The Toxicologist - Society of Toxicology

CYP1A transcription mediated by AHR-estrogen receptor α (ERα) crosstalk.
Taken together, the function of AHRR is conserved in chicken in terms of the negative
regulation of AHR and ERα activities, but its functional mechanism is likely
distinct from those of the mammalian and fish homologues.
354 STABLE OVER-EXPRESSION OF PPARβ/δ AND PPARγ
TO EXAMINE RECEPTOR SIGNALING IN HUMAN
HaCaT KERATINOCYTES.
P. S. Palkar 1 , M. G. Borland 1 , C. Khozoie 1 , B. Zhu 1 , C. Lee 1 , F. J. Gonzalez 2
and J. M. Peters 1 . 1 Veterinary Sciences, Pennsylvania State University, State College,
PA and 2 Laboratory of Metabolism, National Cancer Institute, Bethesda, MD.
Recent evidence suggests that peroxisome proliferator-activated receptor-β/δ
(PPARβ/δ) can cross-talk with other nuclear receptor signaling, including PPARγ
and the retinoic acid receptors (RARs). For example, it was suggested that retinoic
acid may promote tumorigenesis by differentially targeting PPARβ/δ rather than
RAR, or that PPARβ/δ can repress PPARγ activity and inhibit the chemopreventive
properties of PPARγ agonists. To examine PPARβ/δ function and receptor crosstalk
in greater detail, the Migr1 retroviral system was utilized to create stable human
HaCaT keratinocyte cell lines over-expressing PPARβ/δ and PPARγ. Ligand activation
of these receptors in HaCaT keratinocytes over-expressing PPARβ/δ or PPARγ
led to enhanced dose-dependent transcriptional upregulation of a known PPAR
target gene, angiopoietin-like protein 4 (ANGPTL4) as compared to controls. Alltrans
retinoic acid (atRA) increased expression of a known RAR target gene in control
and HaCaT keratinocytes over-expressing PPARβ/δ. However, despite a high
ratio of fatty acid binding protein 5 (FABP5) to cellular retinoic acid binding protein
II (CRABP-II), atRA did not increase expression of a PPARβ/δ target gene,
even in HaCaT keratinocytes expressing markedly higher levels of PPARβ/δ.
Further, while increased efficacy of rosiglitazone to increase ANGPTL4 expression
in HaCaT keratinocytes over-expressing PPARγ was observed, over-expression of
PPARβ/δ did not modulate the efficacy of rosiglitazone activity in HaCaT keratinocytes
as compared to controls. These studies demonstrate that stable over-expression
of PPARβ/δ or PPARγ in HaCaT keratinocytes significantly increases the
efficacy of ligand activation. Despite overexpression of PPARβ/δ, no changes in
retinoic acid-induced, or PPARγ-induced, modulation of target gene expression
were observed in HaCaT cells. (Supported by CA124533, CA126826, CA141029,
CA140369)
355 PPARβ/δ MODULATES AHR SIGNALING IN MOUSE
AND HUMAN KERATINOCYTES.
J. Morales 1 , M. G. Borland 1 , C. Lee 1 , F. J. Gonzalez 2 and J. M. Peters 1 .
1 Department of Veterinary and Biomedical Sciences, The Pennsylvania State
University, University Park, PA and 2 Laboratory of Metabolism, National Cancer
Institute, Bethesda, MD.
Peroxisome proliferator-activated receptors (PPARs) are ligand activated transcription
factors that modulate cell-specific biological functions. Previous evidence has
shown that PPARβ/δ attenuates tumor promotion and skin carcinogenesis, but
PPARβ/δ-dependent modulation of polycyclic aromatic hydrocarbon (PAH) metabolism
or tumor initiation has not been specifically examined. The hypothesis
that PPARβ/δ modulates aryl hydrocarbon receptor (AHR)-dependent signaling
was examined. Surprisingly, Pparβ/δ-null primary keratinocytes exhibited reduced
PAH-mediated induction of cytochrome P450 (Cyp) mRNAs, including Cyp1a1
and Cyp1b1, as compared to wild-type cells. Further functional analyses revealed
that Pparβ/δ-null keratinocytes exhibit decreased AHR occupancy and histone
acetylation at the Cyp1a1 promoter in response to a PAH. To determine whether
this change in occupancy of the Cyp1a1 promoter was due to alterations in promoter
structure, differential DNA methylation was examined in mouse keratinocytes.
Bisulfite sequencing of the Cyp1a1 promoter revealed that Pparβ/δ-null
keratinocytes had increased DNA methylation, which may explain the lower basal
Cyp mRNA expression observed in Pparβ/δ-null keratinocytes. Moreover, studies
using a DNA methylation inhibitor demonstrated that PPARβ/δ modulates AHR
signaling, in part, through DNA methylation. Similar modulation of PAH-mediated
induction of Cyp mRNAs was observed in a human HaCaT keratinocyte cell
line stably expressing shRNA against PPARβ/δ. However, inhibition of DNA
methylation did not alter PAH-induced expression of Cyp mRNAs in this model.
Collectively, these findings demonstrate a novel mechanism in mouse and human
keratinocytes where PPARβ/δ modulates AHR-dependent signaling. Furthermore,
these data suggest that PPARβ/δ may alter the carcinogenic effect of PAHs by modifying
the balance between carcinogen bioactivation and clearance. (Supported by
CA124533, CA126826, CA141029, CA149369, and the NSF GRFP)
76 SOT 2011 ANNUAL MEETING
356 ROLE OF 2, 3, 7, 8-TETRACHLORODIBENZO-P-
DIOXIN-INDUCIBLE POLY(ADP-RIBOSE)
POLYMERASE IN DIOXIN-INDUCED ARYL
HYDROCARBON RECEPTOR SIGNALING.
L. M. MacPherson and J. Matthews. Pharmacology and Toxicology, University of
Toronto, Toronto, ON, Canada.
The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that
mediates the toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). AHR
regulates the expression a hundreds of genes including TCDD-inducible
poly(ADP-ribose) polymerase (TiPARP, PARP-7). TiPARP is a member of the
PARP superfamily, which is an enzyme family that mediates poly(ADPribosyl)ation
of protein targets. Poly(ADP-ribosyl)ation is post-translational modification
associated with a number of biological functions including DNA repair,
transcription, apoptosis and cell division. TiPARP contains a C-terminal PARP catalytic
domain, a conserved WWE (tryptophan-tryptophan-glutamic acid) domain
and a zinc-finger domain. However the biological role of TiPARP and whether
TiPARP plays a functional role in AHR signalling is unknown. The aim of the present
study was to investigate modulation of TCDD-induced AHR activity by
TiPARP. We investigated the effects of loss and overexpression of TiPARP on
TCDD-induced cytochrome P450 1A1 (CYP1A1) and CYP1B1 gene expression
in T-47D human breast carcinoma and HuH-7 hepatoma cell lines. RNAi-mediated
knockdown of TiPARP significantly increased TCDD-induced CYP1A1 and
CYP1B1 expression. TiPARP overexpression decreased TCDD-induced CYP1A1and
CYP1B1-regulated reporter activity in a dose-dependent manner. Chromatin
immunoprecipitation (ChIP) assays showed increased TCDD-dependent recruitment
of AHR to CYP1A1 and CYP1B1 enhancer regions following TiPARP
knockdown. TiPARP knockdown also reduced TCDD-induced AHR protein
degradation following 24 h treatment. TiPARP truncation mutants revealed
TCDD-induced inhibition required the N-terminal (1-234 amino acid residues) as
well as the PARP catalytic domain. Single point mutations of two highly conserved
residues of the PARP catalytic core motif abolished inhibition of TCDD-induced
reporter gene activity. Collectively, these results implicate TiPARP as a negative regulator
of AHR signalling.
357 PPARβ/δ INHIBITS VIRAL HRAS1 (V-HRAS1)-INDUCED
NEOPLASTIC/MALIGNANT TRANSFORMATION OF
MOUSE PRIMARY KERATINOCYTES.
B. Zhu 1 , C. Khozoie 1 , M. T. Bility 1 , N. Blazanin 1 , A. B. Glick 1 , F. J. Gonzalez 2
and J. M. Peters 1 . 1 Molecular Toxicology, University Park, PA and 2 Laboratory of
Metabolism, National Cancer Institute, Bethesda, MD.
Ligand activation of PPARβ/δ inhibits chemically-induced skin tumorigenesis.
Since Harvey sarcoma ras (Hras1) mutation is a critical event in chemical carcinogenesis,
the role of PPARβ/δ in viral Hras1 (v-Hras1)-induced neoplastic transformation
was examined in mouse primary keratinocytes. v-Hras1 infected wild-type
keratinocytes were protected from malignant transformation, while v-Hras1 infected
Pparβ/δ-null keratinocytes underwent malignant transformation. This protection
was due in part to the induction of G2/M arrest by ligand activation of
PPARβ/δ in wild-type keratinocytes. Flow cytometric analysis revealed that
Pparβ/δ-null keratinocytes underwent more active mitosis and adapted to mitotic
stress better than wild-type keratinocytes. Further, while ligand activation of
PPARβ/δ exerts a negative selection against cells expressing higher levels of the v-
Hras1 oncogene by inducing mitosis block in wild-type keratinocytes, Pparβ/δnull
keratinocytes showed a growth advantage in the presence of higher level of v-
Hras1 oncogene. To further investigate the mechanism of G2/M induction by
ligand activation of PPARβ/δ, microarray analysis was performed. Microarray
analysis revealed 62 genes involved in mitosis were induced to a higher level by v-
HRAS1 in Pparβ/δ-null keratinocytes as compared to v-Hras1 infected wild-type
keratinocytes and ligand activation of PPARβ/δ repressed these changes in wildtype
but not Pparβ/δ-null keratinocytes. 33 genes out of these 62 are known to be
regulated by the E2F transcription factors. Interestingly, other known E2F regulated
genes, notably those involved in G1/S phase of the cell cycle, exhibited a similar
pattern of expression as these 62 mitosis-related genes. These results suggest
that PPARβ/δ attenuates Hras1-induced neoplastic/malignant transformation by
inducing G2/M arrest by interfering with E2F transcription factors in primary keratinocytes.
(Supported by CA124533, CA126826, CA141029, CA140369)