The Toxicologist - Society of Toxicology

358 CHARACTERIZATION OF GENOME-WIDE
PEROXISOME PROLIFERATOR-ACTIVATED
RECEPTOR-β/δ (PPARβ/δ) BINDING AND
TRANSCRIPTIONAL REGULATION.
C. Khozoie 1 , M. G. Borland 1 , B. Zhu 1 , F. J. Gonzalez 2 and J. M. Peters 1 .
1 Department of Veterinary and Biomedical Sciences and The Center for Molecular
Toxicology and Carcinogenesis, The Pennsylvania State University, University Park, PA
and 2 Laboratory of Metabolism, National Cancer Institute, Bethesda, MD.
Peroxisome proliferator-activated receptor-β/δ (PPARβ/δ), a member of the nuclear
receptor subfamily of transcription factors, modulates gene expression in response
to activation by endogenous or exogenous ligands. Whether PPARβ/δ promotes
or attenuates carcinogenesis is the subject of much debate. Thus, there is a
need to definitively identify and characterize bona fide target genes of this PPAR
isoform. Recruitment of PPARβ/δ to specific DNA motifs (PPREs) proximal to
target genes results in transcriptional regulation by mechanisms that remain to be
fully elucidated. In this study, PPARβ/δ-bound DNA was isolated from C57BL/6
mouse primary keratinocytes by chromatin immunoprecipitation using a highly
specific anti- PPARβ/δ antibody developed by this laboratory. Next generation sequencing
(ChIP-seq) was performed to map the genomic locations of PPARβ/δ
binding (cistrome). This approach was combined with gene expression profiling
using cDNA microarrays, enabling the characterization of genome-wide PPARβ/δ
cistrome-transcriptome interactions. Novel PPARβ/δ-target genes were identified
and confirmed by qPCR and ChIP-qPCR. Results from this analysis indicate that
DNA-binding dependent and ligand-dependent transcriptional repression, or activation,
together account for over 70% of PPARβ/δ transcriptional regulatory activity.
Moreover, results from these studies suggest that differences in DNA binding
sequence motifs may directly influence the activity of PPARβ/δ between repression
and activation. These findings provide novel insights into the transcriptional regulatory
activities of PPARβ/δ that might explain why targeting PPARβ/δ for chemoprevention/chemotherapy
is feasible. (Supported by CA124533, CA126826,
CA141029, CA140369)
359 FUNCTIONAL CHARACTERIZATION OF PEROXISOME
PROLIFERATOR-ACTIVATED RECEPTOR-β/δ (PPARβ/δ)
EXPRESSION IN COLON CANCER.
J. E. Foreman1 , W. Chang2 , J. L. Williams3 , M. L. Clapper2 , F. J. Gonzalez4 and
J. M. Peters1 . 1Department of Veterinary and Biomedical Sciences and The Center for
Molecular Toxicology and Carcinogenesis, Pennsylvania State University, University
Park, PA, 2Cancer Prevention and Control Program, Fox Chase Cancer Center,
Philadelphia, PA, 3Cancer Prevention Laboratory, State University of New York at
Stony Brook, Stony Brook, NY and 4Laboratory of Metabolism, National Cancer
Institute, Bethesda, MD.
The role of PPARβ/δ in colon carcinogenesis remains uncertain because there are
conflicting reports suggesting that this receptor either promotes or attenuates this
disease. Previous work suggests that PPARβ/δ is up-regulated by the APC/βcatenin/TCF4
signaling pathway and that the chemopreventive effects of nonsteroidal
anti-inflammatory drugs (NSAIDs) are mediated in part by repression of
PPARβ/δ expression and function. However, these ideas are not supported by all
studies. Thus, the present study critically examined the role of PPARβ/δ in human
and mouse colon cancer models. Expression of PPARβ/δ mRNA and protein was
lower and expression of CYCLIN D1 protein, a verified downstream target of the
APC/β-catenin/TCF4 pathway, was higher in human colon adenocarcinomas compared
to control tissue. Similar results were observed in colon tumors from
Apc +/Min-FCCC mice compared to control tissue. Dietary administration of sulindac
to Apc +/Min-FCCC mice had no influence on expression of PPARβ/δ in colon or tumors.
Stable over-expression of PPARβ/δ in human colon cancer cell lines enhanced
ligand activation of PPARβ/δ inhibition of clonogenicity in RKO and
HT29 cells. These studies are the most quantitative to date to demonstrate that expression
of PPARβ/δ is lower in human colon adenocarcinomas and Apc +/Min-FCCC
mouse colon tumors, consistent with the finding that increasing expression and activation
of PPARβ/δ in human colon cancer cell lines inhibits clonogenicity.
(Supported by CA124533, CA126826, CA141029, CA140369)
360 REDEFINING ARYL HYDROCARBON RECEPTOR
ANTAGONISM THROUGH CHARACTERIZATION OF A
NEW LIGAND.
K. J. Smith 1 , I. A. Murray 1 , R. Tanos 1 , Y. Wang 2 , J. E. Tellew 2 , A. E. Boitano 2 ,
M. P. Cooke 2 and G. H. Perdew 1 . 1 Pennsylvania State University, University Park,
PA and 2 Genomics Institute of the Novartis Foundation, San Diego, CA.
The aryl hydrocarbon receptor (AHR), a ligand activated transcription factor, has
been implicated in a variety of physiological functions apart from its classical role in
xenobiotic metabolism. Some of these processes involve the AHR interacting with
the dioxin response element (DRE), but others are mediated through the AHR’s
role in the repression of the acute phase response gene SAA1. The latter activity led
to the classification of a distinct class of ligand, called selective AHR modulators (or
SAhRMs) which bind to the AHR but do not elicit the typical DRE-driven response
while still suppressing SAA1. AHR antagonists have so far been classified
only in the context of inhibiting the DRE-driven function of the AHR. In this
study, we demonstrate that the compound N-(2-(1H-indol-3-yl)ethyl)-9-isopropyl-2-(5-methylpyridin-3-yl)-9H-purin-6-amine
(GNF351) is shown not only
to potently antagonize the DRE-driven transcriptional pathway of the AHR, but
also to block the repression of SAA1, thereby introducing a more complete picture
of AHR antagonism. Through a competitive ligand binding assay, GNF351 was
shown to bind to the AHR with high affinity. It was demonstrated in both human
and murine cells that GNF351 is able to block DRE-driven gene expression in the
nanomolar range, through DRE-driven reporter assays and quantitative PCR.
GNF351 also does not exhibit partial agonist activity for the AHR at higher concentrations,
in contrast to alpha naphthoflavone (αNF). The ability of GNF351 to
block the repression of the acute phase response is demonstrated by quantitative
PCR upon co-treatment with IL-1β, as well as through a mouse ear edema model
upon TPA-induced inflammatory signaling. Therefore, GNF351 offers a more
complete and redefined model of AHR antagonism.
361 COMPARISON OF EFFECTS OF DIFFERENT LIGANDS
AND Y322 ON ARYL HYDROCARBON RECEPTOR
SIGNALING.
M. L. Powis, T. Celius and J. Matthews. Department of Pharm and Toxicology,
University of Toronto, Toronto, ON, Canada.
The aryl hydrocarbon receptor (AHR) mediates the toxic effects of halogenated
aromatic hydrocarbons, but is also activated by a diverse range of compounds.
Classic AHR ligands include 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD),
2,3,4,7,8-pentachlorodibenzofuran (PeCDF) and 2,3,7,8-tetrachlorodibenzofuran
(TCDF). Other compounds, such as omeprazole (Omp), are thought to activate
AHR through phosphorylation of residue Y322. Mutations of the Y322 equivalent
in rat AHR prevented activation by Omp but did not effect TCDD activation. In
this study, we examined the ability of different compounds to activate AHR-dependent
induction of cytochrome P450 1A1 (CYP1A1), and hairy and enhancer of
split 1 (HES1) expression in human breast cancer T-47D and hepatoma HuH7
cells, as well as the role of Y322 in AHR activation by these ligands. Time course
mRNA expression analysis showed that in cells treated with TCDD, PeCDF,
TCDF or Omp CYP1A1 mRNA induction increased throughout the 1.5 to 24h
treatment, while HES1 mRNA levels peaked after 1.5h treatment with no increase
observed after 24h. Chromatin Immunoprecipitation assays revealed similar recruitment
profiles for AHR and coactivator (p300, nuclear receptor coactivator
A1/A3) to the regulatory regions of both genes; although some ligand differences
were apparent. Proteolytic degradation of AHR was evident after treatment with
TCDD, PeCDF, and TCDF, but not with Omp. To investigate the role of Y322 in
AHR activation, AHR deficient MCF-7 AHR100 cells were transfected with
hAHR, Y322F or Y322A mutants. Y322F resulted in 50% reduction of TCDDand
PeCDF-induced CYP1A1 mRNA induction and AHR recruitment to
CYP1A1 relative to wt-hAHR, whereas this mutation ablated activation by TCDF
or Omp. Y322A mutation failed to induce or recruit AHR to CYP1A1 following
exposure to all compounds. Our findings show that (1) AHR regulates its target
genes in temporally distinct manner; (2) different AHR activators recruit similar
coactivator following AHR activation; and (3) Y322 is required for maximal ligand
dependent activation of AHR.
362 IMMUNOMODULATORY EFFECT OF ARYL
HYDROCARBON RECEPTOR ANTAGONISM ALTERS
INFLAMMATORY PHENOTYPE IN HFLS-RA CELLS.
T. S. Lahoti, I. A. Murray and G. H. Perdew. Veterinary Sciences, The
Pennsylvania State University, University Park, PA.
Rheumatoid Arthritis (RA) is a chronic inflammatory disease of unknown etiology
which manifests itself, in part, as inflamed human fibroblast-like synoviocytes
(HFLS). Upon inflammation, FLS in RA aggressively proliferate to form a pannus,
expressing inflammatory mediators such as cytokines, matrix metalloproteinase’s
(MMPs) and cyclooxygenase 2 (COX2). Epidemiological studies identify a correlation
between tobacco smoking and the development and aggressive phenotype of
RA. Tobacco smoke is a rich source of agonistic aryl hydrocarbon receptor (AHR)
ligands. Thus we hypothesize that inhibition of AHR activity with a potent AHR
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