Fyziologie působení farmak a
toxických látek
Přednáška č.6
Jaderné receptory (ER, AR, PR,
GR, TR, RAR/RXR, PPAR) a
jejich ligandy.
JADERNÉ RECEPTORY
The nuclear receptors comprise the largest family of metazoan transcription regulators. These
proteins share an architecture that includes a poorly conserved amino-terminal domain, a highly
conserved DNA-binding domain (DBD), a connecting hinge region and a discrete ligand-
binding domain (LBD). Their ligands include the sex steroids (estrogen, progesterone and
testosterone), as well as related molecules such as glucocorticoids, mineralocorticoids, bile acids
and oxysterols, and more diverse ligands such as vitamin D3, thyroid hormone and retinoids.
JadernJadernéé receptory a enzymy:receptory a enzymy:
Drug Metab. Pharmacokinet. 21: 437-457 (2006)
Metabolic pathways for the acquisition and elimination of nuclear receptor ligands.
With the exception of thyroid hormones and some xenobiotics, all nuclear
receptor ligands are derived from the biosynthetic pathways that generate
cholesterol and fatty acids from acetyl coenzyme A (Acetyl CoA). Ligands (or
their lipid precursors) for the RXR heterodimer receptors are also acquired from
Drug Metab. Pharmacokinet. 21: 437-457 (2006)
FeedbackFeedback andand interactionsinteractions::
Drug Metab. Pharmacokinet. 21: 437-457 (2006)
HepatocytesHepatocytes as modelas model cellscells::
J. Cell. Biochem. Suppls. 32/33:110­122, (1999
DNADNA bindingbinding
We can divide the receptors into
subgroups on the basis of their
pattern of dimerization. One group
consists of the steroid receptors, all
of which appear to function as
homodimers. This group includes
receptors for estradiol (ER),
progesterone (PR), androgens (ARs),
glucocorticoids (GRs) and
mineralocorticoids (MRs). A second
major group contains receptors that
form heterodimers with retinoid X
receptor (RXR) ­ the receptor for 9-
cis retinoic acid. Members of this
group include the receptors for all-
trans retinoic acid (RAR),vitamin D3
(VDR) and thyroid hormone (TR), as
well as liver X receptor (LXR),
peroxisome proliferator activated
receptor (PPAR) and others. A third
group consists of receptors that can
bind DNA as monomers, such as
NGFI-B, RevErb, ROR and SF-1.
A: Unliganded heterodimerizing
receptors, exemplified here by VDR,
exist as weakly associated heterodime
with RXR, presumably bound
nonspecifically to DNA [Haussler et al.,
1998]. Binding of the 1,25(OH)2D3
ligand to VDR (1) promotes high-affinity
heterodimerization with RXR
accompanied by binding of the
heterodimer to its direct repeat VDRE
(2).
B: Unliganded GR, like other receptors
in group (d) (see Fig. 2), exists as a
complex with heat shock proteins in the
cytoplasm. Upon binding its cortisol
ligand (1), GR dissociates from the
cytoplasmic complex, translocates to th
nucleus and forms a homodimer on its
palindromic GRE (2). Triggered by a
ligand-mediated change in GR
conformation, the AF1 and AF2 domain
then synergize to promote a series of
events (3­6) involving the recruitment o
coregulatory complexes similar to those
described for the VDR-RXR
heterodimer, but with some distinctive
features.
Ligand synthesis
NRs
Transactivation
Ligand metabolization
Cell and signaling ­specific context
Functional genes
(metabolism, cell
proliferation,
differrentiation
and cell death)
Toxic compounds, pharmaceuticals
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NNíízkomolekulzkomolekuláárnrníí toxicktoxickéé lláátky nebotky nebo farmakafarmaka mohoumohou
výrazným zpvýrazným způůsobem ovlivnit endokrinnsobem ovlivnit endokrinníí signalizaci.signalizaci.
SteroidnSteroidníí hormonyhormony
HumanHuman chorionicchorionic
somatotrophinsomatotrophin
HumanHuman chorionicchorionic
gonadotropingonadotropin ((hCGhCG))
ProgesteroneOxytocinOxytocin
EstradiolInhibinInhibinFemaleFemale reproductivereproductive
hormoneshormones
Dihydrotestosterone
TestosteroneInhibinInhibinMaleMale reproductivereproductive
hormoneshormones
DHEA
Aldosterone
CortisolAdrenalAdrenal corticalcortical
steroidssteroids
Triiodothyronine (T3)
Thyroxine (T4)ThyroidThyroid hormoneshormones
Amino acid or fatty
acid derived
SteroidPeptide/proteinHormones
FiveFive major steroidmajor steroid familiesfamilies::
Shaded boxes show structural requirements for glucocorticoid and
mineralocorticoid activity. Hatched boxes show additional structural
requirements for specific glucocorticoid or mineralocorticoid
activity.
ˇ Cortisol stimulates the release of amino acids from muscle. These are
taken up by the liver and converted to glucose.
Ťhe increased circulating concentration of glucose stimulates insulin
release. Cortisol inhibits the insulin-stimulated uptake of glucose in muscle
via the GLUT4 transporter.
Čortisol has mild lipolytic effects. These are overpowered by the lipogenic
action of insulin secreted in response to the diabetogenic action of cortisol.
Čortisol also has varied actions on a wide range of other tissues
TheThe glucocorticoidglucocorticoid receptorreceptor andand activationactivation byby cortisolcortisol
1) Unbound, lipophilic cortisol readily crosses
cell membranes and in target tissues will
combine with the glucorticoid receptor (GR).
2) Like the androgen and progesterone
receptors, unliganded GRs are located in the
cytoplasm attached to heat shock proteins
(hsp-90, hsp-70 and hsp-56).
3) When hormones bind to these receptors
hsps are released and the hormone receptor
complexes translocate to the nucleus.
4) These complexes form homo- or
heterodimers and the zinc fingers of their
DNA-binding domains slot into the
glucocorticoid response elements (GREs) in the
DNA helix.
5) Together with other transcription factors,
such as NF-B or c-jun and c-fos, they
initiate RNA synthesis (activation of RNA
polymerase) downstream of their binding.
Diagrammatic outline of the synthesis of cortisol from cholesterol in the
adrenal cortex
Cholesterol is either obtained from
the diet or synthesized from acetate
by a CoA reductase enzyme.
Approximately 300 mg cholesterol is
absorbed from the diet each day and
about 600 mg synthesized from
acetate. Cholesterol is insoluble in
aqueous solutions and its transport
from the main site of synthesis, the
liver, requires apoproteins to form a
lipoprotein complex. In the adrenal
cortex, about 80% of cholesterol
required for steroid synthesis is
captured by receptors which bind
low-density lipoproteins (LDL). The
remaining 20% is synthesized from
acetate within the adrenal cells by
the normal biochemical route.
BiosyntBiosyntéézaza peptidovýchpeptidových
hormonhormonůů::
BiosyntBiosyntééza steroidnza steroidníích hormonch hormonůů::
aromatáza
Total serum concentrations of testosterone - male: 9­25 nmol/l - female: 0.5­2.5 nmol/l
Abbreviations: DHT, dihydrotestosterone; DHEA(-S), dihydroepiandrosterone (-sulfate).
->95<5DHEA-S
-8020DHEA
1030­4545­60Androstenedione
100--5-DHT
50­705­255­25Testosterone
Peripheral
conversion
AdrenalOvaryFemale
-90<10DHEA-S
98<12DHEA
90<120Androstenedione
80<1205-DHT
<5<195Testosterone
Peripheral
conversion
AdrenalTestisMale
Only about 2% of circulating testosterone is in the free form and able to
enter cells. The rest is either bound to albumin (approximately 40%) or to
sex-hormone-binding globulin (SHBG) and is in equilibrium with the free
form. SHBG is synthesized in the liver and its circulating concentration is
increased by estrogen or excess thyroid hormones and decreased by
exogenous androgens, glucocorticoids or growth hormone and by
hypothyroidism, acromegaly and obesity. Most circulating testosterone is
converted in the liver to metabolites such as androsterone and
etiocholanolone that, after conjugation with glucuronide or sulfate are
excreted in the form of 17-ketosteroids. The majority of urinary
ketosteroids are of adrenal origin and, thus, determinations of ketosteroids
do not reliably reflect testicular secretion.
Estradiol, the most important steroid secreted by the ovary because of its
biologic potency and diverse actions, is transported bound to albumin
(approximately 60%) and about 30% to SHBG. It is rapidly converted to
estrone by 17-hydroxy-steroid dehydrogenase in the liver and, whilst some
estrone re-enters the circulation, most of it is further metabolized to
estriol via 16-hydroxyestrone or to 2- or 4-hydroxyestrone (catechol
estrogens) by the action of catecho-O-methyltransferase. The latter
metabolites can be formed in the brain and may compete with receptors for
catecholamines. Metabolites are conjugated with sulfate or glucuronide
before excretion by the kidney.
TyroidnTyroidníí hormonyhormony ššttíítntnéé žžlláázyzy
1) Active uptake of iodide (I-)
in exchange for Na+.
2) Iodide may be discharged
from the follicular cell by
administration of competing
ions such as perchlorate,
bromide or chlorate.
3) Iodide uptake, the main
control point for hormone
synthesis, is stimulated by
TSH.
4) Oxidation of iodide by
hydrogen peroxide (H2O2)
to form active iodine. The
reaction is catalyzed by
thyroid peroxidase (TPO).
5) Active transport of iodine
across the apical surface
of the follicular cell.
6) Incorporation of active
iodine into the tyrosine
residues of thyroglobulin
molecules to form mono-
and di-iodotyrosines (MIT
and DIT).
7) Uptake of the thyroglobulin
into the lumen of the
follicle and lining of
iodinated tyrosine residues.
ThyroidThyroid hormonehormone synthesissynthesis::
1) Under the influence of TSH, colloid droplets
consisting of thyroid hormones within the
thyroglobulin molecules are taken back up into
the follicular cells by pinocytosis.
2) Fusion of colloid droplets with lysosomes causes
hydrolysis of thyroglobulin and release of T3
and T4.
3) About 10% of T4 undergoes mono-deiodination
to T3 before it is secreted. The released
iodide is reutilized. Several-fold more iodide is
reused than is taken from the blood each day
but in states of iodide excess there is loss
from the thyroid.
4) On average approximately 100 g T4 and about
10 g T3 are secreted per day
ThyroidThyroid hormonehormone excretionexcretion::
The iodothyronines are virtually insoluble in water and, once released from
thyroglobulin, they are very rapidly bound to the plasma proteins,
transthyretin (previously called thyroxine-binding prealbumin), thyroxine-
binding globulin (TBG) and albumin. These vary in their capacity and affinity
for T3 and T4); about 70% of circulating thyroid hormones are bound to TBG.
Only a tiny fraction (<0.5%) of released thyroid hormones exist in a free form
in the circulation and this is in equilibrium with the bound forms of thyroid
hormones.
Ťhyroid hormones are metabolized
by a series of deiodinations which
involve three types of deiodinases
(indicated by numbers in brackets)
Šome T4 is metabolised by being
sulfated, decarboxylated,
deaminated or conjugated with
glucuronide (other pathways).
Šome T3 may be sulfated (T3S) or
converted to the acetic acid
derivative triiodoacetic acid
(TRIAC) that is more potent than
its parent T3.
Šerum half lives: T4 -- 7 days, T3
-- 1 day, rT3 -- 4 hours.
Eighty per cent of the total thyroid hormones secreted each
day is T4 but this is relatively inactive at nuclear receptors
and, thus, considered to be a prohormone. Approximately 70­
80% of released T4 is converted by deiodinases to the
biologically active T3, the remainder to reverse-T3 (rT3) which
has no significant biological activity. Deiodinases are unusual
selenium-containing enzymes that are present in a number of
tissues and are responsible for the metabolism of thyroid
hormones.
Removal of an iodine atom from the 5th carbon atom (5) of the
outer tyrosine ring of T4 by Type 1 and Type 2 deiodinases
produces T3 whilst deiodination of the inner (5) tyrosine ring
by Type 1 and Type 3 deiodinases produces rT3. Further
deiodinations at the 3rd and 5th carbon atoms of both outer
and inner tyrosine rings produce increasingly inactive diiodo-
and monoiodo-thyronines and at the same time conserving
iodine. Iodothyronines are excreted in the urine although some
T3 and T4 is conjugated with glucuronide and excreted via the
bile in the feces.
Many of the actions of thyroid hormones are mediated by their binding to
nuclear receptors that have a preferential affinity for T3. T3 receptors are,
like all the steroid hormone receptors, members of a family of nuclear
transcription factors that, in combination with other transcription factors,
regulate gene expression in target cells. Unlike some steroid receptors (i.e.
those for sex steroids and glucocorticoids), thyroid hormone receptors exist in
the nucleus, not the cytoplasm, and may remain bound to DNA in the absence
of hormone binding.
Thyroid hormones are lipid soluble and readily cross cell membranes. Once
inside the nucleus, T3 binds to its receptor. This dimerizes with another T3
receptor (to form a homodimer) or with a different receptor, notably the
retinoid X receptor, to form a heterodimer. In this form, the dimers interact
with DNA. This occurs between recognition sites in the `zinc fingers' of the
DNA-binding domains of the receptors and particular base sequences in the
DNA helix known as hormone response elements (HRE). The location of HREs
determines which genes are regulated by T3.
There is also evidence that thyroid hormones can have rapid, non-genomic
effects on membrane receptors independent of protein synthesis. These
include stimulation of sugar transport, Ca2+ATPase activity and increased Na+
transport in muscle. The receptors for these effects have not been identified.
RetinoidyRetinoidy a jejich receptory:a jejich receptory:
Receptory proReceptory pro retinoidyretinoidy (RAR, RXR)(RAR, RXR)
Struktura a syntStruktura a syntéézaza
kyselinykyseliny retinovretinovéé
Retinoid X Receptors (RXRs) consist of a family of nuclear receptors that
target and regulate multiple signalling pathways. The early evolutionary emergence of RXRs in
comparison to other nuclear receptors may have allowed for the development of unique
properties as transcriptional regulators.
The complexity of these receptors is derived from their ability to activate transcription as
homodimers or as obligate heterodimeric partners of a multitude of other nuclear receptors.
In addition, RXRs can regulate gene expression in a ligand-dependent (forming permissive
heterodimeric complexes) or - independent (forming non-permissive heterodimeric complexes)
manner.
RXRs have a small ligand binding pocket and therefore bind their ligands (such as 9-cis RA)
with both high affinity and specificity. In the presence of ligand, permissive RXR heterodimers
bind coactivators, but nonpermissive complexes can bind coactivators or corepressors depending
on the activation of the RXR's heterodimeric partner.
Physiologically, the temporal and tissue specific pattern of RXRs as well as the presence of
phenotypic abnormalities in receptor knockout studies (most severe in RXRa -/- animals)
demonstrate the important role for these receptors both during development (morphogenesis)
and in adult differentiated tissues (cell proliferation, cell differentiation, cell death). These
receptors also play an important regulatory role metabolic signaling pathways (glucose, fatty
acid and cholesterol metabolism), including metabolic disorders such as type 2 diabetes,
hyperlipidemia and atherosclerosis.
RXRs function as master regulators producing diverse physiological effects through the
activation of multiple nuclear receptor complexes. RXRs represent important targets for
pharmacologic interventions and therapeutic applications.
Receptory aktivovanReceptory aktivovanéé
peroxizperoxizóómovýmimovými proliferproliferáátorytory
(PPAR)(PPAR)
The peroxisome proliferator-activated receptors (PPAR , , ) are activated by
polyunsaturated fatty acids, eicosanoids, and various synthetic ligands. Consistent with
their distinct expression patterns, gene-knockout experiments have revealed that each
PPAR subtype performs a specific function in fatty acid homeostasis.
PPAR is a global regulator of fatty acid catabolism. PPAR activation up-regulates the
transcription of liver fatty acid­binding protein, which buffers intracellular fatty acids
and delivers PPAR ligands to the nucleus. In addition, expression of two members of the
adrenoleukodystrophy subfamily of ABC transporters, ABCD2 and ABCD3, is similarly up-
regulated to promote transport of fatty acids into peroxisomes where catabolic enzymes
promote -oxidation. The hepatocyte CYP4A enzymes complete the metabolic cascade by
catalyzing -oxidation, the final catabolic step in the clearance of PPAR ligands.
PPAR was identified initially as a key regulator of adipogenesis, but it also plays an
important role in cellular differentiation, insulin sensitization, atherosclerosis, and cancer.
Ligands for PPAR include fatty acids and other arachidonic acid metabolites, antidiabetic
drugs (e.g., thiazolidinediones), and triterpenoids. In contrast to PPAR, PPAR promotes
fat storage by increasing adipocyte differentiation and transcription o a number of
important lipogenic proteins.
Ligands for PPAR include long-chain fatty acids and carboprostacyclin. Pharmacological
activation of PPAR in macrophages and fibroblasts results in up-regulation of the ABCA1
transporter, and because of its widespread expression, PPAR may affect lipid metabolism
in peripheral tissues.
EndocrineEndocrine disruptingdisrupting compoundscompounds ((EDCsEDCs))
Hormone Systems That
Can Be Affected
Cell differentiation, Embryonal
development
Retinoids
Brain development, behaviourThyroid
Menstruation cycle, synthesis
of testosterone
Progesterone
Sexual developmentEstrogen, Androgen
Glucose, carbohydrate, lipid,
protein metabolism
Glucocorticoids
FunctionsEndocrine system
The Range ofThe Range of EDCsEDCs
which harm humans or wildlifewhich harm humans or wildlife
Dioxins, PCBs, PAHs,
BFRs
Industrial products
and by-products
Phthalates, Bisphenol
A, Heavy Metals
Plastics and their
additives
Birth control pills,
DES, Cimetidine
Pharmaceuticals
Arsenic, Cadmium,
Lead, Mercury
Metals
DDT, Atrazine, and
many others
Pesticides &
Herbicides