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Enikő Kállay Institut for Pathophysiology and Allergy Research,
Medical University Vienna
NUCLEAR RECEPTORS
Overview
Origin and evolution
Relevance
Structure
Ligands
Mechanisms of action
Resourses:http://www.nursa.org
http://www.receptors.org/NR/
http://www.iuphar-db.org/DATABASE/NHRListForward
Vitamin D receptor
Literature: Aranda A , and Pascual A: Nuclear Hormone Receptors and Gene Expression. Physiol Rev 2001;81:1269-1304
Chronology of the discovery of NRs
Toft and Gorski (PNAS) the first demonstration of binding of a hormone to a receptor in a cell free system. Steroidhormon – Receptors (AR, ER, GR, MR) Brumbaugh and Haussler: discovery of the VDR
TR
RAR PPAR , RXR GCNF, ROR, SF-1; FXR, LXR Orphan receptors
1966
1970
1973
1980
1990
2000
Nuclear Receptors
NR are expressed in most cells of an organism
These cells are targets of NR ligands
Pleiotrop effects on cell / tissue development, homeostasis, metabolism and apoptosis
Nuclear Receptor (NR) Superfamily
Largest Gen-Superfamily, encoding for eukaryotic transcription factors
NR only in animals and sponges
NR common ancestor
NR have similar molecular structure
> 200 related Genes identified:
Caenorhabditis elegans (Nematode) ....... > 270
Drosophila melanogaster......................... 21
Homo sapiens.......................................... 48
Phylogenetic Analysis in Mammals 48 (49) NRs in 6 Subfamilies
NR-Ancestors
RXR (a,b,g)
HNF-4 (a,b,g)
COUP-TF
(a,b,g)
TLX
PNR
TR2 (a,b)
NGFI-B
(a,b,g)
SF-1/FTZ-F1
(a,b)
GCNF
SHP
DAX-1
Known ligands
unknown ligands : Orphan Receptors
TR (a,b)
RAR (a,b,g)
VDR
PPAR
PXR
LXR
FXR
CAR (a,b)
Rev/Erb (a,b)
RZR/ROR
(a,b,g)
UR
GR
AR
PR
MR
ER (a,b)
ERR (a,b,g)
Steroid-Receptors
Definition: Nuclear Receptors Multi-functional ligand-activated transcription factors
Regulate expression of target genes
DNA
Receptor
„Key-Genes“ in
Development
Reproduction
Homeostasis
Metabolism
Cytosol
Nucleus
Ligand
NR: Modular-Structure
conserved DNA-binding domain: 60-70 aa
(Zn2+ fingers )
conserved ligand-binding domain
Variable „Hinge“ Region
(nuclear localization)
variable variable N-terminal
Region
A/B C D E/F
Ligand dependent Activation function
AF-2
Constitutive activation function
AF-1
NR: Modular-Structure
A/B C D E/F
NTD: AF-1 Cofactor binding
DBD: DNA binding Nuclear localisation Dimerisation
LBD: Ligand binding AF-2 Nuclear localisation
Permissive and nonpermissive heterodimers.
Aranda A , and Pascual A Physiol Rev 2001;81:1269-1304
©2001 by American Physiological Society
DNA-Response Elements (RE)
Symmetrical (Palindrom)
GR-GR
PR-PR
AR-AR
ER-ER
MR-MR
RXR-RXR
5‘ AGAACAnnnTGTTCT 3‘
Nuclear receptors bind REs as
Homo-Dimer Hetero-Dimer Monomer
5‘ AGGTCA (n)x AGGTCA 3‘ or
RXR
Direct Repeat or
Inverted Palindrome
RXR-RAR (DR2, DR5)
RXR-VDR (DR3, IP9)
RXR-PPAR (DR1)
RXR-PXR (DR3)
RXR-CAR (DR5).....
RZR/ROR
SF-1 (M, D,H)
Rev-Erb (M,D)
Hinge domain NH2 COO-
Intramolecular cross-talk between LBD and Hinge D
Role in ligand binding, dimerisation
Signal for nuclear localisation
N
H2
Different length and sequence in the different NRs
Conserved among the isoforms of the same NR
D
Ligand-binding domain (LBD) A/B C D E F
NH2 COO-
Compact assembly of 11 helices forming the hydrophobe binding pocket
Upon ligand binding, the amphipathic C-terminal helix 12 (AF-2) would trap the ligand.
Apo-RXR Holo-RAR Aranda & Pascual, 2001
Gomper, Kramer, Tatham (ed): Signal Transduction, 2009
LBD -multifunktional
A/B C D E F
NH2 COO-
Ligand-binding
Dimerisation (H 10,11, 7.8.9)
Regulating transcription by Interaction with „Accessory Factors“
H10
AF-2
Crystallographic structure of the VDR bound to 1,25(OH)2D3
Rochel, N., Wurtz, J.M., Mitschler, A., Klaholz, B. & Moras, D. (2000)
The crystal structure of the nuclear receptor for vitamin D bound to
its natural ligand. Mol. Cell 5, 173–179
Witchel SF and DeFranco DB (2006) Nat Clin Pract Endocrino Metabol 2: 621–631
Figure 1 Subcellular trafficking of the glucocorticoid receptor
CoCh, chaperone–cochaperone complex; CRT,
calreticulin; DBD, DNA-binding domain; GR,
glucocorticoid receptor ; hsp90, heat-shock
protein 90; I7, importin 7; ImP, immunophilin;
NCP, nuclear pore complex; (Ubi)n, multiple
ubiquitin moieties
Integrated model for the subcellular distribution of steroid receptors.
Hager et. Al. 2000. J. Steroid Bioch. Mol. Biol.
Steroid-dependent GFP-GR translocation to the nucleus.
Steroid-dependent GFP-ER translocations in the nucleus.
Hager et. Al. 2000. J. Steroid Bioch. Mol. Biol.
Time Course of GFP-ERα Redistribution HeLa cells were transiently transfected with pEGFP-
C1-hERα, and live cells expressing GFP-ERα were analyzed at 10-min intervals.
Stenoien D L et al. Molecular Endocrinology 2000;14:518-
534
©2000 by Endocrine Society
Colocalization of ERα with RNA Polymerase II and Splicing Domains To determine whether
ER overlapped with sites of nascent RNA transcription or splicing domains, colocalization
studies were performed in MCF-7 cells on endogenous proteins using immunofluores...
Stenoien D L et al. Molecular Endocrinology 2000;14:518-534
©2000 by Endocrine Society
SRm160
Nuclear receptors and their ligands
Receptor Ligands
Steroid hormone receptors
ER GR MR AR PR
Oestrogen receptor Glucocorticoid receptor Mineralocorticoid receptor Androgen receptor Progesterone receptor
Oestradiol Cortisol Aldosterone Testosterone progesterone
Thyroid hormone receptor
TR Thyroid hormone receptor
Triiodothyronine
Retinoid receptors RAR RXR
Retinoic acid receptor Retinoic acid X receptor
All trans retinoic acid 9-cis retinoic acid
Vitamin D receptor VDR Vitamin D receptor Calcitriol (1,25D3)
Lipid sensors LXR FXR
Liver X receptor Farnesoid X receptor
Oxysterols Bile acids
PPAR PPAR Peroxisome proliferator activated receptor
Fatty acids, eicosanoids
Gomper, Kramer, Tatham (ed): Signal Transduction, 2009
NR Ligands
Endogenous metabolites of lipid, steroid, vitamin metabolism
Exogenous compounds
From the diet, environment, xenobiotics, etc.
paracrine
endocrine
autocrine
Effect of Ligands on Gene Expression
Target Gen
Target Gen
Target Gen
Target Gen
Target Gen
Inaktiv
Agonist
Antagonist
Inverse Agonist
Active conformation
Inactive
conformation
Effects of Agonists –Antagonists
NCoA
AF-2
Dimerisation
Agonist: Enables dimerisation and recruiting co-activator complexes (NCoA)
Antagonist: Dimerisation domain missing or modified and/or inability to recrute NCoA-complexes
Enikő Kállay Institut of Pathophysiology und Allergy Research,
Medical University Vienna
Vitamin D Receptor signalling
Schematic Representation of the Major Causes of Vitamin D Deficiency and Potential Health Consequences
Holick M. Nutrition Rev. 2008.
CAUSES CONSEQUENCES
Historical Perspectives
1918: Sir Edward Mellanby induces rickets in dogs and then cures them with cod liver oil
1922: Elmer Verner McCollum – a powerful substance in cod liver oil that can cure rickets “Vitamin D“
1919: Huldshinsky and 1923: Chick et al.: rickets can be cured by sunlight or UV light.
Historical Perspectives
1967: Kodicek: first evidence of the existence of more active polar vitamin D metabolites.
1968: Blunt et al. isolation of 25(OH)D3.
1971: DeLuca group isolated and characterised 1a25(HO)2D3 from intestine.
1972-73: was proved that it is 1a25(HO)2D3 and not 25(OH)D3 the active metabolite of vitamin D.
1973: Brumbaugh and Haussler: discovery of the vitamin D receptor (VDR)
.
Dusso A S et al. Am J Physiol Renal Physiol 2005;289:F8-F28
©2005 by American Physiological Society
50
Bioavailability of Vitamin D Metabolites
Plasma con- Biological Relative
centration Effectiveness Receptor
(molar) in vitro Affinity
Vitamin D3 2 x 10-8 1 1
25(OH)D2 5 x 10-8 15 900
1,25(OH)2D3 8 x 10-11 1000 10000
ANTIPROLIFERATIVE ACTIVITY
Extra renal tissues
Autocrine / paracrine 1a,25(OH)2D3 action
25(OH)D3 Metabolites
24-OHase
24-OHase
1a-OHase
Deeb et al. 2007
.
Dusso A S et al. Am J Physiol Renal Physiol 2005;289:F8-
F28
©2005 by American Physiological Society
Cross et al., Steroid Biochem Mol Biol 62: 21-28, 1997
25-(OH)D3
1,25-(OH)2D3
1a-OHase
1,25-(OH)2D3 (10 nM)
HPLC-TRACINGS OF DIFFERENT COLON
CANCER CELLS
Cac
o-2/
AQ
5 10 15 20 25 30
1,25(OH)2-D3
Cog
a-1
Cac
o-2/
15
Cog
a-13
25-OH-D3
CYP24-Metabolites
CYP24-Metabolites
CYP24-Metabolites
CYP24-Metabolites
1,25(OH)2-3-epi-D3
1,25(OH)2-3-epi-D3
25-OH-D3
25-OH-D3
25-OH-D31,25(OH)2-D3
0
1000
2000
3000
5 10 15 20 25 30
5 10 15 20 25 305 10 15 20 25 30
0
1000
2000
3000
0
1000
2000
3000
0
1000
2000
3000
1,25-(OH)2D3
Effect of 1,25(OH)2D3 on the Proliferation of the Caco-2 Colon Cancer Cell Line
VDR-/-
VDR+/+
Kállay et al. Carcinogenesis. 2001.
The effect of VDR – loss in mouse colon on
PCNA 8-OHdG
8-OHdG PCNA
Proliferation Oxidative stress
Kállay et al. Carcinogenesis. 2001
Current model for the control of vitamin D receptor (VDR)-mediated actions of 1,25(OH)2D3.
Dusso A S et al. Am J Physiol Renal Physiol 2005;289:F8-F28
©2005 by American Physiological Society
Non-classical biological actions of 1,25(OH) 2D3–VDR interaction.
Rojas-Rivera J et al. Nephrol. Dial. Transplant. 2010;25:2850-2865
© The Author 2010. Published by Oxford University Press on behalf of ERA-EDTA. All rights
reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org
Activation and regulation of gene expression of VDR by 1,25(OH)2D3.
Significance of Vitamin D Status for Chronic Diseases
Deficiency Insufficiency Optimal
Short latency diseases:
Rickets
Osteomalacia
Long latency diseases:
Loss of calciotropic effects
Osteoporosis
Muscle pain and fatigue
Hypertension/Cardiovascular diseases
Loss of antiproliferative effects
Cancer (breast, prostate, colon)
Loss of immunomodulatory effects
Diabetes
Multiple sclerosis
Lupus
<10 ng/ml (<25 nmol/l) 10-25 ng/ml (25-50 nmol/l) >31 ng/ml (>78 nmol/l)
NCI: 1970-94 Cancer Mortality Rates by state economic area (age-adjusted for 1970 US population)
Colon cancer
Brest cancer Prostate cancer
Melanoma
Influence of Latitude on Cancer Mortality in the USA
Vitamin D and Cancer
Inhibits cell proliferation
Enhances cell differentiation
Activates apoptosis
Inhibits angiogenesis in tumours
Decreases metastatic potential
Activates the immunsystem
Independence of external growth signals
Loss of sensitivity for growth inhibiting signals
Unlimited growth potential
Insensitivity for active cell death (= Apoptosis)
Continuous neo-angiogenesis
Tissue-invasion and growth in other organs
Characteristics of cancer cells Vitamin D Effects
Effect of 1,25(OH)2D3 on the Proliferation Colon Adenoma Cells
Tong et al, Int. J. Cancer, 1998.
ADENOMA NORMAL MUCOSA
0
2
4
6
8
Vehicle 10 nM 1,25-D3
CP
M /
µg
Pro
t.CPM
/ µ
g Pr
otein
Cancer Research 2008 October 1; 68: (19), 7803-
7810.
„Dietary Induction of Colonic Tumors in a Mouse Model of Sporadic Colon Cancer“ by Kan Yang and her colleagues at the Strang Cancer Research Laboratory.
„Colonic tumors were prevented by elevating dietary calcium and vitamin D3 to levels comparable with upper levels consumed by humans, but tumorigenesis was not altered by similarly increasing folate, choline, methionine, or fiber...“
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