Triiodothyronine and L-thyroxine (T3/T4) , their receptors and liver fibrosis

1. Triiodothyronine and L-thyroxine (T3/T4) , their receptors and liver fibrosis

5. Effect of T3 on liver fibrosis

7. Classic thyroid hormone receptors 461 A/B C D E ligand TR beta 1 DNA 410 82 86 TR alpha 1 Affinity of T3: TR alpha1 >TR beta 1 Two genes, 4 isoforms

8. Thyroid hormone receptor alpha 1 Histone deacetylase (HDAC), inactive chromatin NCoR SMRT SUN-CoR corepressor Corepressor RXR AF-1 DNA binding Ligand Phosphorylation Trip1 TIF1 TRAP CBP ACTR T3 AF-1 RXR Coactivator DNA binding Ligand AF-2 Histone acetylase, active chromatin

9. PEPCK, growth hormone, Myosin a , fatty acid synthetase Collagen I (cardiac fibroblasts), TSH, c-myc, EGFR, prolactin, CD44

11. Phenotypic features of hepatic stellate cell activation

12. Rat hepatic stellate cells expressTRα1, but not TRβ1 TRα1 TRβ1 GAPDH Rat HSC-T6 Human LX-2

13. The second T4/T3 receptor:Integrin avb3 and hepatic stellate cells • avb3 is a receptor for L-thyroxine -T4 • Receptor for vitronectin and fibronectin • Vitronectin induced in liver injury; avb3 present only on activated stellate cells • SiRNA of avb3 apoptosis via induction of MMP-9

14. PDGFR I TGFBR I proliferation II Collagen I Fibrosis pMAPK pSTAT3 TRα1 T3 deiodination T3 pSTAT3 T3 T4 pMAPK PDGFR vitronectin T4 α v 3β fibronectin

15. General aims • Verify our working hypothesis • Role of T4/T3 and their receptors nuclear receptors TRa1 and integrin avb3, in inducing hepatic fibrosis • Resolution of fibrosis by blocking TRa1 and integrin avb3 signaling

16. Specific aims T3/TRa1 • Effect and mechanisms of T3 effect on cell proliferation (cyclin D, c-myc, p27) • Regulation of transcription of collagen I gene by T3 in rat and human HSC • Phenotype of TRa1 and TRb1 overexpressing HSC and of siRNA of TRa1 T4/Integrin avb3 • Regulation of HSC proliferation by T4 • Effect of T4 and ECM on growth factor receptors • Effect of T4 on HSC activation

17. T3 inhibits HSC proliferation-24h T3 inhibits HSC proliferation-48h * * * * Averageof 6 exp Average of 5 exp Does T3 affect HSC proliferation ? T3 in low doses inhibits proliferation of rat HSC at 24 and 48h

18. What about humans?

19. ? Apoptosis Cell cycle arrest How does T3 inhibits proliferation

20. Cell Cycle

21. T3+TRb Transcription of Cyclin D1 Cyclin D1 Promoter Cyclin D1 CKI-p27 CDK2 E2F Rb PP Rb-PPP E2F E2F DP T3+TRb nTRE G1 S Cell proliferation 5’UTR of c-Myc C-Myc In neuroblastoma cells Incubation of Na2-b with T3 leads to: 1.Rapid down regulation of c-myc gene and protein. 2.Decrease of cyclin D1 levels of mRNA protein. 3.Increase levels of p27 half-life. Growth arrest and differentiation Gracia-Silva,JBC 1999/Toxicology 2002

22. * Low dose of T3 inhibits Cyclin D1 in HSC-T6 Average of 6 exp

23. * p27 Average of 5 exp Tub 1 2 3 4 Low dose of T3 induces p27 expression in HSC-T6

24. CyclinD1 Tubulin T3 inhibits CycD1 in LX-2 after 48h * Average of 3 exp

25. * Average of 3 exp P27 Tubulin T3 induces p27 expression in LX-2 after 48h starvation in STD

26. TGF-b C T3 T3 C Collagen I GAPDH 1 2.7 2.4 7 How does T3 affect collagen I expression ?

27. Collagen Promoter Luciferase gene Collagen 1A1 Gene Promoter of Collagen 1A1 TRE Reporter Vector AP-1 Sp1 AP-1 Sp1 TRE TRE AP-1 ATG 5’ UTR LUC LUC LUC LUC LUC The Collagen promoter

28. Transfection procedure Col1A1 promoter-Luc Transfection Reagent Cell

29. Cell Cell LUC LUC LUC LUC Col1A1-Luc Transfection procedure Incubation for 24-48h then lysis and results reading

30. * * T3affects collagen promoter activity

31. Summary Low dose of T3 (10 –11M): • Inhibits proliferation of rat and human hepatic stellate cell lines. • Down regulates cyclin D1 and up regulates cell cycle inhibitor p27. • Inhibits the promoter activity of collagen I.

32. T4/Integrin αvβ3 • Regulation of HSC proliferation by T4 • Effect of T4 and ECM on growth factor receptors • Effect of T4 on HSC activation Aims T3/TRa1 • Mechanisms of T3 inhibition of cell proliferation (cyclin D, c-myc, p27) • Regulation of transcription of collagen I gene by T3 in rat and human HSC • Phenotype of TRa1 and TRb1 overexpressing HSC and of siRNA of TRa1

33. Fibronectin(ECM) T4 T4/T3 Vitronectin(ECM) T3 β3 αV CYTOPLASM Cyclin D p Proliferation Genes activation MAPK p Stat3 Deiodinase RXR TR Col. I transcriptionFibrosis Activation NUCLEUS ? Extracellular receptors likePDGFR, TGFbR1and integrin αVβ3.

34. The integrins are the fastest sense of the cell!

35. T4 induces proliferation of human hepatic stellate cells LX-2 and expression of cyclin D Cyclin D * TCP - Plastic

36. T4 increases phosphorylation of MAPK and decreases phosphorylation of STAT3 pMAPK 42/44, pSTAT3

37. T4 increases expression of integrin αvin LX-2 on TCP and Fibronectin PDGFR TCP Fibronectin Vitronectin - + - + - + ±T4 PDGFR TGFbR1 TGFbR1 α v αv * TCP - Plastic

38. The future T3/TR antagonists • TR antagonists : 250,000 compounds screened using a virtual program, the most potent used to design new compounds that were synthesized and tested • Dronerarone- new TRa1 specific antagonist T4/ avb3 antagonists • Vitaxin- antibody against avb3, clinical trials