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

Without ligand in the cytosol Without ligand in the nucleus

Modular structure of nuclear receptors

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

DNA-Binding-Domain (DBD)

A/B C D E F

DNA 2 Zinc-Fingers

(60-80 aa)

NH2 COO-

Response element

Groups of Nuclear Receptors

Adapted from Jiang et al., 1995, MCB 15: 5131-5143.

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

Action of Nuclear Receptors

Action of Nuclear Receptors

Action of Nuclear Receptors

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

VDR Alopecia, rickets, reproductive

abnormalities

Ordered cofactor recruitment by the ER

Ubiquitin-dependent exchange of corepressors for coactivators

Integration of signalling pathways on nuclear receptor-mediated transcriptional regulation

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

Effect of Vitamin D Receptor Loss

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

1a,25(OH)2D3-mediated Transcription

Deeb et al. 2007 Nature Reviews Cancer

Nuclear import

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)

Bruce W. Hollis Vitamin D summit Meeting 2009

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...“

Mean 25-(OH)D level in healthy adult Austrians

Average intake in Austria (S. Kudlacek at a. 2003)

MCW Block 8 WS 2013/2014