Pharmacological GLI2 inhibition prevents myofibroblast cell-cycle progression and reduces kidney fibrosis
Rafael Kramann, … , Sushrut S. Waikar, Benjamin D. Humphreys
Rafael Kramann, … , Sushrut S. Waikar, Benjamin D. Humphreys
Published July 20, 2015
Citation Information: J Clin Invest. 2015;125(8):2935-2951. https://doi.org/10.1172/JCI74929.
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Research Article Nephrology

Pharmacological GLI2 inhibition prevents myofibroblast cell-cycle progression and reduces kidney fibrosis

Abstract

Chronic kidney disease is characterized by interstitial fibrosis and proliferation of scar-secreting myofibroblasts, ultimately leading to end-stage renal disease. The hedgehog (Hh) pathway transcriptional effectors GLI1 and GLI2 are expressed in myofibroblast progenitors; however, the role of these effectors during fibrogenesis is poorly understood. Here, we demonstrated that GLI2, but not GLI1, drives myofibroblast cell-cycle progression in cultured mesenchymal stem cell–like progenitors. In animals exposed to unilateral ureteral obstruction, Hh pathway suppression by expression of the GLI3 repressor in GLI1+ myofibroblast progenitors limited kidney fibrosis. Myofibroblast-specific deletion of Gli2, but not Gli1, also limited kidney fibrosis, and induction of myofibroblast-specific cell-cycle arrest mediated this inhibition. Pharmacologic targeting of this pathway with darinaparsin, an arsenical in clinical trials, reduced fibrosis through reduction of GLI2 protein levels and subsequent cell-cycle arrest in myofibroblasts. GLI2 overexpression rescued the cell-cycle effect of darinaparsin in vitro. While darinaparsin ameliorated fibrosis in WT and Gli1-KO mice, it was not effective in conditional Gli2-KO mice, supporting GLI2 as a direct darinaparsin target. The GLI inhibitor GANT61 also reduced fibrosis in mice. Finally, GLI1 and GLI2 were upregulated in the kidneys of patients with high-grade fibrosis. Together, these data indicate that GLI inhibition has potential as a therapeutic strategy to limit myofibroblast proliferation in kidney fibrosis.

Authors

Rafael Kramann, Susanne V. Fleig, Rebekka K. Schneider, Steven L. Fabian, Derek P. DiRocco, Omar Maarouf, Janewit Wongboonsin, Yoichiro Ikeda, Dirk Heckl, Steven L. Chang, Helmut G. Rennke, Sushrut S. Waikar, Benjamin D. Humphreys

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Figure 4

Darinaparsin reduces GLI protein levels and induces cell-cycle arrest in vitro, while overexpression of GLI2 rescues this cell-cycle effect of darinaparsin.

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Darinaparsin reduces GLI protein levels and induces cell-cycle arrest in...
(A and B) Darinaparsin induced G0/G1 cell-cycle arrest in the mouse MSC-like, pericyte-like 10T1/2 cell line (BrDU, S phase; 7AAD, 7-aminoactinomycin, DNA content). (C) Representative Western blots of whole-cell lysate from 10T1/2 cells treated with darinaparsin or vehicle in the presence or absence of Shh. Quantification of Western blot analysis for p21 and p-Rb (3 biological replicates) is shown in Supplemental Figure 8. (D and E) Quantification by IOD showing reduced GLI1 and GLI2 protein levels after treatment with with darinaparsin (data are from 2 pooled experiments, with a total of 3 biological replicates). (F and G) qRT-PCR analysis of mRNA expression of Gli1 and Gli2 in 10T1/2 cells after treatment with vehicle or darinaparsin in the presence or absence of Shh (n = 3 replicates). (H and I) Retroviral expression of GLI2 rescued the cell-cycle effect of darinaparsin and drove proliferation of 10T1/2 cells (n = 3 biological replicates; gating is shown in Supplemental Figure 2). (J) 293T cells were transfected with full-length GLI2-myc, cotransfected with GFP-myc as a control protein, and treated with darinaparsin or vehicle. GST agarose beads were added to the cell lysate to bind the glutathione moiety of darinaparsin. The immunoblot for myc suggests that in the presence of darinaparsin, GST is able to pull down GLI2 (myc), indicating darinaparsin binding to GLI2 (IP + DAR). Importantly, the GFP-myc control protein was not detectable, indicating the specificity of the IP. *P < 0.05, **P < 0.01, and ***P < 0.001, by t test (B, D, and F) and by 2-way ANOVA followed by Bonferroni’s post-hoc test (I). Data represent the mean ± SEM. CV, control virus; DAR, darinaparsin; Veh, vehicle.