JBC Papers in Press. Published on July 24, 2003 as ... · Summary Nuclear receptors have been implicated in the transcriptional regulation of expression of a growing number of genes,

Nuclear Receptors CAR and PXR Activate a Drug Responsive Enhancer of the Murine 5-Aminolevulinic Acid Synthase Gene

David J. Fraser, Adrian Zumsteg and Urs A. Meyer

Department of Pharmacology / Neurobiology, Biozentrum of the University of Basel,

Klingelbergstrasse 70, CH-4056 Basel, Switzerland.

Running Title: Drug Induction of Mouse 5-Aminolevulinic Acid Synthase

Corresponding Author: Urs A. Meyer, Division of Pharmacology / Neurobiology, Biozentrum of

the University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland, Phone +41-61-267-

22-21, FAX +41-61-267-22-08, email [email protected]

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Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on July 24, 2003 as Manuscript M306148200 by guest on M

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Summary

Nuclear receptors have been implicated in the transcriptional regulation of expression of a growing

number of genes, including cytochromes P450 and 5-aminolevulinate synthase (ALAS1), the first

and rate-limiting enzyme in the heme biosynthesis pathway. Although drugs that induce

cytochromes P450 also induce ALAS1, the regulatory mechanisms governing these pathways have

not been fully elucidated. We have identified a drug-responsive enhancer in the murine ALAS1

gene. This sequence mediates transcriptional activation by a wide range of compounds including

typical cytochrome P450 pan-inducers phenobarbital and metyrapone, as well as specific activators

of the pregnane X receptor and the constitutive androstane receptor. ALAS1 drug responsive

enhancer sequences were identified by transient transfection of reporter gene constructs in the drug-

responsive leghorn male hepatoma cell line. Using the NUBIScan algorithm, DR4 nuclear receptor

binding sites were identified within the elements and their roles in mediating transcriptional

activation of ALAS1 were confirmed by site-directed mutagenesis. Electrophoretic mobility shift

assays demonstrate clear interactions of mouse pregnane X receptor and constitutive androstane

receptor on the ADRES. Transactivation assays in CV-1 cells implicate the nuclear receptors as

major contributors to transcriptional activation of ALAS1. Moreover, in-vivo studies in knockout

animals confirm the induction of ALAS1 is mediated at least in part by nuclear receptors. These

studies are the first to explain drug induction via drug-response elements for mammalian ALAS1.

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Introduction

The body is under constant assault by drugs and xenobiotics, which can be ingested through diet or

absorbed through lungs or skin. In order to prevent toxicity, these foreign compounds must be

detected by the body and converted to water-soluble metabolites and eliminated. Recent studies

have demonstrated that ligand-dependent transcription factors known as orphan nuclear receptors

(NRs) mediate the induction of cytochromes P450 (CYPs) and chicken ALAS1 in response to

xenobiotic exposure (1-5). NRs regulate genes involved in biotransformation by binding as

monomers, homodimers or heterodimers to specific DNA response elements (6-8). A number of

NRs, including pregnane X receptor (PXR) and the constitutive androstane receptor (CAR),

heterodimerize with retinoid X receptor (RXR) which then bind to cognate DNA recognition

elements (9,10). Transcription rates of target genes are modified by NR binding to these sites,

which normally consist of two hexamer half-sites spaced by a variable number of nucleotides. In

particular, NRs are essential in the induction of members of the CYP superfamily of drug

metabolizing enzymes in chicken (11,12) humans (13-19) and rodents (20,21).

The first and rate-limiting enzyme in the heme biosynthesis pathway is 5-aminolevulinate synthase

(22). Two isoforms of ALAS encoded by distinct genes located on different chromosomes are

found in eukaryotes. The erythroid form ALAS2 is expressed in hematopoietic tissue and is

essential for the generation of functional hemoglobin in erythrocytes whereas ALAS1 is the

housekeeping form that is expressed ubiquitously, providing heme for CYPs and other

hemoproteins. In liver and some other tissues, ALAS1 is inducible by drugs. Inherited defects in

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genes encoding enzymes in the heme biosynthesis pathway are associated with disorders known as

porphyrias, a family of diseases in which increased ALAS1 expression precipitated by drugs have

been associated with neuropsychiatric symptoms (23). Additionally, there are a number of acquired

disorders of heme synthesis including poisoning with lead and other heavy metals (24).

Because ALAS is the rate-limiting enzyme in the heme pathway, it has been the focus of numerous

studies examining the mechanisms of coordinated heme and apocytochrome synthesis during drug

induction of CYPs (25-27). Under normal physiological conditions, free heme levels are low and

tightly regulated, as toxicity can occur with increased cellular concentrations of unincorporated

heme. Following administration of drugs such as phenobarbital (PB) or other prototypical CYP

inducers, heme concentrations are elevated in the liver to accommodate the increased levels of

heme-dependent enzymes (23,27,28). This is achieved by induction of ALAS1 and assures an

adequate and apparently coordinated supply of heme for the generation of functional cytochrome

holoproteins. After accumulation of ALAS1 mRNA and protein, free heme represses hepatic

ALAS1 by a number of negative feedback mechanisms that can inhibit the transport of ALAS1 into

the mitochondria, increase heme degradation by inducing heme oxygenase, and decrease ALAS1

mRNA stability directly (29,30). In this way, the cell can provide an adequate supply of heme when

required while preventing the potentially dangerous accumulation of heme and heme precursors.

In the present work, we characterize a drug responsive element isolated from the 5’-flanking region

of the gene encoding murine ALAS1. This element responds to a wide range of drugs and is

referred to as aminolevulinic acid synthase drug responsive enhancer sequence (ADRES) element,

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similar to those previously identified in avian systems (5). Site-directed mutagenesis data

demonstrate ADRES-mediated drug response to be conferred by DR4-type NR recognition

sequences. Gel-shift assays and transactivations support the hypothesis that both PXR and CAR

can contribute to the transcriptional activation of the ALAS1 gene in a drug-specific fashion. The

observed effects of drugs on ALAS1 mRNA transcription in LMH cells closely mirror the pattern of

induction exhibited by the ADRES elements in response to diverse chemical inducers. In-vivo

studies in wild type and PXR-null mice confirm the role of PXR in ALAS1 transcriptional

activation by NR-linked chemical inducers.

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Experimental Procedures

Reagents. Metyrapone (2-methyl-1,2-di-3-pyridyl-propadone) and 5-pregnene-3β-ol-20-one-

16α-carbonitrile (PCN) were purchased from Sigma Aldrich. Glutethimide was purchased from

Aldrich. Mifepristone (RU-486) was obtained from Roussel-UCLAF. 1,4-Bis[2-(3,5-

dichloropyridyloxy)]benzene (TCPOBOP) was generously provided by U. Schmidt (Institute of

Toxicology, Bayer, Wuppertal, Germany). Propylisopropylacetamide (PIA) was a gift from Dr.

Peter Sinclair (Veterans Affairs Hospital, White River Junction, VT). Phenobarbital sodium salt (5-

ethyl-5-phenyl-barbituric acid sodium salt) was purchased from Fluka. Tissue culture reagents,

media, and sera were purchased from Life Technologies. All other reagents and supplies were

obtained from standard sources.

Plasmids. The pGL3LUC luciferase reporter containing an SV40 promoter was purchased from

Promega and modified to generate the pLucMCS vector as described previously (5). The

pBLCAT5 and pBLCAT6 chloramphenicol acetyl transferase reporter vectors were a kind gift from

Guenter Schuetz, DKFZ, Heidelberg, Germany. Mouse CAR was generated from a mouse liver

cDNA library using a 5’-GGA ATT CAT GAC AGC TAT GCT AAC A-3’ upstream and 5’-CGG GAT CCT C

CAA AAT CTC CCC-3’ downstream primers and cloned into the pSG5 expression vector (Stratagene).

Restriction sites incorporated into the primers to facilitate cloning are indicated in bold type. The

murine PXR expression construct was the generous gift of S. Kliewer, University of Texas

Southwestern Medical Center, Dallas, Texas. The pRSV β-galactosidase vector used for

normalization of transfection experiments was kindly provided by Anastasia Kralli (Biozentrum,

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University of Basel, Basel, Switzerland).

BAC Clone Isolation. A specific probe for the murine ALAS1 gene was generated via PCR using a

mouse ALAS1 cDNA template provided by R. Jover (University Hospital, Valencia, Spain) and

forward primer 5’-GTT CGC AGA TGC CCA TTC-3’ and reverse primer 5’-ATG ATG TCC

TGG AAG TTC TT-3’. The probe was 32P-radiolabeled using the random primer labeling kit

(Roche Molecular Biochemicals) according to the manufacturer’s instructions. A genomic BAC

library generated from adult male C57BL6 mouse liver was purchased from Genome Systems, Inc.

The ALAS1 probe was used to identify two individual BAC clones containing the ALAS1 gene,

which were then obtained from the library manufacturer.

Cloning of Upstream Flanking Region. The BAC clones were used to isolate the 5’-flanking region

of the ALAS1 gene using standard Southern hybridization and chromosome walking procedures.

Briefly, 10µg of BAC clone DNA was digested with a range of common restriction endonucleases

and fragments were separated on 0.7% agarose gels. The DNA was transferred onto Hybond-N+

nucleic acid transfer membranes (Amersham Biosciences) and probed with the same 32P-

radiolabeled ALAS1 fragment used to identify the BAC clones. Initially, an 11kb HindIII fragment

extending 1.2kb upstream from the ALAS1 transcriptional start site was identified and cloned into

the pBluescript SK+ vector (Stratagene). Internal forward 5’-GCT TTC ATA AAT CTG GCC-3’

and reverse 5’-GGT GGC CTC CAA CTT TGG-3’ primers were used to generate a second probe

at the 5’-end of the 11kb clone to identify the upstream 4.4kb XbaI-SmaI fragment. Internal 5’-

AGA ACA GTG TTG GAG GTG G-3’ forward and 5’-GAT TCC ATT ACG GAT GG-3’

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reverse primers were employed to identify the 4.8kb HindIII fragment. The 7.8kb XhoI-EcoRI

fragment was then isolated using a probe generated with 5’-GCT TAA GAA TTT CAA TAC-3’ forward and 5’

GGT TCA CAG CTG ATA CC-3’ reverse primers. The inducible 2.8kb HindIII element extending

from bp –15344 to bp –18184 relative to the transcriptional start site was identified using a probe

amplified with 5’-ACA ATC TTG ATC ACA TGG-3’ forward and 5’-GAG CAA TTG AGT

GTC CAC-3’ reverse primers. DNA fragments were ligated into the pLucMCS reporter vector to

test for in vitro drug response. All clones were confirmed by sequencing using an ABI Prism 310

Genetic Analyzer.

Subcloning. The drug-inducible 2840bp HindIII fragment was reduced to a 369bp element using

PCR and standard subcloning procedures. Initially, the 2.8kb clone was analyzed using PCR-

generated subfragments. Upstream forward primers 5’-TAA ACA AGG CCA CCA CTG-3’, 5’-

TCT GGA ATC AGA CTT GGC TCA-3’ and 5’-TTC CCT CTT TAG ACT CCA GA-3’ were

used in conjunction with a 5’-TAT GTG GTC CCA GGC TGC TG-3’ downstream primer to

generate 2004bp, 1756bp and 1414bp products, respectively. In addition, forward 5’-ATC TGA

TGG CCT CTT CTG-3’ and reverse 5’-GAG CAA TTG AGT GTC CAC-3’ primers were used to

generate a 1094bp fragment encompassing the region between the 2004bp element and the 7.8kb

clone. The 1756bp response element was then reduced to 707bp by excision of the AccI fragment

and ligation into the pLucMCS vector. The 707bp clone was further subdivided into 160bp

PstI/SacI, 170bp SacI/XbaI and 377bp XbaI/AccI fragments which were ligated into the pLucMCS

vector. The 280bp and 328bp clones were generated using 5’-GGG GTA CCA GTC CAG TCA

GAA CCT TC-3’ and 5’-GGG GTA CCG TGG CTG GGT TGG GAT GG-3’ forward primers,

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respectively, along with a 5’-CCG CTC GAG TTT TCT GAG TCC ATG ACA-3’ reverse primer.

The 321bp and 369bp response elements were produced using the 5’-GGG GTA CCA GTC CAG

TCA GAA CCT TC-3’ and 5’-GGG GTA CCG TGG CTG GGT TGG GAT GG-3’ forward

primers in conjunction with a 5’-CCG CTC GAG ATC ATA GGA GGG AGC AGC-3’ reverse

primer. Restriction sites incorporated into the primers to facilitate cloning and are indicated in bold

type. PCR products were then digested with KpnI and XhoI restriction endonucleases and ligated

into the pLucMCS vector. Single copies of the 369bp wild type and mutated elements were cloned

into the pBLCAT5 reporter vector by excising a 387bp fragment with Acc65I and XhoI restriction

endonucleases. Ends were filled in with the Klenow fragment of E. coli DNA polymerase I and

ligated into SmaI digested pBLCAT5 vector. The 2.8kb mutant sequences were generated by

inserting the 166bp SacI-XbaI fragment containing the DR4-1/hs1,2, DR4-2/hs1,2 and DR4-

1,2/hs1,2 mutations into the wild type 2840bp construct in pLucMCS. The natural promoter for the

ALAS1 gene spanning base pairs –1231 to +43 relative to the transcriptional start site was then

isolated from the ALAS1 BAC clone as a HindIII-XhoI fragment and cloned into the promoterless

CAT6 reporter vector. The wild type and mutant 2840bp HindIII fragments were then subcloned

into the CAT6 vector containing a 1.2kb segment of the mouse ALAS1 natural promoter.

Site-directed Mutagenesis. DNA fragments were examined for putative nuclear receptor binding

sites using NUBIScan analysis software, a program which uses positional weighted matrices for the

identification of individual nuclear receptor half sites within an empiric model for dimer binding

(31). For these studies, a Z-score threshold of 4 was used to identify hexamer halfsite direct repeats

with a four-nucleotide spacer. Mutations in the putative DR4 nuclear receptor binding sites were

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introduced into identified the ADRES by PCR using standard overlap techniques. Briefly,

subfragments were amplified with overlapping primers carrying the desired mutations and vector

primers. These subfragments were then combined and used as template in a second PCR using

vector primers to amplify the full-length mutated fragment, which was subsequently digested with

appropriate enzymes and cloned into pBLCAT5. All mutations are shown in bold. The DR4-1/hs1

and DR4-1/hs2 single mutant constructs were made with 5’-GGA GAG CTA AGC TTA CCG

AGT TCG-3’ and 5’-GGG TGA ACC GAA GCT TTT TGC ACT GCC-3’ forward primers in conjunction with

CGA ACT CGG TAA GCT TAG CTC TCC ACC-3’ and 5’-GGC AGT GCA AAA AGC TTC GGT TCA CCC

respectively. DR4-1/hs1,2 double mutation construct was generated with 5’-GGA GAG CTA

AGC TTA CCG AAG CTT TTT GCA CTG CC-3’ forward and 5’-GGC AGT GCA AAA AGC

TTC GGT AAG CTT AGC TCT CCA CC-3’ reverse primers. DR4-2/hs1 and DR4-2/hs2 single

mutants were produced with 5’-CGA GTT CGT TGA ATT CGC CTT GGC C TG-3’ and 5’-GCA CTG CCT C

TGT GGC TTC-3’ forward primers in conjunction with 5’-AGG CCA GGC GAA TTC AAC GAA CTC GGT-

5’-GCC ACA CAC CTG CAG AGG CAG TGC AA-3’ reverse primers, respectively. DR4-2/hs1,2 double

mutants were generated with 5’-AAT TCG CCT CTG CAG GTG TGT GGC TTC-3’ forward and 5’-GCC ACA

CTG CAG AGG CGA ATT CA-3’ reverse primers. DR4-1,2/hs1,2 quadruple mutants were generated with a

5’-GCT AAG CTT ACC GAA GCT TTT GAA TTC GCC-3’ upstream primer and a 5’-GGC GAA TTC AAA

CTT AGC-3’ downstream primer. Following PCR overlap, the products were digested with

appropriate restriction endonucleases, ligated into pLucMCS and pBLCAT5 and verified by

sequencing.

Cell Culture. Leghorn Male Hepatoma (LMH) and CV-1 monkey kidney cells were obtained from

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the American Type Culture Collection. LMH cells were cultivated in 10 cm dishes in Williams E

medium supplemented with 10% fetal bovine serum, 1% glutamine (2mM) and 1%

penicillin/streptomycin (50 IU/ml). Dishes coated with 0.1% gelatin were used for routine culture of

LMH cells in order to facilitate proper seating of the cells onto the plastic plate surface. For

transfections, cells were seeded onto 12-well Falcon 3043 dishes and expanded to 70-80%

confluency. LMH cells were then maintained in serum-free Williams E media for 24 hours and

transfected using the FuGENE 6 transfection reagent (Roche Molecular Biochemicals) according to

the manufacturer’s protocol. CV-1 monkey kidney cells were maintained in Dulbecco’s Modified

Eagle Medium supplemented with 10% FCS, 1% glutamine (2mM) and 1% penicillin/streptomycin

(50 IU/ml) for use in transactivation studies.

Transactivations. Experiments to determine the ability of mouse PXR and CAR to mediate

induction of ALAS1 were done in CV-1 cells according to methods previously described (32,33).

Briefly, cells were expanded for three days on 10cm Falcon 3003 dishes in DMEM/F12 medium

(Gibco BRL) without phenol red supplemented with 10% charcoal-stripped FBS. Cells were then

plated onto 6-well dishes and expanded overnight to approximately 30% density. Cells were then

rinsed with PBS and maintained for transfection in Optimem (Gibco BRL) without further

additions. Transfection of 1µg reporter plus 800ng of pSV β-galactosidase construct and 50ng of

expression vector encoding CAR or PXR was performed using 3µl of LipofectAMINE (Invitrogen)

per well, according to the manufacturers protocol. After 24h incubation, cells were rinsed with PBS

and DMEM/F12 containing 10% delipidated/charcoal-stripped FBS containing either drugs or

vehicle control was added. Because CAR is constitutively active, induction capacity was

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determined by the ability of inducers to overcome the inhibition of CAR activity by 10µM

androstanol. After 16 hours induction, cells were rinsed with PBS and lysed in 600µl CAT lysis

buffer and assayed for CAT enzyme using the CAT-ELIZA kit (Roche Molecular Biochemicals).

CAT levels were then normalized against β-galactosidase levels to compensate for variations in

transfection efficiency.

Analysis of Reporter Gene Expression. Cells were treated with drugs or vehicle for 16h and

harvested. For luciferase assays, lysis was performed with 200µl Passive Lysis Buffer (Promega)

per well and extracts were centrifuged for 1 minute to pellet cellular debris. Luciferase assays were

performed on supernatants using the Luciferase Assay kit (Promega) and a Wallac 1420 Multilabel

Counter. Relative β-galactosidase activities were determined as described (34). For CAT assays,

cells were lysed with 600µl CAT lysis buffer per well and extracts were centrifuged for 1 minute to

pellet cellular debris. Assays were performed using a CAT ELISA kit (Roche Molecular

Biochemicals) according to the manufacturer’s protocol.

Gel mobility-shift assays. Murine CAR, PXR and RXR proteins were expressed using the TNT T7

Quick Coupled Translation System (Promega) according to the manufacturer’s protocol. For DNA

fragment labeling, ends were filled in with the Klenow fragment of E. coli DNA polymerase I in the

presence of radiolabeled [α-32P]ATP and purified over a Biospin 6 chromatography column. A

volume of labeled oligonucleotide corresponding to 50,000 cpm was used for each reaction in

10mM Tris-HCl (8.0) / 40mM KCl / 0.05% Nonidet P40 / 6% glycerol (vol./vol.) / 1mM DTT

containing 0.2µg of poly(dI-dC) and 2.5µl in-vitro synthesized proteins as described previously

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(13,32). The reaction mix was incubated for 20 min at room temperature and electrophoresed on a

6% polyacrylamide gel in 0.5X Tris / borate / EDTA buffer followed by autoradiography.

Experimental Animals. Wild-type 6-8 week old male C57BL6 mice were obtained from Iffa Credo

Laboratories (Strasbourg, France). PXR +/- mice were the kind gift of S. Kliewer and

GlaxoSmithKline, Research Triangle Park, North Carolina, USA. Mice were maintained on

standard laboratory chow and were allowed food and water ad libitum. Eight- to 10-week old

mice were deprived of food for 16 hours prior to treatment with chemical inducers. Wild-type

animals in groups of 4 were injected IP one time with vehicle alone (corn oil plus 5%DMSO), PCN

(40 mg/kg), Phenobarbital (100 mg/kg), TCPOBOP (3mg/kg), Metyrapone (100 mg/kg) or RU-486

(10 mg/kg). Similarly, eight- to 12-week old PXR heterozygous and PXR-null mice were treated

with vehicle, PCN (40 mg/kg) or TCPOBOP (3 mg/kg). After ten hours, animals were killed and

livers harvested and maintained in RNAlater according to the manufacturer’s protocol (Ambion

Inc., Austin, TX, USA ). Liver tissue samples weighing approximately 50-100mg were solubilized

in 1ml TRIzol (Invitrogen Inc, Basel, Switzerland) and homogenized for 5 seconds in FastRNA

tubes using a FastPrep FP120 homogenizer from Qbiogene (Carlsbad, CA, USA).

Quantitative PCR. One microgram of total RNA was reverse transcribed with the Moloney murine

leukemia virus reverse transcriptase kit (Roche Molecular Biochemicals). PCR was performed

using the Taqman PCR core reagent kit (PE Applied Biosystems) and transcript levels quantitated

with an ABI Prism 7700 sequence detection system (PE Applied Biosystems). Relative transcript

levels were determined using the relative quantitation method measuring the ””Ct. The following

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primers and probes were used in these reactions. ALAS1: probe, 5-TTC CGC CAT AAC GAC GTC AAC CAT

CTT-3; forward primer, 5’-GCA GGG TGC CAA AAC ACA T-3’; reverse primer, 5’-TCG ATG GAT CAG AC

ACA-3’. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH): probe, 5’-TGG CGT GCC CAT TGA TCA CA

TTT-3’; forward primer, 5’-GGT CAC GCT CCT GGA AGA TAG T-3’; reverse primer, 5’-GGG CAC TGT CA

GA-3’. Primers and probes were used in rtPCR reactions at concentrations of 9µM and 3µM,

respectively. Transcript levels were measured in separate tubes and GAPDH levels were used for

normalization of ALAS1 levels.

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Results

A BAC clone containing an insert approximately 80kb in length spanning the mouse ALAS1 gene

was isolated and sequenced. Four major subclones were generated from the region upstream of the

transcriptional start site, including a 4374bp XbaI/SmaI and 4824bp HindIII fragments as well as

7802bp XhoI/EcoRI and 2840bp HindIII segments (Fig. 1A). The Xba/SmaI clone extends from -

318bp to -4691bp, whereas the HindIII subfragment spans the region from -3537bp to -8360bp.

The XhoI/EcoRI segment extends from –15755 to –7954 bp while the 2840bp HindIII clone

extends from –18184bp to –15344bp upstream of the transcriptional start site. These subfragments

were cloned into the pLucMCS modified luciferase vector containing an SV40 promoter as

described in Materials and Methods. Drug inducibility was measured in transiently transfected

chicken LMH cells treated with 500µM metyrapone and compared with control values from cells

treated with vehicle alone. The results revealed the 2840bp subfragment to be inducible with

metyrapone, displaying a 6-fold increase in transcriptional activation relative to control values. In

contrast, the 7.8kb, 4.8kb and 4.2kb subfragments exhibited virtually no transcriptional activation in

response to drug treatment (Fig. 1A). The 2840bp subfragment (-18184/-15344) was chosen for

further analysis and was divided into numerous subclones in the pLucMCS reporter vector resulting

in the isolation of the 369bp inducible element (Fig. 1B). This sequence routinely exhibited 10-15

fold induction over control values in reporter gene assays when exposed to metyrapone in LMH

cells (Fig. 1B). Because the 369bp fragment retains high drug response regardless of orientation or

distance from the promoter (data not shown), it is referred to as the aminolevulinic acid synthase

drug responsive enhancer sequence (ADRES) element.

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We next wanted to compare ADRES-mediated induction levels in reporter gene assays after

exposure to a variety of chemical inducers (Fig. 1C). The compounds examined include PB (500

µM) and the PB-like inducers PIA (250µM), glutethimide (500µM), and the potent mouse CYP 2B

inducer 1,4-bis[2-(3,5-dichloropyridyl-oxy)]benzene (TCPOBOP) (10µM). In addition, the

common CYP3A inducers dexamethasone (50µM), metyrapone (500µM), and 10µM mifepristone

(RU-486) as well as the anti-fungal agent clotrimazole (10µM) were employed for comparison.

We were also interested in the effects of 10µM 5-pregnen-3β-ol-20-one-16α-carbonitrile (PCN)

and rifampicin (100 µM) due to their species-specific effects on PXR activation and CYP3A

induction. Metyrapone was the strongest inducer in LMH cells, increasing luciferase expression an

average of 10 fold relative to basal transcript levels (Fig 1C). The general inducers PIA and

glutethimide, as well as the antifungal clotrimazole exhibited marked effects upon the ADRES

elements, while PB was a moderate inducer, eliciting a 3-fold response. In contrast,

dexamethasone, PCN, RU-486, and rifampicin had minor or no effects on either mRNA levels or

ADRES activation. Moreover, the mouse-specific compound TCPOBOP elicited no stimulation of

the ADRES in reporter assays. With the exception of a somewhat muted phenobarbital response,

these experiments indicate a high degree of similarity in the activation profile of the ADRES

element in reporter gene assays when compared to those previously reported for the chicken

ADRES elements (5).

Recent discoveries have implicated NRs in drug mediated enzyme induction (5,12,32) and are

reviewed in (10,35). For this reason, we scanned the responsive element for potential nuclear

receptor response sites using the NUBIScan computer algorithm which is based on a weighted

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nucleotide distribution matrix compiled from published functional hexamer half sites (31). For each

prediction, the Z score was calculated and is presented in Figure 2. The Z score corresponds to the

number of standard deviations from the mean of the similarity scores across the whole sequence.

Thus, a higher Z score indicates a match less likely to happen at random. With a threshold Z score

setting of 4 standard deviations, three potential DR4-type binding sites for orphan NRs were

identified in the ADRES element (Fig. 2). For clarity, the three putative binding sites are labeled

according to their occurrence in the gene, with the furthest upstream from the transcription start site

called DR4-1 and the closest to the start site DR4-3. The putative DR4-1 is in the forward

orientation relative to transcription and is defined by half-sites at –17714GGGTGA-17709 and

–17704AGTTCG-17699 respectively. The DR4-2 and DR4-3 sites are found in the reverse

orientation, with DR4-2 consisting of half-sites –17651AGGCCA-17656 and –17661AGTGCA-17666

and DR4-3 encoding half sites –17417AGTCCA-17412 and –17407AGATCT-17402, respectively.

In order to investigate the roles of PXR and CAR in the activation of the ADRES elements,

transactivation experiments were done in CV-1 monkey kidney cells. These cells express RXR but

exhibit no induction response in the absence of CAR or PXR (data not shown). The wild type 369bp

element was cloned into the pBLCAT5 plasmid containing a thymidine kinase minimal promoter as

described in Materials and Methods. CAT vectors were used for transactivations rather than

luciferase because CAT provided more stable expression and showed higher drug response. The

pSG5 expression vector containing the coding sequence for murine CAR or PXR as well as a β-

galactosidase expression construct to correct for variations in transfection efficiency were

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cotransfected along with the reporter construct. After 24h incubation to allow for the expression of

the NR, induction of the wild type and mutant sequences was tested with a range of inducers

including CAR-specific TCPOBOP and the PXR-specific activators RU-486 and PCN as well as

the CYP broad spectrum inducers phenobarbital and metyrapone. Because CAR is constitutively

active, these experiments were performed in the presence of androstanol, an inverse CAR agonist.

As shown in Figure 3, the 369bp ADRES element is transactivated by both PXR and CAR. PXR-

mediated induction levels of 9- and 14-fold were observed with RU-486 and PCN, respectively,

while metyrapone elicited a modest 3-fold activation. In comparison, PB and TCPOBOP elicited

no effects in cells expressing PXR. CAR-transfected cells mediated a 9-fold induction with

TCPOBOP, as well as 3-fold activation by metyrapone, whereas no CAR-mediated activation was

observed with phenobarbital, RU-486 or PCN (Fig. 3B). These findings are in agreement with

previous studies indicating that TCPOBOP acts in a CAR-dependent fashion while PCN and RU-

486 are PXR-specific activators. Moreover, they demonstrate that the activation of the 369bp

ADRES element is mediated at least in part by the nuclear receptors CAR and PXR.

Because several putative DR4-type NR binding sites were identified in the 369bp ADRES, we next

determined the roles of each site in transcriptional activation. Based on the findings presented in

Figure 1 that the 377bp fragment containing DR4-3 does not respond to drugs whereas the 170bp

minimal element encoding DR4-1 and DR4-2 still retains induction capacity, we concluded that

DR4-3 was inactive and did not warrant further investigation. Site-specific mutagenesis was used

to examine the roles of specific nucleotides within the DR4-1 and DR4-2 recognition sequences in

conferring drug response to the ADRES element (Fig. 4). Mutant constructs of the DR4 core

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recognition sites destroying the putative NR binding sites were generated as described in Materials

and Methods and are shown in figure 4.

To test the effects of the mutations on transcriptional activation, transactivations in CV-1 cells with

PXR and CAR and their respective ligands PCN and TCPOBOP, were performed (Fig. 4). As seen

in Figure 4A, the single mutations in the DR4-1 binding site reduced the induction by PCN to 2.7-

fold and 2.0-fold, respectively, while the double mutant exhibited 1.9-fold activation. The single

mutations in DR4-2 did not significantly reduce induction relative to the wild type value of 5.6-

fold, displaying 5.8- and 5.3-fold activation, respectively, whereas the double mutant was reduced

to 2.5-fold induction. The alteration of both NR binding sites in the 369bp element resulted in the

elimination of PXR-mediated drug response. In Figure 4B, CAR transactivations of the DR4-1

single and double mutants did not exhibit any response to TCPOBOP, with even the basal CAR

activity eliminated. In comparison, the DR4-2/hs1 and DR4-2/hs2 single mutants still exhibited

basal expression that exceeded androstanol-treated levels by 2.5- and 1.9-fold, respectively.

TCPOBOP induction exceeded basal levels in the DR4-2/hs1 construct at a modest 1.4 fold,

whereas no induction was observed for either the DR4-2/hs2 construct or the double mutant. For

the DR4-1,2/hs1,2 quadruple mutant, no basal increase in CAT expression was observed in the

absence of androstanol and the induction capacity was completely eliminated. As depicted in Figure

4, both DR4-1 and DR4-2 sites in the 369bp ADRES element were found to be required for full

activation by either PCN or TCPOBOP. Together, the data indicate that DR4-1 is essential for NR-

mediated induction via CAR or PXR, whereas DR4-2 might contribute in a more indirect fashion to

the overall activation of the 369bp ADRES. These studies demonstrate an essential contribution of

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the sequences within the putative DR4 NR binding sites to drug induction of the ADRES elements.

As both DR4-1 and DR4-2 NR binding sites contribute to the transcriptional activation exhibited

by the ADRES elements in transactivations, gel-mobility shift assays were used to determine where

PXR and CAR might bind within the enhancer (Fig. 5). The 369bp wild type and mutant constructs

were 32P-radiolabeled and examined in the presence of mouse RXR and either mouse PXR (Fig.

5A) or mouse CAR (Fig. 5B). In vitro transcribed/translated mouse RXR or mouse PXR alone did

not interact with the 32P-radiolabeled 369bp ADRES (Fig. 5A, lanes 1 and 2), whereas PXR/RXR

heterodimers bind the wild type drug responsive enhancer (Fig. 5A, lane 3). PXR/RXR binding to

the 369bp ADRES element was eliminated in the DR4-1/hs1 and DR4-1/hs2 single mutants as

well as the DR4-1/hs1,2 double mutant DNA sequences, as demonstrated by the absence of bands

in lanes 4, 5 and 6, respectively. Interestingly, the DR4-2/hs1,2 construct still bound the PXR/RXR

heterodimer, as seen in lane 7. Moreover, the binding of PXR/RXR heterodimers was absent with

the DR4-1,2/hs1,2 quadruple mutant (Lane 8). In a similar fashion, mouse CAR and RXR were

unable to bind as homodimers to the 369bp ADRES, whereas a shift is observed in the presence of

RXR/CAR heterodimers (Fig. 5B, lanes 1-3). CAR/RXR binding is also eliminated in the DR4-

1/hs1 and DR4-1/hs2 single mutants as well as the DR4-1/hs1,2 double mutant (lanes 4-6,

respectively) but still present in the DR4-2/hs1,2 double mutant (lane 7). Not unexpectedly, the

DR4-1,2/hs1,2 quadruple mutant does not bind CAR/RXR heterodimers as observed in lane 8. In

summary, these findings demonstrate interactions of PXR/RXR and CAR/RXR heterodimers with

the DR4-1 but not the DR4-2 binding sites in the 369bp ADRES elements.

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We next tested the capacity of the 2.8kb inducible fragment for drug response in the presence of the

natural ALAS1 promoter. A 1274bp fragment spanning the ALAS1 promoter was cloned into the

CAT6 promoterless reporter vector along with the 2.8kb wt and mutant drug-response sequences as

described in Materials and Methods. The constructs were then transfected in LMH cells and

examined for response to 500µM metyrapone, the best inducer of the mouse ADRES in LMH cells

as reported in Figure 1C. As seen in Figure 6, the metyrapone induction mediated by the natural

ALAS1 promoter is strong for the wild type sequence, eliciting a 5-fold increase over vehicle

alone. Similar to the previous findings, mutations in the DR4-1 eliminated the induction altogether

(0.70-fold), whereas the DR4-2/hs1,2 double mutant still retained some residual drug response

(1.4-fold). As expected, the DR4-1,2/hs1,2 quadruple mutant did not exhibit any response to

metyrapone treatment (1.0 fold). In addition to these experiments, PXR and CAR transactivation

studies were done with PCN and TCPOBOP, respectively, with similar findings (data not shown).

Interestingly, the ALAS1 natural promoter did increase the basal CAT expression relative to that of

the thymidine kinase promoter found in the CAT5 reporter vector, most likely due to the presence

of a more complete array of binding sites mediating basal transcription in the ALAS1 construct.

These studies demonstrate that the presence of a heterologous promoter does not alter the induction

profile of the 369bp ADRES when compared to the activation mediated by the natural promoter.

Studies were then undertaken to confirm that the induction of ALAS1 mediated by the 369bp

ADRES element in vitro were reflected in whole animal systems. As seen in Figure 7A, all of the

compounds tested increased transcription of ALAS1 mRNA, with induction values ranging from 4-

to 8-fold higher when compared to control levels. The strongest effects were observed for the

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broad spectrum inducers phenobarbital and metyrapone, which exhibited 8.3 and 8.1-fold

increases, respectively, while the CAR-specific inducer TCPOBOP displayed a 7.1-fold increase

over control levels. The PXR-specific inducers PCN and RU-486 increased ALAS1 mRNA levels

by 5.8- and 4.1-fold, respectively.

A number of different studies in null mice lacking either CAR or PXR have implicated NRs as

mediators of enzyme induction by endogenous and exogenous chemical compounds (1,2,36,37). As

we were interested in the contributions of NRs on induction in vivo, we then tested for NR-specific

response in knockout animals that lack the PXR gene. As seen in Figure 7B, the fold induction

mediated by TCPOBOP was not altered in the PXR-knockout animals, displaying 4.5-4.7 fold

increases over those observed for control animals. In contrast, PCN-mediated induction was

reduced from 5.3-fold in heterozygotes to 2.2-fold in animals lacking PXR. Interestingly,

expression of ALAS1 also increased 2.3-fold in PXR knockout animals over heterozygotes,

suggesting a role of PXR in the basal expression of this gene. Taken together, these data indicate

that PXR is responsible for the majority, if not all of the ALAS1 induction mediated by PCN,

whereas TCPOBOP works through other NRs or by other mechanisms.

Discussion

Here we report the first mammalian drug-responsive enhancer for the ALAS1 gene. Previous

studies resulting in the identification of upstream enhancer sequences in chicken led us to

hypothesize that similar elements might be found in the flanking region of the ALAS1 gene in other

species (5). The murine ADRES element differs somewhat from those previously described, in that

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only a single responsive region has been identified within the first 18kb of sequence upstream of the

transcriptional start site of the mouse ALAS1 gene. In addition, 369 base pairs are required for full

induction response in in-vitro studies, making the ADRES a relatively large when compared with

other drug responsive enhancers. In contrast, many other genes encoding enzymes involved in

biotransformation, including the chicken ALAS1 gene, contain at least two distinct regions that

exhibit drug-response. Moreover, most inducible enhancers found in the genes encoding CYPs lie

within the first 8kb of transcriptional initiation, whereas activation of ALAS1 appears to be

modulated by elements residing a greater distance from the coding region.

Once identified, analyses of the murine ADRES were focused on the identification of putative DR4

sites that play an active role in transcriptional activation, as previous studies demonstrated a major

role for this type of NR binding site in ALAS1 drug response. Based on a minimum Z-score value

of 4, sequence analyses with the NUBIScan analysis software resulted in the identification of three

distinct DR4-type nuclear receptor binding sites within the 369bp element (31). Interestingly, the

DR4 with the lowest Z score was found to be the major contributor to induction, underscoring the

power of the NUBIScan algorithm in successfully identifying even weakly conserved recognition

sites for DNA binding transcription factors involved in drug induction. These findings demonstrate

that integrated studies combining in silico and traditional laboratory techniques can be a powerful

combination that, when employed in concert, engender a more complete understanding of the

interactions between these transcription factors and the DNA sequences with which they interact.

The data presented here confirm the hypothesis that drugs regulate the induction of ALAS1

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transcription in mice directly via nuclear receptor-mediated interactions with an upstream enhancer

element. In order to define the range of compounds capable of eliciting induction through the

369bp ADRES, an induction profile was generated from reporter gene assays in the drug-

responsive chicken hepatoma cell line LMH treated with a variety of known ALAS1 inducers. The

in vitro induction profile of the ADRES element reported here is similar but not identical to other

drug-responsive enhancers found in genes encoding avian and mammalian CYPs (11-21). The

mouse ALAS1 sequence mediates induction levels in LMH cells mirroring those reported for the

chicken ALAS1 enhancers as previously reported. High induction with PB, glutethimide, PIA, and

metyrapone was observed, with the other inducers listed exhibiting only modest or no effects (Fig.

1C). As expected, PCN, RU-486 and TCPOBOP are extremely weak activators of the mouse

sequence in LMH cells, reflecting the activation pattern of the chicken xenobiotic receptor

CXR(5,12,32).

Transactivation studies demonstrate a role for PXR and CAR in the activation of the ADRES

element in CV-1 cells. The transactivations presented in Figure 3 demonstrate that PCN and RU-

486 can mediate increases in transcriptional activity in the presence of the nuclear receptor PXR.

Moreover, TCPOBOP can derepress the expression of ALAS1 in cells expressing CAR and treated

with the CAR-specific inhibitor androstanol. It is interesting to note that phenobarbital, which

activates transcription through the 369bp element in LMH cells (Figure 1C), does not transactivate

the 369bp element in CV-1 cells via either CAR or PXR. This may suggest a different mechanism

for the activation of ALAS1 transcription by PB in whole mice. Alternatively, these findings could

also reflect differences in the cells used in transactivations and underscore the delicate nature of

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these types of studies. The possibility also exists that there are other drug-responsive enhancers in

mice that mediate the PB effects seen in vivo. Moreover, the transactivations presented in Figure 6

employing a 1.2kb fragment of the ALAS1 natural promoter do not differ significantly from those

performed with the heterologous thymidine kinase promoter. Taken together, these findings

demonstrate the species specificity of the nuclear receptors PXR and CAR as the determinants of

drug induction profiles.

The mutagenesis studies demonstrate the role of the DR4-1 and DR4-2 recognition sites in

conferring drug-response to the ADRES element. The mutation of either of these DR4 sites results

in substantial reductions in transcriptional activation, and mutation of both sites resulted in a

complete loss of induction mediated by both PXR and CAR (Fig. 4). Mutation of individual half-

sites within DR4-1 was sufficient to eliminate induction mediated by either CAR or PXR in

transactivations. In comparison, the alteration of both DR4-2 half-sites was required to reduce

NR-mediated increases in reporter gene expression, whereas the single mutants of DR4-2 had little

effect on the response profile. This may suggest a more indirect role independent of NRs for the

sequence comprising DR4-2 in the induction mechanisms. Because no activation was observed in

the 377bp fragment containing the putative DR4-3 site in Figure 1B, we concluded that this

potential binding site does not contribute to the drug response observed with the fully active 369bp

core ADRES element. These findings are further supported by both transactivation studies and gel

shift data where neither transactivation activity nor binding of NRs, respectively, was observed in

the presence of the intact DR4-3 sequence. As demonstrated in Figure 1B, efforts to reduce the

size of the ADRES element while keeping the DR4 binding sites intact resulted in reductions in

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drug response, indicating an important role of the flanking regions. These data support the general

concept of complex cooperative behavior between proteins at different sites within the ADRES

acting in concert to generate maximal transcriptional activation.

The binding of PXR/RXR and CAR/RXR heterodimers to the ADRES element in EMSAs further

supports a role of these NRs in ALAS1 regulation. The binding of both CAR and PXR to the 369bp

is eliminated when either of the DR4-1 halfsites within this sequence is mutated. In comparison,

double mutant constructs of the DR4-2 NR binding site still bind PXR and CAR heterodimers in

the ADRES. Taken in light of the mutagenesis data, these findings are a strong indication of the

significant role that the DR4-1 plays in drug induction of the ALAS1 gene and also suggest that the

DR4-1 and DR4-2 NR sites might act in a cooperative fashion. We conclude that both CAR and

PXR can indeed bind to the DR4-1 of the 369bp element and that these interactions can be reduced

when the binding motifs are eliminated. These findings are consistent with the concept that both

CAR and PXR activate transcription of ALAS1.

In vivo studies presented here demonstrate that a wide range of compounds that activate CYP

transcription also increase expression of ALAS1 mRNA in C57BL6 mice (Fig. 7A). The data also

indicate that, with the exception of PB, the pattern of transcriptional activation mediated by the

ADRES in reporter gene assays is similar to that observed at the mRNA level in whole liver. The

strong activation by PB in whole animals but not in in vitro assays may reflect weaknesses in the

transactivation systems or could also indicate the presence of other unidentified PB enhancer(s) in

the ALAS1 gene. Moreover, induction by PB, which has been hypothesized to increase gene

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transcription in an indirect fashion, may not reflect the typical pathway for NR-mediated induction

(37). The studies presented here demonstrate that NR-specific inducers such as TCPOBOP and

PCN can work directly through the murine 369bp ADRES via CAR and PXR, respectively, to

modulate ALAS1 transcription, whereas the indirect inducer PB likely functions through different

and as yet undefined mechanisms.

In PXR-heterozygous mice, induction of ALAS1 mRNA by PCN and TCPOBOP closely

resembles that observed in wild type animals (Figure 7). However, it is interesting that the basal

expression of ALAS1 mRNA in PXR-null mice is 3-fold higher than in PXR heterozygote mice.

This may suggest competition between PXR and CAR for the NR binding site, with the

constitutively active CAR dominating in mice lacking PXR, resulting in increased ALAS1 mRNA

transcription. The PXR null mice lack induction of ALAS1 over basal levels after PCN treatment,

clearly indicating an essential role for PXR in ALAS1 drug induction. These results are in

agreement with recent reports that PXR mediates ALAS1 transcription (3). The role of CAR in

TCPOBOP induction is not as clear due to the differences in basal expression of ALAS1 mRNA

between wild type and null mice. Our data indicate a decrease in ALAS1 mRNA expression in

PXR-null mice after TCPOBOP treatment relative to the high basal levels, suggesting a loss of

induction capacity. This would implicate PXR in the TCPOBOP-mediated induction of ALAS1

transcription. In recent studies examining TCPOBOP and PB induction of ALAS1 in CAR

knockout mice, the authors conclude that CAR does not play a role in the regulation of ALAS1

expression by PB (2,37). Additionally, their data indicate marked decreases in ALAS1 mRNA

levels in untreated CAR-null mice relative to wild-type animals, supporting our findings that CAR

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is an important contributor to the basal expression of ALAS1. To our knowledge, the CAR-specific

inducer TCPOBOP has not been tested for its effects on ALAS1 expression in CAR-null mice.

Based on these findings, we propose a model in which CAR and PXR compete for binding to the

enhancers in the ALAS1 gene, with both NRs contributing to the total induction profile observed in

whole animals. Further studies in CAR-null mice are required to fully test this hypothesis.

Moreover, the development of CAR/PXR double knockout mice would be very useful in

determining the contribution of these nuclear receptors to the whole ALAS1 induction profile in

liver. These data support a role for PXR in PCN-mediated induction of ALAS1 mRNA expression

and also suggest that CAR and PXR might co-regulate this gene by competition for the same

binding sites within the enhancer(s).

We conclude that the regulation of murine heme synthesis is mediated through the direct

transcriptional activation of a 369 base pair drug-responsive enhancer sequence in the ALAS1

gene. A wide range of inducer compounds mediate transcriptional activation, closely reflecting the

in vivo induction of ALAS1 mRNA in mouse liver. Our evidence also supports a role for both CAR

and PXR in the transcriptional activation of ALAS1. This relationship may explain the orchestrated

up-regulation of heme and CYPs observed in mammals exposed to xenobiotics. Moreover, because

the upregulation of heme synthesis always accompanies CYP induction, ADRES-mediated

regulation of ALAS1 may serve as an excellent model system for the analysis of new chemicals and

their effects on enzyme induction, including those that initiate acute attacks of porphyria.

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Footnotes

1 The abbreviations used are: ALAS, 5-aminolevulinic acid synthase; ADRES, aminolevulinic acid

drug responsive enhancer sequence; PB, phenobarbital; DR, hexamer half-site direct repeat; h,

hours; bp, base pairs; LMH, leghorn male hepatoma; kb, kilobases; CYP, cytochrome(s) P450;

CXR, chicken xenobiotic receptor; PXR, pregnane X receptor; CAR, constitutive androstane

receptor; RXR, 9-cis-retinoic acid receptor; PIA, propylisopropylacetamide; PCN, 5-pregnen-3β-

ol-20-one-16α-carbonitrile; TCPOBOP, 1,4-bis[2-(3,5-dichloropyridyl-oxy)]benzene; LUC,

luciferase; mifepristone, RU-486; clotrimazole, 1-[ο-chlorotrityl]-imidazole; EMSA,

electrophoretic mobility shift assay; cpm, counts per minute

2 The sequence reported in this publication has been submitted to the GenBankTM data bank

GenBank Accession Number XYYYYY

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Acknowledgements

The authors would like to acknowledge Markus Beer and Renate Looser for their technical support.

We would also like to thank Dr. Mikael Oscarson for critical reading of the manuscript. This work

was supported by grants from the Swiss National Science Foundation

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Figure Legends

Figure 1. Analysis of the 5’-flanking region of the mouse ALAS1 gene. DNA fragments were

cloned into the pLucMCS luciferase reporter vector containing an SV-40 promoter and tested for

induction response. Constructs were transiently transfected together with a transfection-control

construct expressing β-galactosidase into LMH cells and relative luciferase activity was measured

and standardized against cells transfected with vector containing no insert. Experiments were

repeated at least three times and data from a representative experiment tested in triplicate are shown

here. Error bars represent standard deviations. A, isolation of 2840bp drug-responsive region 18kb

upstream of the ALAS1 transcription start site by chromosome walking as described in Materials

and Methods. Transfected cells were induced for 16h with 500µM metyrapone. B, identification of

the 369bp ADRES. Subfragments of the 2840bp construct were generated as described in Materials

and Methods and relative luciferase activities were measured after 16h induction with 500µM

metyrapone. Numbering refers to sequence positions relative to the transcriptional start site of the

mouse ALAS1 gene. C, comparison of ADRES activation by different drugs. The 369bp ADRES

was tested with a battery of known ALAS1 and CYP inducers. Reporter constructs were transfected

into LMH and cells were treated for 16h with the specified compounds as described in Materials

and Methods. Agonist concentrations were 10µM clotrimazole, 50µM dexamethasone, 500µM

glutethimide, 400µM metyrapone, 500µM phenobarbital, 10µM PCN, 250µM PIA, 10µM

rifampicin, 10µM RU-486 and 10µM TCPOBOP.

Figure 2. DNA sequence of the 369bp enhancer. Numbering refers to sequence positions relative to

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the transcriptional start site of the mouse ALAS1 gene. Sequence was analyzed using NUBIScan

analysis software to identify individual nuclear receptor half sites. A Z-score threshold of 4 was

used to identify hexamer halfsite direct repeats with a four-nucleotide spacer. Solid lines identify

putative DR4 NR binding sites. Shaded boxes contain individual half sites. MatInspector V2.2 was

used to identify putative transcription factor binding sites within the 369bp inducible ADRES

element, with hatched lines marking NF1 binding sites.

Figure 3. Transactivation of the ADRES element by mouse CAR and mouse PXR. CV-1 cells were

transfected with the 369bp element cloned into the pBLCAT5 vector containing a thymidine kinase

minimal promoter. Mouse PXR (A) and mouse CAR (B) coding regions cloned into the pSG5

expression vector were cotransfected along with a vector expressing pSV β↑galactosidase as

control. Cells were then treated for 16h with either drugs or vehicle control and cell extracts were

analyzed for CAT expression normalized against β-galactosidase levels as described in Materials

and Methods. 10µM Androstanol (As) was used in part B to block the constitutive activity of CAR,

revealing the induction potential of the drugs. Agonist concentrations were 10µM TCPOBOP,

500µM phenobarbital, 400µM metyrapone, 10µM RU-486 and 10µM PCN. Experiments were repeated at

least three times and error bars represent standard deviations.

Figure 4. Site-directed mutagenesis of the DR4 sites within the 369bp ADRES element. Mutations

in the DR4 half sites of the 369bp sequence were introduced via PCR-based site-directed

mutagenesis. Constructs are labeled in the left column and mutations are depicted with crosses in

the scheme. Specific base pair alterations are described in Materials and Methods. Experiments

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were repeated at least three times and data from representative experiments tested in triplicate are

shown here. Error bars represent standard deviations. A, CV-1 cells were transfected with the wild

type or mutant ADRES constructs along with expression constructs for mouse PXR. A β-

galactosidase expression construct was used as an internal control. The cells were exposed to

vehicle or 10µM PCN for 16h and cell lysates were tested for CAT activity. Relative CAT levels

are standardized against cells treated with vehicle (control set to 1.0) and expressed as fold

induction. B, CV-1 cells were transfected with the wild type or mutant constructs along with

expression constructs for mouse CAR and a β-galactosidase expression construct as an internal

control. The cells were exposed to vehicle (white bars) or the specific CAR inhibitor androstanol

(10µM) in the presence (shaded bars) and absence (black bars) of TCPOBOP (10µM). Cells were

induced for 16h and cell lysates were tested for CAT activity. Relative CAT levels are standardized

against androstanol-treated cells (control set to 1.0) and expressed as fold induction.

Figure 5. Gel-mobility shift assays demonstrating that CAR and PXR bind the ADRES element.

Radiolabelled ADRES wild type (lanes 1-3) and mutant (lanes 4-8) sequences were incubated with

in vitro transcribed / translated PXR (A) and CAR (B) in lanes 1 and 3-8 along with RXR (lanes

2-8), as indicated. Mutant sequence 1,DR4-1/hs1; sequence 2, DR4-1/hs2; sequence 3, DR4-

1/hs1,2; sequence 4, DR4-2/hs1,2; sequence 5, DR4-1,2/hs1,2 as described in Materials and

Methods and presented in Figure 4. Arrows depict the unbound probe and the shifted PXR-RXR-

probe and CAR-RXR-probe complexes.

Figure 6. Effect of the natural ALAS1 promoter on induction. The 2840bp wild type and mutant

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constructs were transiently transfected together with a transfection-control construct expressing β-

galactosidase into LMH cells and relative CAT activity was measured in the presence of 500µM

metyrapone and standardized against cells transfected with vector containing no insert. Experiments

were repeated at least three times and data from a representative experiment tested in triplicate and

presented as fold induction are shown here. Error bars represent standard deviations.

Figure 7. Induction of ALAS1 in wild type, PXR-heterozygous and PXR-null mice. Total RNA

was prepared from the livers of 3-5 animals per group injected i.p. with inducers or vehicle alone

and real-time PCR analyses were performed with an ALAS1-specific probe. Relative mRNA

levels were measured for each sample in duplicate and are presented as fold induction relative to

vehicle controls. A, Induction of ALAS1 in wild type mice by NR-specific and CYP pan-inducers.

Groups of four male C57BL6 wild type mice were deprived of food overnight followed by i.p.

injection with vehicle, 40 mg/kg PCN, 100 mg/kg PB, 3 mg/kg TCPOBOP, 100 mg/kg metyrapone

or 10 mg/kg RU486, respectively. After 10 hours, animals were killed and livers were harvested.

Messenger RNA was isolated from the tissue and reverse transcribed into cDNA as described in

Materials and Methods. B, Induction of ALAS1 in heterozygote and PXR-null mice by PCN and

TCPOBOP. 8-12 week-old male C57BL6 mice that were either heterozygous for the PXR gene or

homozygous PXR-knockout animals were tested in groups of 3-5 animals for response to either

TCPOBOP (3mg/kg) or PCN (40 mg/kg). All values are compared to the basal expression levels

measured in the PXR heterozygote animals treated with vehicle control (corn oil + 5%DMSO).

Error bars represent standard deviations.

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David J. Fraser, Adrian Zumsteg and Urs A. Meyer5-Aminolevulinic acid synthase gene

Nuclear receptors CAR and PXR activate a drug responsive enhancer of the murine

published online July 24, 2003J. Biol. Chem.

10.1074/jbc.M306148200Access the most updated version of this article at doi:

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JBC Papers in Press. Published on July 24, 2003 as ... · Summary Nuclear receptors have been implicated in the transcriptional regulation of expression of a growing number of genes,