Inhibition of Glutaminase 1 Attenuates Experimental Pulmonary Fibrosis

Abstract

It has been increasingly recognized lately that aberrant cellular metabolism plays an important role in the pathogenesis of pulmonary fibrosis. In our previous systemic studies, we found that human lung myofibroblasts undergo glutaminolytic reprogramming, which is mediated by an increased expression of glutaminase (Gls) 1. We showed that augmented glutaminolysis critically regulates collagen production by promoting its stabilization in human lung myofibroblasts. Our study indicates that lung fibroblast Gls1 is a promising therapeutic target for this disease. In this investigation, we primarily focused on delineating the in vivo role of fibroblast Gls1 in mouse models of pulmonary fibrosis and determining the efficacy of Gls1 inhibition in treating this pathology. We now show that fibroblast Gls1 is upregulated in fibrotic mouse lungs. We present evidence that mice with ablation of fibroblast Gls1 are protected from bleomycin-induced lung fibrosis. We show that the Gls1 inhibitor, CB-839, is therapeutically efficacious in treating both bleomycin- and transforming growth factor-β1-induced pulmonary fibrosis. Our study has thus established a solid rationale for advancing Gls1 inhibitors, particularly CB-839, to the next stage of testing in the treatment of this disease.

Keywords: collagen; fibroblast; glutaminase 1; glutaminolysis; pulmonary fibrosis.

Figure 1.

Glutaminase (Gls) 1 is upregulated in lung fibroblasts of mice with bleomycin (BLM)-induced pulmonary fibrosis. (A) Fibroblast tdTomato⁺ mice were instilled intratracheally with saline or BLM. At 3 weeks after instillation, lungs were harvested and single–lung cell suspension prepared. tdTomato⁺ fibroblasts were obtained by FACS sorting and total RNA from these purified cells. The expression of the indicated genes was determined by real-time PCR. n = 3; mean ± SE; *P < 0.05, **P < 0.01, and ***P < 0.001. (B) Fibroblast tdTomato⁺ mice were treated as in A. Lungs were harvested and frozen tissue sections prepared. In situ immunofluorescence assay was performed with anti-Gls1 and anti-RFP primary antibodies. Nuclei were stained by DAPI. Scale bars: 100 μm. Col1A1 = collagen type I α 1 chain; Fn = fibronectin; SMA = smooth muscle actin.

Figure 2.

Transforming growth factor (TGF)-β1 induces Gls1 in mouse lung fibroblasts. (A) Primary normal mouse lung fibroblast line MLg cells were cultured in Eagle’s minimum essential medium (EMEM) supplemented with 1% FBS for 24 hours. The cells were then treated with or without 10 ng/ml mouse TGF-β1 for 24 hours. Total RNA from these cells was purified. The expression of the indicated genes was determined by real-time PCR. n = 4; mean ± SD; **P < 0.01 and ***P < 0.001. (B) MLg cells were treated as in A. The expression of the indicated genes was determined by Western blotting. (C) MLg cells were treated as in A. Gls1 expression was determined by immunofluorescence microscopy. Mitochondria were visualized by MitoTracker Deep Red FM. Nuclei were visualized by DAPI. Scale bars: 50 μm. A representative of two to three independent experiments is shown. Con = control.

Figure 3.

Knockdown of Gls1 inhibits collagen expression at the protein, but not the mRNA, level in TGF-β1–treated mouse lung fibroblasts. (A) Primary normal mouse lung fibroblast MLg cells were transfected with 20 nM control siRNA or Gls1 siRNA, followed by treatment with or without 10 ng/ml TGF-β1 for 48 hours. Levels of the indicated proteins were determined by Western blotting. (B) MLg cells were transfected as in A, followed by treatment with TGF-β1. The expression of the indicated genes was determined by real-time PCR. n = 3; mean ± SD; ***P < 0.001. A representative of two independent experiments is shown. si = siRNA.

Figure 4.

Pharmaceutical inhibition of Gls1 diminishes TGF-β1–induced collagen production in mouse lung fibroblasts. (A) Primary normal mouse lung fibroblast MLg cells were cultured in EMEM supplemented with 1% FBS for 24 hours. The cells were then pretreated with or without 5 μM CB-839 for 1 hour, followed by treatment with or without 10 ng/ml TGF-β1 for 48 hours. Levels of the indicated proteins were determined by Western blotting. (B) MLg cells were treated with TGF-β1 for 24 hours, washed, and treated again with or without 5 μM CB-839 for 24 hours. The cells were then treated with 5 μg/ml cycloheximide (CHX) for 0, 1.5, and 3 hours. Levels of the indicated proteins were determined by Western blotting and densitometric analyses performed using ImageJ. (C) MLg cells were treated as in A. The expression of the indicated genes was determined by real-time PCR. n = 3; mean ± SD. A representative of two to three independent experiments is shown.

Figure 5.

Conditional ablation of Gls1 in fibroblasts attenuates BLM-induced pulmonary fibrosis. (A) Primary mouse lung fibroblasts were isolated from Gls1 fl/fl and fibroblast Gls1^−/− mice. Total RNA was purified and the expression of Gls1 determined by real-time PCR. n = 3 and 4; mean ± SE; **P < 0.01. (B) Gls1 fl/fl and fibroblast Gls1^−/− mice were instilled intratracheally with saline or BLM. At 3 weeks after instillation, lungs were harvested and hydroxyproline levels in the lungs determined. n = 3, 7, 4, 6 for groups Gls1 fl/fl saline, Gls1 fl/fl BLM, fibroblast Gls1^−/− saline and fibroblast Gls1^−/− BLM, respectively; mean ± SE; *P < 0.05 and ***P < 0.001. (C and D) Gls1 fl/fl and fibroblast Gls1^−/− mice were treated as in B. Lungs were harvested and tissue sections prepared. (C) Hematoxylin and eosin and (D) Masson’s trichrome stainings were performed. Scale bars: 500 μm in C and 100 μm in D. (E) Gls1 fl/fl and fibroblast Gls1^−/− mice were treated as in B. Primary mouse lung fibroblasts were isolated and the expression of the indicated genes determined by real-time PCR. n = 3, 4, 4, 4 for groups Gls1 fl/fl saline, Gls1 fl/fl BLM, fibroblast Gls1^−/− saline and fibroblast Gls1^−/− BLM, respectively; mean ± SE; *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 6.

Gls1 inhibitor is therapeutically effective in mice with BLM or active TGF-β1–induced pulmonary fibrosis. (A) Schematic illustration of the experimental design. C57BL/6 mice were instilled intratracheally with saline or BLM. At 7 days after the instillation, the mice were injected intraperitoneally with CB-839 (28 mg/kg body weight dissolved in 30 μl DMSO) or the vehicle DMSO once a day for 2 weeks. At the end of the treatment, mice were killed and lungs harvested. (B) Lung hydroxyproline levels were determined. n = 3, 7, 3, 7 for groups vehicles saline, vehicles BLM, CB-839 saline and CB-839 BLM, respectively; mean ± SE; *P < 0.05 and ***P < 0.001. (C and D) Experiments were performed as in A. Lungs were harvested and tissue sections prepared. (C) Hematoxylin and eosin and (D) Masson’s trichrome stainings were performed. Scale bars: 500 μm in C and 100 μm in D. (E) C57BL/6 mice were instilled intratracheally with control adenovirus (ad-con) or adenovirus expressing active TGF-β1 (Ad-TGF-β1) (6 × 10⁹ pfu/kg body weight). At 7 days after the instillation, the mice were injected intraperitoneally with CB-839 or the vehicle DMSO once a day for 2 weeks. Mice were then killed and lung hydroxyproline levels determined. n = 3 each; mean ± SE; *P < 0.05 and ***P < 0.001. (F) Experiments were performed as in E. Lung tissue sections were prepared and Masson’s trichrome staining performed. Scale bar: 100 μm. (G) Fibroblast Gls1^−/− mice were instilled intratracheally with BLM. At 7 days after the instillation, the mice were injected intraperitoneally with CB-839 or the vehicle DMSO once a day for 2 weeks. Mice were then killed and lung hydroxyproline levels determined. n = 5 and 7; mean ± SE. i.p. = intraperitoneal; i.t. = intratracheal.