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Estrogens. You can’t live without ‘em: Reproductive failure Bone loss Vasomotor disturbances (hot flashes) Some cardiovascular system vulnerabilities Some cognitive declines, mood disorders Skin changes You can’t live with ‘em: Cancer (breast, uterus, colon, pituitary) Blood clots - PowerPoint PPT Presentation
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EstrogensYou can’t live without ‘em:Reproductive failureBone lossVasomotor disturbances (hot flashes)Some cardiovascular system vulnerabilitiesSome cognitive declines, mood disordersSkin changes
You can’t live with ‘em:Cancer (breast, uterus, colon, pituitary)Blood clotsNausea or eating disorders
If you have the wrong ones (xenoestrogens) you’re in big trouble: endocrine disruption of many types
-10
-30
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E1x
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/mL
/ PregL
Age in Years Cycle Phase
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E2
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5 10 15 MP PMEF PO
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pM
Xenoestrogens (XEs) are threats to both sexes
Physiologic E levels vs. sex, age, cycles, & pregnancy
Watson, STKE Dec 1999
How do estrogens and xenoestrogens signal in the cell?
Both:From the nucleus (classical genomic mechanism)
From the membrane(novel, nonclassical nongenomic mechanism)
ERER
GPR30/(ERα36)
Nataliya Bulayeva
Confocal views of E2-peroxidase binding in GH3/B6/F10 pituitary tumor cells
Visualizing Membrane Estrogen Receptors (Binding)
Detection of PROTEIN•PROTEIN epitope interactions – the proximity
ligation assay (PLA)
R Ab
mAb (AB-N09)
mAb
mAb +R Ab -
Either two ERα Abs or Abs for two differenent proteins
Slice 3D
epitope proximity for ER
Two epitopes from within ERα
Guangzhen Hu
ERα partnering with either Gαi or Caveolin
3DSlice
Guangzhen Hu
15 min
Estrogens (either physiologic or environmental) cause rapid disappearance of mERα Ab recognition (H151 hinge region epitope) while other epitope signals are not affected – rapid conformational change.
veh3 min DES(Similar for:E2, dieldrin, endosulfan, nonylphenol)
Such responses are both rapid and potentCeleste Campbell
GH3/B6/F10 pituitary tumor cells
Ever since then we have been examining mechanisms of rapid E and XE actions, related to - functional outcomes – looking at ways XEs can disrupt normal function.
All small molecules ~270-400 MW
We picked these 16 originally to compare because of interests our interests in:
■ life-stage prevalence of physiological estrogens
■ xenoestrogen toxicities and disease associations
■ structural features/groupings
■ therapeutic uses
So many estrogens… so little time!
… and we are adding more compounds all the time to enhance our stucture-activity analyses
Some typical results using these assays:
have taught us important concepts about how these
compounds act via nongenomic mechanisms
Andrea Norfleet/Jennifer Jeng
We see non-monotonic dose-responses for E2-induced PRL release from GH3/B6/F10 pituitary cells at 6 min
(also true for other estrogens)P
RL
sec
reti
on
Prolactin (PRL) secretion
Xenoestrogens also cause PRL release in a nonmonotonic pattern
0
100
200
300
10-12 10-11 10-10 10-9 10-8
PR
L/C
V (
% o
f co
ntr
ol)
Ann Wozniak
* *
*
Bisphenol A
Big news for the toxicology world -- Nonmonotonic responses make it impossible to extrapolate back from a high dose to see what the lowest effective dose would be.
PR
L/C
V (
% o
f C
on
tro
l)
Time (minutes)
10-12M 10-11M 10-10M 10-9M 10-8M
D9 Cell Time Dependent PRL Release at 10-8M E2
D9 Cell Dose Dependent PRL Release at 3 Minutes
Ann Wozniak
E2 Concentration (M)
ERα is necessary for many of these responsesCells selected for very low mERα levels (D9 subclone) ….can’t elicit the PRL
response to estrogens.
To efficiently identify specific receptors involved, we have also used
--siRNA knockdowns of NRs ER primarily responsible, , and GPR30 sometimes modulatory
--receptor-selective agonists --receptor-selective antagonists
To prove action from the membrane we have used:--various impeded ligands (E2-BSA, E2-peroxidase, E2-dendrimers)
--ERα Ab triggering of responses
What signaling mechanisms may be provoked by xenoEs acting on mERs
Ca++
mitogen-activated protein kinases (MAPKs) cAMP and PKA G protein activations other kinases/phosphatases caspases downstream transcription factor post-translational modifications
and how they relate to functional endpoints
Cell signaling is complicated - not just a linear signaling cascade. Many things must be coordinated. It is a web.
Signaling is like a Rube Goldberg Machine http://blueballfixed.ytmnd.com/
created by the Web site Something Awful
Mitogen-activated protein kinases (MAPKs) like ERK:
Are signal integrators. …..many signaling pathways funnel into the ERK “integrator”.
Are associated with major cellular destinies, like cell proliferation
- non-nuclear feeder signaling streams we have tested & found to be involved in E-induced downstream ERK signaling
Signaling starting at the membrane funnels into the downstream kinases ……
and then fans out again to coordinate major complex downstream functions.
The MAP kinases are the integrators that separate these two phases
MAPKsERKsJNKsp38
cell cycle
DNA and proteinsynthesis for replication
Proliferation
new proteins
Differentiationcaspase cascades
Death
2nd messengers and signaling enzymes (kinases, and phosphatases, lipases, NT-cyclases) and scaffolds
receptors
SecretionMigration
One of our most useful techniques is a fixed-cell 96-well plate immunoassay which:
--is optimized for each cell type, epitope-Ab, and cell compartments (based on permiabilization); makes use of the many Abs now available to the activated (usu. p’ated) form of proteins
-- is relatively high throughput allowing us to compare multiple xenoestrogens at a wide range of concentrations acting on multiple antigens
-- allows us to do physiologic Es + XEs in incremental mixtures, the real-life scenarios of XE toxic exposures
E2
DES Endosulfan
Nataliya Bulayeva
0 3' 6' 10' 15' 30'80
90
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150*
Nonylphenol
ER
K a
cti
va
tio
n -
% o
f co
ntr
ol)
time, min
*
0 3' 6' 10' 15' 30'80
90
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140
150
*
0 3' 6' 10' 15' 30'80
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**
0 3' 6' 10' 15' 30'80
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150 *
* * *
DDE
Dieldrin
Coumestrol
E2-Peroxidase
96-well plate immunoassays for phospho-ERK ligands all at 1nM
Responses oscillate with time; XEs cause different phasing than E2
control -14 -13 -12 -11 -10 -9 -8
E2 (3 min)*
* *
control-14 -13 -12 -11 -10 -9 -8
Nonylphenol (30 min)
*
**
control -14 -13 -12 -11 -10 -9 -880
90
100
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150 Coumestrol (6 min)
* *
*
**
*
control -14 -13 -12 -11 -10 -9 -880
90
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150 Endosulfan (30 min)
* **
* *
Concentration Nataliya Bulayeva
Responses are often non-monotonic: U-, V-, or M-shaped dose-responses
Why do MAPK responses oscillate oscillate with time
and concentration.
The sum of pathways A & B is an oscillating curve over time or concentration
Here is a new example of one of the most rapid -- Gi activation (charged with GTP and assayable with an Ab to the charged form).
Because it is so proximal to receptor-binding it is much more rapid (peaks at 15 seconds) than downstream signal integrator responses like for MAPKs (several minutes). Again see differences between E2 and XEs
Guangzhen Hu
Some responses are very rapid – seconds
0 100 200 300 400 500
GTP-bound G
i -- %
of vehicle
40
60
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160E2 NP BPA
*
* *
Seconds
*
Jennifer Jeng
log [M]
Pro
lifer
atio
n (
% o
f co
ntr
ol)
90
100
110
120
130
140E2
BPAEPPPOPNP
Con -15 -14 -13 -12 -11 -10 -9 -8 -7 -6
* *
**
****
*
*
*
******
*
Both physiologic estrogens and xenoestrogens cause pituitary cells to proliferate (just as pituitaries and their tumors grow in response to estrogens)…
Proliferation function downstream of MAPKs are affected by XEs
Rene Vinas
Mechanisms that affect cell number – apoptosis – are elicited by XEs
0 10 20 30 40 50 60
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chol TPA
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daid
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pE
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icle
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chol TPA
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Time (min)0 10 20 30 40 50 60
pA
TF
2 (
% o
f V
eh
icle
)
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Jennifer Jeng
Examples: Elk and ATF2, with some differences in response pattern
…Es and XEs (in this case phytoEs) can also rapidly activate (phosphorylate) transcription factors downstream of ERKs
altered timing (phasing)
altered dose-response patterns and levels
imperfect mimicry or inhibition of multiple estrogenic actions activated via the nongenomic pathways
To summarize the 1st part…endocrine disruption by xenoestrogens includes:
Can a structure/chemical characteristic of an estrogen predict its activity in a nongenomic
response?
Here again are the ones we have studied
They can all be described by their physical characteristic of lipophilicity – the partition coefficient between octanol and water
Short chain alkylphenols are less lipohillic than long chain ones
13 estrogens – their activities in various functional and signaling responses compared to their hydrophobicity
(Chlorinated compounds were left out as they significantly reduced the correlations)
Jennifer JengMikhail Kochukov
The most complicated - both genomic and nongenomic pathway control
Best correlations are for structures that vary in simple ways (side-chain length)
Chemists can use such information to design less estrogenic versions of these compounds
But in real life these estrogens are rarely present by themselves
You have physiologic estrogens on board already – and then you get exposed to xenoestrogens on top of that.
Do xenoestrogens interfere with physiologic estrogens? Do they disturb the normal signaling patterns? How?
In different ways - examples for ERK signaling follow that demonstrate some
principles of estrogenic endocrine disruption.
Temporal phasing of response….XenoEs can delay the response after causing an initial dephosphorylation
0 10 20 30 40 50 60
80
100
120
140
160EP EP+E3
E3
Jennifer JengTime in Minutes
pE
RK
XEs can enhance an estrogenic response at concentrations where it is less effective by itself; inhibit at higher concentrations – this is the
most typical effect of combinations
Jennifer Jeng
(physiologic estrogens all at 1 nM for these studies; 5 min responses)
p-E
RK
(%
of c
on
tro
l)
Log [Concentration (M)]
Have assessed for 3 physiologic estrogens challenged by 5 different XEs
Phospho-activation of ERKE2[10-9M]+ BPA[10-14M] + NP[10-8M]
Time (min)
0 10 20 30 40 50 60
pE
RK
(% o
f ve
hic
le)
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90
100
110
120
130
140
E2[10-9M]
BPA[10-14M ]
NP[10-8M]
E2+BPA+NP
***
*
*
*
**
*
*
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**
*
V
*
*
**
Rene´ Viñas
Extreme Disruption by Multiple XEs - can completely wipe out a physicologic E response – we have seen this now with multiple
combinations of XEs.
Any of these alterations “disrupts” the normal pattern
or extent of physiologic estrogen signaling.
In summary we have learned that Es/XEs acting via nongenomic pathways: ► signal more potently via the membrane versions of estrogen receptors
► activate multiple signaling pathways
► responses often oscillate with time and are non-monotonic (U- V- or even M-shaped dose-responses and XEs alter this to disrupt signaling – a difficult target for regulation
► mediated primarily via ER (depending on tissue); modulated by other ER subtypes
► XEs elicit tissue-specific disfunctions (secretion, differentiation, cell proliferation, enzymatic cascades, components of inflammatory responses, behavior, ROS) in different nontransfected cell types: pituitary, breast, prostate, neuronal, cells of the immune system……
► have particular developmental window / stage-specific vulnerabilities to XEs
► chemical structures of Es/XEs can predict strength/potency of nongenomic responses
► XE mixtures (which is what we are exposed to) dramatically disrupt physiologic E responses
How we would like to use our newly determined principles learned via high throughput signaling assays: Green Chemistry Design
We are currently collaborating with labs who view endocrine disruption at different (increasingly more complicated) levels.
remediation of existing contamin-
ants
animal develop-
ment - later assigned to
a NR
nongenomic cell signaling
- later assigned to
a NR
NR binding and NR-based
genomic response
in silico chemical- receptor dockingREDESIGN
+ -+++
AcknowledgementsWatson lab – over the years working on nongenomic E signaling projects
Todd PappasCeleste Campbell (Finnerty)Andrea NorfleetAdrian JakubasBridget HawkinsGaga ZivadinovicNataliya BulayevaTeresa ReedLeanne LashAnn WozniakRebecca AlyeaYow-Jiun Jennifer JengMikhail KochukovStephanie LaurenceAnannya BangaLuke KoongJune GuptarakGuangzhen HuRene VinasManish Saraf
Acknowledgements
FundingNIEHS (R01s and training grant)NIDA (P20 Center and training grant)Am. Inst. for Cancer ResearchUTMB Center for Addiction ResearchSealy Center for Environ. Health & MedicinePassport Fndn. Innovator Award
Collaborators: Bahiru Gametchu, Mary Thomas, Kathryn Cunningham, Randy Goldblum & Terumi Midoro-Horiuti
Green Chemistry Collaborators: Ruben Abyuan & Fiona McRobb, Bruce Blumberg, Susan Jobling, Terry Collins, John Warner, Pete Myers & Karen O’Brien
Fix cells: various methods to match epitope and cell type -- unpermeabilized measures membrane proteins-- permeabilized (usu. with detergent) measures intracellular proteins
(We have used it for various ERs, DAT, pMAPKs, and p-transcription factors, and GTP-G proteins in many different cell types)
Incubate with 1°Ab; biotinylated 2° Ab; avidin-conjugated alk. phos.
Incubate with pNpp → pNp at 37° in dark, & read at 405 nm Ag quantitation
Wash off reagents
Stain with 0.1% crystal violet, wash, extract, read at 562 nM cell number (normalization for each well)
inside
outsideImmuno-assay for quantitation of membrane vs. intracellular
proteins, their trafficking, and their activation state
molecules- receptors -- cell signaling - organisms- remediation genomics nongenomics
In silico – Ruben Abyuan and Fiona …..Receptor binding and genomic activation – Bruce BlumbergNongenomic cell-type specific signaling – Cheryl WatsonDeveloping organisms – Susan JoblingRemediation – Terry Collins, John WarnerCementing it all together – Pete Myers and Karen O’Brien
In silico cell-free cell dependent development chemical capture of amphib & fishes and degradation
Do TAML activators activate ERK – NO!!!!!
www.secondlife.comGenome Island, Genome (139,145, 37)
CW searching for receptors, wherever they are in the cell
Crucial difference between our studies and past studies on xenoestrogens:
● we look at rapid nongenomic effects
●we use very low (fMnM) concentrations (very relevant to those found commonly in the environment) within very wide concentration ranges ….and sensitive, quantitative cellular response assay systems
● we use non-transfected cell systems to avoid overexpression and heteroexpression artifacts
The assays we plan to do in GH3/B6/F10 pituitary cells for
the Green Chem Center Project
• Signaling assays--G protein activation (proximal signaling)
--ERK (+JNK & p38) kinase activations (downstream integrated signaling)
• Function assays--PRL release (nongenomic functional endpoint)
--Cell proliferation (complex functional endpoint)
If the money got bigger I could add:
• Other cell types in which we have already monitored nongenomic signaling effects (breast cancer, prostate cancer, neuronal, immune system)
• Other myriad signaling pathways (IPs, ions, other kinases, activation of transcription factors)
• Other functional endpoints