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Nuclear Receptors:Estrogen Receptor & SERMs

Outline

Nuclear Receptor structure

Estrogen receptor and Selective Estrogen Receptor Modulators (SERMs)

Hormone Response Elements (DNA binding sites for nuclear receptors):

GlucocorticoidResponse Element:

EstrogenResponse Element:

steroid receptors

receptor is homodimer GR Glucocorticoid R MR Mineralocorticoid R ER Estrogen R PR Progesterone R AR Androgen R

binds to DNA Inverted Repeat (palindromic)

Located in cytoplasm in absence of ligand; ligand binding causes dissociation of heat shock proteins, receptor dimerization, and translocation to nucleus; then binds to DNA and recruits coactivators

Type I Nuclear Receptors: non-steroid receptors

receptor is Heterodimer Usually RXR (retinoid X (9 cis

retinoic acid) receptor) with: TR (Thyroid hormone R) VDR (Vitamin D R) RAR (Retinoic acid R) PPAR (Peroxisome proliferator-

activated R)

binds to DNA Direct Repeat

Repeats are separated by 1-5 nucleotides

Located in nucleus in absence of ligand, often complexed with corepressors

Ligand binding to NR causes dissociation of corepressor and recruitment of coactivator proteins.

Type II Nuclear Receptors:

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Chromosome Immunoprecipitation (ChIP): Another method for Protein-DNA analysis

Used for: mapping the DNA target of

DNA-binding proteins mapping the DNA target of

histone modifying enzymes

Method:

Cross-link the DNA & protein (or leave in its native bound form)

Fragment the chromatin

Precipitate the DNA-Protein complexes with antibody to the protein of interest

Analyze the bound DNA

How do structurally similar NR proteins regulate different genes in different cells?

Ligand:

Different specificity for ligand binding by NR

Metabolic control of ligand availability

Nuclear Receptor

Recognize different DNA sequences (hormone response element)

Different expression patterns of NRs in cells

Activate different coactivators/coregulators

Does ligand specificity explain receptor specificity?The worst case scenario - compare MR and GR

Glucocorticoid

Mineralocorticoid

How do tissues distinguish between Cortisol and Aldosterone?

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How do tissues distinguish between Cortisol and Aldosterone?

11 hydroxysteroiddehydrogenase

Enzyme located in collecting duct of the kidneys

inactive

Glucocorticoid Response Element and Mineralocorticoid Response Elements are identical.

GR and MR both bind glucocorticoids & minerocorticoids with similar affinity [Cortisol] is typically ~10x higher than [Aldosterone].

Chloramphenicol Acetyl Transferase reporter gene assay measures acetylation of chloramphenicol

Luciferase reporter gene assay measures luminescent light emission catalyzed by the firefly enzyme luciferase

Luciferin (substrate)

Luciferase

Studying Nuclear Receptor Function:

Functional Domains of Nuclear ReceptorsHomology among Two Classes of Nuclear Receptors

Class I

Class II

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Functional Domains of Nuclear ReceptorsDNA-binding Domain of Nuclear Receptor: Zinc Fingers

Cartoon view of Zinc fingers of estrogen receptor

Dimerization RegionDNA-binding

Region

Zn Zn

C

C

C

C

C C

C C

Ligand Binding Domain of Nuclear Receptor(Ligand-Binding Domain of estrogen receptor is shown)

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Ligand-binding Domain of Estrogen Receptor

Antagonist Bound:

agonist

antagonist

Helix 12

Helix 12

Co-activator binds here

Co-activators cant bind,Instead co-repressors bind

Agonist Bound:(side view)

No Agonist bound(or Antagonist bound)

Agonist bound

Co-repressors recruited Histones deacetylated Transcription repressed

Co-activators recruited Histones acetylated Transcription enhanced

Estrogen Effects

LH and FSH stimulate estrogen and progesterone production in ovaries

Reproductive function: ovulation, menstrual cycle, pregnancy

Development of female characteristics in puberty

Bone health, prevention of osteoporosis

amount of good HDL cholesterol; bad LDL cholesterol; risk of heart disease

Estrogen-dependent cancers (breast, ovarian); cell proliferation

Thrombosis (blood clots)

Prevent menopausal symptoms (hot flashes, mood swings, fatigue)

Estradiol

September 24, 2002

Womens Health Initiative study on HRT stopped

In July, the National Institutes of Health (NIH) halted a large, in-progress study examining the effects of a widely used type of hormone replacement therapy (HRT) medication called Prempro, which combines the hormones estrogen and progestin.

The study, which is one of five major studies that comprise the large clinical trial called the Womens Health Initiative (WHI), was discontinued because the hormones appear to increase a womans risk of breast cancer as well as heart disease, blood clots and stroke.

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Estrogen ReceptorDrugs that bind to Estrogen Receptor Agonist: Estrogen

Contraceptive Hormone replacement therapy

Selective Estrogen Receptor Modulators (SERM) partial agonist/antagonist Clomiphene

Infertility treatment; blocks estrogen binding in anterior pituitary GnRH stimulates ovulation Tamoxifen

Used to treat estrogen-dependent breast cancer Raloxifene

Used to treat osteoporosis for post-menopausal women estrogen-dependent breast cancer and uterine cancer

Antagonist Fulvestrant

Used to treat metastatic estrogen-dependent breast cancer

Drugs that influence Estrogen Synthesis: Aromatase Inhibitors

Blocks synthesis of estrogen in post-menopausal women Used in treatment of breast cancer and ovarian cancer in post-menopausal women

Estrogen-Induced Proliferation andSpontaneous New Mutations

Normal breast or uterine cell

Increased proliferation

Estrogen stimulation

Mistake in DNA duplication

Aromatase Inhibitors

Estradiol is produced in: Ovaries Adrenal cortex (downstream of DHEA), adipose tissue, (and liver,

brain, placenta) Aromatase enzyme converts androgens to estrogens in peripheral

tissues (e.g. breasts) Aromatase inhibitors block estrogen production in post-menopausal

women, where major source of estrogen is adrenal cortex Aromatase inhibitors not generally used in premenopausal women,

because its effects are reduced by hypothalamus/pituitary feedback: estrogen GnRH LH & FSH estrogen

Antiestrogens

Estrogen receptor

Binding to DNA

Estrogen receptor

Coactivator binds

Genes are activated

Estrogen

Coactivator cannot bind to antiestrogen-bound receptor

No gene activation

Antiestrogen

Binding to DNA

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Selective Receptor Modulator (SRMs)SRMs are partial agonists/antagonists whose action depends on the ligand and the target tissue.

SERMs: ER (estrogen receptor) Used for protection of bones & as anti-cancer agents

SPRMs: PR (progesterone receptor) Under investigation for endometriosis & uterine fibroid tumors

SARMs: AR (androgen receptor) Develop drugs to protect bones and promote muscle without

side effects?

SGCRM: GR (glucocorticoid receptor) Develop an anti-inflammatory agent with fewer side effects?

Other SRMs

Tamoxifen and Breast Cancer Prevention

Cumulative number

of cases (per 1000

women)

Placebo

Tamoxifen

0 1 2 3 4 5 6

40

30

20

10

0

Invasive breast cancer rates in women at high risk

Years

The Need for Better SERMs

Bad effects Increases uterine cancer risk Increases blood clot risk

Tamoxifen

Good effects Reduces breast cancer risk Lowers LDL cholesterol Strengthens bones

Action of SERMS in Target Tissues

Fulvestrant

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Search for the Perfect SERM

The ideal SERM would:

Strengthen bones

Lower LDL cholesterol and raise HDL cholesterol

NOT increase blot clot risk

Relieve hot flashes

Reduce breast cancer risk

Reduce uterine cancer risk

How can SERMs have different effects on different tissues?

Different estrogen receptors. There are two estrogen receptors: ERand ER. They are both widely distributed but with some selectivity for particular tissues. Different SERMs may interact selectively with different ERs.

ERs may adopt multiple conformations, depending on the ligand, and recruit different co-activators or co-repressors. A combination of multiple ER conformations with different tissue distribution of co-activators and co-repressors may be the underlying reason.

Different distribution and abundance of co-activators and co-repressors in different tissues

For the future: Can new and better SERMs be designed with the desired agonist/antagonist properties in different target tissues?

Pharmacology of NR Ligands

SERM-bound ER-ligand binding domains (LBDs) take

intermediate structure

Model: Relative concentration of coactivator /corepressor may determine SERM-bound ERs

action

Difference in cofactor binding surface on these LBDs gives them distinct preference of coregulator partners

This model proposes that SERMs enable ER to bind multiple types of coregulators.

Summary Nuclear receptors contain modular transcription-activation domain, DNA-

binding domain, and hormone-binding domain

Nuclear receptors bind DNA as a dimer, with -helix from first zinc finger of each receptor binding to major groove of DNA to recognize half of the response element

Binding of agonist recruits co-activators (e.g. HATs), while binding of antagonist recruits co-repressors (e.g. histone deacetylases)

Depending on the ligand and the tissue, either agonist or antagonist effects may be produced

SRMs provide opportunities to exploit more beneficial effects of nuclear receptors while avoiding undesired ones.

Selective Estrogen Receptor Modulators (e.g.Tamoxifen, Raloxifene) aim to be estrogen antagonists in breast & endometrium (to cancer), antagonists in blood (to stroke) and estrogen agonists in other tissues (to maintain bone mass, hot flashes, good

HDL cholesterol)