J Clin Invest. 2015;125(8):2935-2951. Speaker: Chung-te Liu

1. Pharmacological GLI2 Inhibition Prevents Myofibroblast Cell-cycle Progression and Reduces Kidney Fibrosis J Clin Invest. 2015;125(8):2935-2951. Speaker: Chung-te Liu Supervisor: Professor Chun-ming Shih, Professor Fen-Yen Lin, Professor Po-hsun Huang

2. Introduction • Kidney fibrosis is the common final pathway for nearly all progressive kidney diseases. • However, there are currently no approved drugs available to treat kidney fibrosis.

3. Myofibroblasts are accepted as the cell type that drives kidney fibrosis J Pathol 2013; 231: 273-289.

4. Pathogenesis of myofibroblast induction J Pathol 2013; 231: 273-289.

5. Hedgehog signaling pathway

6. Hedgehog signaling pathway • In vertebrates, 3 members of GLI transcription factor family exist: • GLI1 – primarily amplifies the transcriptional response. • GLI2 – plays the important role for the activator function in response to Hh signaling. • GLI3 – is the major repressor. • Patched (PTC) – Hh receptor localized around primary cilium. • Smoothened (SMO) – transmembrane protein constitutively inhibited by PTC. Once Hh ligands bind PTC, SMO is released and activates GLI transcription factors.

7. Gli1 and Gli2 were exclusively expressed in interstitial cells in adult kidney Am J Pathol. 2012; 180(4): 1441-1453.

8. Gli1 marked a network of perivascular mesenchymal-stem-cell-like cells that generate myofibroblasts. • These Gli1+ cells play a central role in organ fibrosis after injury. • Genetic ablation of these cells ameliorates heart and kidney fibrosis. Cell Stem Cell 2015; 16: 51-66.

9. Aim of the study • Given the specific expression of GLI1 and GLI2 in myofibroblast and their precursors and the possibility of activation of GLI proteins by known profibrotic pathways, this study investigate the role of GLI1 and GLI2 myofibroblast.

10. Conditional knockout • A technique to generate conditional mutants bearing tissue-specific mutations Nat Rev Immunol. 2001 Oct;1(1):11-9.

12. Rosa26-LacZ reporter system

13. Rosa26-LacZ reporter system Promotor-Cre mice ROSA mice LoxP LoxP Ubiquitinous promotor Stop codon Promotor Cre LacZ Ubiquitinous promotor LacZ β-galactosidase

14. Methods – animals • Gli1-CreERt2 mice (GLI1tm3(cre/ERT2)Alj/J) • When these Gli1-CreERT2 mice are bred with mice containing a loxP-flanked sequence of interest, tamoxifen-inducible, Cre-mediated recombination will result in deletion of the flanked sequences in Gli1 expressing cells.

15. Methods – animals • Gli3T (Gt(ROSA)26Sor tm3(GLI3)Amc /J) • These RosaGli3TFlag c/c mice contain a floxed-neomycin resistance (neo) cassette and polyadenylation signal, cDNA encoding a FLAG-tagged Gli3 repressor gene, an internal ribosome entry site (IRES), and a Venus yellow fluorescent protein (YFP) under control of the ubiquitous Gt(ROSA)26Sor locus.

16. Methods – animals • Gli1 nLacZ • This lacZ reporter mutant mice harbor a β-galactosidase "knock-in" mutation that also abolishes endogenous Gli1 gene function. • Gli2 nLacZ • Mice homozygous for this Gli2lzki allele harbor a β-galactosidase "knock-in" (lzki) mutation that also abolishes endogenous Gli2 gene function. • Gli2 floxed (GLI2 tm6ALj /J) • C57Bl/6J as wild type and background

17. Methods – animals • Gli1-KO mice • generated from homozygous Gli1-CreER t2 mice • by breeding heterozygous Gli1 nLacZ/+

18. Pharmacological inhibition of GLI • Darinaparsin (S-dimethylarsino-glutathione) • Arsenic trioxide (ATO) is used clinically to reat promyelocytic leukemia and was recently known to antagonize both GLI1 and GLI2. • Darinaparsin is a novel arsenic-based drug with favorable systemic toxicity profile and is currently undergoing clinical studies. • GANT61 • GANT61 is a small-molecule inhibitor of GLI that had been shown to ameliorate renal fibrosis in mice.

19. Knockdown of GLI2, but not GLI1, induces G0/G1 cell-cycle arrest in vitro. • To evaluate the role of GLI1 versus GLI2 in cell proliferation in vitro. • siRNA-knockdown was performed in MSC-like cell line 10T1/2.

20. Knockdown of GLI2, but not GLI1, induces G0/G1 cell-cycle arrest in vitro. • Flow cytometric analysis of cell-cycle distribution revealed that knockdown of GLI2 or both GLI1/GLI2 resulted in G0/G1 arrest. • siRNA against GLI1 alone had no effect

21. Overexpression of GLI2 drives cell proliferation • Overexpression of GLI2 was induced by delivery of retrovirus to rescue siRNA knockdown of GLI2.

22. Conditional KO of Gli2 or overexpression of the GLI3 repressor in GLI1+ cells reduces kidney fibrosis, but KO of GLI1 has no effect. • Unilateral ureteral obstruction (UUO) expreriments in Gli1nLacZ and Gli2nLacZ mice costaining for α-SMA and β-gal confirmed that expression of GLI1 and GLI2 is restricted to myofibroblast. • Endogenous GLI1 and GLI2 protein were upregulated after kidney injury.

25. Conditional KO of Gli2 or overexpression of the GLI3 repressor in GLI1+ cells reduces kidney fibrosis, but KO of GLI1 has no effect. • Whether conditional KO of Gli2 in GLI1+ cells would provide further additive protection against fibrosis compared with Gli1 KO alone? • To repress the entire GLI family, Gli1-CreERt2 mice was crossed with R26-Gli3T strain, in which GLI3 repressor is expressed from Rosa26 locus following Cre-mediated recombination (Gli3T).

26. Conditional KO of Gli2 or overexpression of the GLI3 repressor in GLI1+ cells reduces kidney fibrosis, but KO of GLI1 has no effect.

27. Conditional KO of Gli2 or overexpression of the GLI3 repressor in GLI1+ cells reduces kidney fibrosis, but KO of GLI1 has no effect.

28. Conditional KO of Gli2 or overexpression of the GLI3 repressor in GLI1+ cells reduces kidney fibrosis, but KO of GLI1 has no effect.

29. Conditional KO of Gli2 or repression of the GLI3 repressor in GLI1+ cells halts kidney fibrosis progression by inducing myofibroblast-specific cell-cycle arrest. • Whether Gli2 KO or Gli3 T expression reduces fibrosis severity by halting myofibroblst cell-cycle progression in vivo?

30. Conditional KO of Gli2 or repression of the GLI3 repressor in GLI+ cells halts kidney fibrosis progression by inducing myofibroblast-specific cell-cycle arrest. • BrdU: S phase; p-H3: G2/M phase; α-SMA: myofibroblast.

32. Conditional KO of Gli2 or repression of the GLI3 repressor in GLI+ cells halts kidney fibrosis progression by inducing myofibroblast-specific cell-cycle arrest. • Myofibroblasts left the cell cycle at G1 restriction pint before phosphorylation of Rb.

33. The novel organic arsenic darinaparsin reduces GLI1 and GLI2 protein levels and induces cell-cycle arrest of MSC-like cells in vitro. • MSC-like, pericyte-like cell line 10T1/2 was treated with darinaparsin. • Whether darinaprsin inhibits Hh effectors GLI1 and GLI2 and affects the proliferation of MSC-like cells in vitro?

34. The novel organic arsenic darinaparsin reduces GLI1 and GLI2 protein levels and induces cell-cycle arrest of MSC-like cells in vitro. • Whether darinaparsin alters GLI protein levels and the regulator proteins of G1/S cell-cycle transition in a manner similar to siRNA experiments?

35. The novel organic arsenic darinaparsin reduces GLI1 and GLI2 protein levels and induces cell-cycle arrest of MSC-like cells in vitro. • Darinaparsin decreased Gli1 mRNA, but Gli2 mRNA levels remained unchanged, suggesting an effect of darinaparsin in GLI2 protein stability.

36. The novel organic arsenic darinaparsin reduces GLI1 and GLI2 protein levels and induces cell-cycle arrest of MSC-like cells in vitro. • To evaluate the effect of darinaparsin om GLI2 protein stability, human 293T cells was transfected with a full-length GLI2 and treated with darinaparsin over 24 to 72 hours.

37. Overexpression of GLI2 rescue the cell-cycle inhibitory effect of darinaparsin. • Whether overexpression of GLI2 was sufficient to rescue the inhibitory effect of darsinaparsin? • GLI2 was overexpressed in 10T1/2 cells delivered retrovirally.

38. Co-IP suggests that darinaparsin might bind GLI2 in vitro • Arsenic has been proposed to bind to thiol groups on cysteine residues in the GLI zinc finger domains. • Myc-tagged GLI2 (pCS2-MT GLI2 FL) and myc-tagged eGFP (pEGFP-C1-myc, as a control protein) were overexpressed in human 293T cells. • The cells were treated with 0.5μM darinaparsin or vehicle, and the cell lysate was incubated with glutathione-S-transferase (GST) agarosebeads. • GST would bind the glutathione and pull down daripanarsin and proteins binding to it.

39. Co-IP suggests that darinaparsin might bind GLI2 in vitro • Whether darinaparsin directly binds to GLI2? • GST pulled down GLI2 only in the presence of darinaparsin, suggesting that darinaparsin indeed binds to GLI2.

40. Darinaparsin prevents the increase in GLI1 and GLI2 normally observed during fibrosis and ameliorates fibrosis, even when administered after injury. • Whether darinaparsin would reduce the expression of GLI1 and GLI2 in vivo and reduce severity of kidney fibrosis? • WT mice were treated with daripanarsin or vehicle 2 days before UUO surgery.

41. Darinaparsin prevents the increase in GLI1 and GLI2 normally observed during fibrosis and ameliorates fibrosis, even when administered after injury. • lacZ reported mice for GLI1 and GLI2 (Gli1nLacZ, Gli2nLacZ) treated with darinaparsin after UUO.

42. Darinaparsin prevents the increase in GLI1 and GLI2 normally observed during fibrosis and ameliorates fibrosis, even when administered after injury. • Fibrosis was significantly reduced in the darinaparsin treatment group, either before or after UUO surgery.

43. Darinaparsin prevents the increase in GLI1 and GLI2 normally observed during fibrosis and ameliorates fibrosis, even when administered after injury. • Whether darinaparsin preserves kidney function?

44. Darinaparsin induces myofibroblast-specific cell-cycle arrest in vivo • What is the effect of darinaparsin treatment on myofibroblast proliferation in vivo?

45. Darinaparsin induces myofibroblast-specific cell-cycle arrest in vivo • Darinaparsin significantly decreased proliferation of interstitial myofibroblasts, with no appreciable effect on other kidney cells.

46. Darinaparsin induces myofibroblast-specific cell-cycle arrest in vivo • UUO mice treated with darinaparsin showed increased expression of cyclin-dependent kinase inhibitor p21 and upregulation of p-Rb.

47. Treatment with GLI antagonist 61, a specific small-molecule GLI antagonist, ameliorates kidney fibrosis following UUO surgery. • Whether targeting of GLI proteins using specific GLI antagonist 61 (GANT61) might inhibit fibrosis in a manner similar to that of darinaparsin?

48. Treatment with GLI antagonist 61, a specific small-molecule GLI antagonist, ameliorates kidney fibrosis following UUO surgery.

49. Treatment with GLI antagonist 61, a specific small-molecule GLI antagonist, ameliorates kidney fibrosis following UUO surgery.

50. GLI expression is increased in human kidney fibrosis • Fresh tumor nephrectomy specimens were obtained from 10 patients. Histology and RNA extraction were done.