2 Nuclear Hormone Receptor is a ligand-dependent transcriptional factor Classes of hormonesGeneral properties of nuclear hormone receptors superfamilyFunctional characterization of nuclear hormone receptorsThyroid hormones and thyroid hormone receptorsHormone response elements: palindromic (inverted repeat), directed repeat, everted repeat.Mechanism of nuclear hormone action Cofactors in two steps dimerization pathway—Coactivators (CoA)-SRC, Corepressor (CoR)-SMRT
3 Mechanism of transcriptional repression competition for overlapping binding sites 8. Resistance to thyroid hormone (RTH)9. Transgenic model to study the receptor functions.Dominant negative activity10. Thyroid hormone coactivator, corepressor.11. Mutant nuclear hormone has oncogenic potential
4 Binding of extracellular signal molecules Hydrophilic moleculesbind to cell-surface receptorsHydrophobic moleculesbind to intracellular receptorsUnlike polypeptide hormones (e.g., insulin, growth hormone, calcitonin, ACTH), steroid and thyroid hormones and lipid-soluble vitamins do NOT bind plasma-membrane receptors. Instead, they interact with intracellular receptors that are themselves transcriptional activators.*
9 Nuclear Receptor Family is Large but not ubiquitous:mammals have ~50-60 genesflies 21worms 270 (!!!)plants 0yeast 0Only a handful of physiological ligands have been identified,(despite many genes, worms lack any known lipid based endocrine system)
10 For a steroid hormone to turn gene transcription on, its receptor must: bind to the hormonebind to a second copy of itself to form a homodimerbe in the nucleus, moving from the cytosol if necessarybind to its response elementactivate other transcription factors to start transcription.
11 Nuclear receptor family (steroid) Steroid hormone receptors are part of the superfamily of nuclear receptors that contains over 30 members.All members have conserved regions of high homologyHormone binding domain 90% homologous10% difference accounts for specificityDNA binding domain which contains zinc fingersReceptors are found complexed with heat shock proteins (HSP)Unoccupied receptor held in inactive conformation by HSPLigand binding releases HSP and exposes DNA binding domainHormone receptor complex then binds to response elements on gene and allows transcription to occur
12 Structure of Intracellular Receptors Steroid and thyroid hormone receptors are members of a large group ("superfamily") of transcription factors.All of these receptors are composed of a single polypeptide chain that has, in the simplest analysis, three distinct domains: A/B, C, E.
17 EFFECTS T3: - Stimulates protein synthesis. - Stimulates rate of cellular respiration.- Increases metabolic rate.
18 Diseases of the Thyroid HypothyroidDecreased metabolic rate.Weight gain.Decreased ability to adapt to cold.Lethargy.Hyperthyroid:weight loss.Nervousness, irritability (易怒).Intolerance to heat.Bulging eyes.
30 DNA Sequencehalf-sites are arrangedas direct repeats
31 Interaction of Thyroid Hormone Receptors with DNA Thyroid hormone receptors bind to short, repeated sequences of DNA called thyroid or T3 response elements (TREs), a type of hormone response element.A TRE is composed of two AGGTCA "half sites" separated by four nucleotides. The half sites of a TRE can be arranged as direct repeats, palindromes or inverted repeats.
33 In the absence of hormone, some intracellular receptors do bind their hormone response elements loosely and silence transcription,when complexed to hormone, become activated and strongly stimulate transcription.
36 Ligand-bound state: Binding of T3 to its receptor induces a conformational change in the receptor that makes it incompetent to bind the corepressor complex, but competent to bind a group of coactivator proteins.The coactivator complex contains histone transacetylase (HAT) activity, which imposes an open configuration on adjacent chromatin. The coactivator complex associated with the T3-bound receptor functions to activate transcription from linked genes.
51 Resistance to thyroid hormone Childhood:Failure to thrive, growth retardation and attention-deficit hyperactivity disorder.Adults:goitre and tachycardia.
52 Approximately 60 different mutations in TRb have been identified in RTH patients from over 100 families.2. The clinical phenotype can vary among these families that harbor the same mutation and also vary within a family. This suggests that there maybe other genetic modifiers that determine the phenotype.3. The mutant TRs had defective dissociation from this corepressor. In general, the release of SMRT correlated with the T3 -binding affinity of the mutant receptors.
54 Thyroid 4: 485, 1994 Luciferase activity -T3 +T3 Control (no plasmids) 1.84X73.4XTRb1/TRb121.2XTRb1/mTRb1Thyroid 4: 485, 1994
55 Mechanism of Dominant Negative Activity RXRMRXRTRE
56 Mutant Thyroid Hormone Receptor 1. Mutation within 3 regions of hormone binding domain (Reduced T3/T4 binding activity).2. Retain DNA binding.3. Retain dimerization activity.4. Are transcriptional inactive.5. Block normal receptor function (dominant negative activity).
59 TRa1, a2 deletedTransgenic mice in which both TRa-1 and c-erbAa-2 have been deleted(TRa-/-) have a more severe phenotype with hypothyroidism, intestinal malformation, growth retardation, and early death shortly after weaning.
60 TR a-null (-/-) mice 1. Abnormal heart rate (20% lower ) 2. Abnormal body temperature (0.5 degrees C lower ).3. A mild hypothyroidism.EMBO Journal. 17(2):455-61, 1998
61 TR a-null (-/-) mice1. Abnormal intestinal morphology, assessed by a decrease in the number of epithelial cells along the crypt-villus axis and a decrease in proliferating crypt cells.2. No changes occurred in T3Rbeta (-/-) mice.Gastroenterology. 116: , 1999
63 TR b2-null (-/-) mice1. have preserved expression of the TR a1 and TR b 1 isoforms.2. Develop a central resistance to thyroid hormone3. Growth hormone gene expression is marginally reduced.4.TR beta 2-null mice exhibit no evidence of hearing impairment.5. TRb2 may be involved in retinal development.J Clinical Investigation. 104: , 1999
64 A targeted dominant negative mutation of the thyroid hormone alpha 1 receptor causes increased mortality, infertility, and dwarfism in mice.Proc Natl Acad Sci U S A Dec
69 SRC-1: Steroid receptor coactivator-1 Kda; 1440 aa.2. In a reporter assay system, enhanced transactivation (5-10X) by the receptors.3. Contain 3 transferable activation domains.4. Antagonist RU486 abrogates the ability of SRC to interact with and coactivate PR. (Science 270:1354,1995).5. Also call NcoA-1.6. LXXLL structure to interact with NRs (AF-2 domain).7. SRC-3 (AIB-1) is amplified in many cases of breast cancer. Recruit CBP and PCAF.
70 Several common features among these putative coactivators. there are multiple putative nuclear hormone receptor interaction sites that seem to bear a signature LXXLL sequence motif in which X represents any amino acid. This sequence has been shown to be important for coactivator binding to nuclear hormone receptors.In the amino-terminal region, there is a basic helix-loop-helix (bHLH) motif, suggesting that these coactivators may bind to DNA.
74 N-CoR/SMRT(Nuclear receptor corepressor, Silencing Mediator for Retnoid and Thyroid receptor)1. kD. Structural and functional similarity.2. Binding to unliganded TR and RARs3. Hormone binding results in dissociation of the corepressors.4. CoR box in LBD of NR is essential for corepressor binding.5. Play an important role in the syndrome of resistance to thyroid hormone.
75 6. binding to antagonist occupied steroid receptors 6. binding to antagonist occupied steroid receptors (RU 486/PR, tamoxifen/ER), suggesting that active repression is an important part of hormone antagonist action.7. Interact with Sin3A, which in turns interacts with histone deacetylases.
76 Mutant receptor has oncogenic potential Thyroid abnormalities and hepatocellular carcinoma in mice transgenic for v-erbAC Barlow, B Meister, M Lardelli, U Lendahl and B Vennstrom Department of Cell and Molecular Biology, Karolinska Institute, Stockholm, Sweden.The EMBO Journal Vol 13, pp , 1994
78 Thyroid hormone receptor (c-erbA) in cancer Retroviral v-erbA oncogene that contributes to the appearance oferythroleukemia and sarcomas in bird. (Sap. et al., 1986 )2. v-erbA transgenic mice develop hepatocellular carcinomas.(Barlow et al., 1994)A knockin mutant mouse by targeting a mutation (PV) into TRβ gene,spontaneously develop follicular thyroid carcinoma. (Kato et al., 2004)4. v-erbA encodes a highly mutanted chicken TRα1 protein that doesnot bonding T3 and constitutive repressor of TR genes.(Yen et al.,1994) ｌ(c-erbA)
79 4. The evidence of alterations that affect TR genes, their expression and the activity of their protein products in human cancer.These alterations include:● Deletion● Gene rearrangement● Hypermethylation● Aberrant splicing● Point mutation (dominant negative)● Overexpression of inhibitory variant(Jose et al., 2003)
80 AimTo determine the role of thyroid hormone receptor (TR) during hepato-carcinogenesis
81 Pituitary tumor-transforming Gene 1 1. PTTG1 is characterized proto-oncogene that was originally clonedfrom a rat pituitary tumor. (Dominguez et al.1998)2. hPTTG1 cDNA encodes a protein of 202 amino acids.3. PTTG1 is highly expressed in various human tumors, including pituitary,adrenal, kidney, liver, and ovarian tumors. (Molec. Endocr. 13: , 1999)
82 Hypothesis Reduce expression or complete loss of normal TR function (Lin et al, 1999)Out of control in T3-Regulated GeneOverexpressionof PTTG1/FurinHepatocellular carcinoma
83 Effect of T3 on PTTG1 protein expression in HepG2 cell lines
84 To clarify whether Sp family does indeed activated the PTTG1 promoter
85 Sp1 activates PTTG1 transcription by directly binding to the Sp1 sites located between -620 and -512 bp
86 T3 mediates repressed Sp1 by specifically decreasing the PTTG1 expression in HepG2-TRα1 cells
87 The PTTG1 was regulated by TR through Sp1 in human HCC specimensLinear regression analysis(T/N ratios)29/107 (27.1%)
88 Summary1. PTTG1 gene was mediated by Sp1 and indirectly down-regulated by T3.2. T3/TR plays a suppressor role in hepatocarcinogenesis.Cancer Research :68(6), , 2008
89 Furin Furin is a proprotein convertases that process latent precursor proteins into their biologically active products .Furin substrates are: proparathyroid hormone, transforminggrowth factor beta 1 precursor, proalbumin, pro-beta-secretase,membrane type-1 matrix metalloproteinase, PDGF, IGF1,beta subunit of pro-nerve growth factor .This gene is thought to play a role in tumor progression.
90 Activation of furin by T3 in HepG2 cell lines mRNAprotein
96 References and Reviews Brent GA: Mechanisms of disease: The molecular basis of thyroid hormone action. New Eng J Med 331: , 1994.Tsai M, O'Malley BW: Molecular mechanisms of action of steroid/thyroid receptor superfamily members. Annu Rev Biochem 63: , 1994.Zhang J, Lazer MA: The mechanism of action of thyroid hormones. Annu Rev Physiol 62: , 2000.Yen,PM Physiology and molecular basis of thyroid hormone action Physiological Review 81,Annu. Rev. Physiol :315–60Annu. Rev. Physiol :309–33