CYP19 expression is induced by 2,3,7,8-tetrachloro-dibenzo-para-dioxin

in human glioma cells

Wenjuan Tan
b,1
, Tsz Yan Wong
a,1
, Yanfei Wang
b
, Jian Huang
c
, Lai K. Leung
a,b,
a
Food and Nutritional Sciences Programme, School of Life Sciences, Faculty of Science, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong
b
Biochemistry Programme, School of Life Sciences, Faculty of Science, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong
c
Department of Biochemistry, College of Life Sciences, Wuhan University, Wuhan, Hubei Province, PR China
a r t i c l e i n f o
Article history:
Received 11 January 2013
Received in revised form 24 April 2013
Accepted 21 May 2013
Available online 29 May 2013
Keywords:
TCDD
Aromatase
Brain cells
a b s t r a c t
Dioxins are the most concerned environmental pollutants. Recent studies have shown that these
compounds could disrupt the proper functioning of our endocrine system. Estrogen is synthesized in glial
cells of the brain. The hormone has been linked to the maintenance of normal brain operation, ranging
from neurotransmission to synapse formation. Aromatase or CYP19 is the enzyme responsible for
estrogen synthesis. In the present study, we demonstrated that 2,3,7,8-tetrachloro-dibenzo-para-dioxin
(TCDD) stimulated the enzyme activity in human brain cells as low as 1 pM. Increased brain-specic
CYP19 mRNA species was also observed in these cells. Since the brain-specic promoter I.f of CYP19
contains two binding motifs for CCAAT/enhancer binding protein, electrophoretic mobility shift assay
was performed to validate the activation. We further traced the triggering signal and found that the
mitogen-activated protein kinases ERK-1/2 were activated. In summary, TCDD could induce CYP19 tran-
scription in brain cells. Exposure to the pollutant might perturb the hormonal balance in the brain.
2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
Dioxins are a group of polyhalogenated aromatic hydrocarbons
generated from industrial processes such as waste incineration,
paper pulp bleaching and herbicide and pesticide manufacturing.
Because of their stability, they are widespread in our environment
and bio-accumulative in our food chain. These toxicants are biolog-
ically active. In addition to its carcinogenicity and reproductive
toxicity (Birnbaum and Tuomisto, 2000; Steenland et al., 2004),
the neurotoxicity and endocrine disruptive nature of 2,3,7,8-Tetra-
chloro-dibenzo-para-dioxin (TCDD) has also been established after
decades of research. In animal studies, exposure to TCDD may re-
duce food and water intake through increased corticotropin-
releasing factor, arginine vasopressin, and proopiomelanocortin
in the hypothalamus (Moon et al., 2008), blocking prolactin and
increasing luteinizing hormone beta in the hypothalamus and pitu-
itary gland (Cao et al., 2011), and damaging hippocampal-depen-
dent contextual memory (Latchney et al., 2012). Perinatal
exposure to TCDD may affect brain development. Nishijo et al.
(2007) have demonstrated that fetal brain growth in male rats is
compromised upon prenatal TCDD exposure. Decits in behavioral
development, such as hyperactivity, socio-emotional activity,
emotion, loss of aggressiveness in male offspring are also observed
(Nguyen et al., 2013; Haijima et al., 2010; Endo et al., 2012).
Isolated brain analysis indicates that at least one of the mRNA
expressions of oligodendroglial lineage, platelet-derived growth
factor a receptor, and myelin basic protein are changed in cerebel-
lum, medulla oblongata, diencephalon and telencephalon (Fernan-
dez et al., 2010). Whole brain examination also reveals that Cxc14
and 17 mRNA expression is upregulated (Mitsui et al., 2011).
TCDD is an agonist to aryl hydrocarbon receptor (AhR) and dis-
plays weak estrogenic properties (Ohtake et al., 2003), which could
also be the potential signaling pathway of its neurotoxicity. Previ-
ous studies have shown that TCDD interferes with the proliferation
of neuroepithelial stem cells, neural precursor cells (Latchney et al.,
2011) and cerebellar granule cells (Sanchez-Martin et al., 2011)
through an AhR-dependent pathway.
The role of estrogen in the brain has been contradictory. Garcia-
Segura et al. (1999) have observed that reactive astrocytes have in-
creased aromatase expression during brain injury. Since aromatase
is responsible for estrogen bio-synthesis, estrogen is suggested to
be neuroprotective (Saldanha et al., 2009). In contrast, other
researchers have shown that estrogen may intensify brain injury
in a rat stroke model (Bingham et al., 2005). The hormones neuro-
protective effect remains controversial.
Human aromatase is a 55-kDa protein encoded by a single copy
gene with 10 exons (Means et al., 1989; Toda et al., 1990). The cod-
ing region extends from Exon II to IX, and a number of untranslated
0303-7207/$ - see front matter 2013 Elsevier Ireland Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.mce.2013.05.018

Corresponding author. Address: Food and Nutritional Sciences Programme,

School of Life Sciences, The Chinese University of Hong Kong, Rm. 507C, MMW
Bldg., Shatin, N.T., Hong Kong. Tel.: +852 39438137; fax: +852 26037732.
E-mail address: laikleung@cuhk.edu.hk (L.K. Leung).
1
These authors contributed equally to this work.
Molecular and Cellular Endocrinology 375 (2013) 106112
Contents lists available at SciVerse ScienceDirect
Molecular and Cellular Endocrinology
j our nal homepage: www. el sevi er . com/ l ocat e/ mce
promoter regions in Exon I dictate the gene regulation. The tran-
scription starts from any one segment of the promoter in Exon I
and splices to a common site in Exon II. Different tissues can em-
ploy one segment of Exon I for regulation, and tissue-specic reg-
ulation of aromatase expression is achieved through alternate
splicing (Simpson et al., 1997). However, the translated aromatase
is identical in all transcripts since the protein encoding region fall
in the common sequence at Exon II to IX.
TCDD is an endocrine disruptor. Previous studies have shown
that TCDD may alter aromatase expression in different cell types
(Drenth et al., 1998; Moran et al., 2000; Dasmahapatra et al.,
2000). In the present study, we investigated the disruptive effect
of TCDD on aromatase in brain cells. Several glioma cell lines were
used in this project, since glial cells are the major brain cells
expressing aromatase (Garcia-Segura et al., 1999).
2. Materials and methods
2.1. Chemicals
TCDD was obtained from SigmaAldrich (St. Louis, MO, USA).
The ERK inhibitor U0126 was purchased from Calbiochem, San Die-
go, CA, USA. Other chemicals, if not stated, were purchased from
SigmaAldrich.
2.2. Cell culture
The brain cell lines T98G (multiform glioblastoma), DBTRG-
05MG (multiform glioblastoma), U-87 MG (glioblastoma astrocy-
toma) and U251 (glioblastoma astrocytoma) were purchased from
A.T.C.C., Rockville, MD. T98G cells were maintained in MEM med-
ium (Invitrogen Life Technology, Rockville, MD) supplemented
with 10% fetal bovine serum (FBS) from Hyclone Laboratories,
South Logan, Utah, USA. U-87 MG and U251 cells were maintained
in DMEMmedium (Invitrogen Life Technology) supplemented with
10% FBS. DBTRG-05MG cells were cultured in RPMI-1640 (Invitro-
gen Life Technology) supplemented with 10% FBS, 2 mM L-Gluta-
mine and 1 mM sodium pyruvate. The neuroblastoma cell line
LAN-1 (bone marrow) was a gift from Prof. Kwok Nam Leung,
School of Life Sciences, The Chinese University of Hong Kong.
LAN-1 cells were cultured in RPMI-1640 medium supplemented
with 10% FBS.
All cells were incubated at 37 C, 5% carbon dioxide and rou-
tinely sub-cultured when reaching 80% of conuency. Three days
before experiments, the cells were switched to phenol-red free
medium. TCDD at concentrations of 0, 0.001, 0.01, 0.1, 1, 10 nM
were administered in the solvent vehicle dimethyl sulphoxide
(DMSO), and the concentration was limited to 0.1% v/v. Cells were
seeded at a density of 5 10
2
cells/mm
2
in all experiments.
2.3. In-cell aromatase assay
The assays were performed as previously described (Wang
et al., 2005, 2006) with slight modication. In brief, T98G cells
were seeded and allowed 1 day for attachment. After treating with
TCDD for the rst 24 h, assays were started by replacing the culture
medium with serum-free medium containing [1b-3H(N)]-androst-
4-ene-3,17-dione and TCDD. The nal concentration of
androstenedione was controlled at 25 nM, and the reaction was
incubated at 37 C for another 24 h. An aliquot of the medium
was then mixed with equal volume of chloroform, followed by a
13,000g centrifugation at 4 C for 10 min. The aqueous phase was
removed into a new tube containing 500 ll of 5% activated char-
coal suspension. After 30 min incubation, an aliquot of the super-
natant fraction was taken out for scintillation counting. The
protein content of the cells, on the other hand, was determined
by using a bicinchoninic acid (BCA) kit (Thermo Scientic Pierce)
after dissolving the cells in 0.5 mol/l NaOH.
2.4. Quantitative real time reverse transcription-polymerase chain
reaction (RT-PCR) assay on aromatase expression
The protocol for real-time quantitative PCR was carried out as
previously described by our lab (Wang et al., 2008). CYP19 exon-
specic probes for I.1(Ia), I.3, I.4, I.5(2a) and II were previously de-
scribed (Ye et al., 2009). The Exon I.f mRNA species were designed
by Custom-designed Taqman

Gene Expression Assays (Assay-by-

Demand

, Applied Biosystems, Foster City, CA, USA) as shown

below. The expression of glyceraldehyde-3-phosphate dehydroge-
nase (GAPDH) (Taqman probe Cat No. Hs99999905_m1, Assay-on-
Demand

, Applied Biosystems) was used for normalization. The

primer sequences were shown as below. Brain cells were cultured
and treated as described above. After 48 h of treatment, total RNA
was extracted from the cells using TRIzol reagent (Invitrogen,
Carlsbad, CA, USA). The concentration and purity of the isolated
RNA were determined by the absorbance reading observed at
260 and 280 nm. 3 lg of total RNA, oligo-dT, and Moloney murine
leukaemia virus (M-MLV) Reverse Transcriptase (USB Corporation,
Cleveland, Ohio, USA) were used for rst strand synthesis. Target
fragments were quantied by using DNA Engine Opticon 2 sys-
tem (MJ Research, Waltham, MA, USA). Real-time PCR Master
Mix Reagent kit was obtained from Applied Biosystems and PCR
reactions were set up as described in the manual. A typical reaction
contained 1 ll 20 probes, 4 ll cDNA and the nal reaction vol-
ume was 20 ll. The reaction was initiated by preheating at 50 C
for 2 min, followed by heating at 95 C for 10 min. Subsequently,
45 amplication cycles were then carried out with 15 s denatur-
ation at 95 C and 1 min annealing and extension at 58 C. Relative
gene expression data were analyzed using the 2
DDCT
method (Li-
vak and Schmittgen, 2001).
2.5. Electrophoretic mobility shift assay (EMSA)
By using the software TRANSFAC 8.3, two CCAAT/enhancer
binding protein (C/EBP) binding sites were located in the brain-
specic promoter I.f sequence. EMSA was performed on the DNA
binding. Nuclear protein was isolated by using NucBusterprotein
extraction kit (Novagen

, EMD Biosciences, Inc., La Jolla, CA, USA.).

In brief, cells were washed, trypsinized, and packed at 500g at 4 C.
Reagent 1 provided in the kit was added to the packed cells. Nucle-
ar extract was isolated from the cell suspension by vortexing and
mRNA species Forward
primer
Reverse
primer
Reporter sequence
CYP19 I.2 TGAAAAGAGAGCTCTTTAGCAAACACA CCACATGCTAGGATATGATTTAAGTAATGAGA CTGGAGGTGACAAGCTTT
CYP19 Exon I.f GAGAGCCCAGCAACTATGTAACTC CGCTCCTGTGAACAGAGAGTAAT ATCATGCCTCCCTTCCATG
CYP19 GGAGAATTCATGCGAGTCTGGAT GGAACATACTTGAGGACTTGCTGAT TCTGGAGAGGAAACACTC
W. Tan et al. / Molecular and Cellular Endocrinology 375 (2013) 106112 107
centrifugation, and stored at 80 C until assayed. An oligonucleo-
tide encompassing promoter I.f (413/380) was synthesized as
shown below and was labeled with DIG by using DIG Gel Shift
Kit, 2nd Generation (Roche Diagnostics, Indianapolis, IN, USA).
The C/EBP binding site was underlined and at the position
(397/391).
PI:f413= 380 :
5
0
GACTGTAAAGTAGCCCCACAATTCCCACATCTTC-3
0
The nuclear protein was thawed and incubated with the labeled
probe in binding buffer (100 mM HEPES, pH7.6, 5 mM EDTA,
50 mM (NH4)
2
SO4, 5 mM DTT, Tween 20, 1% w/v, 150 mM KCl),
poly(dI-dC) and poly L-lysine for 30 min at room temperature.
The band specicity was veried by co-incubating with antibodies
against C/EBP (Santa Cruz Biotechnology) or competing with 7 C/
EBP unlabeled oligonucleotide. The reaction mix was then sepa-
rated on a 6% non-denaturing gel in 0.5 Trisborate EDTA at
100 V. The labeled oligonucleotide was electro-transferred to a Ny-
lon membrane, xed by UV light, blocked and washed. The shifted
oligonucleotide was detected by autoradiography after incubating
with anti-Digoxigenin-AP conjugate and chemiluminescent sub-
strate CSPD

provided in the kit.

2.6. Western analysis
Cells were washed once by PBS (pH 7.4) and harvested into a
1.5 ml microtube with 0.5 ml lysis buffer (PBS, 1%NP40, 0.5% so-
dium deoxycholate, 0.1% SDS). The lysis buffer contained protease
inhibitors (40 mg/L PMSF, 0.5 mg/L aprotinin, 0.5 mg/L leupeptin,
1.1 mmol/L EDTA and 0.7 mg/L pepstatin). The harvested cells were
then lysed with a cell disruptor (Branson Ultrasonics Corp., Dan-
bury, CT, U.S.A.) on ice for 30 s. The protein concentration of cell ly-
sate was determined by BCA kit (Thermo Scientic Pierce). 50 lg of
lysate protein was separated on 10% SDSPAGE and transferred to
an Immobilon PVDF membrane (Millipore, Bedford, MA, USA).
Anti-aromatase antibody (Abcam, Cambridge, UK), anti-phospho-
p44/42 Mitogen-activated Protein Kinase (MAPK) or ERK 1/2,
anti-phospho-Stress-activated Protein Kinase/JNK, anti-phospho-
p38 MAPK antibodies (Cell Signaling Technology, Danvers, MA,
USA), anti-b-actin primary (Sigma Chem) and secondary antibodies
conjugated with horseradish peroxidase (Santa Cruz Biotechnol-
ogy) were used for protein detection. An ECL Detection Kit (Amer-
sham, Arlington Heights, IL, USA) provided the chemiluminescence
substrate for HRP, and the targeted protein was visualized by auto-
radiography. The optical density readings of the images were ana-
lyzed by using the computer software ImageJ (National Institute of
Mental Health, Bethesda MD, USA).
2.7. Estradiol concentration determination
Estradiol concentrations in culture medium were measured by
using ELISA kits from Cayman Chemical Company (Ann Arbor,
MI). The samples were added into a 96-well plate coated with anti-
body raised against estradiol. After incubating with the respective
tracer and developing at room temperature, the absorbance was
quantitated using a microplate reader (FluroStar

, BMG Labtech-
nologies GmBH, Offenburg, Germany). The amount of estradiol
could be read against a standard curve constructed with the hor-
mone provided in the kits.
2.8. Statistical analysis
Data obtained were analyzed by the software package Prizm5

(GraphPad Software, Inc., San Diego, CA). One Way ANOVA with
Dunnetts post hoc test was used for comparison.
3. Results
3.1. CYP19 expressions in different brain cell lines
CYP19 expressions in the brain cell lines LAN-1, T98G, DBTRG-
05MG (05MG), U-87 MG (U87) and U251 were determined and
compared. Expression in T98G cells was signicantly (p < 0.05)
higher than the other neuroblastoma or glioblastoma cell lines
(Fig. 1). Hence, we chose T98G cell line as the model in our study.
3.2. CYP19 expression was induced by TCDD
In addition to T98G cells, LAN-1 cells were arbitrarily selected
for testing the responses to TCDD exposure. 10 nM TCDD increased
the expression by 14-fold in LAN-1 cells (Fig. 2B), whereas the
same concentration induced 2-fold increase in T98G cells
(Fig. 2A). Doseresponse relationship was demonstrated in both
cell lines. However, aromatase protein and enzyme activity in
LAN-1 cells were not detectable in subsequent experiments.
T98G cells were selected for further study.
3.3. TCDD induced aromatase activity in T98G cells
As the mRNA expression of CYP19 increased, the enzyme activ-
ity should also be induced. We veried that the aromatase activity
in T98G cells was induced by TCDD after 48 h incubation (Fig. 3A)
in a dose-dependent manner. The activity was signicantly
(p < 0.05) increased starting from0.001 nM TCDD, and a vefold in-
crease was observed in cultures treated with 0.01 nM TCDD and
above. Increased estradiol production was also observed in the
presence of testosterone (Fig. 3B), since aromatase catalyzes the
conversion of androgen into estrogen.
3.4. Aromatase protein was increased by TCDD
Increased aromatase protein was also observed in T98G cells
(Fig. 4A). The normalized optical density of the CYP19 was esti-
mated and shown in Fig. 4B. The protein was increased by about
2.7-fold. The result veried that the induced aromatase activity
was regulated at the expression level.
3.5. Specic promoter usage and regulation
By using the Exon-specic probes, we observed that transcripts
from Promoters Ia, I.f, I.2, I.3 and II on Exon 1 were detectable in
L
A
N
-
1
T
9
8
G
0
5
M
G
U
8
7
U
2
5
1
0
1
2
3
30
40
50
60
*
R
e
l
a
t
i
v
e

C
Y
P
1
9
m
R
N
A

E
x
p
r
e
s
s
i
o
n
Fig. 1. Basal CYP19 mRNA expression in various brain cell lines. Brain cells LAN-1,
T98G, DBTRG-05MG (05MG), U-87 MG (U87) and U251 were seeded in 6-well
plates and maintained in culture medium as described in Methods. The amount of
CYP19 mRNA was determined by real time PCR and was normalized by GAPDH
mRNA. Values are means SEM, n = 3. Means labeled with () were signicantly
(p < 0.05) higher than means of the other cell lines.
108 W. Tan et al. / Molecular and Cellular Endocrinology 375 (2013) 106112
this cell line (Fig. 5). Since Promoter I.f is reported as the major reg-
ulatory promoter for CYP19 expression in the brain under normal
condition (Yague et al., 2006), we specically looked into the Exon
I.f expression response. RT-PCR assay result showed that relative
Exon I.f-spliced mRNA was raised by TCDD in a dose dependent
manner with signicant deviation (P < 0.05) occurred beyond 0.1
nM (Fig. 6).
Control 0.001 0.01 0.1 1 10
0
5
10
15
*
R
e
l
a
t
i
v
e

C
Y
P
1
9

m
R
N
A
E
x
p
r
e
s
s
i
o
n

i
n

L
A
N
-
1

c
e
l
l
s
TCDD Conc. (nM)
Control 0.001 0.01 0.1 1 10
0
1
2
* *
R
e
l
a
t
i
v
e

C
Y
P
1
9

m
R
N
A
E
x
p
r
e
s
s
i
o
n

i
n

T
9
8
G

c
e
l
l
s
TCDD Conc. (nM)
A B
Fig. 2. TCDD increased CYP19 mRNA expression in brain cells. Brain cells were seeded in 6-well plates and maintained in phenol-red free RPMI 1640 medium supplemented
with 10% charcoal dextran-treated FBS. Cells were treated with TCDD for 24 h. The amount of CYP19 mRNA was determined by real time PCR and was normalized by GAPDH
mRNA in T98G (A) and LAN-1 (B) cells. Values are means SEM, n = 8. Means labeled with various letters were signicantly (p < 0.05) different.
0 0.001 0.01 0.1 1 10
0
100
200
300
*
*
A
r
o
m
a
t
a
s
e

a
c
t
i
v
i
t
y
(
c
p
m
/
m
g

p
r
o
t
e
i
n
/
h
r
)
TCDD Conc (nM)
0 0.001 0.01 0.1 1 10 0 10
0
5
10
15
20
E
s
t
r
a
d
i
o
l

i
n

c
u
l
t
u
r
e
m
e
d
i
u
m

(
p
g
/
m
l
)
*
*
* *
*
TCDD (nM)
10 nM testosterone
* * *
+ + + + + + - -
b
a
A B
Fig. 3. Aromatase activity was induced by TCDD in T98G cells. T98G cells were seeded in six-well plates and maintained in phenol red-free RPMI medium supplemented with
10% charcoal dextrantreated serum. Aromatase activities were determined in cultures treated with TCDD (A). Estradiol concentrations in culture medium were assayed by
ELISA (B). The data represents the means SEM of 3 samples isolated from independent cultures. () mean values were signicantly (p < 0.05) different from that of the
control cultures.
Control 0.001 0.01 0.1 1 10
0
1
2
3
R
e
l
a
t
i
v
e

O
p
t
i
c
a
l

D
e
n
s
i
t
y
TCDD Conc. (nM)
A
B
Fig. 4. TCDD increased aromatase protein in T98G cells. T98G cells were seeded in
6-well culture dishes and treated with TCDD for 24 h. Amounts of CYP19 in protein
extracts were determined by western blot analysis. The image shown in Fig. 4A is a
representation of three independent experiments, and Fig. 4B display the optical
density readings of the proteins. Values are means SEM, n = 3. Means labeled with
() were signicantly (p < 0.05) different.
G
A
P
D
H I
a
I
.
4
I
.
5
I
.
f
I
.
2
I
.
3 I
I
0.0
0.1
0.2
0.3
0.4
0.95
1.00
R
e
l
a
t
i
v
e

A
l
t
e
r
n
a
t
e
S
p
l
i
c
e
d

m
R
N
A

e
x
p
r
e
s
s
i
o
n
Fig. 5. Exon-specic CYP19 expression in T98G cells. T98G cells were seeded in 6-
well plates and maintained in culture medium as described in Methods. The
amounts of exon-specic spliced mRNA species were determined by real time PCR
and were normalized by GAPDH mRNA. Values are means SEM, n = 3. Means
labeled with () were signicantly (p < 0.05) higher than means of the other cell
lines.
W. Tan et al. / Molecular and Cellular Endocrinology 375 (2013) 106112 109
3.6. Effects of TCDD on C/EBP binding in Exon I.f
The result of Exon I.f-spliced mRNA assay illustrated that TCDD
might activate the transcriptional activity through the correspond-
ing promoter. Subsequently, we scanned the sequence (500/0) of
ExonI.f of CYP19 using TRANSFAC(version8.3), and one C/EBP bind-
ing site was located in the segment (397/391). EMSA assay was
performedtoverifythe functionalityof the binding. Nuclear extracts
isolated from cultures treated with TCDD displayed an increasing
trend of C/EBP binding from 0.001 to 10 nM (Fig. 7). The band spec-
icity was further demonstrated by the weakening of intensity
when co-incubating with 7 C/EBP unlabeled oligonucleotide (SC)
or the antibody of C/EBP (Santa Cruz Biotechnology) (SS).
3.7. Protein kinase signaling pathways
Since the transcriptional factors C/EBP could be activated by
phosphorylation, we examined the activation states of the related
signaling protein kinases by western blot analysis. TCDD appeared
to increase the phosphor- ERK-1 and -2 in the nuclear protein,
while no effect on JNK or p38MAPK was observed (Fig. 8A).
3.8. Effect of the ERK inhibitor U0126 on TCDD-induced mRNA
expression
Since ERK-1/2 was increased by TCDD treatment, effect of ERK
inhibitor on the RNA expression was investigated in a subsequent
experiment. Cultures pre-treated with U0126 (I + TCDD) displayed
a decreased expression of Exon I.f-specic CYP19 mRNA as com-
pared to cells treated with TCDD alone (Fig. 8B). This result illus-
trated that ERK activation was partially responsible for the
induction of CYP19 expression.
4. Discussion
In the present study, an endocrine system of the brain per-
turbed by TCDD was depicted. The toxicant increased CYP19 mRNA
expression in cultured cells and it might induce the Exon I.f trans-
activity through ERK activation.
TCDD is reported to reduce aromatase protein or mRNA in JEG-3
cells (Drenth et al., 1998) and granulosa cells (Moran et al., 2000;
Dasmahapatra et al., 2000). In contrast, other investigators have
shown that the toxicant induces aromatase in human placental
cells (Augustowska et al., 2003), breast cells (Chan et al., 2010)
and primary Sertoli cells (Lai et al., 2005). The conicting results
could be attributed to the species and tissue differences. In the
present study, TCDD consistently increased the expression of
CYP19 in various human brain cells.
ERK appears to be an effector signaling molecule of TCDD. The
toxicant may upregulate or downregulate gene expression through
ERK activation. Studies have shown that the contaminant induces
TNFa and IL-6 expression in synoviocytes (Kobayashi et al., 2008;
Cheon et al., 2007) and THP-1 cells (Cheon et al., 2007), respec-
tively. On the other hand, TCDD suppresses PPAR-c1 expression
in C3H10T1/2 cells (Hanlon et al., 2005). ERK activated by TCDD
also takes part in other physiological pathways, for instance,
Control 0.001 0.01 0.1 1 10
0
1
2
3
R
e
l
a
t
i
v
e

E
x
o
n

I
.
f
-
s
p
l
i
c
e
d
m
R
N
A

e
x
p
r
e
s
s
i
o
n
TCDD Conc. (nM)
Fig. 6. TCDD increased Exon I.f specic CYP19 mRNA expression in T98G cells. T98G
cells were seeded in 6-well plates and maintained in phenol-red free RPMI 1640
medium supplemented with 10% charcoal dextran-treated FBS. Cells were treated
with TCDD for 24 h. The amount of Exon I.f-spliced CYP19 mRNA was determined
by real time PCR and was normalized by GAPDH mRNA. Values are means SEM,
n = 3. Means labeled with various letters were signicantly (p < 0.05) different.
Fig. 7. Effect of TCDD on the binding of C/EBP sequence within promoter I.f. Nuclear extract samples were prepared from TCDD-treated T98G cells and EMSA was performed.
In this gure, lanes labeled with 0, 0.001, 0.01, 0.1, 1 and 10 are samples treated with the respective TCDD concentration (nM); 10-SC and 10-SS are the samples extracted
from 10 nM TCDD and incubated with 7 C/EBP consensus sequence and C/EBP antibody, respectively. Data represent one of two independent experiments with comparable
results. Non-specic binding bands are seen below the C/EBP bands.
110 W. Tan et al. / Molecular and Cellular Endocrinology 375 (2013) 106112
insulin signaling in MCF-10A cells (Park et al., 2004), and apoptosis
in RAW264.7 (Park et al., 2005) and Jurkat (Kwon et al., 2003) cells.
Our laboratory has also demonstrated that TCDD could increase
the stability of CYP19 transcript in cultured MCF-7 cells through
ERK activation (Chan et al., 2010). Contrasting to the present study,
TCDD appeared to increase the CYP19 transcription in brain cells
through a different downstream pathway of ERK.
C/EBPs are a family of transcriptional factors that could be acti-
vated by ERK (Li et al., 2007). The beta isoform of C/EBP is impor-
tant in consolidating long term memory (Carew and Sutton, 2001).
The isoform may also participate in promoting post-ischemic
inammation and cause brain damage (Yi et al., 2007). The delta
isoform of C/EBP, on the other hand, can bind to PI.3/II of CYP19
and initiates the expression in breast cancer cells (Kijima et al.,
2008). In the present study, the C/EBP binding activity within Exon
I.f segment was enhanced upon TCDD treatment. This could be the
underlying mechanism for the induced CYP19 expression.
Other physiological actions of TCDD on the brain have also
been documented, such as inducing apoptosis in cerebellar
granule cells (Sanchez-Martin et al., 2011), changing the neurode-
velopment in fetal mouse brain (Mitsui et al., 2011), and
stimulating the efux transporter at the blood brain barrier (Wang
et al., 2011). However, change in cell number under TCDD treat-
ment as determined by 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl
tetrazolium bromide (MTT) assay was not observed in the present
study (data not shown).
In summary, this study illustrated that TCDD could induce the
transcriptional activity of aromatase in glioblastoma cells. Although
the physiological signicance is not known, the upregulation of
aromatase might perturb the hormonal balance in the brain or exac-
erbate the cellular response after injury.
Acknowledgement
This study was supported by The Chinese University of Hong
Kong.
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