The Toxicologist - Society of Toxicology

that CCNG2 is positively regulated by TCDD via AHR activation, suggesting that
AHR might utilize this pathway to suppress tumor growth. However, the mechanism
by which AHR regulates CCNG2 is unknown. Using chromatin immunoprecipitation
in T-47D human breast cancer cells we report that the prototypical AHR
ligand TCDD (10nM) is able to recruit AHR and nuclear coactivator 3 (NCoA3)
to the enhancer region of CCNG2 (60% and 10% compared to a 5% total) leading
to a 2-fold increase in gene expression after 6h of treatment with TCDD(10nM).
Furthermore, our data reveal that the transcription factor Forkhead box A1
(FOXA1) is recruited to CCNG2 in a TCDD-dependent manner. FOXA1 is
known to be an important pioneer factor controlling the binding of ERα to its target
genes. RNAi-mediated knockdown of FOXA1 abolished TCDD-dependent recruitment
of AHR to CCNG2 and reduced CCNG2 expression levels. Interestingly,
knockdown of FOXA1 also caused a marked decrease in ERα, but not AHR protein
levels. However, RNAi-mediated knockdown of ERα had no effect on TCDDdependent
AHR recruitment to or expression of CCNG2. Our data show that
FOXA1, but not ERα is essential for AHR-dependent regulation of CCNG2, assigning
a role for FOXA1 in AHR action.
372 DIVERGENT ROLES FOR THE ARYL HYDROCARBON
RECEPTOR AND ARYL HYDROCARBON RECEPTOR
NUCLEAR TRANSLOCATOR IN ESTROGEN
SIGNALING.
M. P. Labrecque 1 , K. J. Tam 1 , B. D. Hollingshead 2 , G. G. Prefontaine 1 , G. H.
Perdew 2 and T. V. Beischlag 1 . 1 Faculty of Health Sciences, Simon Fraser University,
Burnaby, BC, Canada and 2 Veterinary and Biomedical Sciences, The Pennsylvania
State University, University Park, PA.
The aryl hydrocarbon receptor (AHR) and the aryl hydrocarbon receptor nuclear
translocator (ARNT) form a heterodimeric transcription factor upon binding a variety
of environmental contaminants, including 2,3,7,8-tetrachlorodibenzo-pdioxin
(TCDD). The activated AHR/ARNT complex (AHRC) regulates the expression
of xenobiotic target genes to combat the effects of environmental
contaminants and evidence has shown that TCDD represses estrogen receptor (ER)
target gene activation through the AHRC. Our data indicates that AHR and
ARNT may act independently of each other at off-target sites so we set out to determine
the specific functions of AHR and ARNT in estrogen dependent signalling.
Human MCF7 breast cancer cells and human ECC-1 endometrial carcinoma
cells were transfected with short inhibitory RNA’s (siRNA) to AHR or
ARNT. Cells were treated with vehicle, 17β-estradiol (10nM), TCDD (2nM) or a
combination of ligands for 16 h and target gene expression and protein levels were
assayed by real-time PCR and Western blotting, respectively. Knockdown of AHR
with siRNA abrogates dioxin-inducible repression of estrogen-dependent gene
transcription. Intriguingly, knockdown of ARNT does not effect TCDD-mediated
repression of estrogen-regulated transcription. Furthermore, basal and activated
levels of cathepsin-D and progesterone receptor mRNA are downregulated in
MCF7 cells but upregulated in ECC-1 cells in response to loss of ARNT. These responses
are mirrored at the protein level with cathepsin-D but progesterone receptor
seems to be refractory. We have obtained experimental evidence demonstrating
a dioxin-dependent repressor function for AHR and a dioxin-independent coactivator/corepressor
function for ARNT in estrogen signalling. These results provide
us with further insight into the mechanisms of transcription factor crosstalk and
putative therapeutic targets in estrogen positive cancers.
373 THE ARYL HYDROCARBON RECEPTOR INTERACTS
WITH ATP5α1, A SUBUNIT OF THE ATP SYNTHASE
COMPLEX, AND MODULATES MITOCHONDRIAL
FUNCTION.
D. M. Tappenden 1, 2 , S. Lynn 1, 2 , R.B.Crawford 3 , N. E. Kamniski 2, 3 , R. S.
Thomas 4 and J. J. LaPres 1, 2 . 1 Biochemistry, Michigan State University, East
Lansing, MI, 2 Center for Integrative Toxicology, Michigan State University, East
Lansing, MI, 3 Department of Pharmacology and Toxicology, Michigan State
University, East Lansing, MI and 4 The Hamner Institutes for Health Sciences,
Research Triangle Park, NC.
Polyaromatic hydrocarbons (PAHs), such as dioxins are prevalent environmental
contaminates produced by both natural and man-made activities. 2,3,7,8 tetrachloro-dibenzo-p-dioxin
(TCDD) is the canonical molecule in this family of compounds.
The aryl hydrocarbon receptor (AHR), a ligand activated transcription factor
and a member of the PAS super-family of environmental sensors, mediates
cellular responses to PAHs. A complete understanding of the signaling pathways involved
in the toxicity induced by ligand activated AHR has not been elucidated. To
explore the potential influences of protein-protein interactions in AHR-mediated
80 SOT 2011 ANNUAL MEETING
toxicity, a tandem affinity purification (TAP) and mass spectrometry strategy was
utilized to isolate and identify proteins that co-purify with the AHR. The known
protein interactions between the AHR, Hsp90, and ARA9 and the ligand-dependent
interaction with ARNT were confirmed, demonstrating the validity of the approach.
Using this method a novel interaction between the AHR and ATP5α1, a
subunit of the ATP synthase complex, has been established. This interaction was
confirmed by co-immunoprecipitation in wild type cells. The ATP synthase complex
is localized to the inner membrane of the mitochondria and is essential for cellular
energetics. Investigation of the interaction between the AHR and ATP5α1 has
revealed that a fraction of the total cellular content of the AHR localizes to the mitochondria.
Furthermore, exposure to TCDD leads to hyperpolarization of the mitochondrial
inner membrane, in an AHR dependent manner, while cellular ATP
levels are maintained. These findings support a role for the AHR in mitochondrial
function and cellular energy homeostasis that is disrupted in TCDD exposure.
374 REGULATION OF CHOLESTEROL BIOSYNTHETIC
GENE EXPRESSION BY AH RECEPTOR THROUGH A
DNA-INDEPENDENT MECHANISM.
R. Tanos, R.D.PatelandG. H. Perdew. Penn State University, University Park, PA.
The aryl hydrocarbon receptor (AHR) is a ligand activated transcription factor.
Activation of AHR by xenobiotic agonists such as TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin)
is known to have toxic consequences through driving the
expression of target genes, namely CYP1A1, by binding to dioxin response element
sequences in their promoter region. Interestingly, Ahr-null mice exhibit a defined
set of physiological phenotypes that indicate an endogenous role for AHR. Recent
microarray studies performed in mice and rats treated with TCDD have shown a
decrease in the expression of lipid metabolism genes. In our effort to understand
the mechanisms of this regulation, we conducted a microarray analysis on liver
samples from ligand-treated transgenic mice expressing a DNA-binding mutant
AHR (A78D) on an Ahr-null background and showed that AHR DNA-binding is
not essential for suppression of genes involved in cholesterol synthesis. To alleviate
the phenotype of Ahr-null mice, we established a mouse line expressing a liver specific
DNA-binding mutant AHR on a CreAlb/AhrFx/Fx background. Real-time
qPCR performed on these mice as well as wild-type mice confirmed repression of
cholesterol biosynthesis genes namely HMGCR, SQLE and LSS following receptor
activation in both mouse lines. Conversely, using real-time qPCR and western blot
analysis, we showed that absence of the receptor in liver-specific CreAlb/AhrFx/Fx
mice leads to upregulation of those genes in the absence of an exogenous ligand.
These data firmly establish the role of AHR as regulator of cholesterol biosynthesis
independently of its DNA-binding ability and suggest that AHR may be a previously
unrecognized therapeutic target.
375 MULTI-SPECIES ANALYSES OF DIRECT ACTIVATORS
OF THE CONSTITUTIVE ANDROSTANE RECEPTOR.
C. Omiecinski 1 , D. M. Coslo 1 , T. Chen 1 and R. C. Peffer 2 . 1 Center for Molecular
Toxicology, Penn State University, University Park, PA and 2 Syngenta Crop Protection,
Inc., Greensboro, NC.
The constitutive androstane receptor (CAR; NR1I3) is a member of the superfamily
of nuclear receptors that functions as an important xenochemical sensor in
mammalian cells. Upon chemical activation, CAR undergoes nuclear translocation
and heterodimerization with the retinoid X receptor to subsequently interact with
DNA targets to transcriptionally modulate gene expression. CAR is unusual among
nuclear receptors in that it possesses a high level of constitutive activity in cell based
assays, obscuring the detection of direct ligand activators. However a human splice
variant of CAR, termed hCAR3, exists that exhibits negligible constitutive activity;
rather, CAR3 is activated by ligands with similar specificity as the reference form of
the receptor, CAR1. In this study, we hypothesized that similar hCAR3 variant receptors
could be constructed across various mammalian species’ forms of CAR,
thereby preserving species specific ligand responses yet enabling their detection in a
much more sensitive ligand-dependent mode. Therefore, in this study, a battery of
CAR3 variant receptors were produced for the respective native CARs of mouse, rat
and dog, and comparatively evaluated with selected ligands in mammalian cell reporter
assays. The results demonstrate that the 5 amino acid insertion that typifies
human CAR3 also creates ligand activated receptor function in other species’
CARs, while maintaining signature responses in each species to select CAR ligands.
These variant constructs permit evaluation of differential effector chemical responses
across species and facilitate, for example, comparative study of tumor promotional
properties of suspected hepatocarcinogens on direct CAR activation responses
in different species.