【关键词】 study
division of environmental health and occupational medicine (t.-c.e.l., s.-c.c., l.-a.l.), national health research institutes, zhunan, miaoli 350, taiwan, republic of china
department of safety, health
environmental engineering (s.-c.c., p.-c.h.), national kaohsiung first university of science and technology, kaohsiung 811, taiwan, republic of china
abstract
although polychlorinated biphenyls (pcbs) have been shown to accumulate in the adrenal, little is known about the effects of these endocrine disruptors on adrenal steroidogenesis. our previous studies showed that high concentrations of pcb126 stimulated cyp11b1 and cyp11b2 mrna expression and consequently raised cortisol and aldosterone synthesis in the human adrenocortical h295r cells, respectively. in this study, we further investigated the mechanism underlying the pcb126-induced steroidogenic alterations. we first examined the role of the pcb126 nuclear receptor aryl hydrocarbon receptor (ahr) using a potent antagonist 3',4'-dimethoxyflavone (3',4'-dmf). although 3',4'-dmf abolished ahr-dependent transcriptional activity, it could not block pcb126-stimulated cyp11b1 and cyp11b2 induction. conversely, 3',4'-dmf synergistically increased the stimulatory effects of pcb126. furthermore, pcb39, -77, -132, -156, and -169, whether ahr ligands or not, all could increase cyp11b1 and cyp11b2 mrna accumulation. promoter analyses demonstrated that pcb126 had little effects on the transcription rate of both genes, whereas rna degradation assays showed that pcb126 protected both transcripts from degradation. in contrast, 3',4'-dmf exhibited positive effects on transcription but no influence on transcript stability. the synergistic induction of cyp11b1 and cyp11b2 mrna levels by the pcb126/3',4'-dmf cotreatment might result from the combination of transcriptional regulation by 3',4'-dmf and posttranscriptional regulation by pcb126. this study also demonstrated that an internal region of cyp11b1 mrna (nucleotides 881-1285) was important for pcb126-mediated transcript stabilization. from these findings, we concluded that pcb126 up-regulated steroidogenic cyp11b1 and cyp11b2 mrna expression not via ahr-mediated transcriptional activation but by increasing posttranscriptional mrna stability.
introduction
polychlorinated biphenyls (pcbs) had been widely used in various industries for decades before their use was banned. however, their degradation resistance and long-term use make them remain ubiquitous today (1, 2, 3). people are frequently exposed to these persistent chemicals through ingestion of contaminated foods and accumulate them in the fat-rich tissues (4). therefore, pcbs constitute a continuing threat to human health. in 2001, delegates from the globe in a united nations stockholm conference approved pcbs as one of the 12 most dangerous persistent pollutants.
exposure to pcbs causes a range of adverse effects on the endocrine system. pcbs have been demonstrated to reduce thyroid hormone levels in humans and animals, thus injuring normal growth and development (5, 6). pcbs also interfere with gonadal sex steroid production (7, 8). normal levels of sex steroid hormones are critical for gamete maturation, conception, and live birth rate. pcbs also accumulate in the adrenals (9, 10), where three major classes of steroid hormones, cortisol, aldosterone, and androgen, are synthesized. our previous studies have shown that pcb126 reduces androgen synthesis dose-dependently in human adrenocortical h295r cells as observed in the gonadal cells. on the other hand, high concentrations of pcb126 stimulate cortisol and aldosterone synthesis in the h295r cells. cotreatment with potassium, angiotensin ii, and camp amplifies aldosterone production, whereas camp reverses the stimulatory effect of pcb126 on cortisol synthesis (11, 12).
these changes in steroid hormone levels, particularly in the cortisol and aldosterone levels, may be associated with the high incidence of diabetes mellitus and cardiovascular mortality found among highly exposed people (13, 14, 15, 16, 17). cortisol and aldosterone modulate insulin sensitivity and glucose tolerance. elevated levels of circulating cortisol impair the action of insulin on suppression of glucose production and stimulation of glucose utilization (18, 19), whereas excess aldosterone diminishes insulin secretion and sensitivity (20). research has suggested that insulin resistance and hyperglycemia alter cardiac and vascular functions, greatly increasing risk for cardiovascular diseases (21). in addition to the diabetes-related cardiovascular risk, cortisol and aldosterone have pharmacological effects on vascular tone. excess cortisol or aldosterone induces hypertension. moreover, aldosterone impairs vascular matrix, damages endothelial function, and causes vascular smooth muscle cell hypertrophy and hyperplasia (22). chronic high circulating aldosterone leads to myocardial fibrosis (23).
pcb126 elevates cortisol and aldosterone synthesis in the h295r cells mainly by increasing the conversion of cortisol from 17-oh-progesterone and aldosterone from progesterone. the increases in the cortisol and aldosterone conversions are accompanied by increased mrna expression of cytochrome p450 enzymes cyp11b1 and cyp11b2 that catalyze the rate-limiting final conversion steps in the respective cortisol and aldosterone synthesis pathways (11, 12). pcb126 is a well-recognized ligand for aryl hydrocarbon receptor (ahr). its binding commences the translocation of ahr from the cytoplasm to the nucleus. after heterodimerizing with ahr nuclear translocator (arnt), ahr interacts with specific upstream dna elements named dioxin-responsive elements (dres) and consequently activates transcriptional expression of a battery of target genes, including cytochrome p450 enzymes cyp1a1 and cyp1b1 (24). is it possible that pcb126 also acts through ahr to induce cyp11b1 and cyp11b2 expression as well as associated steroidogenic changes the current study is aimed to investigate the molecular mechanism underlying the pcb126-induced cyp11b1 and cyp11b2 up-regulation. our results show that pcb126 regulates cyp11b1 and cyp11b2 expression through an ahr-independent posttranscriptional pathway.
materials and methods
cell culture and reagents
human adrenocortical h295r cells were cultured in phenol red-free dmem/f12 medium (sigma-aldrich co., st. louis, mo) containing 10% charcoal/dextran-treated fetal bovine serum (bioclot gmbh, aidenbach, germany) at 37 c under 5% co2. pcb126 (3,3',4,4',5-pentachlorobiphenyl), pcb77 (3,3',4,4'-tetrachlorobiphenyl), pcb156 (2,3,3',4,4',5-hexachlorobiphenyl), pcb169 (3,3',4,4',5,5'-hexachlorobiphenyl), pcb39 (3,4',5-trichlorobiphenyl), pcb132 (2,2',3,3',4,6'-hexachlorobiphenyl) (accustandard inc., new haven, ct). and 3',4'-dimethoxyflavone (3',4'-dmf) (indofine chemical co., hillsborough, nj) were dissolved in dimethyl sulfoxide (dmso) (sigma) and added to the medium to the indicated concentrations. the dmso concentration in all treatments was 0.1%.
rna extraction and real-time rt-pcr
rna was extracted using the rezol reagent (protech technology, tainan, taiwan, roc) following the supplemented protocol, except that nuclease-free dnase i digestion (takara bio inc., otsu, shiga, japan) was performed to eliminate genomic dna contamination. purified rna was reverse transcribed using oligo-deoxythymidine as a primer. gene-specific cdna was amplified from first-strand cdna using a pair of gene-specific primers and the lightcycler-faststart dna master sybr green i system (roche diagnostics gmbh, mannheim, germany) as described previously (11). gene expression was relatively quantified by calibration against standard curves generated with serial dilutions of first-strand cdna mix. to compensate the variations of input and pcr efficiency between samples, the abundance of a target gene transcript in a sample was normalized to the abundance of a housekeeping gene transcript with a matching expression status. in this study, low-expressing cyp11b1 and cyp11b2 were normalized to low-expressing housekeeping gene porphobilinogen deaminase, whereas high-expressing cyp1a1 was normalized to high-expressing -actin. gene-specific primer pairs were designed using software probe design (roche). the primer sequences are listed in table 1. the pcr product of each primer pair was verified by sequencing.
transfection analysis of ahr activity
to assess the transactivation activity of ahr, an ahr reporter, 2xdre-tata-luc, was constructed by cloning two copies of dre (25) into tata-luc plasmid. tata-luc was derived from pgl2-promoter (promega, madison, wi) by replacing sv40 promoter with a simple tata sequence. each of the 2xdre-tata-luc and tata-luc (2 μg) was cotransfected with sv40-gal (0.5 μg) into h295r cells (around 70% confluence) using lipofectamine (invitrogen, carlsbad, ca). after 1, 2, and 3 d of treatment with 0.1% dmso, 10 μm pcb126, or 10 μm pcb126 plus 10 μm 3',4'-dmf, the transactivation activity of ahr was determined in the luciferase to -galactosidase activity ratio, i.e. the normalized luciferase activity. the luciferase activity encoded by luc was measured following an established method (26), whereas the -galactosidase activity encoded by gal was detected using the -galactosidase enzyme assay system (promega).
transfection analysis of cyp11b1 and cyp11b2 promoter activities
human cyp11b1 and cyp11b2 upstream sequences were pcr amplified from genomic clones rp11-706c16 and rp11-304e16 (children’s hospital oakland-bacpac resources, oakland, ca), respectively. after sequence verification, the cyp11b1 and cyp11b2 upstream sequence fragments were assembled into varying lengths and cloned in front of the luciferase reporter gene of pgl2-enhancer (promega) and pgl2-basic (promega), respectively. to examine the effects of pcb126 on these two series of promoters, each promoter-luciferase plasmid (3 μg) along with sv40-gal (0.1 μg) was lipofectamine transfected into h295r cells at approximately 70% confluence. the transfected cells were then grown 3 d under the indicated treatments. the promoter activity was determined based on the luciferase activity after normalization to the -galactosidase activity.
whole-cell rna degradation assay by actinomycin d (act d) chase
act d (sigma) was dissolved in dmso and added to h295r cells at 5 μg/ml after the cells were grown under 0.1% dmso or 10 μm pcb126 for 7 d. total rna was extracted from the cells at hour intervals after 2 h act d incubation. cyp11b1 and cyp11b2 mrna levels were measured by real-time rt-pcr as described above except that rt was performed using random primers rather than oligo-deoxythymidine, and that 18s rrna, not porphobilinogen deaminase, was used as a normalization control.
s100 cytoplasmic extract preparation
cytoplasmic proteins were extracted by modification of a method by moallem et al. (27). cells were washed twice and harvested in cold pbs (sigma). after centrifugation at 1000 x g for 5 min at 4 c, the cells were resuspended in 2 vol of cold homogenization buffer (10 mm tris-hcl, ph 7.4; 10 mm kcl; 1.5 mm mgcl2; 0.5 mm dithiothreitol; and 20% glycerol) with a protease inhibitor cocktail (roche) freshly added at 40 μl/ml buffer. cells were homogenized with 20 strokes in dounce (wheaton science products, millville, nj) on ice, and 0.1 vol of 1.5 m kcl was added to the homogenate. the s100 extract was acquired by centrifugation at 6000 x g for 5 min followed by 100,000 x g for 1 h at 4 c. the s100 extract was stored at 70 c in aliquots. an aliquot of the extract was used for protein concentration determination by micro bca protein assay (pierce, new york, ny).
rna probe preparation
human cyp11b1 cdnas including full-length and nucleotides 1880, 11338, and 12862092 were assembled from pcr-amplified cdna fragments and cloned into psp64 polya (promega) between the hindiii and xbai sites. the plasmid was linearized by digestion at the single ecori site located downstream of the poly(a) tailing signal. 32p-labeled transcripts were synthesized in vitro from the linear template using the sp6 riboprobe system (promega) and [-32p]utp (nen life science products, boston, ma) according to the manufacturer’s protocol. in addition, m7g5'pppg (promega) was included in the reaction for capping of the 5' ends of the in vitro synthesized transcripts. the transcripts were cleaned up by gel filtration through a microspin g-50 sephadex column (amersham biosciences europe gmbh, freiburg, germany). radioactivity was measured using a scintillation -counter (packard, haverhill, ma).
cell-free rna degradation assay
transcripts (2 x 105 cpm) were incubated at room temperature with 10 μg cytoplasmic proteins and 80 u/ml rnasin (promega) in a total volume of 50 μl. at each time point, 10 μl rna was purified through a qiaquick pcr purification column (qiagen gmbh, hilden, germany). the transcripts from each time point were run on a 1% tris-acetate-edta agarose gel. after the gel was dried under vacuum at 80 c for 1 h, the undegraded transcripts were autoradiographed and quantified using software imagej (http://rsb.info.nih.gov/ij).
statistical analysis
all data are expressed as means ± se. the numbers of replicate experiments (n) are indicated in the corresponding figure legends. the significance of a treatment vs. control was analyzed by student’s t test in excel 2000 (microsoft corp., redmond, wa).
results
cyp1a1, cyp11b1, and cyp11b2 exhibited differential expression patterns in response to pcb126 stimulation
our previous study demonstrated that pcb126 at 10 μm stimulated cyp11b1 and cyp11b2 mrna expression in human adrenocortical h295r cells (12). it is also known that pcb126 can activate cyp1a1 gene expression by activating ahr to recognize upstream dres (25). however, the cyp11b1 and cyp11b2 genes exhibited extremely different responses to the pcb126 stimulation from the cyp1a1 gene in terms of time and intensity. the real-time rt-pcr analysis showed that when the h295r cells were treated with 10 μm pcb126 for 1 d, the cyp1a1 mrna level rapidly rose to 28.61 ± 2.84-fold of the vehicle control. the cyp1a1 induction continued linearly as the treatment was extended to 6 d (fig. 1a). in contrast, the cyp11b1 and cyp11b2 mrna levels only slightly increased after 1 d of treatment. the increase of the latter even lacked statistical significance. expression levels of cyp11b1 and cyp11b2 were remarkably elevated after 3 and 6 d of the pcb126 treatment but to a much lesser extent compared with cyp1a1 (fig. 1, b and c). in view of the delayed reaction of cyp11b1 and cyp11b2 to pcb126, a question arose in us whether pcb126 regulated cyp11b1 and cyp11b2 in a mechanism different from cyp1a1. it seemed possible that pcb126 altered cyp11b1 and cyp11b2 mrna expression via an ahr-independent mechanism. to resolve this doubt, we examined the involvement of ahr using an antagonist.
3',4'-dmf inhibited pcb126-induced ahr activity in h295r cells
3',4'-dmf has been shown to be a potent ahr antagonist in human breast cancer cells (28). to check whether 3',4'-dmf can successfully block pcb126 from inducing transcription of the ahr target gene cyp1a1 in the adrenocortical h295r cells, we added increasing concentrations of 3',4'-dmf to the cells with and without 10 μm pcb126 for 24 h. although 3',4'-dmf alone caused minor changes in cyp1a1 mrna levels, high concentrations of 3',4'-dmf suppressed pcb126-activated cyp1a1 induction. the 10 μm 3',4'-dmf removed approximately 90% of pcb126-induced cyp1a1 mrna expression (fig. 2a). the inhibition went on at least 6 d (fig. 2b).
resveratrol, a polyphenol found in grapes, also inhibited dioxin-induced cyp1a1 mrna expression in the breast cancer cells at 10 μm. however, resveratrol repressed cyp1a1 gene expression via an ahr-independent pathway because 10 μm resveratrol could not hinder dioxin from activating an ahr-dependent reporter (29). for that reason, we also examined the inhibition of 3',4'-dmf on pcb126-induced ahr activity using a reporter assay. 2xdre-tata-luc, an ahr-dependent luciferase reporter, was transiently transfected into the h295r cells. in contrast to the numb response of tata-luc (data not shown), 10 μm pcb126 significantly activated the luciferase activity of 2xdre-tata-luc. cotreating the transfected cells with 10 μm 3',4'-dmf abolished the pcb126-induced luciferase activity, and the inhibition continued throughout a 3-d course (fig. 2c).
3',4'-dmf enhanced pcb126-induced cyp11b1 and cyp11b2 mrna expression
next, we examined the impacts of ahr blockage on pcb126-induced cyp11b1 and cyp11b2 mrna expression. the h295r cells were treated with vehicle, 10 μm 3',4'-dmf, 10 μm pcb126, or both drugs together for 1, 3, and 6 d. the 10 μm 3',4'-dmf significantly induced cyp11b1 and cyp11b2 mrna expression in 1 d, but the power of the induction diminished when the treatment was prolonged. in contrast, pcb126 exhibited a comparatively slow but persistent stimulation on both genes. the induction for cyp11b1 and cyp11b2 after a 6-d pcb126 treatment was 2.60 ± 0.23-fold and 6.98 ± 0.61-fold, respectively. 3',4'-dmf did not reduce pcb126-stimulated cyp11b1 and cyp11b2 mrna expression. on the contrary, 3',4'-dmf enhanced the stimulation of pcb126. the synergistic stimulation was particularly notable 3 d after the cotreatment started (fig. 3, a and b). the results revealed that ahr was not necessary for pcb126-induced cyp11b1 and cyp11b2 expression.
pcbs with different structures exhibited the capability to stimulate cyp11b1 and cyp11b2 mrna expression
do other pcbs possess similar stimulatory effects on these two genes to answer this question, pcbs with different structures have been examined. among them, pcb77, -156, and -169 are coplanar pcbs like pcb126, but they are weaker ahr ligands compared with pcb126. pcb39 also contains a coplanar structure but lacks affinity for ahr. pcb132 is an ortho-substitutive non-coplanar pcb. treating the h295r cells with each of these pcbs at 10 μm for 3 d significantly elevated cyp11b1 and cyp11b2 mrna levels (fig. 4, a and b). these results further confirmed that ahr was irrelevant to pcb stimulation on cyp11b1 and cyp11b2 mrna expression. up-regulation of cyp11b1 and cyp11b2 expression seemed to be a common effect among pcbs. however, the strength of stimulation varied with congeners and genes. although pcb39 raised cyp11b1 and cyp11b2 mrna levels around 2- to 3-fold, pcb132 drastically increased cyp11b1 expression to 16.95 ± 2.12-fold and cyp11b2 expression to 4.39 ± 0.62-fold (fig. 4, a and b).
pcb126 lacked effects on cyp11b1 and cyp11b2 promoter activation
to investigate the effects of pcb126 on the transcription rate of cyp11b1 and cyp11b2 genes, we cloned varying lengths of human cyp11b1 and cyp11b2 upstream sequences before a luciferase reporter gene. transient transfection experiments showed that each promoter-luciferase plasmid of both genes produced similar levels of luciferase activity when the transfected cells were treated 3 d with vehicle and 10 μm pcb126 (fig. 5, a and b). in contrast, 10 μm 3',4'-dmf increased luciferase expression from the cyp11b1 promoters. cotreating with pcb126 did not significantly boost the 3',4'-dmf-mediated induction (fig. 5a). obviously, pcb126 did not elevate cyp11b1 and cyp11b2 mrna levels by enhancing transcription of these two genes.
pcb126 treatment increased cyp11b1 and cyp11b2 mrna stability
because pcb126 had little effect on the cyp11b1 and cyp11b2 promoters, we speculated that pcb126 increased cyp11b1 and cyp11b2 mrna expression by increasing mrna stability. to test this hypothesis, we added the rna synthesis inhibitor act d (5 μg/ml) to vehicle- and pcb126-treated h295r cells so that the stability of transcripts in cells could be monitored without the interference of mrna neosynthesis. the amounts of stable 18s rrna in samples were used for normalization in this analysis. as shown in fig. 6, a and b, the cyp11b1 and cyp11b2 transcripts appeared degraded in a slower pace in the pcb126-treated cells.
we also conducted cell-free rna degradation assays to assess the stability of the full-length human cyp11b1 mrna in incubation with the s100 cytoplasmic extracts of different treatments. the autoradiographs of three independent experiments all showed that more 32p-labeled cyp11b1 mrna remained intact after 40 min of incubation with the cytoplasmic extracts of pcb126-treated h295r cells than the vehicle control (fig. 7a). the half-life of cyp11b1 mrna was estimated based on the percentage of undegraded transcripts remaining against time (fig. 7b). a 3-d treatment with 10 μm pcb126 lengthened the half-life of cyp11b1 mrna from 14.4 to 31.1 min. cotreating with 10 μm 3',4'-dmf did not further stabilize cyp11b1 mrna. the mrna half-life for the cotreatment was about 31.4 min. taking the results of figs. 3a, 5a, and 7 together, we demonstrated that pcb126 increased cyp11b1 mrna expression by increasing transcript stability, whereas 3',4'-dmf did so by increasing the transcriptional rate of the promoter. the synergistic increase in the mrna level stimulated by the cotreatment appeared to be a joint result of transcriptional and posttranscriptional regulation.
the region containing the nucleotides 881-1285 of cyp11b1 mrna was important for pcb126 stabilization
furthermore, we mapped the region in the human cyp11b1 mrna important for the transcript stabilization by pcb126. three fragments of cyp11b1 mrna (nucleotides 1880, 11338, and 12862092) were in vitro synthesized and labeled in the same manner as the full-length mrna (nucleotides 12092). fragment 12862092 degraded rapidly in the cell-free degradation assays, whereas fragment 1880 exhibited high stability. however, both fragments had similar degradation patterns when incubated with the cytoplasmic extracts from either vehicle- or pcb126-treated h295r cells. in contrast, the mrna stability of fragment 11338 was largely increased by treating the cells with 10 μm pcb126 for 3 d (fig. 8, a and b). this mapping analysis suggested that the sequence(s) mediating pcb126-induced cyp11b1 mrna stabilization was located within nucleotides 881-1285.
discussion
historically, endocrine toxicity research has focused on estrogenicity and antiestrogenicity of chemicals with concerns focusing on reproduction and development. in fact, the adrenal cortex is the most vulnerable target for endocrine toxins because of its massive blood supply, high lipid content, and steroidogenic capacity for all major steroids. dysregulation of adrenal steroidogenesis is vitally dangerous to health. elucidation of the underlying toxicological mechanisms has been recommended to be relevant for human risk assessment (30). despite limited information of adrenal toxicology, several drugs, pesticides, and phytochemicals have been reported to inhibit steroidogenic enzymes and cause adrenocortical insufficiency (31, 32, 33). our study shows that pcb126, on the contrary, stimulates cyp11b1 and cyp11b2 and increases cortisol and aldosterone production (11, 12).
pcb126 is a coplanar aromatic hydrocarbon that can induce cyp1a1 and cyp1b1 through the ligand-dependent transcription factor ahr. the cyp1a1 and cyp1b1 enzymes metabolize xenobiotic and endogenous aromatic hydrocarbons to reactive intermediates that interact with dna and protein macromolecules, causing genotoxicity and carcinogenesis (34, 35). knockout of the ahr gene makes mice resistant to acute toxicity and teratogenicity induced by its classic ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (tcdd) (36, 37). in addition, ahr is a key mediator for the antiestrogenic effects of its aromatic hydrocarbon ligands. tcdd cannot suppress estradiol-induced uterine changes in the ahr knockout mice as in the wild-type mice (38).
however, pcb126 does not elevate cyp11b1 and cyp11b2 mrna expression and associated steroid synthesis by acting through ahr. although we successfully antagonized ahr-mediated reporter activation and cyp1a1 expression using 3',4'-dmf, the ahr antagonist could not block pcb126-induced cyp11b1 and cyp11b2 mrna accumulation. furthermore, we demonstrated that pcbs with weaker or little affinity to ahr were also capable of up-regulating cyp11b1 and cyp11b2 mrna expression.
some recent studies have suggested that pcbs exert certain endocrine-disrupting effects through ahr-independent pathways. andric et al. (7) showed that pcb mixture aroclor 1248 rapidly inhibited in vitro testosterone production by rat testicular interstitial cells in 1015 min. the authors concluded that the acute inhibition exhibited by aroclor 1248 could not be mediated through ahr. they speculated that aroclor 1248 had rapid negative actions directly on androgenic enzyme activities in a manner similar to the feedback regulation of testosterone on its synthesis (7). another study by gore et al. (39) revealed that aroclor 1221 and 1254 had little effect on the nuclear nascent transcript levels of gnrh but differentially altered the cytoplasmic mrna levels in the hypothalamic gt1-7 cells. the results suggested that pcbs regulated hypothalamic gnrh gene expression at the posttranscriptional level (39). our promoter analyses also showed that pcb126 had little effect on the transcription rate of steroidogenic cyp11b1 and cyp11b2 promoters. the observed up-regulation of cyp11b1 and cyp11b2 mrna expression appeared not to act at the transcriptional level. the act d chase analysis further showed that pcb126 increased cyp11b1 and cyp11b2 mrna accumulation by decreasing the degradation rate of the transcripts.
a number of 3'-untranslated region elements have been identified to modulate mrna stability via interacting with specific rna-binding proteins (40, 41). our cell-free degradation assays demonstrated that deletion of the c terminus of cyp11b1 mrna increased stability. however, neither the 3' region (nucleotides 12862092) nor the 5' region (nucleotides 1880) was associated with pcb126-induced mrna stabilization. the pcb126 stabilization seemed likely to involve machinery distinct from what had been known. identifying the responsible cis-acting elements and trans-acting proteins will be our next goal.
growing evidence indicates that regulation of transcript stability is an important mechanism in the steroid hormone system. steroid hormones use this mechanism to control expression levels of their own receptors, thus autoregulating their hormonal actions. estradiol has been shown to increase the half-life of estrogen receptor- (er) mrna and consequently its expression in the endometrium (42), but it decreases er mrna stability and accumulation in the breast cancer cells (43). dihydrotestosterone exhibits comparable stabilizing and destabilizing regulation on androgen receptor mrna in the prostate and breast cancer cells, respectively (44).
pituitary gonadotropin and acth also modulate steroid hormones at the posttranscriptional level. lh desensitizes the estradiol responsiveness of the preovulatory follicular granulosa cells by down-regulating er mrna stability (45). in contrast, acth stabilizes its receptor mrna and boosts its steroidogenic stimulation in bovine adrenocortical cells (46). in addition, acth raises cyp11a1 mrna half-life 5-fold in bovine adrenocortical cells (47). cyp11a1 mrna encodes an enzyme catalyzing the side-chain cleavage of cholesterol, initiating the steroidogenic process (48).
it is likely that environmental chemicals employ a similar posttranscriptional mechanism to disrupt steroid homeostasis. this study provides evidence that pcb126 can modulate the stability of key steroidogenic gene transcripts to influence cortisol and aldosterone biosynthesis. the perturbation can be magnified when combined with other endocrine modulators, such as 3',4'-dmf. despite lack of direct proof that the increasing metabolic and cardiovascular risk among exposed people (13, 14, 15, 16, 17) is caused by chronic cortisol and aldosterone excess, our study coupled with the well-established knowledge of adrenal dysfunction raises a warning that the adrenal pcb accumulation as a consequence of constant exposure may lead to the above hazards. more mechanistic information of how pcb126 regulates the stability of steroidogenic gene transcripts may help to reduce the risk.
acknowledgments
the sv40-gal plasmid is a kind gift from dr. tsui-chun tsou, national health research institutes, taiwan, roc.
footnotes
this work was supported by eo-093-pp-02 from national health research institutes, taiwan, roc.
first published online january 5, 2006
abbreviations: act d, actinomycin d; ahr, aryl hydrocarbon receptor; 3',4'-dmf, 3',4'-dimethoxyflavone; dmso, dimethyl sulfoxide; dre, dioxin-responsive element; er, estrogen receptor; pcb, polychlorinated biphenyl.
accepted for publication december 9, 2005.
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