Carcinogenesfcs vol.13 no.4 pp.651-655, 1992

The stability of dioxin-receptor ligands influences cytochrome P450IA1 expression in human keratinocytes

Anna Berghard, Katarina Gradin and Rune Toftgard Center for Biotechnology, Karolinska Institute, NOVUM, S-141 57 Huddinge, Sweden

Three dioxin-receptor ligands were analyzed for their effect on cytochrome P450IA1 mRNA expression in normal human keratinocytes. Although a 2 h pulsed treatment with the receptor agonists 2,3,7,8-tetrachlorodibenzofuran (TCDF) and /3-naphthoflavone (BNF) gave the same maximal induction response, the effect of BNF was transient compared to effect of TCDF. This was most likely due to metabolism of BNF as exemplified by the fact that a P450IA1 enzyme suicide-inhibitor, 1-ethynylpyrene, could prolong the induction response following a short BNF treatment. The TCDF induction of a reporter gene construct under the control of the -1140 to +2435 part of the CYPIA1 gene transiently transfected into HK was effectively inhibited by the dioxinreceptor antagonist a-naphthoflavone (ANF). In addition, ANF inhibited the accumulation of TCDF-activated nuclear receptors with capacity to bind to a xenobiotic response element. Interestingly, ANF could also suppress already maximally induced P450IA1 mRNA levels. The data demonstrate that the stability of the ligand influences the longterm effects on gene expression and that the effect of stable ligands may be masked due to receptor antagonist presence. In addition, the results support the hypothesis that a constant low level of activated nuclear receptors is required to maintain induced P450IA1 expression.

Introduction The epidermis is exposed to a variety of genotoxic and tumorpromoting compounds. Cytochrome P450IA1* has the capacity to catalyze the activation of polycyclic aromatic hydrocarbons (PAH) like 7,12-dimethylbenz[a]anthracene and benzo[a]pyrene to the ultimate carcinogenic species (1 and refs therein) and this monooxygenase-associated enzyme is present in the epidermis (2) as well as in cultured keratinocytes (3). PAHs such as 3-methylcholanthrene and /3-naphthoflavone (BNF), as well as a number of polyhalogenated PAHs typified by 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD), regulate P450IA1 gene expression on the transcriptional level (4). The CYPIA1 gene, encoding the cytochrome P450IA1 product, has been used as a model gene to characterize the molecular mechanisms of action of the environmental contaminant TCDD (5). TCDD binds to an intracellular protein, termed the dioxin or Ah receptor, that was recently purified (6). The importance of the ligand in receptor activation has been thoroughly investigated •Abbreviations: P45OIAI, cytochrome P450IA1; PAH, polycyclic aromatic hydrocarbons; BNF, 0-naphthoflavone; TCDD, 2,3,7,8-tetrachlorodibenzo-pdioxin: XRE, xenobiotic response element; TCDF, 2,3,7,8-tetrachlorodibenzofijran; ANF, a-naphthoflavone; HK, human keratinocytes; EP, 1-ethynylpyrene; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; CAT, chloramphenicol acetyl transferase.

© Oxford University Press

(7). The cytoplasmic inactive receptor is associated with the 90 kDa heat-shock protein (hsp90; refs 8,9) and binding of ligand results in enrichment of the activated ligand —receptor complex in the nucleus. Recently a factor important for the nuclear translocation of the receptor was cloned by a functional complementation assay (10). In the nucleus, the activated, hsp90 free ligand-receptor complex binds specifically to DNA sequences, 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 quite resistant to metabolism and is not in itself genotoxic (14 and refs therein). Besides acute toxic effects, like lethality, thymic involution, teratogenicity, liver and kidney damage, TCDD can act as an effective tumor promotor in vivo in rat liver (15) and hairless mouse skin (16) and in vitro in C3H/10T1/2 cells (17). The dioxin receptor is necessary but not sufficient for the tumor promotion response (18). The mechanism(s) underlying the tumor-promoting effect is not known, but it has been suggested to involve a change in the gene expression pattern in the affected tissue. The biological effects of dioxin-receptor ligands show considerable species and tissue variation. In humans the most characteristic effect is persistent hyperkeratotic lesions involving the epidermis (19). Thus, keratinocytes represent relevant target cells for the study of the effects of this class of compounds. In this study we have compared the effect of 2,3,7,8tetrachlorodibenzofuran (TCDF) to that of two naphthoflavones, BNF and a-naphthoflavone (ANF), on P450IA1 mRNA expression in normal human keratinocytes (HK). TCDF is almost as potent as TCDD in binding to the receptor and as a tumor promotor (16). BNF is widely regarded, though not firmly established, as being ineffective as a tumor promotor (20). In addition, TCDF is a stable ligand (21) as opposed to BNF (20). ANF has been shown to inhibit both TCDD binding to the receptor (22) and P450IA1 activity by reversible binding to the enzyme. Here we demonstrate the effect of a stable ligand (TCDF) on P450IA1 expression as compared to two unstable ligands (BNF and ANF). The necessity of activated dioxin receptors for persistent effects on gene expression and the mechanism underlying ANF's antagonistic effect are also addressed. Materials and methods Cell culture and treatments All chemicals, medium and growth factors were purchased from Sigma, unless stated otherwise. HK were isolated from adult or newborn donors as described previously (23), with modifications (24). To allow for maximal growth, the cells were grown in MCDB 153 medium with a final Ca 2+ 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, bovine pituitaries purchased from Pel-Freeze), 100 /jg/ml streptomycin, 100 IU/ml penicillin (Nordcell) and 0.25 /ig/ml fungizone (Nordcell) until near confluence when they were subcultured and plated onto fibronectin and collagen-I coated cell-culture plastic. Cells at the third to fourth passage were routinely used for experiments. In order to maximize the P450IA1 induction response in HK the medium was switched to contain 2 mM Ca2"1" and bovine pituitary extract was

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A.Berghard, K.Gradin and R.Toflgard 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 kind gift from Dr W.L.Alworth, Tulane University, New Orleans. Each experiment was 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). RNA was fractionated through formaldehyde-agarose gels, blotted onto nylon membranes, then UV cross-linked, and the filters were subsequently prehybrklized, hybridized and washed according to standard procedures (27) prior to autoradiography. The insert of phPl-4503' (28) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 29) were isolated and P-labeled using a random priming procedure. Transfeaion and transient expression assays HK (25 cm2) were incubated overnight in 70 fiM Ca 2+ medium with 30 yX of cationic liposomes (Hpofectin, Bethseda Research Laboratories), 0.5 jig RSVluc and 5 pgpRNHUC. pRNHHC contains the -1140 to +2435 part of the human CYP1A1 gene directly fused to the bacterial chloramphenicol acetyl transferase (CAT) reporter gene (30). RSV-luc is a construct where the Rous sarcoma virus long terminal repeat drives the expression of the luciferase reporter gene (31). The medium was changed to contain 5% serum and 2.0 mM Ca 2+ for the following 24 h, and the cells were subsequently treated with either 50 nM TCDF 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) with the modification that the cells were lysed on ice by a brief treatment using a sonicator (Branson Sonic Power Co.). Luciferin was purchased from BioThema and [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 for 1 h before harvest. Nuclei were prepared and protein extracted as previously described (32). DNA-binding reactions were assembled in 20 /J with 10 /ig of nuclear protein at a final concentration of 25 mM HEPES (pH 7.9), 0.2 mM EDTA, 75 mM KC1, 2 mM MgCI 2 , 1 mM dithiothreitol, 0.1 mM phenylmethylsulphonyl 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 a double-stranded, 32P-labeled oligonucleotide (5'-CTCCGGTCCTTCTCACGCAACGCCTGGGCA-3' corresponding to -969 to - 9 9 8 of the human CYPIA1 gene) were used. Competition with an oligonucleotide corresponding to XRE1 of the rat CypIAI (11) was indistinguishable from competition with the unlabeled probe DNA (unpublished data). As unspecific competitor DNA a double-stranded oligonucleotide (5 '-TCTAGTGTTGGAGAACGAATCAGCATCTGAGTAC-3') was used, whereas unlabeled probe DNA served as specific competitor. DNA-protein complexes were separated under non-denaturing conditions in a 5% polyacrylamide gel (29:1) run in 0.35 x TBE (1 x TBE is 0.09 M Tris-borate, 0.001 M EDTA) at 4°C.

Results Maximal induction of P450IA1 mRNA steady-state levels was obtained at a dose of ~50 ^M BNF (Figure 1) which was comparable to that induced by 5 nM TCDF. When inducer was continuously present, induction by both compounds showed a similar time dependency. As can be seen in Figure 2(A), the

P450IA1

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P4KHA1

GAPDH 0 I B 16 24 32 40 46 72 961201 6 16 24 32 40 48 72 9612olfh) BNF

TCDF

P450IA1

0 I 6 16 24 32 40 48 72 961201a 16 24 32 40 48 72 961201 (h) 2 h PULSE BNF

2 h PULSE TCDF

Fig. 2. Time course of induction of P450LA1 mRNA levels by constant or pulsed treatment with TCDF and BNF. Northern analysis showing P450IA1 and GAPDH mRNA levels after treatment with 50 nM TCDF or 50 /iM BNF (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 cells were harvested at the indicated time-points.

P450IA1

GAPDH

GAPDH in o

p o Io

in

p o

in o

p m

BNF (\1M)

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4 h of treatment the level of nuclear receptors has returned to a constant low level (22). When TCDF was allowed to exert its effect for 2 h before ANF was added, the increase in P450IA1 mRNA was nevertheless inhibited. This suggests that the initial peak of activated nuclear dioxin-receptors is not sufficient to induce a longer-lasting P450IA1 induction response. ANF treatment, at a time-point after TCDF treatment when the level of activated nuclear dioxin receptors is low, led to suppression of already maximally induced mRNA levels. This indicates that these low levels of nuclear receptors may in fact be important for maintaining the effect on gene expression. Given the difficulty of establishing a ligand exchange of already activated nuclear receptors (39), ANF is unlikely to displace TCDF from the already activated receptor. However, ANF does compete with TCDF for cytosolic binding sites (22). ANF is also a weak receptor agonist and can induce gene expression through an XRE in a human hepatoma cell line (40). The data obtained could be explained if continuous new activation of receptors is needed in order for TCDF to induce P450IA1 and if ANF inhibits the accumulation of TCDF-receptor complexes in the nucleus. In addition, a model for the inhibitory effect of the ANF-bound receptor has to include the possibilities that it is either inefficient in the activation step or slow in nuclear translocation, or alternatively is not readily retained in the nucleus. It will be important to define the biochemical properties of the ANF-occupied receptor in order to understand the mechanism of action of the ligand-regulated dioxin receptor.

Stability of dioxin-receprtor ligands

Acknowledgements We 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 the pRNHUC. This work was supported by the Swedish National Board of Environmental Protection and the Swedish Work Environment Health Fund.

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The stability of dioxin-receptor ligands influences cytochrome P450IA1 expression in human keratinocytes.

Three dioxin-receptor ligands were analyzed for their effect on cytochrome P450IA1 mRNA expression in normal human keratinocytes. Although a 2 h pulse...
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