1. Dec. 2001 Steroid Hormone/Nuclear Receptors Don DeFranco, Ph.D., dod1@pitt.edu Steroid Hormone Structure Steroid Hormone Structure Nuclear Receptors Nuclear Receptors -Description -Structure -Signal Transduction -DNA-Binding Properties -Regulation of Gene Expression -Diseases -Drug Targets

2. Dec. 2001 Steroid Hormone/Nuclear Receptors: Clinical Relevance Endocrine Disorders (Hormone Insensitivity Syndromes; Cancer) Endocrine Disorders (Hormone Insensitivity Syndromes; Cancer) Regulators of Various Metabolic Processes Regulators of Various Metabolic Processes Therapeutic Use to Reduce Inflammatory Diseases Therapeutic Use to Reduce Inflammatory Diseases

3. Dec. 2001 Reduce inflammation and immune responses In clinical practice since 1948 $10,000,000,000./year market size in US GLUCOCORTICOIDS

4. Dec. 2001 Glucocorticoids: Side Effects

5. Dec. 2001 Steroid Hormones: Derived from Cholesterol Lipid Soluble: Able to cross plasma membrane by passive diffusion

6. Dec. 2001 SUMMARY OF INITIAL STEPS IN STEROID HORMONE SIGNAL TRANSDUCTION 1.Stimulation of hormone production by releasing hormones or factors that are synthesized and secreted from neuroendocrine cells (e.g. Adrenocorticotrophic hormone [ACTH] from specialized cells in the anterior pituitary stimulates glucocorticoid production from specialized adrenal cortical cells). 2.Transport of hormone to its target cell via plasma transport proteins in the bloodstream. 3.Dissociation from the plasma transporter proteins and diffusion of the free hormone across the plasma cell membrane. 4.Hormone encounters receptor either in the cytoplasm or the nucleus.

7. Dec. 2001 Hypothalamic-Pituitary-Adrenal (HPA) Axis: Regulation of Cortisol Synthesis and Secretion

8. Dec. 2001 Nuclear Localization of Steroid Receptors Unliganded GR Liganded GR Unliganded/ Liganded ER NOTE: Steroid Receptors Shuttle Between the Nucleus & Cytoplasm

9. Dec. 2001 Domain Organization of Steroid Receptors

10. Dec. 2001 HormoneReceptor (s) AndrogensAR MineralocorticoidsMR EstrogensER  ER  ProgesteronePR A PR B GlucocorticoidsGR GR  Steroid Hormone Receptors: Limited Forms

11. Dec. 2001 TWO RECEPTORS FOR ESTROGEN ER  & ER  Note: Both types of ER can form homodimers (e.g. ER  /ER  ) or heterodimers (i.e. ER  /ER  )

12. Dec. 2001 Estrogen Receptor Distribution Within the Body

13. Dec. 2001 Differential Actions of ER  and ER 

14. Dec. 2001

15. Vitamin D Ligands for Some Nuclear Receptors

16. Dec. 2001 Ligands for Various Orphan Receptors

17. Dec. 2001 Metabolic Pathways of Nuclear Receptor Ligands Chawla et al Science 294:1866, 2001

18. Dec. 2001 Active ingredient of GugulipidActive ingredient of Gugulipid Gum resin of Commiphor muklGum resin of Commiphor mukl In use since 600 BCIn use since 600 BC Antagonist of FXRAntagonist of FXR (Farnesoid or Bile Acid Receptor)(Farnesoid or Bile Acid Receptor) Lowers cholesterol and triglycerideLowers cholesterol and triglyceridelevels

19. Dec. 2001 Active ingredient in St. John’s WartActive ingredient in St. John’s Wart Extract of Hypericum perforatumExtract of Hypericum perforatum In use over 2000 yearsIn use over 2000 years Ligand for PXRLigand for PXR (Pregnane X or Xenobiotic Receptor) Induction of CYP3A4Induction of CYP3A4

20. Dec. 2001 NR Classification Nuclear receptors can be classified by: Their DNA binding specificity Their hormone binding specificity Their dimerization properties

21. Dec. 2001 DNA Binding Configurations of NRs HOMODIMER MONOMER HETERODIMER Steroid hormone receptors bind primarily as homodimers Retinoid X Receptor (RXR; 9-cis RA) is a common dimerization partner for various nuclear receptors including thyroid hormone receptor, retinoic acid receptor (all-trans RA), vitamin D receptor and others

22. Dec. 2001 Domain Structure of Nuclear Receptors and Dimerization and DNA Binding PropertiesEFDCA/B LBD AF-2 LBD AF-2hingeDBDAF-1 1180263304554595 ER LBD DBD Homodimeric Binding to Inverted Repeats Steroid Receptors ER, GR, MR, AR, PR Some Orphan Receptors LBD RXR DBD Heterodimeric Binding to Direct Repeats With RXR Partner Non-Steroid Receptors TR, RAR, VDR, PPAR LBD DBD Monomeric Binding to Half Site Orphan Receptors Ligand Binding Unknown NGF1-B, ERR, COUP ?

23. Dec. 2001

24. Glucocorticoid Response Unit of the PEPCK Gene CEBP/ß TBP -27 -90 -325 COUP GR -380 HNF3 HNF4 -410-445 +1 Accessory DNA-binding Factors Required!! ( Can impart tissue-specificity) NOTE: Phosphoenolpyruvate carboxykinase (PEPCK) gene is glucocorticoid regulated in liver only

25. Dec. 2001 Estrogen Regulated Promoters Association of other Transcription Factors All EREs on chromosomes 21 & 22 From: Myles Brown laboratory (Dana Farber Cancer Institute), 2005, 2006 Genome wide analysis

26. Dec. 2001 GR +1 GR GRE Transcriptional Activation Transcriptional Repression I (Direct DNA-binding) HH +1 nGRE GR H cGRE GR H Transcriptional Repression II (Co-occupancy) +1 GR H NF  B Transcriptional Repression III (Tethering) AP1 Basic Mechanisms of Transcriptional Activation/Repression by the Glucocorticoid Receptor

27. Dec. 2001 Glucocorticoid Side Effects: Molecular Mechanisms

28. Dec. 2001 CHROMATIN: Higher Order DNA Compaction Within the Nucleus Histone Core: 2 copies each of H2A, H2B, H3, H4 (all basic proteins-rich in Arg, Lys residues)

29. Dec. 2001 Histone Acetylation/Deacetylation

30. Dec. 2001 (From Glass and Rosenfeld) Other transcription factors NOTE: Rubinstein-Taybi Syndrome caused by mutations in CBP. NOTE: Overexpression of some coactivators (e.g. Amplified in Breast Cancer-1 [AIB-1]) associated with hormone-independent breast cancer

31. Dec. 2001 (From Glass and Rosenfeld) Other transcription factors NOTE: HDAC inhibitors in clinical trials for cancer treatment

32. Dec. 2001

33. Nuclear Receptors as Drug Targets Reproductive or Endocrine Disorders Reproductive or Endocrine Disorders Hormone-dependent Cancers (ER-breast cancer, AR- prostate cancer) Hormone-dependent Cancers (ER-breast cancer, AR- prostate cancer) Metabolic Diseases (PPAR-  for Type 2 Diabetes and Metabolic Syndrome) Metabolic Diseases (PPAR-  for Type 2 Diabetes and Metabolic Syndrome) NOTE: Ligands for nuclear receptors are small ligands that are cell permeable CELL OR TISSUE-SPECIFIC LIGANDS POSSIBLE?

34. Dec. 2001 Thiazolidinediones (TZD) PPAR-  Ligands Endogenous PPAR-  ligand Activation of PPAR-  in fat cells increases fatty acid and triglyceride uptake and storage as well as impacts adipokine gene expression (e.g. downregulates the insulin resistance factor resistin while upregulating the insulin sensitizing factor adiponectin)

35. Dec. 2001 Steroid Hormone Action raloxifene N HO O estradiol 4-hydroxytamoxifen (Z-OHT) Z-pseudo diethylstilbestrol HO OH OH HO HO S OH O O N Estrogen receptorligands elicit different tissue-specific responses Estrogen target tissues Breast Uterus Bone agonist antagonist agonist partial agonistantagonist partial agonist Liver CNS agonist ???? antagonist partial agonist antagonist Selective Estrogen Receptor Modulators (SERMs )

36. Dec. 2001 Estrogen Receptor Ligand-binding Domain: Ligand-induced Conformational Change Helix 12 Contacts: Coactivator proteins!

37. Dec. 2001

38. SERM as Agonist Recruitment of Coactivators! (e.g. Tamoxifen in uterus) MECHANISM OF SERM ACTION SERM as Antagonist Recruitment of Corepressors! (e.g. Tamoxifen in breast)

39. Dec. 2001 Summary or Nuclear Receptor Action: SPECIFICITY DETERMINANTS 1.Hormone binding 2.DNA binding 3.Cooperation with DNA-bound accessory factors (tissue-specificity) 4.Recruitment of coactivator and/or corepressor complexes (i.e. histone modifications-alterations in chromatin structure) 5.Ligand-induced conformational changes