Dec. 2001

Steroid Steroid Hormone/Nuclear Hormone/Nuclear

ReceptorsReceptorsDon DeFranco, Ph.D., dod1@pitt.eduDon DeFranco, Ph.D., dod1@pitt.edu

• Steroid Hormone StructureSteroid Hormone Structure• Nuclear ReceptorsNuclear Receptors

-DescriptionDescription-StructureStructure-Signal TransductionSignal Transduction-DNA-Binding PropertiesDNA-Binding Properties-Regulation of Gene ExpressionRegulation of Gene Expression-DiseasesDiseases-Drug TargetsDrug Targets

Dec. 2001

Steroid Steroid Hormone/Nuclear Hormone/Nuclear

Receptors:Receptors:Clinical RelevanceClinical Relevance

• Endocrine Disorders (Hormone Endocrine Disorders (Hormone Insensitivity Syndromes; Cancer)Insensitivity Syndromes; Cancer)

• Regulators of Various Regulators of Various Metabolic ProcessesMetabolic Processes

• Therapeutic Use to Reduce Therapeutic Use to Reduce Inflammatory DiseasesInflammatory Diseases

Dec. 2001

• Reduce inflammation and immune responses

• In clinical practice since 1948

• $10,000,000,000./year market size in US

GLUCOCORTICOIDSGLUCOCORTICOIDS

Dec. 2001

Glucocorticoids: Side EffectsGlucocorticoids: Side Effects

Dec. 2001

Steroid Hormones: Derived from CholesterolSteroid Hormones: Derived from CholesterolLipid Soluble: Lipid Soluble: Able to cross Able to cross plasma plasma membrane by membrane by passive passive diffusiondiffusion

Dec. 2001

SUMMARY OF INITIAL STEPS IN STEROID SUMMARY OF INITIAL STEPS IN STEROID HORMONE SIGNAL TRANSDUCTIONHORMONE SIGNAL TRANSDUCTION

1.1. Stimulation of hormone production by releasing hormones or Stimulation of hormone production by releasing hormones or factors that are synthesized and secreted from neuroendocrine factors that are synthesized and secreted from neuroendocrine cells (e.g. Adrenocorticotrophic hormone [ACTH] from cells (e.g. Adrenocorticotrophic hormone [ACTH] from specialized cells in the anterior pituitary stimulates specialized cells in the anterior pituitary stimulates glucocorticoid production from specialized adrenal cortical glucocorticoid production from specialized adrenal cortical cells).cells).

2.2. Transport of hormone to its target cell via plasma transport Transport of hormone to its target cell via plasma transport proteins in the bloodstream.proteins in the bloodstream.

3.3. Dissociation from the plasma transporter proteins and Dissociation from the plasma transporter proteins and diffusion of the free hormone across the plasma cell membrane.diffusion of the free hormone across the plasma cell membrane.

4.4. Hormone encounters receptor either in the Hormone encounters receptor either in the cytoplasm cytoplasm or the or the nucleusnucleus..

Dec. 2001

Hypothalamic-Pituitary-Adrenal (HPA) Axis: Hypothalamic-Pituitary-Adrenal (HPA) Axis: Regulation of Cortisol Synthesis and Regulation of Cortisol Synthesis and SecretionSecretion

Dec. 2001

Nuclear Localization of Steroid ReceptorsNuclear Localization of Steroid Receptors

Unliganded Unliganded GRGR

Liganded Liganded GRGR

Unliganded/Unliganded/Liganded ERLiganded ER

NOTE: Steroid Receptors Shuttle Between the Nucleus & Cytoplasm

Dec. 2001

Domain Organization of Steroid ReceptorsDomain Organization of Steroid Receptors

Dec. 2001

HormoneHormone Receptor (s)Receptor (s)

AndrogensAndrogens ARAR

MineralocorticoidsMineralocorticoids MRMR

EstrogensEstrogens ERERERER

ProgesteroneProgesterone PRPRAA

PRPRBB

GlucocorticoidsGlucocorticoids GRGRGRGR

Steroid Hormone Receptors: Limited FormsSteroid Hormone Receptors: Limited Forms

Dec. 2001

TWO RECEPTORS FOR ESTROGENTWO RECEPTORS FOR ESTROGENERER & ER & ER

Note: Both types of ER can form homodimers Note: Both types of ER can form homodimers (e.g. ER(e.g. ER/ER/ER) or heterodimers (i.e. ) or heterodimers (i.e. ERER/ER/ER))

Dec. 2001

Estrogen Receptor Distribution Within the BodyEstrogen Receptor Distribution Within the Body

Dec. 2001

Differential Actions of ERDifferential Actions of ER and ER and ER

Dec. 2001

Dec. 2001

Vitamin D

Ligands for Some Nuclear ReceptorsLigands for Some Nuclear Receptors

Dec. 2001

Ligands for Various Orphan ReceptorsLigands for Various Orphan Receptors

Dec. 2001

Metabolic Pathways of Nuclear Receptor Ligands

Chawla et al Science 294:1866, 2001

Dec. 2001

•Active ingredient of GugulipidActive ingredient of Gugulipid•Gum resin of Gum resin of Commiphor muklCommiphor mukl•In use since 600 BCIn use since 600 BC•Antagonist of FXR Antagonist of FXR •(Farnesoid or Bile Acid Receptor)(Farnesoid or Bile Acid Receptor)•Lowers cholesterol and triglycerideLowers cholesterol and triglyceridelevelslevels

Dec. 2001

•Active ingredient in St. John’s WartActive ingredient in St. John’s Wart•Extract of Extract of Hypericum perforatumHypericum perforatum•In use over 2000 yearsIn use over 2000 years•Ligand for PXR Ligand for PXR (Pregnane X or Xenobiotic Receptor)(Pregnane X or Xenobiotic Receptor)•Induction of CYP3A4Induction of CYP3A4

Dec. 2001

NR ClassificationNR Classification

Nuclear receptors can be classified by:

• Their DNA binding specificity• Their hormone binding specificity

• Their dimerization properties

Dec. 2001

DNA Binding Configurations of NRsDNA Binding Configurations of NRs

HOMODIMERHOMODIMER MONOMERMONOMER HETERODIMERHETERODIMER

• Steroid hormone receptors bind primarily as homodimers

• Retinoid X Receptor (RXR; 9-cis RA) is a common dimerization partner for various nuclear receptors including thyroid hormone receptor, retinoic acid receptor (all-trans RA), vitamin D receptor and others

Dec. 2001

Domain Structure of Nuclear Receptors and Dimerization and DNA Binding

Properties

EE FFDDCCA/BA/B

LBD AF-2LBD AF-2hingehingeDBDDBDAF-1AF-111 180180 263263 304304 554554 595595

ER

LBD LBD

DBDDBD

Homodimeric Bindingto Inverted RepeatsSteroid ReceptorsER, GR, MR, AR, PR

Some Orphan Receptors

LBD RXRRXR

DBDDBD

Heterodimeric Bindingto Direct RepeatsWith RXR Partner

Non-Steroid ReceptorsTR, RAR, VDR, PPAR

LBD

DBDDBD

Monomeric Bindingto Half Site

Orphan ReceptorsLigand Binding Unknown

NGF1-B, ERR, COUP

?

Dec. 2001

Dec. 2001

Glucocorticoid Response Unit of the PEPCK GeneGlucocorticoid Response Unit of the PEPCK Gene

CEBP/ß

TBP

-27

-90

-325

COUPGR GR

-380

HNF3HNF4

-410-445

+1

Accessory DNA-binding Accessory DNA-binding Factors Required!!Factors Required!!

((Can impart tissue-Can impart tissue-specificity)specificity)NOTE: Phosphoenolpyruvate carboxykinase NOTE: Phosphoenolpyruvate carboxykinase

(PEPCK) gene is glucocorticoid regulated (PEPCK) gene is glucocorticoid regulated in liver onlyin liver only

Dec. 2001

Estrogen Regulated PromotersEstrogen Regulated PromotersAssociation of other Transcription FactorsAssociation of other Transcription Factors

All EREs on All EREs on chromosomes 21 chromosomes 21 & 22& 22

From: Myles From: Myles Brown Brown laboratory laboratory (Dana Farber (Dana Farber Cancer Cancer Institute), Institute), 2005, 20062005, 2006

Genome wide Genome wide analysisanalysis

Dec. 2001

GRGR +1GRGR

GREGRE Transcriptional ActivationTranscriptional Activation

Transcriptional Repression ITranscriptional Repression I((Direct DNA-bindingDirect DNA-binding))

HH HH

+1

nGREnGRE

GRGRHH

+1

cGREcGRE

GRGRHH

Transcriptional Repression IITranscriptional Repression II((Co-occupancyCo-occupancy))

+1GRGR

HH

NFNFBB

Transcriptional Repression IIITranscriptional Repression III((TetheringTethering))

AP1AP1

Basic Mechanisms of Transcriptional Activation/RepressionBasic Mechanisms of Transcriptional Activation/Repressionby the Glucocorticoid Receptorby the Glucocorticoid Receptor

Dec. 2001

Glucocorticoid Side Effects: Molecular MechanismsGlucocorticoid Side Effects: Molecular Mechanisms

Dec. 2001

CHROMATIN: Higher Order DNA Compaction Within the Nucleus

Histone Core:Histone Core:

2 copies each of H2A, H2B, H3, H4 (all basic proteins-rich in Arg, Lys residues)

Dec. 2001

Histone Acetylation/DeacetylationHistone Acetylation/Deacetylation

Dec. 2001 (From Glass and Rosenfeld)(From Glass and Rosenfeld)

Other transcription factors

NOTE: Rubinstein-NOTE: Rubinstein-Taybi Syndrome Taybi Syndrome caused by mutations caused by mutations in CBP.in CBP.NOTE: Overexpression of NOTE: Overexpression of some coactivators (e.g. some coactivators (e.g. Amplified in Breast Amplified in Breast Cancer-1 [AIB-1]) Cancer-1 [AIB-1]) associated with hormone-associated with hormone-independent breast cancerindependent breast cancer

Dec. 2001 (From Glass and Rosenfeld)(From Glass and Rosenfeld)

Other transcription factors

NOTE: HDAC NOTE: HDAC inhibitors in inhibitors in clinical trials for clinical trials for cancer treatmentcancer treatment

Dec. 2001

Dec. 2001

Nuclear Receptors as Drug TargetsNuclear Receptors as Drug Targets

• Reproductive or Endocrine DisordersReproductive or Endocrine Disorders

• Hormone-dependent Cancers (ER-breast Hormone-dependent Cancers (ER-breast cancer, AR- prostate cancer)cancer, AR- prostate cancer)

• Metabolic Diseases (PPAR-Metabolic Diseases (PPAR- for Type for Type 2 Diabetes and Metabolic Syndrome)2 Diabetes and Metabolic Syndrome)

NOTE: Ligands for nuclear NOTE: Ligands for nuclear receptors are small ligands that receptors are small ligands that are cell permeableare cell permeable

CELL OR TISSUE-SPECIFIC LIGANDS POSSIBLE?CELL OR TISSUE-SPECIFIC LIGANDS POSSIBLE?

Dec. 2001

Thiazolidinediones (TZD) Thiazolidinediones (TZD) PPAR-PPAR- Ligands Ligands

Endogenous PPAR-Endogenous PPAR- ligand ligand

Activation of PPAR-Activation of PPAR- in fat cells increases in fat cells increases fatty acid and triglyceride uptake and storage fatty acid and triglyceride uptake and storage as well as impacts adipokine gene as well as impacts adipokine gene expression (e.g. downregulates the insulin expression (e.g. downregulates the insulin resistance factor resistin while upregulating resistance factor resistin while upregulating the insulin sensitizing factor adiponectin)the insulin sensitizing factor adiponectin)

Dec. 2001

Steroid Hormone ActionSteroid Hormone ActionSteroid Hormone Action

raloxifene

N

HO

O

estradiol4-hydroxytamoxifen

(Z-OHT) Z-pseudodiethylstilbestrol

HO

O HO H

HO

HOS

O HO

O N

Estrogen receptor ligands elicit different tissue-specific responses

Estrogentarget tissues

Breast

Uterus

Bone

agonist agonist

agonist agonist

antagonist

agonist

agonist

partial agonist antagonist

antagonist

partial agonist

partial agonist

Liver

CNS

agonist

agonist

agonist

agonist

????

antagonist

partial agonist

antagonist

Selective Estrogen Receptor Modulators (SERMsSelective Estrogen Receptor Modulators (SERMs))

Dec. 2001

Estrogen Receptor Ligand-binding Domain: Estrogen Receptor Ligand-binding Domain: Ligand-induced Conformational ChangeLigand-induced Conformational Change

Helix 12 Contacts:Helix 12 Contacts:Coactivator proteins!Coactivator proteins!

Dec. 2001

Dec. 2001

SERM as SERM as AgonistAgonist Recruitment Recruitment of of CoactivatorsCoactivators!!(e.g. (e.g. Tamoxifen in Tamoxifen in uterus)uterus)

MECHANISM OF SERM ACTIONMECHANISM OF SERM ACTION

SERMSERM as as AntagonistAntagonist Recruitment of Recruitment of Corepressors! Corepressors! (e.g. Tamoxifen (e.g. Tamoxifen in breast)in breast)

Dec. 2001

Summary or Nuclear Receptor Summary or Nuclear Receptor Action: SPECIFICITY DETERMINANTSAction: SPECIFICITY DETERMINANTS

1.1.Hormone bindingHormone binding2.2.DNA bindingDNA binding3.3.Cooperation with DNA-bound Cooperation with DNA-bound

accessory factors (tissue-accessory factors (tissue-specificity)specificity)

4.4.Recruitment of coactivator Recruitment of coactivator and/or corepressor complexes and/or corepressor complexes (i.e. histone modifications-(i.e. histone modifications-alterations in chromatin alterations in chromatin structure)structure)

5.5.Ligand-induced conformational Ligand-induced conformational changeschanges