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

Effect of T3 on liver fibrosis Pathological score (0-3) TimeTreatment 06 weeksTAA 2.7+/ weeks 0.85+/ weeksTAA+PTU 0.55+/ weeksTAA+MMI 0.10+/ weeksTAA+surgical thyroid removal 1.75+/ weeksTAA+T3 (Eltroxin) 2.75+/ weeksTAA+T3 (Eltroxin)

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

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

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

Phenotypic features of hepatic stellate cell activation

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

The second T4/T3 receptor:Integrin  v  3 and hepatic stellate cells  v  3 is a receptor for L-thyroxine -T4 Receptor for vitronectin and fibronectin  v  3Vitronectin induced in liver injury;  v  3 present only on activated stellate cells  v  3 apoptosis via induction of MMP-9SiRNA of  v  3 apoptosis via induction of MMP-9

3β3β α v vitronectin fibronectin T4 T3 TRα1 T3 T4 deiodination Collagen I Fibrosis pMAPK pSTAT3 proliferation PDGFR TGFBRI I II pSTAT3 pMAPK

General aims Verify our working hypothesisVerify our working hypothesis Role of T4/T3 and their receptors nuclear receptors TR  1 and integrin  v  3, in inducing hepatic fibrosisRole of T4/T3 and their receptors nuclear receptors TR  1 and integrin  v  3, in inducing hepatic fibrosis Resolution of fibrosis by blocking TR  1 and integrin  v  3 signalingResolution of fibrosis by blocking TR  1 and integrin  v  3 signaling

Specific aims T3/TR  1 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 TR  1 and TR  1 overexpressing HSC and of siRNA of TR  1 T4/Integrin  v  3 Regulation of HSC proliferation by T4 Effect of T4 and ECM on growth factor receptors Effect of T4 on HSC activation

Does T3 affect HSC proliferation ? Average of 5 exp * * * * T3 inhibits HSC proliferation-24hT3 inhibits HSC proliferation-48h Average of 6 exp T3 in low doses inhibits proliferation of rat HSC at 24 and 48h

What about humans?

How does T3 inhibits proliferation Cell cycle arrest Apoptosis

Cell Cycle

In neuroblastoma cells Gracia-Silva,JBC 1999/Toxicology 2002 CDK2 Cyclin D1 RbE2F PP Rb-PPP E2F DP G1  S Cell proliferation CKI-p27 C-Myc nTRE 5’UTR of c-Myc T3+TR  Cyclin D1 Promoter Transcription of Cyclin D1 T3+TR  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

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

Low dose of T3 induces p27 expression in HSC-T6 p27 Tub Average of 5 exp *

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

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

How does T3 affect collagen I expression ? C TGF-  T3 Collagen I C T3 GAPDH

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

Transfection procedure Cell Col1A1 promoter-Luc Transfection Reagent

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

T3affects collagen promoter activity T3 affects collagen promoter activity **

Summary Low dose of T3 (10 –11 M): 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.

Aims T3/TR  1 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 TR  1 and TR  1 overexpressing HSC and of siRNA of TR  1 T4/Integrin αvβ3 Regulation of HSC proliferation by T4 Effect of T4 and ECM on growth factor receptors Effect of T4 on HSC activation

CYTOPLASM NUCLEUS RXRTR T3 Col. I transcription Fibrosis Activation αV β3 Extracellular receptors like PDGFR, TGFbR1 and integrin αVβ3. Proliferation Genes activation T4/T3 Vitronectin (ECM) Fibronectin (ECM) T4 Deiodinase Stat3 MAPK Cyclin D p p

The integrins are the fastest sense of the cell!

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

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

T4 increases expression of integrin in LX-2 on TCP and Fibronectin T4 increases expression of integrin αv in LX-2 on TCP and Fibronectin PDGFR TGFbR TCP FibronectinVitronectin α v PDGFR TGFbR1 αv ±T4 * TCP - Plastic

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 TR  1 specific antagonist T4/  v  3 antagonists Vitaxin- antibody against  v  3, clinical trials