steroid receptors & steroid actions kina m.m. mcdougall 15 july, 2014

STEROID RECEPTORS & STEROID ACTIONS

Kina M.M. McDougall15 July, 2014

OBJECTIVES

Review structure and function of steroid receptors

Review molecular signaling pathway Discuss common steroid hormones & their

controlled pathways

TYPES OF HORMONES

Tyrosine derivatives Steroids Peptides Proteins

Tyrosine derivatives

Steroids Peptides(<20 amino acids)

Proteins(>20 amino acids)

Epinephrine Testosterone Oxytocin Insulin

Norepinephrine Estradiol Vasopressin Glucagon

Dopamine Progesterone Angiotensin ACTH

T3 Cortisol MSH TSH

Thyroxine Aldosterone Somatostatin Prolactin

Vitamin D TRH Motilin

Gastrin LH

CCK GnRH

Growth Hormone

secretin

CRH

GHRH

PTH

Calcitonin

HCG

STEROID HORMONES - OVERVIEW

Hydrophobic/Lipophilic (lipid soluble) Require transporter proteins in the blood Receptors located inside cells Diffuse into cells and binds to respective

receptor Receptor has a DNA binding domain & interacts

with specific response elements associated with certain genes

Lipid soluble hormones control gene expression (action that requires time)

STEROID HORMONES - OVERVIEW

Steroid hormone receptors are intracellular Steroid hormones diffuse into the cell and bind to

nuclear receptors (NRs)

2 main classes of nuclear receptors Type I: most steroid hormones Type II: Vit D, thyroid hormone

NUCLEAR RECEPTORS

Structure Large protein complexes Made up of at least 2 protein subunits

Ligand-binding subunit Heat shock protein 90 (hsp90) Other proteins:

FKBP52, Cyp40, hsp70, p23

NUCLEAR RECEPTORS

Ligand binding subunit: 6 segments (A-F) from N-terminal to C-terminal A/B domain C domain (DNA binding domain, DBD) D domain (“hinge”) E domain (ligand binding domain, LBD) F domain

NUCLEAR RECEPTORS

N-terminal A/B domain Most variable region among all members of

NR super family in terms of length and sequence

Function not well defined In some receptors, the A/B domain is

believed to contain a positive transcriptional activation factor that is ligand independent

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NUCLEAR RECEPTORS

C domain Contains DNA binding domain (DBD) Includes 2 subunits known as zinc fingers

Loop containing 4 cysteines stabilized by bonds with Zn2+ ions

Amino acid sequence between 1st & 2nd fingers is responsible for establishing specific contacts with DNA

Overall structure similar throughout NR superfamily Crucial for recognizing DNA sequences regulated by

the receptor Binds hormone response element (HRE) on DNA

NUCLEAR RECEPTORS

D domain Flexible segment known as the “hinge” May be important for spatial configuration of

the protein

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NUCLEAR RECEPTORS

E domain Contains the ligand binding domain (LBD) LBD consists of 12 α helical segments (H1-

H12) Carries transcription-activating function AF-2,

which is strictly dependent on hormone binding

Discriminates between respective ligand molecules

NUCLEAR RECEPTORS

F domain Weakly conserved (variable) No known function

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NUCLEAR RECEPTORS

Hsp90 Is present as a dimer Attached to C (DBD), D, and E (LBD) domains Passively prevents the hormone-binding

component from binding DNA However, can actively detach hormone-binding

subunit from DNA Also has protective function

Disruption of hsp90 activity leads to loss of hormone-binding or receptor function

NUCLEAR RECEPTORS

Ligand binding Binds within a hydrophobic pocket created by

the α helical segments Major structural change induced by ligand

binding is an internal folding of the C-terminal helix H12, which forms a cap on the ligand binding pocket

NUCLEAR RECEPTORS

Receptor Activation Hormone-binding subunit is released

from remainder of “native complex”

DNA-binding Binding occurs at hormone response elements (HREs) HREs consist of two hexa- or pentanucleotides (“half-

sites”) organized as direct or inverted repeats separated by a spacer

Each half-site interacts with one molecule of the receptor

NUCLEAR RECEPTORS

Dimerization Dimerization of NRs is essential for transcriptional

activation of gene expression NR association with DNA promotes dimerization However, dimer formation can occur when NR

receptors are not bound to DNA NR dimerize as homodimers (Type I NRs) or

heterodimers (Type II NRs) Homodimers: bind half-sites organized as inverted repeats Heterodimers: bind half-sites organized as direct or

inverted repeats

TYPE I NUCLEAR RECEPTORS

TYPE II NUCLEAR RECEPTORS

Olefsky J M J. Biol. Chem. 2001;276:36863-36864

STRUCTURE OF NUCLEAR RECEPTORS

NUCLEAR RECEPTORS

Mechanisms of regulation Ligand-dependent activation

Ligand-bound receptor increases transcription of a target gene to which it is bound

Transcriptional activation itself is mediated primarily by the LBD

Ligand-independent repression of transcription

Ligand-dependent negative regulation

NUCLEAR RECEPTORS

Factors modulating receptor activity Receptor concentration Ligand concentration Ligand function Concentrations and types of coactivators and

corepressors Phosphorylation state of NR

STEROID HORMONE - PRODUCTION

Formed enzymatically through a series of modifications of their common precursor cholesterol

Minimal storage of steroid hormones in their cells of origin

Synthesis and secretion are aspects of the same process Steroid hormones diffuse across the plasma

membrane as soon as they are formed

STEROID HORMONES

Main steroids in humans: Adrenal steroids

Glucocorticoids Mineralocortictoids Androgens

Gonadal steroids Ovarian Testicular

Vitamin D

Mineralocorticoids

Glucocorticoids

Androgens

Catecholamines

ADRENAL STEROIDS

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GLUCOCORTICOIDS

HYPOTHALAMIC-PITUITARY-ADRENAL AXIS

CIRCADIAN RHYTHM OF ACTH & CORTISOL

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GLUCOCORTICOID PRODUCTION

ACTH acts by binding to a specific cell-surface receptor, the melanocortin receptor-2 (MCR-2)

ACTH up-regulates expression of these receptors, thereby increasing the steroidogenic response to further ACTH stimulation

ACTH binding to its receptors activates adenylyl cyclase, increasing cyclic AMP (cAMP) production, which in turn stimulates cAMP-dependent protein kinase (protein kinase A) and phosphorylation of a number of proteins.

STIMULATION OF STEROIDOGENESIS BY ACTH

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EXAMPLE OF HORMONAL IMBALANCE

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FIGURE 4.25

CORTICOSTEROID TRANSPORT More than 90% of cortisol is bound

Predominantly to cortisol-binding globulin (CBG) Small amount to albumin Free cortisol is biologically active (only small fraction of

total) CBG is made in liver and has high affinity for

cortisol CBG levels are increased by:

Estrogen (2-3 fold in pregnancy) Chronic hepatitis

CBG levels are reduced by: Glucocorticoids Cirrhosis, nephrosis, hyperthyroidism

CORTICOSTEROID METABOLISM

Cortisol half-life is 70-120 minutes Free cortisol is excreted through kidneys

1% of total cortisol secretion

Most of cortisol metabolized in liver and kidney to inactive metabolites

GLUCOCORTICOID METABOLISM

Thyroid hormone Hyperthyroidism increases cortisol metabolism and

clearance and hypothyroidism causes the converse Effect on hepatic HSD11B1 and 5α/5β-reductases

IGF-1 Increases cortisol clearance by inhibiting hepatic

HSD11B1 Cortisol

Increases metabolism by inducing 6β-hydroxylase Drugs

Rifampin and phenytoin induce 6β-hydroxylase

SYSTEMIC GLUCOCORTICOID EFFECTS

MINERALOCORTICOIDS

MINERALOCORTICOIDS

Aldosterone is the main mineralocorticoid in humans, with 100-150 mcg/day produced

Mineralocorticoids have a much more restricted role compared to glucocorticoids Fluid (sodium) balance Potassium balance

RENIN-ANGIOTENSIN SYSTEM

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MINERALOCORTICOID SYNTHESIS

Have same precursor as cortisol Produced in adrenal glomerulosa

Low concentration of CYP17 (17-hydroxylase) Only zone that has CYP11B2 (aldosterone synthase)

Production and release are stimulated by Angiotensin II Rise in serum potassium Decline in serum sodium (minor role)

ALDOSTERONE SECRETION

Angiotensin II and hyperkalemia act on the zona glomerulosa

Promote conversion of cholesterol to pregnenolone and corticosterone to aldosterone via stimulation of aldosterone synthase Chronic sodium restriction leads to a 10-fold increase in

messenger RNA for aldosterone synthase and in aldosterone synthase activity

Zona glomerulosa is extremely sensitive to change in K+

Aldosterone secretion rises linearly in response to increasing plasma K+ concentration above 3.5 meq/L

ALDOSTERONE ACTION

Aldosterone diffuses into cells and binds the nuclear receptor, resulting in RNA transcription

Similarly to glucocoticoids, aldosterone binds to mineralecorticoid receptors which dissipates the heat shock protein receptor complex allowing dimerization

Translocation of the hormone into the nucleus results in enhanced transcription of the epithelial sodium channel (ENaC)

ALDOSTERONE ACTION

Na+/Cl- reabsorption and K+ excretion Increases open Na+ channels (ENaC) in

luminal membrane Inserting new channels Opening previously silent channels

Enhances basolateral membrane Na+/K+-ATPase activity

ALDOSTERONE ACTION

H+ excretion H+ is mainly excreted in intercalated cells of

cortex and tubular cells of outer medulla Occurs via H+-ATPase

Acid/base status does not affect aldosterone release

ALDOSTERONE ACTION

Extra-renal effects Reduces concentration of Na+ and raises that

of K+ in colonic and salivary secretions and in sweat

Generally of limited physiologic importance Colonic secretion/elimination of K+ can become

important in patients with end-stage renal disease

MINERALOCORTICOID EFFECT OF CORTISOL

The MR and GR share considerable homology 57% in the steroid binding domain 94% in the DNA binding domain

There is dual binding/overlap – with aldosterone binding to the GR and cortisol binding to the MR

Cortisol binds to the aldosterone receptor with equal affinity

MINERALOCORTICOID EFFECT OF CORTISOL

Cortisol circulates in much higher concentrations than aldosterone

However, cortisol does not act as a major mineralocorticoid Target tissues (eg aldosterone-sensitive cells in the

collecting tubules and salivary glands) possess enzymes, such as 11-beta-hydroxysteroid dehydrogenase (11β-HSD)

11β-HSD converts cortisol to cortisone and other inactive metabolites

MINERALOCORTICOID EFFECT OF CORTISOL

Inactivation or saturation of 11β-HSD leads to manifestations of hyperalodsteronism

FOR EXAMPLE???

THE CORTISOL-CORTISONE SHUTTLE

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ADRENAL ANDROGENS

ADRENAL ANDROGENS

Androgens are 19-carbon steroids

Most abundant adrenal steroid (>20 mg/day)

Produced mainly in the zona reticularis in the adrenal gland

Zona reticularis develops shortly before puberty

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ADRENAL ANDROGEN PRODUCTION

DHEA & DHEA-S produced from 17-hydroxypregnelonone

Zona reticularis does not have 3β-HSD Minimal testosterone production in adrenal glands

Androstenedione is converted to testosterone by

17-ketosteroid reductase in peripheral tissues

ADRENAL ANDROGEN SECRETION

DHEA demonstrates circadian rhythm Androgen secretion appears to be dissociated

from glucocorticoid secretion Chronic high-dose dexamethasone suppresses cortisol,

but decreases DHEA by 20% Aging reduces DHEA production but not cortisol Anorexia nervosa leads to decrease in DHEA but not

cortisol

ANDRENAL ANDROGEN SECRETION Prolactin

Abundant receptors on adrenocortical cells In women with prolactinomas, serum DHEAS concentrations

are weakly correlated with serum prolactin concentrations

T-lymphocytes Lymphocytes are present in zona reticularis Lymphocytes increase DHEAS four-fold in vitro Adrenal androgen secretion is reduced in patients receiving

T-lymphocyte suppressive drugs

ACTH Increase in ACTH can result in excessive production of

androgen precursors leading to excess androgen production

GONADAL STEROIDS

Androgens Estrogens Progestogens

TESTICULAR ANDROGENS

Testosterone is the major testicular steroid, comprising more than 99% of testicular steroid hormone production

Testosterone production is mainly regulated by pituitary luteinizing hormone (LH)

TESTOSTERONE METABOLISM Testosterone is converted to dihydrotestosterone

(DHT) by 5α-reductase Testosterone:DHT ratio in plasma 10-15:1

Testosterone is aromatized to estradiol by aromatase 85% of estradiol is synthesized in peripheral tissues, mostly in

adipose tissue Estradiol production increases with weight gain and age

Additionally, through the action of a series of 5α- and 5β-reductases, 17β-HSDs & hydroxylases, testosterone is converted into a number of inactive metabolites that are conjugated and excreted in the urine.

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GONADAL STEROID TRANSPORT Transported in plasma largely bound to albumin and sex

hormone-binding globulin (SHBG) Free testosterone - 2% SHBG - 44% (1000 times more affinity for testosterone than

albumin) Albumin or other proteins – 54% (much higher concentration

than SHBG) Nearly all of the albumin-bound testosterone is

available for tissue uptake bioavailable testosterone in plasma is sum of free plus albumin-bound hormone

Concentration of SHBG in men is about one-third to one-half that in women

Prepubertal boys and hypogonadal men have higher SHBG levels than normal men

GONADAL STEROID TRANSPORTSHBG concentration is decreased by:

Androgen administration Hypothyroidism

SHBG concentration is increased by: Estrogen administration Hyperthyroidism

Alterations in the SHBG concentration do not affect androgen physiology in the steady state HPA responds to acute changes in concentrations of bioavailable

testosterone Testosterone synthesis reestablishes a normal serum level of

bioavailable testosterone

ANDROGEN ACTION

Testosterone and DHT In androgen target tissues, both testosterone and DHT

bind to the androgen receptor (AR) and regulate gene expression

Testosterone binds to the androgen receptor with half the affinity of DHT, although the maximal binding capacity is similar for both androgens Main role of DHT is to amplify the androgen signal

ANDROGEN ACTION

Androgens promote nitrogen retention and stimulate muscle protein synthesis and hence increase muscle mass

Testosterone inhibits the differentiation of preadipocytes into adipocytes

ANDROGEN SIGNALING

OVARIAN STEROIDS

OVARIAN STEROIDS

The ovary secretes a variety of C19 steroids, including DHEA, androstenedione, and testosterone

They are produced by the thecal cells and to a lesser degree by the ovarian stroma

Androstenedione can be converted to estrogen or testosterone in the ovary and in extraglandular tissues.

OVARIAN STEROIDS

The principal estrogens (C18 steroids) are: Estradiol (most potent) (E2) Estrone (E1)

Secreted by the ovary Another important source of estrone is extraglandular

conversion of androstenedione in peripheral tissues Estriol (16-hydroxyestradiol) (E3)

Most abundant estrogen in urine and is produced by the metabolism of estrone and estradiol in extraovarian tissues

Aromatase: key enzyme for production of estrogens in ovary

ESTROGEN ACTION

Estradiol Regulates gonadotropin secretion Promotes development of the secondary sexual

characteristics of women Promotes uterine growth, thickening of vaginal

mucosa, thinning of cervical mucus Linear growth of ductal system of breast

OVARIAN PROGESTOGENS

The principal progestogens (C21 steroids) are: Pregnenolone

Progesterone

17-hydroxyprogesterone

Pregnenolone is of primary importance in the ovary because of its key position as precursor of all steroid hormones

Progesterone is the principal secretory product of the corpus luteum and is responsible for the progestational effects (i.e., cell differentiation and induction of secretory activity in the endometrium of the estrogen-primed uterus)

PROGESTOGEN ACTION

Progesterone is required for implantation of the fertilized ovum and maintenance of pregnancy

It also induces decidualization of the endometrium, inhibits uterine contractions, increases the viscosity of cervical mucus, promotes lateral (alveolar) development of the breast glands, and increases basal body temperature

17-hydroxyprogesterone, also secreted by the corpus luteum, has little, if any, biologic activity

SUMMARY

Steroid hormones include adrenal (glucocorticoids, mineralorticoids, androgens), gonadal (androgens, estrogens, progestogens) and vitamin D

All steroids are derived from cholesterol Steroids are hydrophobic and need transport

proteins Steroids bind nuclear receptors and modulate

gene expression

REFERENCES

Williams Textbook of Endocrinology Endocrinology, 4th Edition. by DeGroot and Jameson Companion site for Basic Medical Endocrinology, 4th

Edition. by Dr. Goodman UpToDate