Glycogen Synthase Kinase-3β Inhibition with 9-ING-41 Attenuates the Progression of Pulmonary Fibrosis

Abstract

Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease with a median survival of 3 years after diagnosis. Although the etiology of IPF is unknown, it is characterized by extensive alveolar epithelial cell apoptosis and proliferation of myofibroblasts in the lungs. While the origins of these myofibroblast appear to be diverse, fibroblast differentiation contributes to expansion of myofibroblasts and to disease progression. We found that agents that contribute to neomatrix formation and remodeling in pulmonary fibrosis (PF); TGF-β, Factor Xa, thrombin, plasmin and uPA all induced fibroblast/myofibroblast differentiation. These same mediators enhanced GSK-3β activation via phosphorylation of tyrosine-216 (p-Y216). Inhibition of GSK-3β signaling with the novel inhibitor 9-ING-41 blocked the induction of myofibroblast markers; α-SMA and Col-1 and reduced morphological changes of myofibroblast differentiation. In in vivo studies, the progression of TGF-β and bleomycin mediated PF was significantly attenuated by 9-ING-41 administered at 7 and 14 days respectively after the establishment of injury. Specifically, 9-ING-41 treatment significantly improved lung function (compliance and lung volumes; p < 0.05) of TGF-β adenovirus treated mice compared to controls. Similar results were found in mice with bleomycin-induced PF. These studies clearly show that activation of the GSK-3β signaling pathway is critical for the induction of myofibroblast differentiation in lung fibroblasts ex vivo and pulmonary fibrosis in vivo. The results offer a strong premise supporting the continued investigation of the GSK-3β signaling pathway in the control of fibroblast-myofibroblast differentiation and fibrosing lung injury. These data provide a strong rationale for extension of clinical trials of 9-ING-41 to patients with IPF.

Conflict of interest statement

D.M. Schmitt and F.J. Giles are employees of Actuate Therapeutics Inc. and have equity interest in Actuate Therapeutics Inc. No potential conflicts of interest were disclosed by the other authors.

Figures

Figure 1

Lung tissue sections from TGF-β and bleomycin injured mice were stained for GSK-3β (red) and nuclei (blue) and imaged by confocal microscopy. GSK-3β expression was increased in TGF-β (A) and bleomycin-injured (B) mice compared to controls. Images are representative of 30 fields/slide and n = 4–6 samples/condition. Images were taken at 25X optical zoom. Bar indicates 100 µm.

Figure 2

Mediators implicated in pulmonary organization induce myofibroblast differentiation of normal and IPF fibroblasts. Serum starved human fibroblasts were treated with various mediators to induce myofibroblast differentiation (TGF-β, FXa, thrombin (THB), plasmin (PLN) and uPA; see Materials and Methods). Cell lysates and conditioned medias, collected after 48 h, were then resolved by SDS-PAGE and western blotted for α-SMA, total GSK-3β, tyrosine 216 phosphorylated GSK-3β (pTyr-GSK-3β) and collagen 1 (Col-1), in NF (A) and IPF cells (C). β-actin was the loading control. α-SMA and collagen 1 expression were then quantified by densitometric analyses. Plotted data are the mean ± SEM of n = 3 independent experiments. Collagen was most prominently induced by TGF-β and FXa. Images are representative of three independent experiments. NF (B) and IPF (D) cells were treated PBS, TGF-β, Xa, thrombin, plasmin and uPA for 24 h incubation. RNA was then collected, and qPCR analyses were then performed for α-SMA and collagen 1 expression. GAPDH was the loading control. Plotted data are the mean ± SEM of n = 3–4 independent experiments.

Figure 3

IPF fibroblasts demonstrate increased GSK-3β nuclear localization. Normal and IPF fibroblasts were seeded on glass coverslips. Serum-starved cells were then treated with TGF-β for 48 h. Cells were then fixed, permeabilized and immunostained for GSK-3β. GSK-3β (green) and nuclei (red) were then visualized by confocal microscopy. Images are representative of 10 fields/slide and n = 3 samples/condition. Images were taken at 40X optical zoom. The Mean Fluorescence Intensity (MFI) of nuclear GSK-3β was graphed as mean ± SEM. *Denotes p 

Figure 4

9-ING-41 blocks TGF-β mediated fibroblast to myofibroblast transition. Fibroblasts (normal, A,B and IPF, C,D) were treated with various doses of 9-ING-41 (10-0.5 µM) in serum free media. Cells were then treated with TGF-β for 48 h. Conditioned medias and cell lysates were then resolved by SDS-PAGE and immunoblotted for collagen (Col-1), α-SMA, GSK-3β, and tyrosine-216 phosphorylated GSK-3β. Under control conditions, TGF-β induced Col-1 and α-SMA protein in NF and IPF fibroblasts (A and C). 9-ING-41 (10 and 5 µM) significantly blocked TGF-β mediated induction of α-SMA and Col-1 in NF and IPF cells. Tyr-216 phosphorylation of GSK-3β (pGSKt) was likewise reduced by pretreatment with 9-ING-41 in both NF and IPF fibroblasts. Graphed data are the means of n = 3 independent experiments. Images are representative of 3–4 independent experiments. Total RNA was isolated from TGF-β treated cells in the presence or absence varying doses 9-ING-41 (10–0.5 µM). Changes in α-SMA and collagen 1 expression were then determined by qPCR analyses (B and D). GAPDH was used as the reference gene. Data are expressed as mean ± SEM. n = 3 independent experiments. *Denotes p < 0.05 compared to TGF-β control. Normal (E) and IPF (F) fibroblasts were treated with varying doses of TDZD-8 (40-5 µM) prior to the addition of TGF-β. Cell lysates and conditioned media were then resolved via SDS-PAGE and probed for changes α-SMA and collagen. β-actin was the loading control. TDZD-8 modestly reduced α-SMA and collagen induction by TGF-β at the highest dose (40 µM). Images are representative of two independent experiments.

Figure 5

9-ING-41 reverses established fibroblast to myofibroblast transition. Serum-starved NF and IPF cells were treated with TGF-β for 24 hours. Varying doses of 9-ING-41 (10-5 µM) were then added to the TGF-β–treated cells and allowed to incubate for 48 hours. Conditioned media and lysates from NF (A) and IPF (C) were resolved by SDS-PAGE and immunoblotted for collagen (Col)-1 and α-smooth muscle actin (α-SMA). Lysates were also immunoblotted for GSK-3β phosphorylation at tyrosine 216. β-Actin was used as loading control. For quantitative PCR (qPCR) analyses varying doses of 9-ING-41 (10 to 0.5 µM) were added to TGF-β–treated normal (B) and IPF (D) cells and then allowed to incubate for 24 hours. Total RNA was then isolated and transcribed into cDNA. α-SMA expression was determined by qPCR analyses. GAPDH served as the reference gene. Data are expressed as means ± SEM. n = 3 independent experiments. ∗p < 0.05 versus TGF-β treatment.

Figure 6

9-ING-41-treated mice demonstrate reduced TGF-β mediated PF. Mice were intratracheally administered TGF-β adenovirus to induce pulmonary fibrosis. After 7d, mice received daily intraperitoneal injections of 9-ING-41 (30 mg/kg) for the next 7d. At the completion of the 14d time course, lung compliance and volumes were determined. A, Significant decrements in lung compliance and volume were reversed by 9-ING-41 treatment. Data are expressed as a mean ± SEM. n = 6 mice/condition. *Indicates a p $Denotes p < 0.05 compared to GFP adenovirus 9-ING-41 treatment. B. Lung tissue sections from DMSO and 9-ING-41 treated mice were Trichrome stained to show areas of injury and collagen (blue). Images were taken at 20X optical zoom. Images are representative of 30 fields/slide/condition. n = 6–7 mice. Lung injury score data are expressed as means ± SEM. (C) Lung tissue sections from TGF-β adenoviral mice treated with vehicle and 9-ING-41 mice were immunostained to visualize collagen (Col-1) deposition (red) and nuclei (blue) by confocal microscopy. Images were taken at 25x optical zoom. Collagen injury score data are expressed as mean ± SEM. n = 6 mice/condition. Bar indicates 100 µm. (D) Lung tissue sections from GFP and TGF-β adenovirus infected mice were stained for apoptosis by fluorescent TUNEL stain (green) and nuclei (blue). Images were taken at 20X optical zoom. Images are representative of 15 fields/slide/condition and n = 5–6 samples/condition. Data are expressed as mean ± SEM. p denotes a p < 0.05. Bar indicates 50 µm.

Figure 7

9-ING-41-treated mice demonstrate reduced bleomycin mediated PF. (A) Anesthetized C57Bl/6 J mice were intratracheally administered 0.8U bleomycin/kg. After 14d mice were administered vehicle control (DMSO) or 9-ING-41 (30 mg/kg) by daily intraperitoneal injection for the next 14d. At the completion of the 28d time-course lung compliance and volumes were determined with the Scireq flexivent and by chest CT imaging, respectively. 9-ING-41 treated mice demonstrated significantly better lung compliance and lung volumes than DMSO-treated controls. *Indicates a p < 0.05 compared to saline control. $Denotes p < 0.05 compared to bleomycin/saline treated mice. #Denotes a p < 0.05 compared to bleomycin/DMSO treated mice. (B) Lung tissue sections from DMSO and 9-ING-41 treated mice were Trichrome stained to show areas of injury and collagen (blue). Images were taken at 20X optical zoom. Images are representative of 30 fields/slide/condition and n = 6 mice. Lung injury score data are expressed as means ± SEM. (C) Lung tissue sections from vehicle and 9-ING-41 treated mice were immunostained to visualize collagen deposition (red) by confocal microscopy. Images were taken at 10x optical zoom. Bar indicates 100 µM. (D) Lung tissue sections from saline, bleomycin and bleomycin/9-ING-41 treated mice were stained for apoptosis by fluorescent TUNEL stain (green). Images were taken at 20X optical zoom. Images are representative of 15 fields/slide/condition. Data are expressed as mean ± SEM. p denotes a p < 0.05. n = 5–6 samples/condition. Bar indicates 50 µm.