Transcription Regulation and Gene Expression in Eukaryotes FS08 NUCLEAR HORMONE RECEPTORS

Transcription

1 Transcription Regulation and Gene Expression in Eukaryotes FS08 NUCLEAR HORMONE RECEPTORS RG. Clerc, April

2 Transcription Regulation and Gene Expression in Eukaryotes NUCLEAR HORMONE RECEPTORS Brief history of steroid signaling (100 years on) Starling refers to bioactive chemicals extracted from glands as hormones Kendall crystallizes thyroid hormone Kendall and Reichstein complete structural analysis of cortisol from adrenal cortex 1946 Selye coins the term glucocorticoid, needed for survival and response to stress 1949 Hench administers cortisone to arthiritic patients with dramatic effects 1950 Kendall, Hench and Reichstein get the Nobel Prize Classical model of steroid hormone action 1986 today - Receptor identification : reverse endocrinology GR, ER, TR, RAR, RXR RG. Clerc, May

3 The family of nuclear hormone receptors steroid receptors adopted nuclear receptors orphan receptors Members Type I Type II Type III/orphan Glucocorticoid receptor Estrogen receptor Androgen receptor Mineralocorticoid receptor. Progesteron receptor Retinoic acid receptor (RAR) Retinoid X receptor (RXR) Vitamin D3 receptor (VD3R) Thyroid hormone receptor (T3R) Peroxisome proliferator activated receptor (PPAR) Liver X receptor (LXR) Farnesol X receptor (FXR) Pregnane activated X receptor (PXR) Steroid+xenobiotic X receptor (SXR) Benzoate X receptor (BXR) Androstane receptor(car) nuclear NGFI-B ELP Nurr77 SHP Localisation Cytoplasma nuclear nuclear (HSP90 complexation) GR Dimerisation homo hetero (RXR) hetero (RXR) Binding IR 3 DR Single Repeat + extension Mode of action Transactivation Transactivation? systemic AP-1 antagonism DAX LRH1 ERR (48 in human; 230 in C. elegans; ; 21 in D. melanogaster)

4 Nuclear Receptors and Small Lipophilic Ligands R.G. Clerc 4

5 Nuclear Hormone Receptors are Modular in Nature (operationally defined from A-F) Ligand independent trx AD AF-1 function DNA binding dimerization Hinge NLS Hsp90 Ligand dependent trx AD dimerization AF-2 function co-regulator recruitment NLS Hsp90

6 The family of nuclear hormone receptor: unified nomenclature (based on the two well conserved domains C and E) (48 in human)

7 Steroid Signaling Pathway hormone Membrane receptor? steroid GR GR aporeceptor complex hsp90 hsp90 molecular chaperones dissociation dimerization GR GR active receptor Transfer into nucleus coregulator GR GR GRE POL2 G TF s eg. glucocorticoid responsive genes nucleus cytoplasm Induction of steroid responsive genes involves hormone dependent dissociation of the receptor from hsp90 (type I nuclear receptors)

8 LBD of GR Mediates Translocation to the Nucleus in presence of Hormone

9 PPAR/RXR-dependent nuclear signaling (type II nuclear receptors) PPAR - Peroxisome Proliferator Activated Receptor RXR - Rexinoid Receptor Fibrates TZDs Prostaglandins PUFAs PPAR RXR Rexinoids PPAR PGC-1 RXR POL2 apo A-I, II apo C-III aco P450 LPL PPRE NHR are the final effectors of a complex cytoplasmic/nuclear transduction cascade

10 C domain (DBD): 2 cys-cys Zinc Fingers eg. ERα Cooperative Binding (homodimer/heterodimer)

11 Homodimer/heterodimer (NR/RXR) formation involves both the C and E domains

12 E domain: canonical structure of the LBD Characteristical sandwich architecture with 3 layers built in by 12 alpha antiparallel helices and 1 antiparallel beta sheet Structure-AA sequence relationship for NHR LBD s Ligand binding dependent pocket remodeling Receptor dimerization surface Co-regulator proteins interface Control of agonist vs. antagonist modes of action

13 Nuclear Hormone Receptor Superfamily: Well Conserved DBD, Poorly Conserved LBD DNA-Binding Domain Ligand Binding Domain Progesterone Receptor N 100% 100% C NGF1B N 40% 18% C Retinoid X Receptor N 35% 18% C Thyroid Hormone ReceptorN 28% 16% C well conserved at sequence level well conserved at structural level poorly conserved at sequence level well conserved at structural level

14 NHR Ligand Binding pocket Delineated by H3, H5, H7, H11, H12 α-helices and β-strands

15 Three Different Conformational Changes for RXRα

16 Nuclear Hormone Receptors are Transcriptonal Regulators

17 Corepressor vs. Coactivator Interfaces Structure Remodeling (eg,. RXR) mouse trap unliganded co-repressor binding liganded co-activator binding

18 Protein : Protein Interaction in vivo Screen: Two Hybrid-Screen

19 Coactivator CBP/p300, Corepresssor Ncor, etc Combinatorial roles of multiple cofactor complexes are required to switch between transcriptional repression and activation functions

20 PGC-1 Coactivator Functions in Energy Homeostasis Cold exposure Fasting Physical exercise NR PGC-1 PGC-1 txn other NRs TR/ PPARγ? GR /HNF4 PPARα gluconeogenesis fatty acid oxidation Hormones mitochondrial biogenesis respiration

21 Coactivator and Corepressor Complexes with the Basal Machinery are Involved in the Regulation of NHR Transcriptional Activity Remodelling Complexes Acetylase (HAT) Complexes Mediator Complexes ACTIVATION REPRESSION Deacetylase (HDAC) Corepressor Complexes

22 Nuclear Receptor Coregulator Interaction Motifs

23 Non Genomic Actions of Steroid Receptors Some criteria for defining non genomic actions: A non genomic action is defined as any action that does not influence directly gene expression as done by steroid receptors Effects that are not blunted by inhibitors of transcription (Actinomycin D) cannot involve gene expression directly. Insensitivity to protein synthesis inhibitors Short time frame: in the seconds to minutes range Membrane initiated steroid signalling

24 Receptor Exemplary Nuclear Hormone Receptors and Target Therapeutic Application Target Therapeutic Application NURR1 RZRb RORa NOR-1 Rev-ErbAb Tlx NGFI-Bb HZF-2a COUP-Tfa Nur77 LXRa COR ERa GR PPARα PPARβ PPARγ RAR RXR Parkinson`s Disease Rev-ErbAa Sleep Disorders HNF4a Arthritis, Dislipidaemia TOR CNS Disorders, Cancer SHP CNS Disorders FXR CNS Disorders SF-1 CNS Disorders LXRb CNS Disorders GCNF CNS Disorders TR2-1a,b CNS Disorders TR4 Hypercholesterolaemia ERRa,b Hypercholesterolaemia DAX-1 Endocrinology PXR Inflammation, Diabetes CAR Dislipidaemia, Diabetes Diabetes, Skin, Cancer Diabetes Type II Skin disorders, Emphisaema Skin diseases, Metabolic Disorders Obesity Diabetes Immune Disorders Metabolic Disorders Dislipidaemia Metabolic Disorders Metabolic Disorders Infertility Infertility, Contraception Infertility, Contraception Diabetes/Obesity Adrenal Hypoplasia Xenobiotic response Xenobiotic response

25 Metabolic Syndrome and Tissue-Tissue Cross Talk Defective insulin secretion Increased glucose output Dyslipidemia HYPERGLYCEMIA Obesity Decreased glucose disposal Insulin resistance Fat

26 Integrated Physiology by PPAR Isoforms β

27 Nuclear Hormone Receptors, Natural and Synthetic PPAR ligands

28 Ligands and Functions of the PPARα and -γ Isoforms PPARα PPARγ POLYUNSATURATED FATTY ACIDS Docohexaenoic acid, eicosapentaenoic acid, linoleic acid, linolenic acid and arachidonic acid FIBRATES Gemfibrozil, Benzafibrate, Fenofibrate THIAZOLIDINEDIONES Rosiglitazone, Pioglitazone Ciglitazone HDL RAISE, LIPID CATABOLISM PEROXISOME PROLIFERATION CONTROL OF INFLAMMATION GLUCOSE HOMEOSTASIS LIPID STORAGE ADIPOCYTE DIFFERENTIATION CONTROL OF INFLAMMATION

29 Ligands and Functions of PPARβ PPARβ/δ POLYUNSATURED FATTY ACIDS PROSTAGLANDINS GW DIFFERENTIATION of oligodendrocytes, epithelial cells, keratinocytes and adipocytes LIPID METABOLISM IN THE BRAIN EMBRYO IMPLANTATION AND DECIDUALIZATION TUMORIGENESIS IN THE COLON REVERSE CHOLESTEROL TRANSPORT WOUND HEALING

30 Reverse Cholesterol Transport and Bile Acid Metabolism macrophages LXR ABC1,8 OxC LXR liver A-1 A-1 HDL SR-BI C CYP7A1 CYP8B1 BA BSEP BA VLDL LDL R SHP LRH-1 FXR NTCP OATP-1 Enterohepatic circulation gut

31 Role of Cyp7A1 in Bile Acid Synthesis LXR FXR SHP Repa and Mangelsdorf Annu Rev Cell Dev Biol 16,459-81

32 LXRα Null Mice Shows Defects in Cholesterol Disposal Gross morphology of livers of LXRα wt vs -/. Mice on 2% cholesterol diets Appears due to failure to upregulate Cyp7A1 expression Changes in Bile Acid Composition of LXRα wt vs -/- Mice on 2% cholesterol diets Peet et al Cell 93,

33 Adrenal Gland and Steroidogenesis regulated by RAAS driven by ACTH driven by LH & ACTH

34 Steroids and the Adrenal Cortex GLUCOCORTICOID MINERALOCORTICOID The adrenal cortex is responsible for production of 3 major classes of steroid hormones: glucocorticoids, which regulate carbohydrate metabolism; mineralocorticoids, which regulate the body levels of sodium and potassium;blood pressure and androgens, whose actions are similar to that of steroids produced by the male gonads ESTRADIOL/TESTOSTERONE

35 Mineralocorticoid Receptor and Its Target Genes

36 Estrogen : The Feminine Homone With Ambivalent Functions

37 Effects of Estrogen at Various Sites in the Body Tissue Effect of Estrogen Stimulation Clinical Effect of Stimulation Clinical Effect of Absence of Stimulation Bone Increased deposits of calcium into bone Increased bone density Osteoporosis Brain Blocks the release of ovarian estrogen None Hot flashes, sleep disorders, mood changes, problems with memory? Alzheimer s disease?? Breast Blood Clotting Blood Fats Stimulates growth of breast tissue Bigger breasts,? Increased risk of breast cancer, increased sensitivity of the breast, Increased risk of blood clots Increased HDL, decreased LDL, decreased Cholesterol, Smaller breasts No change in clotting Decreased HDL, increased LDL, increased Cholesterol Skin Increased fat deposits in skin Softer skin Thinner skin, liver spots, dry skin Uterus Increased stimulation of uterine lining and muscle Heavier cycles, increased risk of uterine cancer No periods Vagina Increased thickening of skin, better blood supply to tissue Vaginal discharge, feelings of pelvic congestion Dryness, vaginal infections, painful sex, incontinence of urine, pelvic weakness

38 Molecular Action of Estradiol Adapted from Howell A, Osborne CK, Morris C, Wakeling AE. ICI 182, 780 (Faslodex ), development of a novel, "pure" antiestrogen. Cancer 2000; 89: 819.

39 Molecular Action of SERM (Tamoxifen) Adapted from Howell A, Osborne CK, Morris C, Wakeling AE. ICI 182, 780 (Faslodex ), development of a novel, "pure" antiestrogen. Cancer 2000; 89: 819.

40 Molecular Bases for Agonism vs, Antagonism: ERα Co-regulator box LXXLL

41 Selective Estrogen Receptor Modulators Estrogens SERMs Phytoestrogens SERMs- designed to act in specific ways at each of the estrogen receptor sites in different tissues Anti Estrogens ERDR

42 Selective Estrogen Receptor Modulator The ideal SERM is one that prevents bone loss, has no risk of uterine or breast cancer, no adversed effect on lipids & cardiovascular system, relieves PMS and maintains cognitive function of the brain Adopted from Rita de Cassia M Dardes & V Craig Jordan

43 Selective Modulator Concept Gave boost to the continued research for SERMs. Concept of profiling selective modulators has been established since then eg. (SFXRM s, SPPARM s, SLXRM s) Ligand dependent selective co-regulator recruitment likely to elicit specific target gene repertoire linked to desirable pharmacological effects, (eg: LXR ligands which promote reverse cholesterol transport but do not induce hypertriglyceridaemia (SREBP1c gene pathway induction)