Vitamin D Receptor Activation Enhances Benzo[a]pyrene ...dmd.aspetjournals.org/content/dmd/early/2012/07/25/dmd.112.046839.full.pdf · DMD #46839 4 Abstract Benzo[a]pyrene (BaP) activates

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Vitamin D Receptor Activation Enhances Benzo[a]pyrene Metabolism via CYP1A1

Expression in Macrophages

Manabu Matsunawa, Daisuke Akagi, Shigeyuki Uno, Kaori Endo-Umeda, Sachiko

Yamada, Kazumasa Ikeda, and Makoto Makishima

Division of Biochemistry, Department of Biomedical Sciences, Nihon University School

of Medicine, 30-1 Oyaguchi-kamicho, Itabashi-ku, Tokyo 173-8610, Japan (M.M., D.A.,

S.U., K.E.-U., S.Y., M.M.); Department of Applied Biological Science, Nihon University

College of Bioresource Sciences, Fujisawa, Kanagawa 252-8510, Japan (D.A., K.I.)

DMD Fast Forward. Published on July 25, 2012 as doi:10.1124/dmd.112.046839

Copyright 2012 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on July 25, 2012 as DOI: 10.1124/dmd.112.046839

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Running title: Enhanced BaP metabolism by VDR activation

Address correspondence to: Makoto Makishima, Division of Biochemistry, Department

of Biomedical Sciences, 30-1 Oyaguchi-kamicho, Itabashi-ku, Tokyo 173-8610, Japan.

E-mail address: [email protected].

Number of text pages: 42

Number of figures: 5

Number of tables: 0

Number of references: 51

Number of words in Abstract: 239

Number of words in Introduction: 717

Number of words in Discussion: 987

Abbreviations: BaP, benzo[a]pyrene; AHR, aryl hydrocarbon receptor; CYP, cytochrome

P450; 1,25(OH)2D3, 1α,25-dihydroxyvitamin D3; VDR, vitamin D receptor; RXR,

retinoid X receptor; ER, everted repeat; PXR, pregnane X receptor; CAR, constitutive

androstane receptor; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; ADTT,

(25R)-25-adamantyl-1α,25-dihydroxy-2-methylene-22,23-didehydro-19,26,27-trinor-20

-epi-vitamin D3; ADMI3,


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(25S)-26-adamantyl-1α,25-dihydroxy-2-methylene-22,23-didehydro-19,27-dinor-20-epi

-vitamin D3; PCR, polymerase chain reaction; siRNA, small interfering RNA; EMSA,

electrophoretic mobility shift assay; ChIP, chromatin immunoprecipitation; LCA,

lithocholic acid; XRE, xenobiotic responsive element


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Abstract

Benzo[a]pyrene (BaP) activates the aryl hydrocarbon (AHR) and induces the expression

of genes involved in xenobiotic metabolism, including cytochrome P450 (CYP) 1A1.

CYP1A1 is involved not only in BaP detoxification but also in metabolic activation,

which results in DNA adduct formation. Vitamin D receptor (VDR) belongs to the NR1I

subfamily of the nuclear receptor superfamily, which also regulates expression of

xenobiotic metabolism genes. We investigated the cross-talk between AHR and VDR

signaling pathways and found that 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3], a potent

physiological VDR agonist, enhanced BaP-induced transcription of CYP1A1 in human

monocytic U937 cells and THP-1 cells, breast cancer cells and kidney epithelium-derived

cells. 1,25(OH)2D3 alone did not induce CYP1A1 and 1,25(OH)2D3 plus BaP did not

increase CYP1A2 or CYP1B1 mRNA expression in U937 cells. Combination of

1,25(OH)2D3 and BaP increased CYP1A1 protein levels, BaP hydroxylation activity and

BaP-DNA adduct formation in U937 cells and THP-1 cells more effectively than BaP

alone. The combined effect of 1,25(OH)2D3 and BaP on CYP1A1 mRNA expression in

U937 cells and/or THP-1 cells was inhibited by VDR knockdown, VDR antagonists, and

α-naphthoflavone, an AHR antagonist. Electrophoretic mobility shift assays and

chromatin immunoprecipitation assays showed that VDR directly bound to an everted

repeat 8 motif in the human CYP1A1 promoter. Thus, CYP1A1 is a novel VDR target gene

involved in xenobiotic metabolism. Induction of CYP1A1 by the activation of VDR and


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AHR may contribute to BaP-mediated toxicity and the physiological function of this

enzyme.


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Introduction

Polycyclic aromatic hydrocarbons such as benzo[a]pyrene (BaP) are environmental

pollutants produced by the combustion of cigarettes, creosote railroad ties, and coke

ovens (Miller and Ramos, 2001; Uno and Makishima, 2009). BaP is implicated as a

causative agent in malignancies, such as lung and head-and-neck cancers, and

atherosclerosis as a consequence of cigarette smoking (Alexandrov et al., 2010; Shimada

and Fujii-Kuriyama, 2004). BaP inhalation activates the aryl hydrocarbon receptor

(AHR), which forms an active transcription factor heterodimer with the AHR nuclear

translocator, and induces expression of a group of genes called the [Ah] gene battery,

which includes the phase I enzymes [cytochrome P450 (CYP) 1A1 (gene symbol,

CYP1A1), CYP1A2, CYP1B1, and NAD(P)H:quinone oxidoreductase 1] and the phase II

enzymes (glutathione S-transferase A1 and UDP glucuronosyltransferase 1A6) (Nebert et

al., 2000).

BaP mediates carcinogenic, mutagenic and cytotoxic effects after conversion to

toxic metabolites through an AHR-dependent mechanism of metabolic activation (Miller

and Ramos, 2001; Shimada and Fujii-Kuriyama, 2004). BaP is first oxidized by CYP1A1

and CYP1B1 to phenols, such as 3-hydroxy-BaP and 9-hydroxy-BaP, and epoxides, such

as BaP-7,8-epoxide (Shimada, 2006; Uno and Makishima, 2009). BaP-7,8-epoxide is

then metabolized by epoxide hydrolase to BaP-7,8-diol, which serves as substrate for a

second CYP-dependent oxidation, generating the toxic compound


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BaP-7,8-diol-9,10-epoxide. Among the metabolites in BaP-treated cells,

(+)-BaP-7,8-diol-9,10-epoxide-2 is the most reactive carcinogen (Alexandrov et al.,

2010; Shimada, 2006).

Original studies on mutant Hepa-1 cells that are resistant to BaP-induced growth

suppression have shown that BaP resistance is associated with mutations in the Cyp1a1

gene and dysfunction of the AHR transcription factor (Hankinson et al., 1991).

Expression of exogenous CYP1A1 in CYP1A1-deficient cells restores the formation of

BaP-induced DNA adducts (Maier et al., 2002). These findings indicate that metabolic

activation of BaP requires the AHR-CYP1A1 cascade. In contrast, BaP-induced DNA

adducts are increased in the liver and BaP clearance from the blood is slower in

CYP1A1-null mice (Uno et al., 2001). Overexpression of CYP1A1 in hepatocytes

suppresses BaP-induced DNA adduct formation and AHR transactivation (Endo et al.,

2008). CYP1A1 may be involved in both metabolic activation and detoxification of BaP

depending on conditions (Uno et al., 2006).

The active form of vitamin D3, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], binds to

the vitamin D receptor (VDR; NR1I1) and regulates numerous physiological and

pharmacological processes, including bone and calcium metabolism, cellular growth and

differentiation, immunity, and cardiovascular function (Choi and Makishima, 2009;

Nagpal et al., 2005). Natural and synthetic VDR ligands inhibit the proliferation and/or

induce the differentiation of various types of malignant cells, including myeloid leukemia


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(Hozumi, 1998; Nagpal et al., 2005). On ligand binding, VDR undergoes conformational

changes that result in dynamic interaction with the heterodimer partner retinoid X

receptor (RXR; NR2B) and exchange of cofactor complexes (Makishima and Yamada,

2005). Corepressors bind to the VDR-RXR heterodimer in the absence of ligand and

ligand binding reduces the affinity of corepressors and increases the affinity for

coactivators, a structural transition that induces transcription of specific genes. The

VDR-RXR heterodimer binds preferentially to a vitamin D response element that consists

of a two hexanucleotide (AGGTCA or a related sequence) direct repeat motif separated

by three nucleotides. An inverted palindrome of the hexanucleotide motif, also called

everted repeat (ER) element (Mangelsdorf and Evans, 1995), separated by six, seven,

eight, or nine nucleotides has been also identified as vitamin D response elements in

genes including CYP3A4 (Choi and Makishima, 2009; Thummel et al., 2001). VDR has

been found to act as a receptor for secondary bile acids, including lithocholic acid (LCA)

and 3-ketocholanic acid, and to induce the expression of CYP3A enzymes (Makishima et

al., 2002). CYP3A enzymes catalyze the metabolic conversion of a wide variety of

xenobiotics and endogenous substrates, including bile acids, to more polar derivatives

(Xie and Evans, 2001).

VDR belongs to the NR1I nuclear receptor subfamily along with pregnane X

receptor (PXR; NR1I2) and constitutive androstane receptor (CAR; NR1I3), both of

which play a role in the regulation of xenobiotic metabolism. These findings suggest that


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VDR may be involved in the regulation of xenobiotic-metabolizing enzymes other than

CYP3A. Recently, CAR has been found to induce CYP1A1 and CYP1A2 expression by

binding to a common regulatory element in the human CYP1A1 and CYP1A2 genes in

hepatocytes (Yoshinari et al., 2010). In this study, we report that VDR activation enhances

AHR-induced CYP1A1 expression in human macrophage-derived cells.


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Materials and Methods

Chemical Compounds. 1,25(OH)2D3, BaP and 2,3,7,8-tetrachlorodibenzo-p-dioxin

(TCDD) were purchased from Wako Pure Chemical Industries (Osaka, Japan), and

α-naphthoflavone was from Sigma-Aldrich (St. Louis, MO). LCA acetate, ADTT

[(25R)-25-adamantyl-1α,25-dihydroxy-2-methylene-22,23-didehydro-19,26,27-trinor-2

0-epi-vitamin D3] and ADMI3

[(25S)-26-adamantyl-1α,25-dihydroxy-2-methylene-22,23-didehydro-19,27-dinor-20-ep

i-vitamin D3] were synthesized in the Sachiko Yamada laboratory (Igarashi et al., 2007;

Ishizawa et al., 2008; Nakabayashi et al., 2008).

Cell Culture. Human monocyte/macrophage-derived cells (U937 and THP-1;

RIKEN Cell Bank, Tsukuba, Japan) and human breast cancer MCF-7 cells (American

Type Culture Collection, Rockville, MD) were cultured in RPMI 1640 medium

containing 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin

at 37°C in a humidified atmosphere containing 5% CO2. Human kidney HEK293 cells

(RIKEN Cell Bank) were cultured in Dulbecco's modified Eagle medium containing 5%

fetal bovine serum. Cell viability after all of the treatments was more than 90%, as

determined by exclusion of trypan blue.

Reverse Transcription and Real-time Quantitative Polymerase Chain Reaction.

Total RNAs from samples were prepared by the acid guanidine

thiocyanate-phenol/chloroform method (Matsunawa et al., 2009; Tavangar et al., 1990).


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cDNAs were synthesized using the ImProm-II Reverse Transcription system (Promega

Corporation, Madison, WI). Real-time polymerase chain reaction (PCR) was performed

on the ABI PRISM 7000 Sequence Detection System (Applied Biosystems, Foster City,

CA) using Power SYBR Green PCR Master Mix (Applied Biosystems). Intron-spanning

primers were as follows: CYP1A2 (GenBank accession no. NM_000761), 5'-AGT GGG

AGA TCT TCC TCT TC-3' and 5'-GGT GTC TTC TTC AGT TGA TG-3'; VDR

(GenBank accession no. NM_000376), 5'-CAC GTC ACT GAC GCG GTA CTT-3' and

5'-GCT GAC CTG GTC AGT TAC AGC A-3'; AHR (GenBank accession no.

NM_001621), 5'-GTA AGT CTC CCT TCA TAC C-3' and 5'-AGG CAC GAA TTG GTT

AGA G-3'. Other primers were reported previously (Matsunawa et al., 2009). The RNA

values were normalized to the amount of β-actin mRNA, or mRNA copy numbers were

determined using expression plasmids for a standard curve in real-time PCR reactions

(Uno et al., 2006).

Western Blotting Analysis. For VDR and AHR expression, nuclear extracts were

prepared as previously described (Inaba et al., 2007; Schreiber et al., 1989). For CYP1A1

expression, microsomes (S9 fraction) from cells were prepared as previously described

(Endo et al., 2008; Uno et al., 2001). Western blot analysis was performed using a

monoclonal anti-VDR antibody (Santa Cruz Biotechnology, Santa Cruz, CA), a

polyclonal anti-AHR antibody (R&D Systems Inc., Minneapolis, MN), a polyclonal

anti-CYP1A1 antibody (Daiichi Pure Chemicals, Tokyo, Japan) and a monoclonal


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anti-β-actin antibody (Sigma-Aldrich), visualized with an enhanced chemiluminescence

detection system or an alkaline phosphatase conjugate substrate system (Endo et al.,

2008).

Enzyme Activity Assays. Extracts from microsomal fractions were subjected for

enzyme activity assays. BaP hydroxylation was assayed as previously reported (Endo et

al., 2008; Nebert and Gelboin, 1968). Briefly, 200 μg of microsomal protein was

incubated in 100 mM potassium phosphate buffer (pH 7.4) containing 1 mg/ml bovine

serum albumin, 80 μM BaP and 0.5 mM NADPH at 37°C for 20 minutes. The reaction

was stopped by the addition of acetone/n-hexane (1:3). After the organic phase was

extracted with NaOH, the concentration of 3-hydroxy-BaP was measured

spectrofluorometrically with activation at 396 nm and fluorescence at 522 nm.

Measurement of DNA Adducts. BaP-induced DNA adducts were determined by a

32P-postlabeling method (Endo et al., 2008; Talaska et al., 1996; Uno et al., 2001). After

DNA extraction from cells, hydrolysis to 3'-phosphodeoxynucleotides with micrococcal

endonuclease and spleen phosphodiesterase, and removal of non-adducted

3'-phosphodeoxynuleotides with n-butanol extraction, the 3'-phosphodeoxynucleosides

were labeled at the 5' positions with [32P]ATP and T4 polynucleotide kinase.

Two-dimensional thin-layer chromatography on polyethylenimine cellulose sheets was

used to resolve the 32P-labeled DNA adducts (Randerath and Randerath, 1964), which

were then visualized and quantified by scintillation counting (Packard 1900 CA,


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Downers Grove, IL). Specific activity of isotope-labeled ATP was expressed as relative

adduct labeling values normalized with amounts of DNA in each sample.

RNA Interference. Small interfering RNAs (siRNAs) directed against VDR

(Dharmacon M-003448) and control siRNA (Dharmacon D-001206) were purchased

from Thermo Fisher Scientific (Waltham, MA). siRNA oligonucleotides were transfected

into cells using DharmaFECT1 Reagent (Thermo Fisher Scientific) according to the

manufacturer's instructions.

Electrophoretic Mobility Shift Assays. Electrophoretic mobility shift assays

(EMSAs) were performed as reported previously (Endo-Umeda et al., 2012; Yoshikawa

et al., 2001). Briefly, receptor proteins were in vitro translated with a TNT Quick Coupled

Transcription/Translation System (Promega Corporation). Sequences for double stranded

oligonucleotides are shown in Fig. 5A. Binding reactions were performed in a buffer

containing 10 mM Tris-HCl (pH 7.6), 50 mM KCl, 0.05 mM EDTA, 2.5 mM MgCl2,

8.5% glycerol, 1 mM dithiothreitol, 0.5 μg/ml poly(dI-dC), 0.1% Triton-X100, and

nonfat milk. Unlabeled probes and anti-VDR antibody (Santa Cruz Biotechnology) were

used for competition experiments and supershift experiments, respectively. Samples were

separated on 5% polyacrylamide gels and were visualized with autoradiography.

Chromatin Immunoprecipitation. Chromatin immunoprecipitation (ChIP) was

performed as reported previously (Matsunawa et al., 2009; Shang et al., 2000). After

nuclear proteins were cross-linked to DNA in 1% formaldehyde for 15 min, cells were


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washed and lysed in lysis buffer (50 mM Tris-HCl, pH 8.1, 1% SDS, 10 mM EDTA).

After sonication and removal of cellular debris, the lysates were diluted in ChIP dilution

buffer (16.7 mM Tris-HCl, pH 8.1, 0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 167

mM NaCl). ChIP was performed with control IgG antibody, anti-AHR antibody, or

anti-VDR antibody (Santa Cruz Biotechnology). DNA was purified with MonoFas DNA

Purification Kit (GL Sciences, Torrance, CA). PCR was performed using GoTaq Master

Mix (Promega) with the following primers: 5'-GAA CGC TGG GCG TGC AGA TGC

CTC-3' and 5'-CAC TAA GGC GAT CCT AGA GGC TG-3', detecting the region -375 to

-693 in CYP1A1 promoter as shown in Fig. 5A. The PCR products were separated by

electrophoresis in 2% agarose gel.

Statistical Analyses. All values are shown as means ± S.E.M. The two-tailed,

unpaired Student’s t test was performed to assess significant differences.


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Results

1,25(OH)2D3 Enhances the Expression of CYP1A1 induced by BaP. We

previously reported that AHR activation by BaP and TCDD enhances the expression of

CYP24A1 in human monocyte/macrophage-derived THP-1 cells treated with

1,25(OH)2D3 (Matsunawa et al., 2009). To further investigate the reciprocal effects of

VDR and AHR signaling, we treated monocyte-derived U937 cells and THP-1 cells with

BaP and/or 1,25(OH)2D3 and examined the expression of CYP1A1, an AHR target gene

that has been shown to be involved in metabolic activation and detoxification of BaP

(Uno and Makishima, 2009). Treatment with BaP (0.3 μM) induced the expression of

CYP1A1 in U937 cells and THP-1 cells, while 1,25(OH)2D3 (30 nM) did not show

significant induction (Fig. 1A). Interestingly, CYP1A1 mRNA expression induced by BaP

was further increased by co-administration of 1,25(OH)2D3 in these cells (Fig. 1A).

Combined BaP and 1,25(OH)2D3 treatment also increased CYP1A1 mRNA expression to

a higher degree than by BaP alone in breast cancer MCF-7 cells and in kidney

epithelium-derived HEK293 cells (Fig. 1A). Previous reports have demonstrated

expression of functional VDR and AHR proteins in U937 cells, THP-1 cells, MCF-7 cells

and HEK293 cells (Amano et al., 2009; Campbell et al., 2000; Hayashi et al., 1995; Inaba

et al., 2007; Ishizawa et al., 2008; Zhang et al., 2008). We compared mRNA and protein

levels of VDR and AHR in these cell lines. VDR mRNA expression levels in U937 cells,

THP-1 cells, MCF-7 cells and HEK293 cells were 1,008 ± 126 copies, 51 ± 9 copies, 690


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± 88 copies and 526 ± 28 copies/μg total RNA, respectively, while those for AHR were

856 ± 244 copies, 107 ± 19 copies, 7,232 ± 922 copies and 1,900 ± 217 copies/μg total

RNA, respectively (Fig. 1B). We next examined expression of nuclear VDR and AHR

protein levels. As reported previously (Amano et al., 2009), VDR protein expression was

observed in U937 cells and, to a lesser extent, in THP-1 cells in the absence of ligand

(Fig.1C). BaP plus 1,25(OH)2D3 increased VDR protein levels in these cells as well as in

MCF-7 cells and HEK293 cells (Fig. 1C). AHR protein expression was observed in all

cell lines both with and without combined BaP and 1,25(OH)2D3 treatment (Fig. 1C).

There were some discrepancies between mRNA and protein levels of VDR and AHR (Fig.

1, B and C). It may be due to translational or post-translational regulation of these

proteins. As reported previously (Ishizawa et al., 2008; Matsunawa et al., 2009),

1,25(OH)2D3 treatment effectively induced expression of the VDR target CYP24A1 in

U937 cells and THP-1 cells (Fig. 1D), indicating that VDR functions in these cells. BaP

(1 μM) did not induce CYP1A2 or CYP1B1 expression in U937 cells, and the combination

of BaP and 1,25(OH)2D3 had no effect on expression of these genes (Fig. 1E). These

findings indicate that combined BaP and 1,25(OH)2D3 treatment effectively induces

CYP1A1 mRNA expression.

Next, we examined the effects of several concentrations of BaP in combination with

1,25(OH)2D3 on CYP1A1 mRNA expression in U937 cells. In the absence of

1,25(OH)2D3, BaP at 0.1 μM increased CYP1A1 mRNA expression, an effect not seen at


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0.01 μM or 0.03 μM (Fig. 2A). Interestingly, in combination with 10 nM 1,25(OH)2D3,

BaP at 0.01 μM effectively induced CYP1A1 expression. While BaP alone exhibited

maximal CYP1A1 induction at 0.1 μM, the combination of 1,25(OH)2D3 further enhanced

CYP1A1 expression in U937 cells treated with 0.1 μM, 0.3 μM and 1 μM BaP (Fig. 2A).

1,25(OH)2D3 alone up to 100 nM did not increase CYP1A1 expression. 1,25(OH)2D3 at 3

nM to 30 nM increased CYP1A1 expression induced by 0.3 μM BaP (Fig. 2B). Thus,

enhanced BaP-induced CYP1A1 expression by 1,25(OH)2D3 is concentration-dependent.

The effects of combined BaP and 1,25(OH)2D3 on CYP1A1 protein levels and

enzyme activity were examined. BaP treatment induced CYP1A1 protein expression in

U937 cells and the addition of 1,25(OH)2D3 further increased CYP1A1 protein level,

while 1,25(OH)2D3 alone caused modest induction (Fig. 3A). Whereas CYP1A1 proteins

were detected only at low levels in THP-1 cells treated with BaP or 1,25(OH)2D3 alone,

combined BaP and 1,25(OH)2D3 treatment effectively induced CYP1A1 protein

expression in THP-1 cells (Fig. 3A). We examined BaP hydroxylation activity by

detecting conversion of BaP to 3-hydroxy-BaP. CYP1A1, but not CYP1A2 or CYP1B1,

mediate this cellular reaction (Endo et al., 2008). Cells were treated with BaP and/or

1,25(OH)2D3 for 24 hours, and microsomes were isolated for enzyme activity assays. BaP

treatment effectively increased BaP hydroxylation activity in U937 cells (Fig. 3B).

Interestingly, 1,25(OH)2D3 slightly increased BaP hydroxylation activity in U937 cells

(Fig. 3B), consistent with CYP1A1 protein expression (Fig. 3A) but not with mRNA


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expression (Fig. 1A and Fig. 2). 1,25(OH)2D3 may increase CYP1A1 protein levels and

enzymatic activity through an unknown post-translational mechanism. Combined BaP

and 1,25(OH)2D3 treatment further increased BaP hydroxylation activity in U937 cells

(Fig. 3A). BaP treatment also increased BaP hydroxylation activity in THP-1 cells, but

the enzyme activity levels were very weak when compared to U937 cells (Fig. 3B). This

difference is likely due to lower CYP1A1 protein levels in THP-1 cells (Fig. 3A).

Although 1,25(OH)2D3 alone was not effective, it enhanced BaP hydroxylation activity in

THP-1 cells treated with BaP (Fig. 3B). Exogenous CYP1A1 expression increases and

decreases BaP-DNA adduct formation in Hepa-1 cells and HepG2 cells, respectively

(Endo et al., 2008; Maier et al., 2002). We next examined the effect of 1,25(OH)2D3 on

BaP-DNA adduct formation. BaP-DNA adduct formation was detected in BaP-treated

U937 cells and THP-1 cells, and combined 1,25(OH)2D3 treatment further increased

BaP-DNA adducts in these cells (Fig. 3C). Therefore, BaP and 1,25(OH)2D3 co-treatment

increases CYP1A1 protein levels, BaP hydroxylation activity and BaP-DNA adduct

formation in U937 cells and THP-1 cells.

Both VDR Activation and AHR Activation are Involved in CYP1A1

Transcription. VDR is activated by LCA and its derivatives, such as LCA acetate, as

well as 1,25(OH)2D3 (Ishizawa et al., 2008). LCA acetate alone did not induce CYP1A1

mRNA expression in U937 cells (Fig. 4A). Similarly to 1,25(OH)2D3, LCA acetate


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further increased CYP1A1 mRNA levels induced by BaP (Fig. 4A), suggesting that

CYP1A1 transcription is regulated by VDR activation. To examine the VDR dependency

of CYP1A1 induction, we knocked down VDR using siRNA. VDR knockdown

attenuated CYP1A1 induction by combination of BaP and 1,25(OH)2D3 in U937 cells and

THP-1 cells (Fig. 4B). In addition, we examined the effect of VDR antagonists, ADTT

and ADMI3 (Igarashi et al., 2007; Nakabayashi et al., 2008). Both ADTT and ADMI3

effectively reduced CYP1A1 expression induced by BaP plus 1,25(OH)2D3 in THP-1 cells

(Fig. 4C). Thus, VDR activation is involved in effective CYP1A1 induction.

The effect of 1,25(OH)2D3 on CYP1A1 expression was examined in U937 cells

treated with TCDD. TCDD is a high-affinity AHR ligand that is virtually not metabolized

in cells (Bock and Kohle, 2006). 1,25(OH)2D3 increased CYP1A1 expression induced by

TCDD (Fig. 4D). Next, we examined the effect of α-naphthoflavone, an AHR antagonist

(Gasiewicz and Rucci, 1991), on CYP1A1 expression induced by BaP plus 1,25(OH)2D3

in U937 cells. The addition of α-naphthoflavone inhibited CYP1A1 induction by BaP

alone and combined treatment of BaP and 1,25(OH)2D3 (Fig. 4E). These findings indicate

that both AHR and VDR are necessary for CYP1A1 induction by BaP plus 1,25(OH)2D3.

VDR-RXR Binds to the CYP1A1 Promoter. VDR, PXR and CAR belong to the

NR1I subfamily of the nuclear receptor superfamily and regulate common target genes,

such as CYP3A4 (Makishima et al., 2002; Thummel et al., 2001; Xie et al., 2000). The


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CAR-RXR heterodimer binds to an ER8 element in the human CYP1A1 promoter

(Yoshinari et al., 2010). We performed EMSAs using oligonucleotide probes containing

the ER8 element and a known xenobiotic-responsive element (XRE) (WT in Fig. 5A).

The VDR-RXR heterodimer bound to isotope-labeled XRE-ER8 (Fig. 5B). Complex

formation was inhibited by addition of unlabeled XRE-ER8 (Fig. 5B). Mutation of the

XRE and a proximal half-site of ER8 (MT2 in Fig. 5A) inhibited binding of VDR-RXR to

isotope-labeled wild-type XRE-ER8 but mutation of both hexanucleotides of ER8 (MT1

in Fig. 5A) failed to exhibit competition (Fig. 5B). Addition of anti-VDR antibody

induced a supershift of the VDR-RXR complex with XRE-ER8 (Fig. 5B). The results

indicate that VDR-RXR directly binds to ER8 in the CYP1A1 promoter.

Finally, we performed ChIP assays to examine direct VDR binding to the CYP1A1

promoter in cells using anti-AHR or anti-VDR antibodies and PCR for the -379 to -693

CYP1A1 promoter region, which contains the XRE (-489 to -495) and the ER8 (-506 to

-525) (Fig. 5A). Six hours after ligand addition, BaP and 1,25(OH)2D3 recruited AHR and

VDR, respectively, to the CYP1A1 promoter in U937 cells, and combination of these

compounds did not further increase the recruitment of AHR or VDR (Fig. 5C). At 24

hours, VDR recruitment was slightly higher with BaP plus 1,25(OH)2D3 compared to

treatment of 1,25(OH)2D3 alone (Fig. 5C). BaP and 1,25(OH)2D3 also induced

recruitment of AHR and VDR, respectively, to the CYP1A1 promoter in THP-1 cells (Fig.

5D). Therefore, VDR regulates CYP1A1 transcription by direct binding to the promoter in


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monocyte/macrophage-derived cells.


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Discussion

We show here that VDR activation enhanced the CYP1A1 expression and activity

induced by AHR ligands in monocyte/macrophage-derived U937 cells and THP-1 cells.

EMSA and ChIP assays demonstrate that VDR binds to the ER8 motif located in the

proximal promoter of the human CYP1A1 gene. CAR, another NR1I subfamily nuclear

receptor, also binds to the same motif and its activation induces expression of both

CYP1A1 and CYP1A2 in hepatocytes (Yoshinari et al., 2010). In contrast to CYP1A1

induction, the combined treatment of BaP and 1,25(OH)2D3 did not induce CYP1A2

expression in U937 cells (Fig. 1). While administration of BaP to mice induces Cyp1a1

mRNA levels in liver, small intestine, spleen and bone marrow, it induces Cyp1a2 mRNA

expression in liver, small intestine and to a lesser degree in spleen, but not in bone

marrow (Uno et al., 2006). Although the human CYP1A1 and CYP1A2 genes are located

in a head-to-head orientation on chromosome 15 and share a common regulatory region

(Ueda et al., 2006), transcription of these genes may be regulated by additional tissue- or

cell-type-specific mechanisms. Bone marrow-derived cells may be less responsible for

CYP1A2 induction.

While CAR activation induces expression of CYP1A1 and CYP1A2 independent of

AHR (Yoshinari et al., 2010), use of an AHR antagonist, α-naphthoflavone, shows that

the effect of 1,25(OH)2D3 on CYP1A1 induction requires AHR activation (Fig. 4). These

findings indicate that VDR signaling is only effective in augmenting CYP1A1


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transcription activated by AHR in monocyte/macrophage-derived cells. VDR siRNA and

VDR antagonists decreased CYP1A1 expression induced by BaP plus 1,25(OH)2D3 (Fig.

4), suggesting that VDR bound to the CYP1A1 promoter is functionally active. The

combination of BaP and 1,25(OH)2D3 did not further increase the recruitment of AHR or

VDR to the CYP1A1 promoter (Fig. 5). Although AHR activation modifies the

transcriptional activity of estrogen receptor through direct association, a direct interaction

between AHR and VDR has not been detected (Ohtake et al., 2003). These findings

suggest that AHR and VDR bind independently to the CYP1A1 promoter. The

combination of ligands may effectively induce CYP1A1 mRNA expression through

formation of a multimeric complex where AHR interacts indirectly with VDR via

coregulatory proteins. Further studies are needed to elucidate the molecular mechanisms

of CYP1A1-selective action of VDR on a regulatory region of the CYP1A1_CYP1A2

gene locus.

Increased expression of CYP1A1 by BaP plus 1,25(OH)2D3 resulted in enhanced

BaP-DNA adduct formation (Fig. 3). BaP is first oxidized by CYP1A1 and CYP1B1 to

phenols, such as 3-hydroxy-BaP and 9-hydroxy-BaP, and epoxides, such as

BaP-7,8-epoxide (Shimada and Fujii-Kuriyama, 2004). We observed enhanced BaP

hydroxylation to 3-hydroxy-BaP in cells treated with BaP and 1,25(OH)2D3 (Fig. 3), a

finding consistent with increased CYP1A1 expression. BaP-7,8-epoxide is then

metabolized by epoxide hydrolase to BaP-7,8-diol, which serves as a substrate for a


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subsequent CYP-dependent oxidation, generating the toxic compound

BaP-7,8-diol-9,10-epoxide (Shimada and Fujii-Kuriyama, 2004). BaP-7,8-diol is also

metabolized to BaP-7,8-dione by aldoketoreductase, and BaP-1,3-, 1,6-, and 3,6-diones

are thought to be formed by metabolism of BaP to phenols by CYP enzymes (Shimada,

2006). These BaP quinones are also involved in DNA adduct formation. Glutathione

S-transferase suppresses BaP-induced DNA adduct formation by conjugation of reactive

BaP metabolites (Uno and Makishima, 2009). The formation of BaP-induced DNA

adducts is decreased in Hepa-1 c37 cells, a CYP1A1-deficient mutant clone of Hepa-1

cells (Maier et al., 2002). Stable transfection of a Cyp1a1 expression plasmid restores

adduct formation to the level of parent Hepa-1 cells (Maier et al., 2002). Exogenous

expression of the oncogenic AML1-ETO fusion protein in U937 cells up-regulates

CYP1A1 expression and increases BaP-DNA adduct formation (Xu et al., 2007). These

findings are similar to our result showing that CYP1A1 induction by BaP plus

1,25(OH)2D3 was associated with BaP-DNA adduct formation (Fig. 3C). By contrast,

DNA adduct formation is increased in CYP1A1-deficient mice (Uno et al., 2004), and

overexpression of CYP1A1 in hepatocytes suppresses BaP-induced DNA adduct

formation (Endo et al., 2008). CYP1A2 is also involved in the suppression of BaP-DNA

adduct formation but it does not cause 3-hydroxy-BaP formation (Endo et al., 2008).

CYP1A2 expression was not increased in U937 cells treated with BaP plus 1,25(OH)2D3

(Fig. 1E). Insufficient expression of CYP1A2 or other detoxifying enzyme(s) may


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contribute to the accumulation of BaP metabolites and to DNA adduct formation.

Monocytes and monocyte-derived leukemia cells exhibit antimicrobial activity by

producing reactive oxygen species in response to 1,25(OH)2D3 (Levy and Malech, 1991;

Sly et al., 2001). BaP enhances differentiation of monocytic THP-1 cells induced by

1,25(OH)2D3 (Matsunawa et al., 2009). Increased reactive oxygen species associated

with monocyte/macrophage differentiation may modify BaP metabolism and promote

DNA adduct formation. In addition to monocytic differentiation, AHR activation

enhances VDR-dependent expression of CYP24A1, which stimulates 1,25(OH)2D3

inactivation (Matsunawa et al., 2009). Thus, combined administration of BaP and

1,25(OH)2D3 enhances BaP-DNA adduct formation and 1,25(OH)2D3 catabolism through

CYP1A1 and CYP24A1, respectively. These signaling pathways may be related to

BaP-induced toxicities.

CYP1A1 is involved in the phase I metabolism of xenobiotics, such as BaP, and

endogenous compounds, including estradiol and eicosanoids (Nebert and Karp, 2008;

Uno and Makishima, 2005). Induced differentiation of myeloid leukemia cells by

1,25(OH)2D3 is associated with increased expression of enzymes involved in eicosanoid

metabolism, such as arachidonate 5-lipoxygenase and prostaglandin-endoperoxide

synthase 1 (Amano et al., 2009; Matsunawa et al., 2009). Prostaglandin production plays

a role in inducing differentiation of myeloid leukemia cells (Rocca et al., 2004). CYP1A1

induced in monocyte-derived cells may be involved in monocyte/macrophage function


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by altering eicosanoid metabolism. Recently, AHR has been implicated in the regulation

of immunity and inflammation (Kerkvliet, 2009). TCDD pretreatment enhances

cholestasis-induced liver damage and proinflammatory cytokine production, phenotypes

that are further exaggerated in CYP1A1/CYP1A2-null mice (Ozeki et al., 2011). The

AHR-CYP1A cascade may regulate inflammatory responses in immune cells, including

monocytes and macrophages. The enhancing effect of 1,25(OH)2D3 on CYP1A1 mRNA

expression was also observed in breast cancer- and kidney epithelium-derived cells.

Further studies are needed to elucidate the physiological role of CYP1A1 in

monocytes/macrophages and other cell types.


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Acknowledgements

The authors thank members of the Makishima laboratory for technical assistance

and helpful comments, and Dr. Andrew I. Shulman for editorial assistance.


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Authorship Contributions

Participated in research design: Matsunawa, and Makishima

Conducted experiments: Matsunawa, Akagi, Uno, and Endo-Umeda

Contributed new reagents or analytic tools: Uno, Endo-Umeda, and Yamada

Performed data analysis: Matsunawa, Akagi, Uno, and Endo-Umeda

Wrote or contributed to the writing of the manuscript: Matsunawa, Akagi, Ikeda, and

Makishima


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Footnote

This work was supported in part by the Ministry of Education, Culture, Sports, Science,

and Technology of Japan [Grant-in-Aid for Scientific Research on Priority Areas

18077005] (to M.M.) and Nihon University [Nihon University Multidisciplinary

Research Grant for 2005 and 2006] (to M.M.).

Current address of Manabu Matsunawa: Cancer Genomics Project, Graduate School of

Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan


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Legends for Figures

Fig. 1. Effect of BaP and 1,25(OH)2D3 treatment on mRNA expression of CYP1 enzymes.

(A) Combined effect of BaP and 1,25(OH)2D3 on CYP1A1 mRNA expression in

monocytic leukemia-derived U937 cells, THP-1 cells, breast cancer MCF-7 cells, and

kidney epithelium-derived HEK293 cells. Cells were cultured with 0.3 μM BaP and/or 30

nM 1,25(OH)2D3 for 24 hours. (B) Real-time quantitative reverse transcription-PCR

analysis of VDR and AHR in in U937 cells, THP-1 cells, MCF-7 cells and HEK293 cells.

(C) Western blotting analysis of VDR and AHR in in U937 cells, THP-1 cells, MCF-7

cells and HEK293 cells. Cells were cultured with vehicle control or 1 μM BaP plus 30 nM

1,25(OH)2D3 for 24 hours. Each lane was loaded with 50 μg of nuclear proteins. (D)

Effect of 1,25(OH)2D3 on expression of the VDR target gene CYP24A1 in U937 cells and

THP-1 cells. Cells were cultured in the absence or presence of 30 nM 1,25(OH)2D3 for 24

hours. (E) BaP and 1,25(OH)2D3 are not effective in inducing CYP1A2 and CYP1B1

expression in U937 cells. Cells were cultured with 1 μM BaP and/or 30 nM 1,25(OH)2D3

for 24 hours. The values represent means ± S.E.M. of triplicate assays. *p < 0.05; **p <

0.01.

Fig. 2. Concentration-dependent effects of BaP (A) and 1,25(OH)2D3 (B) on CYP1A1

mRNA expression in U937 cells. (A) Cells were treated with the indicated concentrations

of BaP in the absence or presence of 10 nM 1,25(OH)2D3 for 24 hours. *p < 0.05; **p <


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0.01 compared with vehicle control. #p < 0.05; ###p < 0.001 compared with 1,25(OH)2D3

alone. †p < 0.05; ††p < 0.01. (B) Cells were treated with the indicated concentrations of

1,25(OH)2D3 in the absence or presence of 0.3 μM BaP for 24 hours. *p < 0.05; **p <

0.01. The values represent means ± S.E.M. of triplicate assays.

Fig. 3. Effect of BaP and 1,25(OH)2D3 treatment on CYP1A1 protein expression (A), BaP

hydroxylation activity (B) and DNA adduct formation (C). For Western blotting and BaP

hydroxylation assays, cells were cultured with 0.3 μM BaP and/or 30 nM 1,25(OH)2D3

for 24 hours. Western blotting was repeated with similar results. For DNA adduct

detection, U937 cells and THP-1 cells were cultured with 0.3 μM BaP and/or 30 nM

1,25(OH)2D3 for 24 hours and 48 hours, respectively. *p < 0.05; **p < 0.01; ***p < 0.001.

ND, not detected. The values represent means ± S.E.M. of triplicate assays.

Fig. 4. CYP1A1 mRNA induction requires activation of VDR and AHR. (A) CYP1A1

expression is induced by BaP plus LCA acetate. U937 cells were cultured with 0.3 μM

BaP and/or 30 μM LCA acetate for 24 hours. (B, C) CYP1A1 induction by BaP plus

1,25(OH)2D3 is decreased by VDR siRNA (B) and VDR antagonists (C). (B) U937 cells

or THP-1 cells were transfected with control siRNA (Cont-si) or VDR siRNA (VDR-si)

and treated with vehicle control or 1 μM BaP plus 30 nM 1,25(OH)2D3. (C) THP-1 cells

were cultured with vehicle control or 1 μM BaP plus 30 nM 1,25(OH)2D3 in combination


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with vehicle control (Cont), 1 μM ADTT or 1 μM ADMI3. (D) CYP1A1 expression is

induced by TCDD plus 1,25(OH)2D3. U937 cells were cultured with 0.1 nM TCDD

and/or 10 nM 1,25(OH)2D3 for 24 hours. (E) CYP1A1 induction by BaP plus

1,25(OH)2D3 is inhibited by α-naphthoflavone. U937 cells were cultured with 1 μM BaP

and/or 10 nM 1,25(OH)2D3 in the absence or presence of 10 μM α-naphthoflavone (aNF)

for 24 hours. The values represent means ± S.E.M. of triplicate assays. *p < 0.05; **p <

0.01; ***p < 0.001.

Fig. 5. An ER8-type vitamin D response element in the human CYP1A1 promoter. (A)

Schematic illustration of the human CYP1A1 promoter. An ER8 motif (-506 to -525) is

located near a XRE (-489 to -495). Mutations in oligonucleotides used for EMSAs are

shown as MT1 and MT2, compared with the wild-type sequence (WT). PCR primers for

ChIP assays amplify fragments (-379 to -693) containing the XRE and ER8 motifs. (B)

EMSAs show direct binding of VDR-RXR to the human CYP1A1 ER8 motif. WT

oligonucleotides (WT) shown in (A) were labeled and incubated with VDR and/or RXRα

proteins. Preincubation with 100-fold molar excess of unlabeled WT, MT1 or MT2

oligonucleotides, shown in (A), and anti-VDR antibody was performed for competition

and supershift, respectively. (C) U937 cells were cultured with 0.3 μM BaP and/or 30 nM

1,25(OH)2D3 for 6 hours and 24 hours and were subjected to ChIP analysis. PCR results

using control IgG, anti-VDR antibody and anti-RXRα antibody were shown in agarose


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DMD #46839

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gel electrophoresis. (D) THP-1 cells were cultured with 0.3 μM BaP and/or 30 nM

1,25(OH)2D3 for 6 hours and were subjected to ChIP analysis. EMSAs and ChIP assays

were repeated with similar results.


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Vitamin D Receptor Activation Enhances Benzo[a]pyrene ...dmd.aspetjournals.org/content/dmd/early/2012/07/25/dmd.112.046839.full.pdf · DMD #46839 4 Abstract Benzo[a]pyrene (BaP) activates