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林光輝(KH Lin): 生物醫學研究所 教授兼副院長 醫學大樓 Room 0861 Tel: 5177, 5975

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Presentation on theme: "林光輝(KH Lin): 生物醫學研究所 教授兼副院長 醫學大樓 Room 0861 Tel: 5177, 5975"— Presentation transcript:

1 林光輝(KH Lin): 生物醫學研究所 教授兼副院長 醫學大樓 Room 0861 Tel: 5177, 5975

2 Nuclear Hormone Receptor is a ligand-dependent transcriptional factor
Classes of hormones General properties of nuclear hormone receptors superfamily Functional characterization of nuclear hormone receptors Thyroid hormones and thyroid hormone receptors Hormone 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 activity 10. Thyroid hormone coactivator, corepressor. 11. Mutant nuclear hormone has oncogenic potential

4 Binding of extracellular signal molecules
Hydrophilic molecules bind to cell-surface receptors Hydrophobic molecules bind to intracellular receptors Unlike 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. *


6 Small molecule coactivators of transcription

7 Small molecule coactivators of transcription
Lipid-Soluble Vitamins & Thyroid Hormone Vitamin A Thyroid Hormone

8 Thyroid hormone receptors

9 Nuclear Receptor Family
is Large but not ubiquitous: mammals have ~50-60 genes flies 21 worms 270 (!!!) plants 0 yeast 0 Only 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 hormone bind to a second copy of itself to form a homodimer be in the nucleus, moving from the cytosol if necessary bind to its response element activate 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 homology Hormone binding domain 90% homologous 10% difference accounts for specificity DNA binding domain which contains zinc fingers Receptors are found complexed with heat shock proteins (HSP) Unoccupied receptor held in inactive conformation by HSP Ligand binding releases HSP and exposes DNA binding domain Hormone 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.

13 Thyroid hormone receptors


15 Thyroid Thyroid just below the larynx.
Follicular cells secrete T3, T4 (which are “assembled” in colloid). Parafollicular cells secrete calcitonin, which regulates blood calcium levels.

16 Thyroid

17 EFFECTS T3: - Stimulates protein synthesis.
- Stimulates rate of cellular respiration. - Increases metabolic rate.

18 Diseases of the Thyroid
Hypothyroid Decreased metabolic rate. Weight gain. Decreased ability to adapt to cold. Lethargy. Hyperthyroid: weight loss. Nervousness, irritability (易怒). Intolerance to heat. Bulging eyes.

19 CTE: JBC:277(28),25115–25124, 2002

20 TR isoforms


22 Nuclear hormone receptor: domain structure
Zinc finger DNA binding domain… inserts into major groove of DNA


24 Steroid hormone receptors family




28 Hormone response elements


30 DNA Sequence half-sites are arranged as 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.

32 Half site 5‘-AGGTCA-3’ n IR: inverted repeat n DR: directed repeat n ER: everted repeat

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.

34 “3– 4–5” rule: VDR: DR3 TR: DR4 RAR: DR5 Endocr Rew: 15:391, 1994.

35 The updated hormone action-Two steps model

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.

37 Thyroid hormone action
Deiodinase T4 T4 T3

38 Thyroid hormone action
Transcription machinery T3 Pol II TR RXR Transcriptional start site Thyroid responsive element (TRE)

39 A two-step model for TR/RXR heterodimer receptor activation

40 Coactivator Annu. Rev. Physiol. 2004. 66:315–60

41 Corepressor Annu. Rev. Physiol. 2004. 66:315–60

42 Nuclear receptor function
-Ligand +Ligand


44 Transcriptional Repression Mechanisms

45 Mechanisms of Transcriptional Repression

46 Histone acetylation is reversible
HDACs (histone deacetylase complexes) remove acetyl groups from N-terminal lysines, restoring their positive charge Histone deacetylation correlates with transcriptional inactivation.

47 Key repression step: Deacetylation of
N-terminal lysines Acetyl group no positive charge transcriptional inactivation


49 Resistance to thyroid hormone (RTH)
TRb1 mutation Autosomal dominant disorder Elevated free thyroid hormones in the serum TSH elevated or inappropriated normal

50 Mutation sites for TRb1

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)
1.84X 73.4X TRb1/TRb1 21.2X TRb1/mTRb1 Thyroid 4: 485, 1994

55 Mechanism of Dominant Negative Activity

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).


58 Knockout and transgenic mice

59 TRa1, a2 deleted Transgenic 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 (-/-) mice 1. 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

62 TR b-null (-/-) Elevated thyroid hormone levels
Abnormal central regulation of thyrotropin Develop profound hearing loss J Clinical Investigation. 104: , 1999

63 TR b2-null (-/-) mice 1. have preserved expression of the TR a1 and TR b 1 isoforms. 2. Develop a central resistance to thyroid hormone 3. 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

65 Thyroid hormone action


67 Coactivator action Annu. Rev. Physiol :309–33


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.

71 RID RID PAS: Per-Arnt-Sim homology domain RID: receptor interacting domain HAT:histone acetyltransferase

72 Corepressor

73 Corepressor action Annu. Rev. Physiol. 2005. 67:309–33

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 RARs 3.        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-erbA C 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 of erythroleukemia 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 does not 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 Aim To 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 cloned from 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 Gene Overexpression of PTTG1/Furin Hepatocellular 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 specimens Linear regression analysis (T/N ratios) 29/107 (27.1%)

88 Summary 1. 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, transforming growth 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
mRNA protein

91 The experimental protocol

92 Furin over-expression induced by T3 o r TGF-β
enhances HepG2-TRα1 cell invasion in SCID mice


94 Co-operative model for T3-and TGFβ-activated
furin gene expression

95 T3 directly regulates furin gene expression, thereby enhancing
Conclusion T3 directly regulates furin gene expression, thereby enhancing its invasiveness. Endocrinology, 149, 3817, 2008.

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 :315–60 Annu. Rev. Physiol :309–33

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