Steroid/Intracellular Receptor Steroid/Intracellular Receptor PharmacologyPharmacology

Therapeutic uses of agonist/antagonistsTherapeutic uses of agonist/antagonists Replacement therapyReplacement therapy

• adrenal steroids for Addison’s diseaseadrenal steroids for Addison’s disease• estrogen and progesterone for menopauseestrogen and progesterone for menopause• thyroid (hypothyroid disorders)thyroid (hypothyroid disorders)

Pharmacologic (non-physiological) uses Pharmacologic (non-physiological) uses • glucocorticoids as anti-inflammatory agentsglucocorticoids as anti-inflammatory agents• estrogen/progesterone for contraceptionestrogen/progesterone for contraception• androgens for increased muscle mass (abused by athletes)androgens for increased muscle mass (abused by athletes)• mifepristone (RU486) for pregnancy terminationmifepristone (RU486) for pregnancy termination

Cancer chemotherapyCancer chemotherapy• tamoxifen, anastrozole for breast cancertamoxifen, anastrozole for breast cancer• flutamide/bicalutamide, leuprolide for prostate cancerflutamide/bicalutamide, leuprolide for prostate cancer

LXR oxysterolsFXR farnesoidsPXR zenobioticsCAR phenobarbital

AF1AF1 AF2AF2

Vitamin D, thyroid hormone,Vitamin D, thyroid hormone,Retinoic Acid ReceptorsRetinoic Acid Receptors& Adopted Orphan Receptors& Adopted Orphan Receptors

Classical Steroid Hormone Receptors: these are a small subset Classical Steroid Hormone Receptors: these are a small subset of the 48 Nuclear/Intracellular Receptor Family members found of the 48 Nuclear/Intracellular Receptor Family members found in the human genome.in the human genome.

There are two genes coding for estrogen receptors in thehuman genome: ER and ER

18% 97% 60%homology

They bind the same DNA sequences, dimerize with themselvesand each other. Are they just redundant or do they have specificphysiological roles?

(there appears to be no AF-1 function in human ER(there appears to be no AF-1 function in human ER

Expression patterns of ERsExpression patterns of ERs

• ERER– breastbreast

– uterusuterus

– cervixcervix

– vaginavagina

– brain regionsbrain regions

• ERER– ovaryovary

– prostate, testisprostate, testis

– spleen, thymusspleen, thymus

– lunglung

– hypothalamus, other hypothalamus, other brain regionsbrain regions

Knockout phenotype:female sterility, no mammarygland development, obesity, male sterility, epididymal dysfunction,

testicular degeneration

Knockout phenotype:male--fertile, female--reducedfertility, ovarian dysfunction,vascular problems/develophypertension as they age

Structure of the DNA binding domain

agonistsagonists

antagonists or partial agonistsantagonists or partial agonists

The classic view of estrogen action: The classic view of estrogen action: E binds to ER (displacesE binds to ER (displacesa complex of chaperone proteins), ER forms dimers and interacts a complex of chaperone proteins), ER forms dimers and interacts with the ERE to activate genes by recruitment of co-activators. with the ERE to activate genes by recruitment of co-activators.

How does estrogen act in classical mode to induce geneexpression from genes that have EREs in their promoter?

However, a number of However, a number of observations do not fit this observations do not fit this

modelmodel some genes induced by E do not have a recognizable some genes induced by E do not have a recognizable

EREERE

tamoxifen can inhibit E induction of genes in breast but tamoxifen can inhibit E induction of genes in breast but can be stimulatory of E responsive genes in uteruscan be stimulatory of E responsive genes in uterus

some of the effects of E are too fast to be transcriptional some of the effects of E are too fast to be transcriptional (e.g. rapid activation of the MAP kinase pathway in <5 (e.g. rapid activation of the MAP kinase pathway in <5 min.)min.)

pharmacological actions of agonists and antagonists do pharmacological actions of agonists and antagonists do not make sense in the classical modelnot make sense in the classical model

new observation: ER does not need to bind to an ERE in order to induce gene expression. Induction can occur when ER binds indirectly to other transcription factors such AP1 and then recruits coactivators.

classic pathway

indirect gene activationpathway

(fos/jun)

The regulation of ERE versus AP1-activated promotersThe regulation of ERE versus AP1-activated promotersdepends on the receptor subtype, the ligand, and the cell type depends on the receptor subtype, the ligand, and the cell type

Promoter E TAM E TAMER ER

ERE luciferase

luciferaseAP1

A Structural basis for the differences in ER activity whenA Structural basis for the differences in ER activity whenagonist or antagonist ligands are boundagonist or antagonist ligands are bound

the C-terminal trans-activation helix (H12)is in green

AF2

Steroid Receptors also signal throughSteroid Receptors also signal throughnon nuclear mechanisms to activate MAP non nuclear mechanisms to activate MAP kinase kinase

Summary and prospects for the futureSummary and prospects for the future

1. estrogen receptor ligands can act through multiple 1. estrogen receptor ligands can act through multiple mechanisms:mechanisms:(1) classical ligand dependent induction thru an ERE (1) classical ligand dependent induction thru an ERE

(2) indirect ligand dependent induction by interaction with (2) indirect ligand dependent induction by interaction with AP1AP1 or perhaps other DNA bound transcription or perhaps other DNA bound transcription factors. factors.

(3) ligand-dependent interactions with Src kinase in the (3) ligand-dependent interactions with Src kinase in the cytoplasm and the activation of Map kinase cytoplasm and the activation of Map kinase

signalingsignaling

2. ligands can have agonist or antagonist properties 2. ligands can have agonist or antagonist properties depending on whether the receptor is ERdepending on whether the receptor is ER or ER or ER and the and the tissue complement of co-regulatorstissue complement of co-regulators

3. the complexity of biological responses can be utilized 3. the complexity of biological responses can be utilized pharmacologically by the design of SERMs (selective pharmacologically by the design of SERMs (selective

estrogen receptor modulators --other nuclear receptors estrogen receptor modulators --other nuclear receptors such as AR, GR may also be approached this way.such as AR, GR may also be approached this way.

genistein: a phytoestrogen that is a selective estrogen receptor modulator (SERM):

every morning with breakfast!

Results from the 2002 Women’s Health Initiative Study on Hormone Replacement Therapy in Postmenopausal Women

WHI subject profileWHI subject profile

note: note:

many subjects had many subjects had been E deficient for 10 been E deficient for 10 yrsyrs

high percentage high percentage were overweight. were overweight.

Conjugated equine Conjugated equine estrogen (a mixture of estrogen (a mixture of estrogens from estrogens from pregnant horse urine) pregnant horse urine) and and medroxyprogesterone medroxyprogesterone were given orally--the were given orally--the liver sees highest conc.liver sees highest conc.

LXR oxysterolsFXR farnesoidsPXR zenobioticsCAR phenobarbital

AF1AF1 AF2AF2

Vitamin D, thyroid hormone,Vitamin D, thyroid hormone,Retinoic Acid ReceptorsRetinoic Acid Receptors& Adopted Orphan Receptors& Adopted Orphan Receptors

Corticosteroid Receptors

cortisol binds to both GR and MR

aldosterone specific for MR (mineralcorticoid receptor)

Therapeutic use of adrenal Therapeutic use of adrenal steroidssteroids

1) Replacement therapy1) Replacement therapyAddison’s disease: administer a glucocorticoid (e.g. cortisol)Addison’s disease: administer a glucocorticoid (e.g. cortisol)

and a mineralocorticoid (e.g. fludrocortisone)and a mineralocorticoid (e.g. fludrocortisone)Congenital adrenal hyperplasiaCongenital adrenal hyperplasia

21 21 -hydroxylase deficiency is the most common cause-hydroxylase deficiency is the most common cause2) Diagnosis of Cushing’s syndrome 2) Diagnosis of Cushing’s syndrome 3) Cancer chemotherapy (especially lymphoma/leukemia) 3) Cancer chemotherapy (especially lymphoma/leukemia) 4) Anti-inflammatory agents4) Anti-inflammatory agents

The HPA axis regulates The HPA axis regulates the immune system, the the immune system, the musculoskeletal system musculoskeletal system and many tissues and many tissues involved in overall involved in overall metabolism like liver metabolism like liver and fat.and fat.

Physiological effects of glucocorticoidsPhysiological effects of glucocorticoids

Metabolicgluconeogenesis (liver)release of amino acids (muscle)release of fatty acids-lipolysis (fat)plasma glucoseinsulin secretion (pancreas-in response to glucose)glucose uptake (muscle)bone resorption fibroblast proliferationcollagen synthesischanges in mood and excitability

altered leukocyte functions (anti-inflammatory)altered leukocyte functions (anti-inflammatory)

Glucocorticoid anti-inflammatory Glucocorticoid anti-inflammatory mechanismsmechanisms

leukocyte traffic controlleukocyte traffic control leukocyte functionleukocyte function inhibition of the inhibition of the

prostaglandin/leukotriene pathwayprostaglandin/leukotriene pathway

RECGC

GC blocks NF-kB function and may also induce inhibitors of the NF-kB pathway like IkB

GC

Glucocorticoids block neutrophil migration out of blood vessels by inhibiting response to chemotactic molecules and by preventing passage of neutrophils through endothelial gap junctions

Glucocorticoids inhibit macrophage-Tcell interactions by blocking interleukin induction and preventing macrophage activation

Glucocorticoids inhibit tissue destruction by macrophage by blocking both macrophage activation and subsequent release of proteases like collagenase, elastase, and plasminogen activator

Toxic effects of chronic glucocorticoidsToxic effects of chronic glucocorticoids

gluconeogenesis (liver)--gluconeogenesis (liver)--hyperglycemiahyperglycemiarelease of amino acids-catabolism (muscle)--release of amino acids-catabolism (muscle)--muscle weaknessmuscle weaknessrelease of fatty acids-lipolysis (fat)--together with increase in release of fatty acids-lipolysis (fat)--together with increase in

insulin, leads to insulin, leads to inappropriate fat deposition, obesityinappropriate fat deposition, obesityinsulin secretion (pancreas-in response to glucose) insulin secretion (pancreas-in response to glucose)

hyperinsulinemiahyperinsulinemiabone resorption--leading to bone resorption--leading to osteoporosis, fracturesosteoporosis, fracturesfibroblast proliferation--fibroblast proliferation--thin skin, bruising, poor wound healingthin skin, bruising, poor wound healingcollagen synthesiscollagen synthesisgrowth retardationgrowth retardation (in children) (in children)changes in mood and excitability--changes in mood and excitability--euphoria, restlessnesseuphoria, restlessnessaltered leukocyte functionsaltered leukocyte functions (anti-inflammatory)--may (anti-inflammatory)--may

mask underlying symptomsmask underlying symptomssuppression of the HPA axis: suppression of the HPA axis: acute withdrawl can lead to deathacute withdrawl can lead to death

LXR oxysterolsFXR farnesoidsPXR zenobioticsCAR phenobarbital

AF1AF1 AF2AF2

Other members of the nuclear receptor family

LXR and FXR: regulate cholesteroland bile acid homeostasis

PXR regulates drug metabolic (cyp)genes