Download KorbSpr08

Synergistic Action of 17-b estradiol and Bisphenol A in a Pituitary Cell Line
Sarah Korb and Winnifred Bryant, Ph. D.
Department of Biology
University of Wisconsin, Eau Claire
*
*
*
*
600
*†
300
ALU/mg protein
*
350
ALU/mg protein
INTRODUCTION
The gonadal steroid 17 β-estradiol (E2) is produced
primarily by the ovaries and regulates gene regulation,
growth and differentiation in a number of target
tissues, including the pituitary gland, where E2
regulates the production and/or release of hormones
such as prolactin and luteinizing hormone.
250
*
200
*
150
*
100
50
E2
10-15M
10-12M
Bisphenol A
10-9M
*
400
*
*
300
200
100
0
Vehicle
*
500
vehicle
Vehicle
10-6M
E2
E2
G 10-12
G 10-9
0
G 10-6
10-12M 10-9M
Genistein
10-6M
vehicle
Vehicle
E2
E2
G 10-12
10-12M
Kepone
G 10-9
10-9M
G 10-6
10-6M
Figure 1. Bisphenol A (BA, xenoestrogen), genistein (G, a phytoestrogen), and kepone (K, a xenoestrogen) stimulate transcriptional activity of the pGL3 reporter
in a dose-related fashion. Cells were transfected with ERa and the pGL3 reporters. Administration of physiological doses of BA, G, and K significantly increased
transcriptional activity (as compared to vehicle treated controls). There was a positive correlation between increasing concentrations and transcriptional activity
*Significantly different from vehicle controls, p<.01 or p<.05., † significantly different from 10-9M and 10-12M G
1C
*
1600
*
1400
http://www.healthsystem.virginia.edu/uvahealth/adult_gyneonc/images/ei_0282.jpg
*
ALU/mg protein
1200
Estrogen receptor alpha (ERα) is a ligand-inducible
transcription factor that mediates the physiological
effects of E2. In a target cell, ERa occupancy by E2
results in receptor activation and nuclear localization.
Subsequent binding to a regulatory site (estrogen
response element (ERE)) in a target gene allows
genes to be activated, and ultimately, produces
proteins are produced that elicit physiological effects
*
1000
800
*
600
400
200
0
Vehicle
Vehicle
ICI
ICI
E2
E2/ICI
E2
E2/ICI
G
BA
G/ICI
BA/ICI
BA
G
BA/ICI
G/ICI
D
D/ICI
D
D/ICI
K
K
K/ICI
K/ICI
Figure 2. Stimulatory effects on transcriptional activity in GH3 cells are mediated by ERa. Cells were transfected with ERa and pGL3 reporter, then stimulated
with E2 (10-8M) , or a panel of xeno/phytoestrogens (10-6M) in the absence and presence of the pure ERa antiestrogen, ICI 182,780. Transcriptional effects of the
estrogenic compounds were ameliorated in the presence of ICI 182,780, indicating that these effects are receptor mediated. *Significantly different from vehicle
controls, p<.001
18000
*
MATERIALS AND METHODS
Cells
GH3 cells (a rat pituitary cell line secreting both prolactin
and growth hormone) were cultured in DMEM with
growth serum; cells were maintained at 37oC in
95%O2/5%CO2.
Gene Expression in Mammalian Cells
100 ng rat ERa and 200ng pGL3 reporter (ERE gene
fused to firefly luciferase gene) were transiently
transfected into GH3 cells. For all studies, cells were
treated for 24 hours with 10nM E2, and/or the estrogen
mimics bisphenol A (BA), genistein (G), daidzein (D),
and kepone (K) at doses indicated in respective figures.
In antagonist studies, the ERa antagonist ICI 182,780
was used at a concentration of 1mM. Cells were
collected in 200 ul 1X Promega lysis buffer.
To measure transcriptional activity, 50ul luciferin was
added to an equal volume of lysate and luciferase
activity assessed using a Turner Biosystems 20/20n
luminometer. Data are expressed as arbitrary light units
(ALU)/mg protein.
Statistical Analysis.
Transfections were performed in triplicate. Data were
analyzed by one way ANOVA and a Bonferroni post-hoc
test using GraphPad Prism software.
*
2500
12000
10000
*
8000
6000
2000
1500
1000
*
4000
500
*
2000
0
0
Untransfected Reporter Only
E2 alone
BA alone
E2/BA
untransfected
reporter
Untransfected Reporter Only
receptor/reporter
E2 alone
30000
ALU/mg protein
14000
ALU/mg protein
ALU/mg protein
16000
The actions of endogenous E2 can be mimicked by
environmental estrogens that are produced in plants
(phytoestrogens) or produced commercially as synthetic
compounds (xenoestrogens). These studies examine
changes
in
gene
transcription
following
coadministration of E2 and a variety of E2 mimics
3000
*
receptor/reporter
K alone
*
25000
*
20000
15000
10000
5000
receptor/reporter
E2/K
0
Untransfected Reporter Only
E2 alone
D alone
E2/D
Figure 3. The xenoestrogens kepone acts synergistically with E2 to stimulate transcriptional activity of the pGL3 reporter. GH3 cells were transfected with ERa and
pGL3 reporter. Transfected cells were treated with vehicle, E2 alone, xenoestrogen alone, or E2 plus xenoestrogen. White bars represent vehicle treated cells in
each group.*Significantly different from vehicle controls, p<.01
SUMMARY AND CONCLUSIONS
The steroid hormone E2 targets the pituitary gland and the brain where it regulates, among other things, the growth and release of reproductive hormones. It is clear that the
presence of one hormone may be required for another hormone to have its designated physiological effect, as is the case in priming. Moreover two hormones may act at their
respective receptors to achieve a controlled physiological response. The sensitivity of the pituitary gland to E2 and the ability of non-steroidal compounds to bind/activate the
ERE have led us to examine interactions between estrogenic compounds and their subsequent effect on gene transcription on a model promoter (pGL3).
Cells exhibited moderate (1.5 fold) to robust (7 fold) dose-related transcriptional responses to single treatments of E2, xenoestrogens, or phytoestrogens. As indicated in
Figure 1, the stimulating concentrations of xeno/phytoestrogens were well within the physiological range that most endogenous hormones circulate. Antagonist studies (Figure
2) indicate that the panel of xeno/phytoestrogens used in the studies utilize the same receptor as E2, Thus, as we consider the greater than additive (E2 and BA) or synergistic
(E2 and kepone) effects on transcription that are observed with are co-administered, it is possible that results are due to differential recruitment of cofactors in the nucleus
during gene activation. Further studies will examine this hypothesis.
ACKNOWLEGMENTS
These studies were supported by ORSP-UWEC