptors and potential in vivo signal transduction pathways. Of particular interest are xenoestrogens because of the broad (pleiotropic) effects of estrogens and their analogues in all vertebrates.
In the last ten years, complete genome sequences have become available for C. elegans, C. briggsae (nematodes), Drosophila melanogaster (insect), Saccharomyces cerevisiae (yeast), Fugu rubripes (teleost fish), human and mouse. Several authors have compared these genomes, focusing on the NR family (Enmark and Gustafsson, 2001; Sluder and Maina, 2001, Maglich et al., 2001, 2003) with surprising results. Thus, dramatically different numbers of NR genes have been described: 284 in C. elegans, versus only 21 in Drosophila, 68 in fugu and approximately 50 in humans (Sullivan and Thummel, 2003; Maglich et al., 2003; Gissendanner et al., 2004). Phylogenetic analysis of the vertebrate NRs, based on the human genome, showed seven groups (0 to VI) of nuclear receptors, with several subgroups in each group (Laudet, 1997; NucleaRDB: http://www.receptors.org/NR/, Horn et al., 2001). In C. elegans, fifteen of the NR genes have apparent homologs in insects and vertebrates, and can be assigned to 5 of the large groups of NR. However, the other NRs in C. elegans cannot be placed into any of the seven major NR subfamilies and are labeled "divergent nematode NRs" (Sluder and Maina, 2001). Despite the large number of NRs in the C. elegans genome, the steroid hormone receptor (NR3) group is not represented in this species. In contrast, in Drosophila at least one member (dERR) of the NR3 subfamily has been described, confirming the ancient metazoan origin of this family. Recently, and consistent with this, the isolation of an estrogen receptor ortholog from a representative group of Protostomes, the mollusk Aplysia californica has been described (Thornton et al., 2003). After reconstruction, synthesis, and experimental characterization of the functional domains of this ancestral ER ortholog, the authors suggest that this gene was lost in the Ecdysozoan (Nematode and Arthropod) lineage. The finding of the ‘ER’ ortholog in a protostome (mollusk) suggests that several classes of invertebrates may be subject to endocrine disruption through xenoestrogen activated gene pathways and networks as in vertebrates, and will explain the observed effects after estradiol exposure in some invertebrate species (see Di Cosmo et al., 2002; Osada et al., 2003). However, other signal transduction pathways, possibly involving invertebrate receptors not immediately identified as homologs of vertebrate nuclear receptors for estrogens and xenoestrogens, remain to be identified.
In our prior studies (Custodia et al., 2001) of C. elegans responses to vertebrate steroids (estradiol, progesterone and testosterone), using vitellogenin as biomarker, we examined the possibility that this end point, the prototypical estrogen response for all vertebrates except mammals, might be responsive to estradiol in C. elegans. These studies showed a clear dose response relationship between vitellogenin synthesis and estradiol exposure in culture. Further, using DNA microarrays, we showed that at least two C. elegans vitellogenin genes (vit-2 and vit-6) were significantly up regulated by estradiol, in a manner reminiscent of the effect of the hormone in vertebrates. Thus, the possibility exists that estrogens, xenoestrogens and other related steroids/sterols may be involved in the regulation of this important metabolic pathway in both invertebrates and vertebrates. Furthermore, heavy metals, such as cadmium, are known to interact with steroid transcription factors (such as GR and ER) and have been shown to block/antagonize estrogen action (Simons et al., 1990; Stoica et al., 2000). It is possible that responses of C. elegans to cadmium may provide more information about genomic effects of this metal.
In this paper, we further examine C. elegans NR genes that may be associated with the up-regulation of vtg genes. It is possible (likely?) that an incipient network of genes that can be activated by internal and external environmental signaling via (xeno)estrogens exists and is associated with this important metabolic pathway and others at this level of animal organization. Preliminary characterization of the NRs family proteins in C. elegans and related species (C. briggsae, B. malayi) suggests that high numbers of NR genes are expressed (Sluder et al., 1999), and are functional (Miyabayashi et al., 1999; Kim, 2001). However, all nematode nuclear receptors belong to the orphan class of NRs, since ligands have not yet been identified. DNA sequence analysis suggests that NRs have the structural potential for ligand binding. In order to explain the unprecedented abundance and diversity of C. elegans divergent NR genes, confirmed in other nematode genomes (C. briggsae, B. malayi) (Sluder and Maina, 2001), several related hypotheses have been formulated. It is suggested that proliferation and diversification of NR sequences have continued through nematode evolution, with distinct NRs contributing to specific adaptations for particular lifestyles (Sluder et al., 1999, see review Van Gilst et al., 2002). It is also postulated, for several receptors, that the NRs originally evolved from proteins that mediate signals from environmental compounds or nutrients (Yamamoto, 1997).
It is well documented that C. elegans requires sterol, usually supplied as cholesterol, and this can be metabolized to unusual 4-alpha-methyl sterols (4MSs) (Merris et al., 2003). In addition, it has been shown that sterols such as campesterol and stigmasterol are metabolized in C. elegans (Lozano et al., 1985). Other studies in C. elegans show that sterols can be found in excretory gland cell, amphid and phasmid socket cells, spicule socket cells, pharynx and intestine, suggesting that sterols are not only required for cell membranes, but may be required as hormone precursor and/ or developmental effectors (Merris et al., 2003). Here, we suggest that sterol metabolites might serve as ligands to NR. Recently, the finding in C. elegans that daf-9 encodes a cytochrome P450 and daf-12 a nuclear receptor, provides substantial evidence for hormonal signaling by lipophilic molecules in C. elegans (Gerisch et al., 2001). Also, a xenobiotic sensing function has been described for nhr-8 (Lindblom et al., 2001). Thus, it is possible that some of these proteins serve as ligand-independent transcription factors, the expression of which might be regulated by environmental factors such as temperature, metal ions or pH (Enmark and Gustafsson, 2000). For other C. elegans NRs, ligands may be found among the many natural chemicals and xenobiotics. The natural environment of C. elegans and other free-living nematodes is the soil-water interface, a location in which the organisms would be exposed to all water-soluble xenobiotics. Thus, it is likely that these animals would have evolved a wide variety of pathways for detection and detoxification of xenobiotics, explaining the large number of orphan NRs.
The development of simple animal models for high throughput testing of the effect of multiple xenobiotics and mixtures on gene expression is important for an understanding of the dangers of environmental exposure. Our previous studies showed that C. elegans genes other than those for vitellogenin are also sensitive to vertebrate steroid hormones. We observed changes in the expression of members of the cytochrome P450 family, which typically can metabolize steroid hormones, fatty acids, and xenobiotics, as well as genes associated with oxidation-reduction (such as the glutathione-s-hydrogenase family). The present study is an extension of the previous one; here we describe expression changes of the NR family in C. elegans by DNA microarray analysis after exposure to steroid hormones (progesterone, cholesterol and estradiol) as well as cadmium. Additional information on vitellogenin gene expression with regard to cholesterol and cadmium exposure is presented.
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MATERIALS AND METHODS |
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Culture of C. elegans
Adult N2 strain of C. elegans were grown in 250 ml of liquid S medium with Escherichia coli OP50/1 (grown in 2XTY media) according to Sulston and Hodgkin (1988)
. Triplicate nematode cultures were exposed for 4 days to cholesterol or to different vertebrates steroids. All steroids, purchased from Fisher Biotech (Fair Lawn, NJ), estrogen (17ß-estradiol), progesterone (4-pregnene-3,20-dione) and cholesterol (20-hydroxycholesterol) were prepared in absolute ethanol, and added to cultures in amounts necessary to achieve final concentrations of 10–5 (estradiol), 10–5 and 10–7 (progesterone), and 10–9 (cholesterol) M in culture. Control cultures received an equal volume of absolute ethanol. For cadmium experiments short-term (4 days) and long-term (7 days) exposure experiments were performed. CdCl2 was purchased from Sigma (St Louis, MO), and the stock solutions were prepared in sterile water, and control cultures for these experiments received sterile water. Two independent experiments were performed with CdCl2. In the first experiment, three different concentrations of CdCl2 were tested 1, 10 and 100 µM. In the second experiment, due to the high toxicity observed with 100 µM of CdCl2 we tested 0.1, 1 and 10 µM CdCl2. For all the different treatments, cultured nematodes were harvested after 4 (or 7) days treatment, washed and stored at –70°C pending analysis.
RNA isolation
Total RNA was isolated from C. elegans liquid cultures using Trizol reagent following a modified method of the protocol specified by the manufacturer (Life Technologies, Rockville, MD). Adult worm populations were suspended in Trizol, 4 ml/ml of compacted worms. The RNA was separated using chloroform, precipitated with isopropyl alcohol and washed with 75% ethanol. The RNA pellet was dried under vacuum for 5–10 minutes and dissolved in DEPC-treated water. The poly (A+) RNA was subsequently isolated using Separator Kit (Clontech, Palo Alto, CA) according to the manufacturer's instructions.
DNA microarray preparation and analysis
Poly (A+) mRNA was sent to the Stanford Microarray Database (SMD; http://genome-www.stanford.edu/microarray/; Gollub et al., 2003), for DNA microarray hybridization and data analysis. The microarray data was analyzed in order to identify gene expression affected by hormone and cadmium treatments. The normalized values used were: G/R ratio >2.6 for up-regulation and R/G ratio >2.6 for down-regulation. G: green color that corresponds to the treated samples, and R: red color, corresponding to the control sample. Data were filtered for spot flag = 0, regression correlation >0.6, and Ch1 Net (red) and Ch2 Normalized Net (green) = 150 to eliminate either flagged spots or if the PCR did not work (no band or a doublet), or if there was something abnormal about the hybridization.
Yolk protein extraction
Compacted worms were homogenized using Tris-HCl buffer according to Sharrock (1983). Yolk proteins were extracted from the homogenates using the homogenizing buffer plus 0.1% (W/V) NP-40. Protein concentration was determined by Bradford assay using bovine serum albumin (Calbiochem, La Jolla, CA) as standard (Bradford, 1976), and 20 µg total protein was loaded for each treatment group.
Electrophoresis and western blotting analysis
Yolk protein extracts were resolved in a 7.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; Laemmli, 1970). Proteins were transferred to nitrocellulose membrane using a mini-trans-blot (Bio-Rad, Hercules, CA). For immunodetection we used, as primary antibodies, anti-YP-88, and YP-170 antibodies kindly provided by Dr. Thomas Blumenthal, and as a secondary antibody an anti-rat IgG conjugated to alkaline phosphatase (Sigma, St. Louis, MO). The membranes were developed using NBT-BCIP substrate system (Promega, Madison, WI).
Gene annotation
Different databases of C. elegans genetic information exist. We preferentially used the database WormPD and Gene Ontology to make the annotations for biological function and expression of the genes studied (https://www.incyte.com/proteome/WormPD; www.geneontology.org, respectively). WormPD is a sub-library of the BioKnowledge Library created and run by Proteome, Inc. In WormPD, the genes of C. elegans are categorized according to cellular roles and the functions of corresponding proteins. For each gene, there is information about predicted and experimental biological and molecular function, regulation, sequence, GenBank accession number and expression linked to references to original research papers.
Hierarchical clustering and gene classification for CdCl2 experiments
The raw data were uploaded into the Stanford Microarray Database (SMD). Normalized data (log (base2) (control/experimental)) were downloaded using the following filter criteria: flag = 0 and red or green intensity >2.5 fold of the background intensity, regression correlation >0.6, and the channel intensity of red or green channel were >350. Using these criteria 19838 SUIDS (unique identifying number within the SMD which is specific for a single arrayed clone or PCR-amplified region of genomic DNA) passed filters. The data value-based filter used was: cutoff (only select genes whose is absolute value >2 at least 1 array); so filter removed 19823 SUIDS. The data quality filter (only using genes with >80% good data) removed 4 SUIDS. With this number of genes we did hierarchical clustering followed by visual inspection to subdivide the nodes. Most gene annotations are from (WormPD: https://www.incyte.com/proteome/WormPD) and are manually annotated.
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RESULTS AND DISCUSSION |
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Changes in NRs gene expression (Table 1)
The complete list of genes from NRs family in C. elegans that show alterations of gene expression after exposure to different steroids and cholesterol is presented in Table 1. To define an alteration in gene expression we used normalized values: G/R ratio >2.6 for up-regulation and R/G ratio >2.6 for down-regulation. Using these criteria, 25 NRs genes were identified, representing 9% of total NR genes. Having defined the NRs list of genes that are altered after exposure to vertebrate steroids and cadmium, we subdivided them into genes that appeared to be "steroid-specific." It is of interest that each steroid activated or inhibited a totally different subset of genes, therefore no overlap was observed between the NR genes that had altered expression associated with different steroid treatments. Of 25 NR genes, expression of 11 was altered after estradiol exposure, 10 after progesterone and 4 after cholesterol.
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Using more relaxed cut off criteria (<2 fold change), we are able to identify 2 members of NRs family that were down regulated after cadmium exposure. These two NRs belong to the group of nhr-58 (T13F3.2 and nhr-58). Cadmium has been shown to influence transcription of several vertebrate nuclear receptors previously (Simons et al., 1990
; Stoica et al., 2000), and is therefore likely to influence the biological properties of these proteins.
NRs genes regulated by estrogen
Previous studies have shown that exogenous estrogen can induce vitellogenin mRNA levels (Custodia et al., 2001; Kohra et al., 1999) in C. elegans, and at concentration of 5 µM stimulated growth, having a cholesterol-like potency for C. elegans (Tominaga et al., 2001). In our study, 10 µM estradiol induced over-expression of the nhr-47 member of the NR family (Table 1). By gene ontology, of the under-expressed genes, the majority is predicted to be associated with transcriptional regulation, and others are involved in development and lipid storage. It is of interest to speculate that these genes may be part of an estrogen sensitive gene network related to vitellogenesis, since vitellogenin is dependant upon the availability of lipid stores to the gastrointestinal cells involved in vtg synthesis. One of these NR genes (unc-55) has an important role in neurogenesis. Specifically unc-55 is implicated in neural differentiation and control of post-embryonic remodeling of the synaptic specificity of particular motor neurons (Zhou and Walthall, 1998) in C. elegans. In the absence of unc-55 function, animals exhibit locomotion defects due to defects in the synaptic connections of the VD motor neurons (Walthall, 1990). The unc-55 NR gene is a member of the conserved COUP NR group (NR2F). It is of interest that vertebrate members of these families of NR are implicated in neurogenesis and regulation of eye and neural development (Fjose et al., 1993; Pereira et al., 1995). This suggests that neural specification could be an ancient function of this particular NR group. A role of estrogen in vertebrate neurogenesis is an area of intense research and significance.
NRs genes regulated by progesterone
The two different concentrations of progesterone tested (0.1 and 10 µM) in cultures of C. elegans provoked over-expression of 4 NRs and down-regulation of 6 of NRs family (Table 1). The effect on NRs mRNA expression was not dose related, because only one of the NR (R11G11.1, Table 1) was down regulated by high doses of progesterone (10 µM). Of particular interest, nhr-69 (up-regulated by progesterone, Table 1) is a member of the conserved NR2A group (the human paralog is HNF4), and is expressed in gut, hypodermis and uterus (Gissendanner et al., 2004). Vertebrate members of this NR family (NR2A) are involved in cholesterol and amino acid metabolism, as well as aspects of carbohydrate, lipid and xenobiotic metabolism, as well as other liver specific genes (Giguere, 1999). In nematodes the gut exerts many of the functions of vertebrate hepatic tissues, such as vitellogenesis.
Response of C. elegans to cholesterol
C. elegans requires small amounts of exogenous cholesterol for growth and as a structural component of membranes and key metabolic intermediates. It is also suggested that its availability has an important role in molting and induction of a specialized non-feeding larval stage (see review in Kurzchalia and Ward, 2003). Supporting an important role, in this study we show that low doses of cholesterol provoke changes in expression of a large number of genes (Fig. 1) reflecting potential interference in a wide range of pathways. In more detail, we will focus on genes related to lipid metabolism, transport, storage and regulation of transcription (Tables 1 and 2). Of particular interest to the biology of C. elegans, as well as its use as an environmental bioindicator species, are genes implicated in: (a) regulation of transcription such as NR and other transcription factors, (b) cholesterol metabolism to steroid signaling molecules similar to vertebrate steroids, and (c) cholesterol as an important component of egg (oocyte) yolk.
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(a) Genes implicated in transcription (NRs):
We show that low concentrations of cholesterol (1 nM) changed the expression patterns of 4 members of NRs in C. elegans. These are nhr-66, F10G2.9, nhr-68 and K06B4.8. The spatial and temporal expression patterns of nhr-66 have been described by examining transgenic animals (Miyabayashi et al., 1999
), showing that it is expressed in neuronal and non-neuronal cells (hypodermal seam cells). No expression pattern has been described for the other three regulated NR members: nhr-68, K06B4.8, F10G2.9 but it is predicted that they are implicated in biological processes such as steroid metabolism, neurogenesis, and transcription (see WormPD database). In this study, nhr-68 and K06B4.8 are down regulated by cholesterol. Studies using RNAi showed that nhr-68 increased body fat in C. elegans (Ashrafi et al., 2003) and K06B4.8 was implicated in regulation of growth (Simmer et al., 2003). In addition, cholesterol treatment caused over-expression of several transcription factors (Table 2). By gene ontology and sequence comparison, these genes are predicted to have diverse functions in morphogenesis, lipid storage and vulval development, reflecting the wide range of pathways in which cholesterol is involved. Of special importance are the changes in expression of G-protein-coupled receptors (GPCRs) suggesting that cholesterol may be involved in neuroendocrine pathways in C. elegans.
(b) Potential genes implicated in cholesterol metabolism to steroid signaling molecules similar to vertebrate steroids:
Treatment of C. elegans with low concentrations of cholesterol provoked over-expression of two genes of the P450 family: K10D2.6 and H02I12.8, and down regulation of gst genes (Table 2). Cytochrome P450 enzymes are monooxygenases that metabolize many endogenous and exogenous lipophilic compounds including steroid hormones, xenobiotics and fatty acids (Mansuy, 1998). These cytochrome P450's may participate in synthesizing, modifying or degrading putative hormonal derivatives of cholesterol. Although there is evidence that cholesterol metabolites, steroid hormones or ecdysones can serve as candidate ligands for nematode NR (see review Kurzchalia and Ward, 2003), there is no clear chemical identification of these molecules in worms. Previous studies (Custodia et al., 2001), demonstrated that C. elegans respond to vertebrate steroids by altering expression patterns of vitellogenin, cyp450, gst and hsp genes. In particular, gst-4 has been shown to be down regulated by progesterone and estradiol in our previous studies, and in this study by cholesterol. Further, we show here that the expression of certain members of NRs in C. elegans change after exposure to vertebrate steroids and cholesterol, suggesting that NRs respond to a wide variety of exogenous compounds. In this regard, it is of interest to speculate that nutrient cholesterol can be metabolized and serves as a precursor for ligands that bind to NR (orphan receptors, OR) in C. elegans as in vertebrates. In vertebrates, OR are considered potential lipid sensors (Chawla et al., 2001). In C. elegans, indirect evidence of a sterol metabolic pathway exists. Thus, blocking sterol metabolism by 25-azacoprostane-HCl treatments causes serious defects in germ cell development, growth, cuticle development and motility (Choi et al., 2003). However, while cholesterol-derived steroids have not yet been identified in C. elegans, the genome of C. elegans contains approximately 80 cytochrome P450s and 17 estradiol dehydrogenases that are candidate enzymes for modification of cholesterol to form steroid hormones (Mansuy, 1998; Nelson, 1998). It is possible that these vertebrate OR lipid sensors represent part of a conserved network of genes which were organized at the outset of prokaryotic cellular organization (around 950 Myr before present). Thus, ligand binding to the receptor may activate a metabolic cascade (gene network) that maintains nutrient homeostasis and all subsequent metabolic pathways.
Studies involving the biological effects of vertebrate steroids upon parasitic nematodes have been performed, and observations from these studies indicate that nematodes are responsive to vertebrate host steroids. These studies indicate that vertebrate steroids affect reproduction, growth, molting, feeding, embryogenesis and movement (reviewed in Chitwood, 1999), suggesting the existence of a hormonal signaling pathway.
(c) Vitellogenin genes and others:
Recent studies of cholesterol distribution and transport indicate that the process of cholesterol transport in C. elegans (Matyash et al., 2001) has similarities to that of vertebrates particularly with regard to the overall process of vitellogenesis (see Duggan et al., 2001). The studies provide support for the concept that macromolecular transport of cholesterol in association with triglycerides, phospholipids and proteins may have co-evolved in association with the process of oocyte yolk deposition. Vitellogenins are a primary macromolecular transporter of cholesterol to the oocyte in C. elegans (Matyash et al., 2001). In contrast, in vertebrates vitellogenin per se does not transport cholesterol to the oocyte, this function being provided by the well-described LDL pathway (Brown and Goldstein, 1986), which also delivers cholesterol to somatic cells that do not take up vitellogenin. Non-mammalian vertebrate oocytes express both the vitellogenin receptor (Bujo et al., 1994) and the LDL-receptor. The argument that vitellogenin is a primordial macromolecular transporter of cholesterol is strengthened by observations of the homology between the primary protein of the LDL particle (apolipoprotein B) and vitellogenin (Baker, 1988; Perez et al., 1991; Mann et al., 1999). Clearly, other mechanisms for cellular cholesterol accumulation, such as the LDL pathway, occur in C. elegans, because cholesterol uptake starts before yolk protein expression, and males do not express vitellogenin but accumulate cholesterol in sperm (Matyash et al., 2001). In connection with this, Matyash et al. (2001) identified a 37 KDa cholesterol binding protein in males and hermaphrodites which is a candidate cholesterol transporter. The studies of Matyash et al. (2001) have also shown that cholesterol is accumulated in the pharynx, nerve ring, excretory gland cell and gut of L1–L3 larvae and later in oocytes and sperm. It is of interest that in previous studies (Custodia et al., 2001), using western-blot analysis we showed that low doses of 20-hydroxycholesterol (1 nM) increased expression of yolk proteins YP-170s. This suggests that substrate availability (cholesterol) may influence the process of yolk protein synthesis. In the present study, DNA microarrays confirmed up-regulation of the majority of members of vtg gene family (Table 2), including vit-3 (Table 2).
In addition, one oocyte-enriched gene was up regulated after cholesterol treatment, ptr-2 (Table 2). Studies using RNAi showed that ptr-2 has an important role in embryogenesis, embryonic cleavage, and energy metabolism in C. elegans (Piano et al., 2002).
Response of C. elegans to cadmium
In two experiments, C. elegans cultures were exposed to CdCl2. In experiment 1, changes in gene expression were analyzed by cDNA microarrays and vitellogenin protein levels by western blot, after 0.1, 1 and 10 µM CdCl2 exposure for 7 days. No changes in vitellogenin protein expression (data not shown) were seen as assessed by western-blot analysis. In experiment 2, cultures of C. elegans were exposed to 1, 10 and 100 µM CdCl2 for 4 days and changes of yolk protein expression were assessed by western blot analysis (Fig. 2); microarray analysis was not done for this experiment. By western-blot analysis the expected number and size of vitellogenin translation products were detected in both experiments. High concentrations of cadmium (100 µM CdCl2) resulted in inhibition of translation of the three vitellogenin proteins (Fig. 2), and clear toxicity effects were observed (mortality > 70%), suggesting the inhibition observed in vitellogenin protein levels is due to toxicity. By western blot analysis, exposure to 10 µM CdCl2 resulted in a slight inhibition of yolk proteins YP170s and YP115, but was without effect on YP88.
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The differences in observed vitellogenin protein levels in response to cadmium in the two different experiments may be explained by differences in exposure time (4 day versus 7 day), and the high levels of expression of mtl-2 and cdr-1 genes (see below) observed in experiment 1. Expression of these genes would protect the organism from cadmium toxicity, and prevent the inhibition of vitellogenin gene expression observed when exposed to cadmium for a longer time. However, microarray analysis will be necessary to assess this possibility. In support of a protective role of metallothionein gene expression and cadmium sequestration in vitellogenin gene expression, de novo induction of vitellogenin synthesis has been shown to occur in the rainbow trout once metallothionein has begun to sequester cadmium (Olsson, 1995
). Cadmium triggered expression changes in a small number of genes in a dose dependant manner (Fig. 1). To summarize the data for microarrays and the changes in gene expression induced by cadmium treatment, we used a hierarchical clustering algorithm to sort the genes according to their similarity in expression pattern during the exposure time to different doses of CdCl2. The hierarchical clustering sorted the genes into two main nodes (Fig. 3). Almost all the genes in the first node (cluster A) are over-expressed in relation to the dose of cadmium, while almost all the genes in the second node (cluster B) are under-expressed in relation to CdCl2 exposure. 50% of all the genes in the first node (cluster A) have peak over-expression in adults in the 10 µM CdCl2 treatment, and the other 50% of the genes have almost the same level of gene expression irrespective of the concentration of cadmium used in the experiment.
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To identify cellular functions that can be affected by cadmium treatment, we determined which functional classes of genes were over-expressed in this study (Fig. 4). Thus, up regulated genes were placed into one of the following putative molecular function classes: cell communication, stress signaling, GPCR receptors, metabolism, immunoglobulin signaling, collagens, detoxification; genes which could not be assigned to a functional class were placed into the Unknown class.
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In more detail, to protect against cadmium-induced damage, cells respond by increasing the expression of genes encoding stress-responsive proteins implicated in detoxification. In this study, two genes that are specific for the cadmium-induced response have been identified: mtl-2 (member of metallothionein family) and cdr-1. The ability of cadmium to induce metallothionein gene expression in a variety of species has been documented (Liao and Freedman, 1998
; Waisberg et al., 2003) and a detailed discussion of the effect of cadmium on C. elegans metallothionein gene transcription can be found in Freedman et al. (1993). The other gene, cdr-1, recently identified, is a cadmium inducible lysosomal protein required for resistance to cadmium (Liao et al., 2002). These two genes are expressed in intestine, and are transcriptionally regulated by cadmium through a specific metal-responsive element (MRE) found in the promoter region of both genes. High levels of expression of these two genes suggest the possibility that C. elegans can survive and avoid the negative effects of cadmium by sequestration of metal ions. In addition, other genes that respond to metal stress have been identified (Fig. 4). These are: Y73F8A.H, Y1055A.C, Y105C5A.D, F19G12.7; according to their predicted function these genes are involved in repair processes after stress (based on sequence similarity, WormPD). The mechanism by which cadmium affects the expression of these genes remains unknown, but possibly involves the presence of MREs in promoter regions of these genes.
An important functional class of genes which are related to cuticle formation and collagen are over-expressed after cadmium exposure. The effect of cadmium on collagen genes is well documented by both in vivo and in vitro studies. Cadmium acts as a pro-fibrinogenic agent in liver, and it is suggested that oxidative stress may stimulate lipid peroxidation and collagen synthesis (del Carmen et al., 2002; Liao and Freedman, 1998).
We also observed that cadmium induced over-expression of C09H5.2 gene, which is a member of the E1–E2 ATPase family. This predicted gene, according to sequence similarity and domain content, may be implicated in cation transport and metal ion homeostasis. Cadmium may displace metal ions from proteins by altering the homeostasis of metals such as Zn and Ca, possibly explaining the effect of cadmium on gene expression. In turn, signal transduction pathways are impacted which then can influence the expression of myriad genes. 50% of genes over-expressed after cadmium treatment have unknown functions (Fig. 4). Of these, many have ubiquitin transferase domain that is involved in the degradation of unfolded proteins.
Relevance to endocrine disruption
C. elegans has been used as biosensor in environmental monitoring studies since the late 1980s (see review in Custodia et al., 2001). Previous studies from this laboratory demonstrated that C. elegans responds to vertebrate steroids by altering expression patterns of vitellogenin, cyp450, gst and hsp genes suggesting that this organism may be useful laboratory model for screening of endocrine disruptor compounds (EDCs) (Custodia et al., 2001). In this study, we show that the expression of certain members of NRs in C. elegans change after exposure to vertebrate steroids and cholesterol, suggesting that NRs can respond to a variety of exogenous steroids. This may influence the regulation of metabolic and developmental pathways, allowing nematodes to adapt and exploit changing environments.
That steroids, particularly estrogen, widely impact gene expression in C. elegans suggest that this model may be used to assess the actions of xenoestrogens and other environmental contaminants in the laboratory at the genome level. In this regard, cadmium, as a representative of the toxic heavy metal group, is of particular interest, as it may act in an estrogen-like manner. Cadmium is a heavy metal with no known biological function and it is one of the more serious environmental pollutants. It has been classified as a group 1 human carcinogen (IARC, 1993) and it is listed by the US EPA as one of 126 priority pollutants. Recently it has been reported that cadmium can provoke direct inhibition of DNA mismatch repair (Jin et al., 2003), thus cadmium toxicity can represent a new mechanism by which genomes can be destabilized with profound implications for human health, risk assessment and biological understanding of environmental mutagens (see review McMurray and Tainer, 2003). Furthermore, it has been shown that cadmium has potent estrogen-like activity in vivo (Johnson et al., 2003), adding a new dimension to currently used risk-assessment protocols for this environmental chemical. In vivo and in vitro studies have shown that dysregulation of gene expression is a major factor that can explain the multiple effects of cadmium exposure. A survey of genes that are induced by cadmium (reviewed by Waisberg et al., 2003), classifies them into four different groups: (1) Immediate early response genes (IEGs) such as c-fos, p53, c-jun; (2) Stress response genes such as mtl, hsp, and genes controlling glutathione and related proteins; (3) transcription and translation factors; (4) miscellaneous genes such as collagen, rRNAs, pyruvate carboxylase. Thus, many cellular processes can be disrupted by this heavy metal.
C. elegans is a powerful animal model for the study of functional genomics (see review Kim, 2001). DNA microarray experiments can be used to determine expression changes after different exposures, in different mutants with different reproductive backgrounds to provide new insights into invertebrate and vertebrate biology. A combination of microarray and functional data from web-based databases will make it possible to analyze gene network pathways implicated in endocrine disruption. It will be important to develop C. elegans and other species such as Danio rerio into high through-put models in which a wide variety of xenobiotics may be tested in low concentrations for genome wide-responses. Comparative genomic studies have shown that 83% of C. elegans genes have human homologs (Lai et al., 2000), thus such studies will aid in our comprehension of the impact of xenobiotics on the human genome. Beyond direct implications for human biology and health, to understand the effects of EDC on reproduction and development is of cardinal importance for maintenance of biodiversity and invertebrate species, which comprise approximately 95% of all terrestrial and aquatic animal species (Wilson, 1999).
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ACKNOWLEDGMENTS |
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This study was Supported by NIH ES 07381 to IPC. Apolonia Novillo was supported by a postdoctoral fellowship from Ministry of Science and Technology of Spain.
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FOOTNOTES |
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1 From the Symposium EcoPhysiology and Conservation: The Contribution of Endocrinology and Immunology presented at the Annual Meeting of the Society for Integrative and Comparative Biology, 5–9 January 2004, at New Orleans, Louisiana.
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SYNOPSISINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONReferences |
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