The stability of dioxin-receptor ligands influences cytochromeP450IA1 expression in human keratinocytes
Anna Berghard, Katarina Gradin and Rune Toftgard
Center for Biotechnology, Karolinska Institute, NOVUM, S-141 57Huddinge, Sweden
Three dioxin-receptor ligands were analyzed for their effecton cytochrome P450IA1 mRNA expression in normal humankeratinocytes. Although a 2 h pulsed treatment with thereceptor agonists 2,3,7,8-tetrachlorodibenzofuran (TCDF)and /3-naphthoflavone (BNF) gave the same maximal induc-tion response, the effect of BNF was transient compared toeffect of TCDF. This was most likely due to metabolism ofBNF as exemplified by the fact that a P450IA1 enzymesuicide-inhibitor, 1-ethynylpyrene, could prolong the induc-tion response following a short BNF treatment. The TCDFinduction of a reporter gene construct under the control ofthe -1140 to +2435 part of the CYPIA1 gene transientlytransfected into HK was effectively inhibited by the dioxin-receptor antagonist a-naphthoflavone (ANF). In addition,ANF inhibited the accumulation of TCDF-activated nuclearreceptors with capacity to bind to a xenobiotic responseelement. Interestingly, ANF could also suppress alreadymaximally induced P450IA1 mRNA levels. The datademonstrate that the stability of the ligand influences the long-term effects on gene expression and that the effect of stableligands may be masked due to receptor antagonist presence.In addition, the results support the hypothesis that a cons-tant low level of activated nuclear receptors is required tomaintain induced P450IA1 expression.
IntroductionThe epidermis is exposed to a variety of genotoxic and tumor-promoting compounds. Cytochrome P450IA1* has the capacityto catalyze the activation of polycyclic aromatic hydrocarbons(PAH) like 7,12-dimethylbenz[a]anthracene and benzo[a]pyreneto the ultimate carcinogenic species (1 and refs therein) and thismonooxygenase-associated enzyme is present in the epidermis(2) as well as in cultured keratinocytes (3). PAHs such as3-methylcholanthrene and /3-naphthoflavone (BNF), as well asa number of polyhalogenated PAHs typified by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), regulate P450IA1 geneexpression on the transcriptional level (4). The CYPIA1 gene,encoding the cytochrome P450IA1 product, has been used as amodel gene to characterize the molecular mechanisms of actionof the environmental contaminant TCDD (5).
TCDD binds to an intracellular protein, termed the dioxin orAh receptor, that was recently purified (6). The importance ofthe ligand in receptor activation has been thoroughly investigated
(7). The cytoplasmic inactive receptor is associated with the90 kDa heat-shock protein (hsp90; refs 8,9) and binding of ligandresults in enrichment of the activated ligand —receptor complexin the nucleus. Recently a factor important for the nucleartranslocation of the receptor was cloned by a functionalcomplementation assay (10). In the nucleus, the activated, hsp90free ligand-receptor complex binds specifically to DNAsequences, termed xenobiotic response elements (XREs),upstream of the CypIAl (11), glutathione S-transferase Ya (12)and NAD(P)H:quinonereductase (13) target genes. TCDD is quiteresistant to metabolism and is not in itself genotoxic (14 and refstherein). Besides acute toxic effects, like lethality, thymic involu-tion, teratogenicity, liver and kidney damage, TCDD can act asan effective tumor promotor in vivo in rat liver (15) and hairlessmouse skin (16) and in vitro in C3H/10T1/2 cells (17). The dioxinreceptor is necessary but not sufficient for the tumor promotionresponse (18). The mechanism(s) underlying the tumor-promotingeffect is not known, but it has been suggested to involve a changein the gene expression pattern in the affected tissue.
The biological effects of dioxin-receptor ligands showconsiderable species and tissue variation. In humans the mostcharacteristic effect is persistent hyperkeratotic lesions involvingthe epidermis (19). Thus, keratinocytes represent relevant targetcells for the study of the effects of this class of compounds.
In this study we have compared the effect of 2,3,7,8-tetrachlorodibenzofuran (TCDF) to that of two naphthoflavones,BNF and a-naphthoflavone (ANF), on P450IA1 mRNA expres-sion in normal human keratinocytes (HK). TCDF is almost aspotent as TCDD in binding to the receptor and as a tumorpromotor (16). BNF is widely regarded, though not firmlyestablished, as being ineffective as a tumor promotor (20). Inaddition, TCDF is a stable ligand (21) as opposed to BNF (20).ANF has been shown to inhibit both TCDD binding to thereceptor (22) and P450IA1 activity by reversible binding to theenzyme. Here we demonstrate the effect of a stable ligand(TCDF) on P450IA1 expression as compared to two unstableligands (BNF and ANF). The necessity of activated dioxinreceptors for persistent effects on gene expression and themechanism underlying ANF's antagonistic effect are alsoaddressed.
Materials and methodsCell culture and treatmentsAll chemicals, medium and growth factors were purchased from Sigma, unlessstated otherwise. HK were isolated from adult or newborn donors as describedpreviously (23), with modifications (24). To allow for maximal growth, the cellswere grown in MCDB 153 medium with a final Ca2+ concentration of 70 jiM,supplemented with 0.1 mM ethanolamine, 0.1 mM phosphoethanolamine,0.4 /tg/ml hydrocortisone, 10 ng/ml epidermal growth factor, 5 /ig/ml insulin,100 fig/ml bovine pituitary extract (prepared as described in ref. 25, bovinepituitaries purchased from Pel-Freeze), 100 /jg/ml streptomycin, 100 IU/mlpenicillin (Nordcell) and 0.25 /ig/ml fungizone (Nordcell) until near confluencewhen they were subcultured and plated onto fibronectin and collagen-I coatedcell-culture plastic. Cells at the third to fourth passage were routinely used forexperiments. In order to maximize the P450IA1 induction response in HK themedium was switched to contain 2 mM Ca2"1" and bovine pituitary extract was
substituted with 5% fetal bovine serum (Hyclone) 48 h before treatments (24).Cultures were treated with TCDF (Cambridge Isotope Laboratories), BNF (Serva)and ANF (Serva) dissolved in DMSO while control cultures received solvent only,not exceeding a final concentration of 0.2%. 1-Ethynylpyrene (EP) was a kindgift from Dr W.L.Alworth, Tulane University, New Orleans. Each experimentwas performed at least twice using cells derived from different donors
RNA isolation and Northern analysis
Total RNA was isolated using acid—phenol extraction as described (26). RNAwas fractionated through formaldehyde-agarose gels, blotted onto nylonmembranes, then UV cross-linked, and the filters were subsequently prehybrklized,hybridized and washed according to standard procedures (27) prior toautoradiography. The insert of phPl-4503' (28) and glyceraldehyde-3-phosphatedehydrogenase (GAPDH; 29) were isolated and P-labeled using a randompriming procedure.
Transfeaion and transient expression assays
HK (25 cm2) were incubated overnight in 70 fiM Ca2+ medium with 30 yX ofcationic liposomes (Hpofectin, Bethseda Research Laboratories), 0.5 jig RSV-luc and 5 pgpRNHUC. pRNHHC contains the -1140 to +2435 part of thehuman CYP1A1 gene directly fused to the bacterial chloramphenicol acetyltransferase (CAT) reporter gene (30). RSV-luc is a construct where the Roussarcoma virus long terminal repeat drives the expression of the luciferase reportergene (31). The medium was changed to contain 5% serum and 2.0 mM Ca2+
for the following 24 h, and the cells were subsequently treated with either 50 nMTCDF and/or 10 nM ANF or DMSO alone for an additional 40 h prior to harvest.The luciferase and CAT assays were performed as described previously (27) withthe modification that the cells were lysed on ice by a brief treatment using asonicator (Branson Sonic Power Co.). Luciferin was purchased from BioThemaand [l4C]chloramphenicol from Amersham.
Nuclear extract preparation and gel-shift assay
Cells were treated with 50 nM TCDF and/or 10 jtM ANF or DMSO alone for1 h before harvest. Nuclei were prepared and protein extracted as previouslydescribed (32). DNA-binding reactions were assembled in 20 /J with 10 /ig ofnuclear protein at a final concentration of 25 mM HEPES (pH 7.9), 0.2 mMEDTA, 75 mM KC1, 2 mM MgCI2, 1 mM dithiothreitol, 0.1 mMphenylmethylsulphonyl fluoride, 5% glycerol, 4% Ficoll, 0.1 ^g/fil poly(dI-dC)(Pharmacia) and 12.5 ng/jd poly(dA—dT) (Pharmacia). Two femtomoles of adouble-stranded, 32P-labeled oligonucleotide (5'-CTCCGGTCCTTCTCACGCA-ACGCCTGGGCA-3' corresponding to -969 to -998 of the human CYPIA1gene) were used. Competition with an oligonucleotide corresponding to XRE1of the rat CypIAI (11) was indistinguishable from competition with the unlabeledprobe DNA (unpublished data). As unspecific competitor DNA a double-strandedoligonucleotide (5 '-TCTAGTGTTGGAGAACGAATCAGCATCTGAGTAC-3')was used, whereas unlabeled probe DNA served as specific competitor.DNA-protein complexes were separated under non-denaturing conditions in a5% polyacrylamide gel (29:1) run in 0.35 x TBE (1 x TBE is 0.09 MTris-borate, 0.001 M EDTA) at 4°C.
ResultsMaximal induction of P450IA1 mRNA steady-state levels wasobtained at a dose of ~50 ^M BNF (Figure 1) which wascomparable to that induced by 5 nM TCDF. When inducer wascontinuously present, induction by both compounds showed asimilar time dependency. As can be seen in Figure 2(A), the
maximal P450IA1 mRNA level was obtained after 8 h and thislevel of expression remained stable for 5 days after TCDF treat-ment and at least for 4 days after treatment with BNF.
To investigate if a brief, pulsatile treatment of the culture wouldbe enough to elicit a persistent effect on gene expression thefollowing experiment was performed. TCDF or BNF wasincluded in the medium for 2 h. The medium was then changed,and the cells were harvested at different time points. These agentsare not easily washed out from the cells and a pulsed treatmenthereafter refers to a 2 h presence of the compound in the medium.A pulse of TCDF treatment led to induction of P450IA1 mRNA,which was observed up to 5 days after exposure, whereas theeffect of BNF was much more short-lived (Figure 2B). A pulseof 50 )M BNF gave rise to maximal P450IA1 mRNA levels at8 h that declined to a low level already 30 h after BNF had beenexcluded from the medium. Forty-eight hours after a pulse ofBNF the mRNA level was very low; however, it was still possibleto induce P450IA1 to maximal levels by one additional treatment(Figure 3, compare, for example, the 72 h time points).
Next we investigated the involvement of the P450IA1 enzymein the metabolism of BNF. HK were treated with a pulse of BNF
P4KHA1
GAPDH
•••••*••-••###!§#•
0 I B 16 24 32 40 46 72 961201 6 16 24 32 40 48 72 9612olfh)
Fig. 2. Time course of induction of P450LA1 mRNA levels by constant orpulsed treatment with TCDF and BNF. Northern analysis showing P450IA1and GAPDH mRNA levels after treatment with 50 nM TCDF or 50 /iMBNF (A) throughout the indicated time period or (B) only for the first 2 h(time 0 - 2 h), then the inducer was excluded from the medium and the cellswere harvested at the indicated time-points.
P450IA1
P450IA1 GAPDH
GAPDH
in ino in o inp p in p <-, p p in p o
o I o o o m in | o o o m in I
BNF (\1M) TCDF (nM)
Fig. 1. Dose-response of P450IA1 mRNA induction by TCDF and BNF.Northern analysis of P450IA1 mRNA levels in comparison to levels of acontrol gene, GAPDH. Confluent HK cultures were treated with theindicated concentrations of TCDF and BNF respectively for 25 h beforeharvest.
Fig. 3. The capacity of HK to respond to consecutive dioxin-receptorligand treatments: Northern analysis of P450IA1 and GAPDH mRNAlevels. At time 0 h, HK were treated with a 2 h pulse of 50 pM BNF. Notethat this treatment led to maximal induction of P450IA1 mRNA at 24 h thathad declined by 48 h. At time 48 h the cells were either treated with a 2 hpulse of 50 nM TCDF, a 2 h pulse of BNF or BNF until harvest. Cellswere harvested at 72 h.
652
Stability of dioxin-rereptor Ugands
and then one or two times with EP, a suicide-inhibitor of theP450IA1 enzyme (33). As can be seen in Figure 4, EP treat-ment prolonged the P450IA1 induction response produced byBNF (compare the 48 h time points). EP treatment also resultedin a subtle, albeit reproducible lowering of the EC50 for induc-tion of P450IA1 mRNA by BNF (unpublished data). ANF couldinhibit P450IA1 mRNA induction by both TCDF and BNF. Inorder to obtain maximal inhibition of TCDF induction, at leasta 200-fold excess of ANF was required, whereas a 2-fold excessof ANF was sufficient to inhibit BNF-dependent induction ofP450IA1 mRNA levels (Figure 5). This is in agreement withthe previously published relative affinities of these compoundsfor the receptor (34). Moreover, inhibition of P450IA1 mRNAinduction by ANF could be reversed by increasing the concen-tration of the agonist (unpublished data). At high concentrationsANF treatment in itself caused a small but reproducible induc-tion of P450IA1 mRNA levels (Figure 5), suggesting that ANFis only a partial antagonist. ANF was effective not only ininhibiting the induction of P450IA1 mRNA, but effectively
f|«P450IA1
GAPDH
BNF (0-2 h)EP (2- h)EP (16-h)Total time (h)
Fig. 4. Inhibition of P4501A1 activity by EP prolonged the effect of apulsed BNF treatment on P450IA1 mRNA expression. HK were given a 2 hpulse of 50 /iM BNF, then the medium was changed and the cells harvestedat the indicated time-points. As indicated in the figure, cells represented inlanes 7-11 received 1 ^M EP at time 2 h and in lanes 12-14 anadditional EP treatment at time 16 h.
suppressed already induced mRNA levels. In the experimentshown in Figure 6 the cells had received TCDF for 18 h priorto addition of ANF. Already after 8 h of ANF treatment theP450IA1 mRNA levels had declined to a low level.
To test whether the inhibition by ANF was reversible, HK weretreated with a pulse of TCDF and subsequently with either a cons-tant level or a pulse of ANF and were then analyzed for P450IA1mRNA levels at different time points (Figure 7). Although TCDFwas given 2 h to exert its effect, ANF could still inhibit P450IA1mRNA to reach detectable levels. This was observed at earlytime-points, both if ANF was given as a pulse or wasadministered throughout the experiment. However, when the cellshad been cultured in medium lacking both TCDF and ANF foralmost 4 days Qane 5), P450IA1 mRNA was detectable andremained elevated for the subsequent 4 days of the experiment.This shows that the effect of a stable ligand may be masked dueto the presence of an antagonist. Interestingly, in the culturesthat were continuously treated with ANF, the same pattern wasseen, though the P450IA1 level was much lower.
To determine whether the inhibitory effect of ANF was at thelevel of transacting factors, the following experiments wereperformed. A CAT construct containing the -1140 to +2435part of the human CYPIA1 gene was transiently transfected into
P450IA1
GAPDH
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TCDF(nM) - 50 50^50 50 50 5 0 '
BNF (nM) . . . . . .
ANF (uM) - ~ 1 *€K lOteOlOO^-
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1 &3 10S3&100%-- 3 10 30100
Fig. 5. The dose of ANF required to inhibit P450IA1 induction by TCDFand BNF. HK were simultaneously treated with the indicated concentrationsof either TCDF or BNF and increasing concentrations of ANF. Cells wereharvested after 24 h and RNA prepared for subsequent Northern analysis.
Fig. 7. Time course of inhibition of TCDF-induced P450IA1 mRNA byconstant or pulsed treatment with ANF. Following a 2 h pulsed treatmentwith 50 nM TCDF the cells were treated for 2 h or throughout theexperiment with 10 /iM ANF. The medium was changed every other dayand to the cultures receiving constant ANF treatment, ANF was added eachtime. Note that d denotes days and that 2 h - denotes that the cultures hadreceived constant ANF until harvest
AcCM* * *
P450IA1
GAPDH
TCDFANF
18 19 21 23 25 42 64 (h)- 2 4 6 8 25 47 (h)
Fig. 6. ANF suppression of maximally induced P450IA1 levels. HK weretreated with 50 nM TCDF for 18 h before 10 ftM ANF was added and thecells were harvested at different time-points. Note that the length of ANFtreatment, indicated in the figure denotes hours before harvest. P450IA1remained maximally induced throughout the experiment if ANF was notincluded (compare with Figure 2A).
CM
TCOF (nM)
ANF (nM)
T T ? T50 50
10 10
Fig. 8. ANF inhibition of TCDF-induced expression of a transfectedCYPIAI/CAT plasmid construct. A representative thin-layer analysis of theCAT activity obtained from pRNHl 1C after a 40 h treatment with 50 nMTCDF and/or 10 nM ANF is shown. Cell extracts were normalized to theluciferase activity obtained from a co-transfected control plasmid, RSV-luc.AcCM denotes the product, i.e. acetylated chloramphenicol, and CMdenotes chloramphenicol.
653
A.Berghard, K.Gradin and R.Toflgfird
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Fig. 9. ANF inhibition of TCDF-induced accumulation of an XRE-specificretarded DNA-protein complex. Nuclear extracts were prepared from cellstreated with 50 nM TCDF and/or 10 pM ANF for 1 h. The extracts wereanalyzed for presence of nuclear proteins showing specific binding to anXRE sequence. Both unspecific and specific competitor oligonucleotideswere added in x 100 molar excess (see Materials and methods). Note that inthe DMSO lane with specific competitor the background bands areexaggerated due to overloading. DNA-protein complexes 1 and 2 showedspecific binding. Complex 1 was induced by TCDF treatment and wasinhibited in the extract from TCDF and ANF treated cells. F denotes themigration of the free oligonucleotide probe.
HK. This part of the gene can mediate the induction by TCDF(24,30). As shown in Figure 8, ANF completely inhibited thisinduction, leaving the basal level of expression unaffected. Toinvestigate the possibility that this was a direct effect on TCDF-induced accumulation of activated receptors with capacity to bindto an XRE sequence, a gel shift experiment was performed.Nuclear extracts from TCDF-treated HK showed a retardedDNA-protein complex that could not be detected in controlextracts (Figure 9, complex 1), consistent with the properties ofthe bona fide dioxin receptor. In comparison, extractssimultaneously treated with TCDF and ANF showed a dramaticdecrease in the level of complex 1. A constitutively present XRE-specific factor has previously been described (35,36), which islikely to be responsible for the formation of the invariablecomplex 2.
Discussion
We have shown that a short extracellular treatment of HK witha stable dioxin receptor ligand such as TCDF, as opposed to BNF,can exert long-term effects on P450IA1 gene expression. Incontrast, both compounds could induce P450IA1 mRNA to thesame maximal level. When the cells were exposed to BNF for2 h the subsequent induction showed a time dependency similarto that observed in vivo after a single dose of BNF (37). Thispulsed treatment might therefore be more accurate than constantexposure when assessing differences in biological effects in vitrobetween different dioxin-receptor ligands. The dose —responserelationship for induction by BNF in relation to TCDF did notcorrelate to what has been described for their ability to competefor [3H]TCDD binding to the receptor in cytosolic extracts (34)
in that a relatively higher concentration of BNF was needed toachieve a maximal response. This might be due to rapidmetabolism of BNF by the cells or that the efficiency of uptakeis much lower for BNF. Treatment with the P450IA1 enzymesuicide-inhibitor EP prolonged the time a BNF pulse was effec-tive in inducing P450IA1 mRNA, indicating that BNF is partlymetabolized by the P450IA1 enzyme, as has previously beensuggested (38). The cells were still capable of responding to treat-ment at a time-point when the BNF-induced mRNA had declinedto a low level. This result suggests that the ligand is importantto maintain activated dioxin-receptors at high enough levels tohave a prolonged effect on gene expression.
The effect on P450IA1 expression by an initial exposure toTCDF was inhibited by ANF until this antagonist was presumablydegraded. Thereafter an increase in the P450IA1 mRNA wasobserved. This finding emphasizes that it could be important tostudy the effect on gene expression over a prolonged period oftime when investigating the effects of complex mixtures of dioxin-receptor ligands. A characteristic feature of tumor promotion isthat a persistent biological effect(s) has to be achieved by thetumor-promoting compound. Even very infrequent exposures tocompounds like TCDD and TCDF could lead to persistent effectson gene expression by virtue of these compounds being poorlymetabolized and excreted. This might be an important aspect ontheir relative potencies as tumor promotors.
The TCDF induction of a CAT reporter gene under the controlof a part of the CYPIA1 gene involved in transcriptional regula-tion was inhibited by ANF. This was most likely due to the factthat accumulation of TCDF-activated nuclear receptors, with thecapacity to bind specifically to an XRE sequence, was effectivelyblocked by simultaneous treatment with ANF. In line with thismodel, ANF has been shown to inhibit the nuclear accumula-tion of [3H]TCDD-activated receptors (22).
TCDD has been reported to give a transient increase inactivated nuclear dioxin receptors, which reach maximal levelsafter 1 h of treatment. After >4 h of treatment the level ofnuclear receptors has returned to a constant low level (22). WhenTCDF was allowed to exert its effect for 2 h before ANF wasadded, the increase in P450IA1 mRNA was neverthelessinhibited. This suggests that the initial peak of activated nucleardioxin-receptors is not sufficient to induce a longer-lastingP450IA1 induction response. ANF treatment, at a time-point afterTCDF treatment when the level of activated nuclear dioxinreceptors is low, led to suppression of already maximally inducedmRNA levels. This indicates that these low levels of nuclearreceptors may in fact be important for maintaining the effect ongene expression.
Given the difficulty of establishing a ligand exchange of alreadyactivated nuclear receptors (39), ANF is unlikely to displaceTCDF from the already activated receptor. However, ANF doescompete with TCDF for cytosolic binding sites (22). ANF is alsoa weak receptor agonist and can induce gene expression throughan XRE in a human hepatoma cell line (40). The data obtainedcould be explained if continuous new activation of receptors isneeded in order for TCDF to induce P450IA1 and if ANF inhibitsthe accumulation of TCDF-receptor complexes in the nucleus.In addition, a model for the inhibitory effect of the ANF-boundreceptor has to include the possibilities that it is either inefficientin the activation step or slow in nuclear translocation, or alter-natively is not readily retained in the nucleus. It will be impor-tant to define the biochemical properties of the ANF-occupiedreceptor in order to understand the mechanism of action of theligand-regulated dioxin receptor.
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AcknowledgementsWe thank S.Bohm and Dr L.Poellinger for critical comments on the manuscript.Dr W.L.Alworth kindly provided the 1-ethynyIpyrene and Dr R.N.Hines thepRNHUC. This work was supported by the Swedish National Board ofEnvironmental Protection and the Swedish Work Environment Health Fund.
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Received on September 30, 1991; revised on January 2, 1992; accepted on January2, 1992.
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