Margaret E' Wierman, MD

1
Sex steroid effects at target tissues mechanism
of action
Margaret E. Wierman, MD Professor in Medicine,
Physiology and Biophysics University of
Colorado, Denver Health Science Center
2
Hypothalamic-Pituitary-Gonadal Axis
Hypothalamus

(-)
(-)
Gonad
Gonadal peptides
Sex Steroids E, P, T
Gametogenesis
Secondary Sex Characteristics
3
Sex Hormone Targets

• Breast
• Female Reproductive Tract uterus, ovary
• Bone
• Vascular System
• Central Nervous System
• Other GU, Male Reproductive Tract, Muscle, GI,
  Immune System, Skin, Kidney, Lung

4
Sex Hormone Ligands

• Androgens testosterone, adrenal prohormones
  (DHEA, DHEAS)
• Estrogens estradiol, estrone, estriol
• Progestins progesterone

5
Sex Hormone Ligands

• Testosterone (T) is a prohormone
• Converted by 5-a reductase to dihydrotestosterone
  (DHT)
• Converted by aromatase to estradiol (E)
• DHEA is a prohormone that is converted to T and
  then DHT and/or E

6
Steroid Hormones in the Vasculature
Fig 1 from M. Mendelsohn and R. Karas Molecular
and Cellular Basis of Cardiovascular Gender
Differences. Science 308 1583-1587, 2005.
7
Sex Hormone Ligands

• Agonists act like the natural ligands
• Altered receptor affinity or half life (eg.
  phytoestrogens, synthetic estrogens)
• Antagonists
• Partial or mixed ER tamoxifen, raloxifene
• Pure ER ICI 182,780
• Aromatase and 5-a reductase inhibitors
• Block production of E or A ligands

8
Sex Steroid Receptors

• Estrogen receptors ERa, ERß
• Progesterone receptor PRA, PRB
• Androgen receptor AR
• Glucocorticoid receptor GR

9
Nuclear Receptors
Steroid Receptors
Class ll Receptors
Orphan Receptors
GR Glucocorticoid PR Progesterone AR Androgen
ER Estrogen
NGFI-B SF-I ERR ReVERB
VDR, PPAR TR, FXR RXR, LXR RAR, PXR
NR
SR
SR
NR
RXR
Palindrome HREs
Halfsite HREs
Direct Repeat HREs
AGAACA-N3-TGTTCT GRE/PRE AGGTCA-N3-TGACCT ERE
AAA-ACGGTCA NBRE TCA-AGGTCA SFRE
ACGGTCA-N1-5-AGGTCA
10
Nuclear Receptor Family
Human
Drosophila C. elegans
48 receptors 21 receptors 250
receptors 23-ligands 1-ligand
0-ligands 25-orphans 20-orphans
250- orphans
11
Modular Structure of Steroid Receptors
variable sequence/length
C
D
E
F
A/B
C
DBD
LBD
N-domain
hinge
AF-1
AF-2
DI
DI
AF-1/AF-2 transcription activation functions or
domains DIdimerization interfaces DNA
dependent(DBD) and independent DBD-DNA binding
domain LBD-ligand binding domain
Heat shock proteins /chaperones
12
Comparison of Human ER? and ERß
hERa
1
595
N
C
A/B
DBD
LBD
F
hinge
AF1
AF2
hERß
N
C
60
22
29
14
96
1
530
13
ER? vs. ERß Tissue Distribution

• Breast ? gtgt?
• Female reproductive axis ? gtgt?
• Bone both ? and ?
• Vascular System both ? and ?
• Central Nervous System both ? and ß but in
  different areas

14
Model Systems to Study Steroid Hormone Action

• Initial biochemical reconstitution studies in
  nonphysiologic cell lines
• Tissue specific cell systems
• Primary cultures of relevant cell types
• Animal models rodent, primate
• Transgenic model systems overexpression of
  ß-gal, GFP
• Knockouts generalized then tissue specific and
  conditional
• Human studies both normal physiology and
  experiments of nature

15
Steroid Receptor Action Direct DNA Binding
Cytoplasm
Ligand
Nucleus
Cell specific Response
SR
1
Protein
HRE

• Direct DNA Binding

mRNA
16
Steroid Receptor Action Protein/Protein
Cytoplasm
Ligand
Nucleus
Cell specific Response
SR
2
1
Protein
TF
TF

• Direct
• Indirect protein-protein interactions

HRE
mRNA
17
Steroid Receptor Action
E
E
Classical HRE
ER
ER
Estrogen response element
E
ER
Nonclassical HRE
Fos
Jun
AP-1 element (or Sp-1)
18
Ligand Receptor Interactions
DNA- Binding
Ligand Binding
Domain (DBD)
Domain (LDB)
A/B C D
E
F
N-Terminal Hinge

C-Terminal

Domain
Region
Domain
Transactivation










Transactivation
Function 1 (AF-1)









Function 2 (AF-2)
N
O
OH
OH
OH
OH
( )
10
N
O




Estradiol

Tamoxifen
ICI-164,384
AF-1











-
AF-2







-



-



Agonist

Mixed Agonist/
Pure Antagonist
APS 2006 Refresher Course








Antagonist
19
Agonist
Antagonist
Fig 3 from AM Brzozowski et al. Molecular basis
of agonism and antagoinsm in the oestrogen
receptor. Nature 389 753-758, 1997
20
Steroid Receptor Coregulators

• Proteins that interact with unliganded or
  liganded steroid receptors to modulate their
  action
• Coactivators
• Corepressors

21
Estrogen Receptor Coactivators mediate
transcription
pCAF
CBP
histone acetylation
SRC
SRC
ER? ???????
ER? ??????
transcription
TFII-B
TBP
RGGTCA
RNA Pol.
ACTGGR
22
Modular Structure of Nuclear Receptors
variable sequence/length
C
D
E
F
A/B
C
DBD
LBD
N-domain
hinge
AF-2
AF-1
DI
DI
Multiple serine/threonine phosphorylations
Heat shock proteins /chaperones
AF-1/AF-2 transcription activation functions or
domains DIdimerization interfaces DNA
dependent(DBD) and independent DBD-DNA binding
domain LBD-ligand binding domain
Signals from intracellular pathways
23
Structural domains of P160 coactivators
SR interaction Box
Activation domains
PAS
bHLH
AD1
AD2
N-
C
LXXLL
(? helix)
1 2 3
LXXLL motif - interacts with hydrophobic pocket
of SR in AF-2
AD1 interacts with p300/CBP (HAT) AD2 interacts
with CARM1(HMT) bHLH/PAS interacts with CoA
(co-activator) BAP57 (ATP-chromatin remodeling
complex)
24
Coactivators direct Estrogen receptor mediated
transcription
pCAF
CBP
histone acetylation
SRC
SRC
ER? ???????
ER? ??????
transcription
TFII-B
TBP
RGGTCA
RNA Pol.
ACTGGR
25
Diverse Functions of Coregulators
26
Corepressors Inhibit Gene Transcription
mRPD3
Sin3
histone deacetylation No Transcription
NCoR/SMRT
SR
SR
27
Structural Domains of Corepressors

1
2473
305
783
RD1
RD2
RD3
RID1
RID2
DAD

• SMRT and NCOR are coregulators that activate
  histone deacyetalases (HDACs)
• RD1-3 Transcription repression domains. Sites
  of
• interaction and recruitment of HDACs. Some HDACs
  are recruited directly( HDAC 3,4,5,7), others
  (HDAC1,2) indirectly through Sin3.
• RID1,2 Steroid Receptor interaction domains
  (CoRNR boxes) IXXI/VI similar to LXXLL but
  extended sequence
• DAD deacetylase activation domain (HDAC3 is
  inactive until it binds to SMART/NCOR and
  activation is dependent on DAD domain)

28
Nuclear signal transduction mediated by many
coregulatory proteins
Fig 6 from DY Lee et al. Role of
protein Methylation in the regulation of
transcription. Endocr. Rev.26147-170, 2005.
29
Activation/Repression
ACTIVATION
pCAF
CBP
SRC
SRC
chromosome remodeling histone acetylation Transcri
ption
SR
SR ??????
REPRESSION
Sin3
mRPD3
mRPD3
Sin3
chromosome condensation histone deacetylation
NO transcription
NCoR/SMRT
NCoR/SMRT
SR
SR
30
Role of Coactivators and Corepressors in Disease

• Tamoxifen is an E antagonist in the presence of
  corepressors in reconstitution systems
• Decreased levels of N-CoR are detected in
  Tamoxifen-resistant MCF-7 breast cancer cells
• Suggests the levels of the coactivators or
  corepressors may modulate phenotype
• Some preliminary clinical data to support

31
Change in Complement of Coactivators and/or
Corepressors?
ACTIVATION
pCAF
CBP
SRC
SRC
chromosome remodeling histone acetylation Transcri
ption
SR
SR ??????
REPRESSION
mRPD3
Sin3
NCoR/SMRT
chromosome condensation histone deacetylation
NO transcription
SR
SR
32
Membrane versus Nuclear Signaling by Steroid
Receptors
Fig 1 from J. Lorenzo A new hypothesis for how
sex steroid hormones regulate bone mass.J
Clinical Investigation 111 1641-1664, 2003.
from J Lorenzo, JCI 1111642, 2003
33
Nongenomic actions of Sex steroids

• Nonreceptor mediated actions at the plasma
  membrane (membrane fluidity ligand at uM
  concentrations)
• Steroid activation of non-nuclear receptors at
  plasma membrane (ion channels, GPRs
  insensitivity to antagonists)
• Rapid signaling through membrane-bound steroid
  receptors (2 of pool, ERa or ß, PR)

34
Nongenomic effects of E via GPRs
Fig1 from ACB Cato et al. Rapid actions of
steroid receptors in cellular signaling pathways.
Science STKE 138 2002
35
Liganded AR and ER differentially interact with
Src
Fig 2 from ACB Cato et al. Rapid Actions of
steroid receptors in cellular signaling pathways.
Science STKE 138 2002
APS 2006 Refresher Course
36
Role of Phosphorylation of Steroid Receptors
C
D
E
F
A/B
C
DBD
LBD
N-domain
hinge
AF-1
AF-2
DI
DI
Multiple serine/threonine phosphorylations
Heat shock proteins /chaperones
AF-1/AF-2 transcription activation functions or
domains DIdimerization interfaces DNA
dependent(DBD) and independent DBD-DNA binding
domain LBD-ligand binding domain
Signals from intracellular pathways
37
Tissue Specific Effects of Sex Steroids

• Ligand availability estrogens, androgens
  progestins
• Receptor expression tissue specific expression
  of different steroid receptors
• Promoter specificity HRE or not?
• Co-regulator expression ubiquitous or cell
  specific?
• Nongenomic specificity of signaling systems in
  each cell type

38
Nongenomic Effects of Progesterone in Oocyte
maturation
2
3
1
Fig3 from ACB Cato et al. Rapid actions of
steroid receptors in cellular signaling pathways.
Science STKE 138 2002
39
ERß Splice Variants