The Toxicologist - Society of Toxicology

These results confirmed our hypothesis that by inhibitory interactions at the
3’IgHRR, AhR suppresses Ig expression during TCDD treatment. Interestingly,
basal expression of both the 3’IgHRR-regulated transgene and IgA was increased in
the AhR-knockdown cell population suggesting a role of AhR independent of ligand-activation.
Ongoing studies are focused on determining the DRE target of
TCDD-activated AhR in the 3’IgHRR and elucidating the involvement of AhR in
basal Ig expression which could reveal a novel physiological role of AhR.
(Supported by NIEHS R01ES014676 and undergraduate research supplement
R01ES014676-04S1)
1122 TCDD-INDUCED MODULATION OF THE hs1, 2
ENHANCER WITHIN THE 3’IgHRR IN MOUSE AND
HUMAN.
J. Liu, T. Fernando, S. Ochs and C. E. Sulentic. Pharmacology and Toxicology,
Boonshoft School of Medicine, Wright State Unversity, Dayton, OH.
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a potent environmental toxin
known to suppress immunoglobulin (Ig) gene expression and antibody secretion. It
has been suggested that the inhibition of B cell function by TCDD is transcriptionally
mediated through the aryl hydrocarbon receptor (AhR) and binding to
dioxin response elements (DRE). Transcriptional regulation of the Ig heavy chain
(IgH) involves the 3’IgH regulatory region (3’IgHRR) and its enhancers (hs3,
hs1,2, and hs4), which contain DNA binding sites for several transcription factors
including NF-κB and the AhR. Previously our lab has demonstrated TCDD-induced
binding of the AhR complex to a DRE in both hs1,2 and hs4 enhancers as
well as a marked inhibition of the mouse 3’IgHRR in a CH12.LX B-cell line.
Interestingly, in humans, a polymorphism of the hs1,2 enhancer results in a varying
number of tandem repeats of a 53 bp sequence which has been correlated with autoimmune
diseases such as IgA nephropathy and Celiac disease. The human hs1,2
enhancer has binding sites for κB and DRE; however, Pax5 and NF-αP, which are
important regulators for the mouse hs1,2 enhancer, are not present. The purpose of
our study is to comparatively evaluate the effect of TCDD and LPS on human and
mouse hs1,2 enhancers in a mouse (CH12.LX) and a human (IM-9) B cell line.
Using transient luciferase assays, TCDD markedly enhanced human hs1,2 activity
and even augmented LPS-induced activation in LPS-stimulated CH12.LX mouse
B cells. This starkly contrasted with the TCDD-induced inhibition of the mouse
hs1,2 enhancer and 3’IgHRR. Moreover, we observed a similar activation of the
human hs1,2 enhancer by TCDD in the IM9 human B-cell line. Mutational analysis
is underway to evaluate the impact of different binding sites within the human
and mouse hs1,2 enhancers on TCDD-induced transcriptional modulation. Taken
together, our data suggest that the hs1,2 enhancer is a significant target of TCDD
and support species differences in transcriptional regulation of the
hs1,2.(Supported by NIEHS R01ES014676,R01ES014676-03S2)
1123 PAX5 MAY MEDIATE TCDD-INDUCED DIFFERENCES
IN TRANSCRIPTIONAL REGULATION OF THE MOUSE
AND HUMAN hs1, 2 ENHANCER.
S. Ochs, J. Liu and C. Sulentic. Pharmacology and Toxicology, Wright State
Unversity, Dayton, OH.
Immunoglobulin heavy chain (IgH) expression and Ig secretion is inhibited by the
environmental contaminant 2,3,7,8-tetrachlorodibenzo-ρ-dioxin (TCDD).
Within the IgH gene, the 3’ IgH regulatory region (3’IgHRR) has been identified
as a transcriptional target of TCDD. The 3’IgHRR, which in part regulates transcription
of the IgH gene, is composed of four enhancers in the mouse: hs3a; hs1,2;
h3b; hs4 and three enhancers in the human: hs3a; hs1,2; hs4. Interestingly, a repeating
polymorphism, approximately 53 bp in length, within the human hs1,2 enhancer,
has been correlated with several autoimmune disorders. TCDD induces a
decrease in transcriptional activity of the mouse hs1,2 enhancer, while the human
polymorphic hs1,2 enhancer is increased. The purpose of the current study is to determine
if this opposing result may be partially due to Pax5, a regulator of B-cell
maturation that is down-regulated at the plasma cell stage to allow IgH transcription
and Ig secretion. Two Pax5 binding sites are present in the mouse hs1,2
whereas none are present in the human hs1,2 and TCDD has been shown to interfere
with the down-regulation of Pax5. Utilizing site-directed mutagenesis, a Pax5
binding site identical to the mouse was inserted into a human hs1,2 enhancer reporter
plasmid. Transient transfection studies using a mouse (CH12.LX) and
human (IM-9) B cell line expressing functional AhR signaling demonstrated a decrease
in overall TCDD-induced transcriptional activity with the insertion of a
Pax5 site within the human hs1,2 enhancer. These results suggest Pax5 may be in
240 SOT 2011 ANNUAL MEETING
part responsible for the differing transcriptional regulation between the mouse and
human hs1,2 enhancer following TCDD treatment. Additionally, species specific
sequence differences may complicate the translation of mouse studies to humans.
(Supported by NIEHS R01ES014676)
1124 ROLE OF THE 3’IgHRR IN TCDD-INDUCED
SUPPRESSION OF THE IMMUNOGLOBULIN HEAVY
CHAIN.
J. L. Panchal, E. Romer, T. Fernando and C. Sulentic. Pharmacology and
Toxicology, Boonshoft School of Medicine, Wright State University, Dayton, OH.
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) inhibits antibody secretion and immunoglobulin
heavy chain (IgH) expression. Our previous work has shown that a
possible mechanism for inhibition of IgH expression could be inhibition of the
3’IgH regulatory region (3’IgHRR). The 3’IgHRR has four enhancer regions (hs3a;
hs1,2; hs3b; hs4) which are purported to control IgH gene expression. Previously
we demonstrated a sensitive inhibition by TCDD of LPS-induced 3’IgHRR activity
which is correlated with an inhibition of LPS-induced hs1,2 enhancer activity;
however, the hs4 enhancer showed TCDD-induced activation following the cotreatment
of TCDD and LPS. Therefore, the objective of the current study was to
determine if the hs1,2 enhancer mediates the inhibitory effect of TCDD on
3’IgHRR activation. We generated a CH12.LX cell line stably expressing a γ2b
transgene under the regulation of the 3’IgHRR and containing loxp sites flanking
either the hs3a/hs1,2 or the hs3b/hs4 enhancer pairs. Transient transfection with
Cre recombinase results in recombination at the loxp sites and deletion of the appropriate
enhancer pair. Cells expressing Cre recombinase were sorted and 96
clones from each parental (i.e., loxp flanking hs3a/hs1,2 or hs3b/hs4) were analyzed
by PCR for successful recombination. We found 8 clones and 6 clones with successful
deletion of the hs3a/hs1,2 and hs3b/hs4 respectively. Preliminary analysis
showed inhibition of the hs3a/hs1,2 enhancer pair and no effect to inhibition of the
hs3b/hs4 enhancer pair. These results suggest that the hs1,2 is the primary mediator
of TCDD-induced 3’IgHRR inhibition and that the hs4 enhancer in combination
with the hs3b enhancer is affected differently than when the hs4 is analyzed in
isolation or by transient transfection. A polymorphism of the hs1,2 enhancer is correlated
with autoimmune diseases like IgA nephropathy and Celiac disease. Hence
modulation of hs1,2 enhancer activity by TCDD may influence the initiation or
progression of these diseases. (Supported by NIEHS R01ES014676)
1125 NF-κB/REL AND THE AhR IN MODULATING THE
3′IgHRR.
R. Salisbury and C. Sulentic. Pharmacology and Toxicology, Boonshoft School of
Medicine, Wright State University, Dayton, OH.
Transcriptional regulation of the immunoglobulin heavy chain (IgH) gene involves
several regulatory elements including the 3′IgH regulatory region (3′IgHRR). The
mouse 3′IgHRR is composed of at least four enhancers (hs3A; hs1,2; hs3B; hs4)
and contains binding sites for several transcription factors including NF-κB/Rel
proteins and dioxin responsive elements (DRE). 2,3,7,8-Tetrachlorodibenzo-pdioxin
(TCDD), a disrupter of B-cell differentiation and Ig production, induces
binding of the aryl hydrocarbon receptor (AhR) to a DRE motif within both the
hs1,2 and hs4 enhancers. Interestingly, TCDD profoundly inhibits 3′IgHRR activation
induced by the B-cell activator lipopolysaccharide (LPS), but enhances the
activation of the hs4 enhancer in transient luciferase reporter studies. Within the
hs4 enhancer, the DRE overlaps an NF-κB/Rel binding site suggesting that both
the AhR and the NF-κB together play a role in overall modulation of the 3’IgHRR.
The objective of the current study was to evaluate the role of NF-κB/Rel and the
AhR following LPS or CpG (activators of the NF-κB pathway) stimulation and
TCDD treatment on 3′IgHRR and hs4 activity. In our studies we utilized the
CH12.LX B cell line, the CH12.IκBαAA cell line which expresses an inducible
IκBα super repressor (IκBαAA), and the CH12.γ2b-3′IgH cell line that stably expresses
a γ2b-3′IgHRR reporter. Utilizing transiently expressed luciferase reporters,
we found that induction of IκBαAA expression partially attenuated both LPS and
CpG-induced activation of the 3’IgHRR and hs4 and partially reversed the effects
of a TCDD and LPS/CpG co-treatment on the activity of the 3’IgHRR and hs4.
Furthermore, the addition of an AhR antagonist, CH223191, markedly reversed
TCDD-induced inhibition of the LPS-activated 3’IgHRR and inhibited the synergistic
activation of the hs4. Co-immunoprecipitation assays demonstrated an interaction
between the AhR and NF-κB/Rel. These results suggest that the NF-κB/Rel
proteins, perhaps through an interaction with the AhR, are partially responsible for
3′IgHRR modulation by LPS/CpG and TCDD. Supported by NIEHS
R01ES014676)