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. 2016 Jan;54(1):51-9.
doi: 10.1165/rcmb.2014-0445OC.

Novel Mechanisms for the Antifibrotic Action of Nintedanib

Sunad Rangarajan  1 , Ashish Kurundkar  1 , Deepali Kurundkar  1 , Karen Bernard  1 , Yan Y Sanders  1 , Qiang Ding  1 , Veena B Antony  1 , Jianhua Zhang  2 , Jaroslaw Zmijewski  1 , Victor J Thannickal  1
Affiliations

Novel Mechanisms for the Antifibrotic Action of Nintedanib

Sunad Rangarajan et al. Am J Respir Cell Mol Biol. 2016 Jan.

Abstract

Idiopathic pulmonary fibrosis (IPF) is a disease with relentless course and limited therapeutic options. Nintedanib (BIBF-1120) is a multiple tyrosine kinase inhibitor recently approved by the U.S. Food and Drug Administration for the treatment of IPF. The precise antifibrotic mechanism(s) of action of nintedanib, however, is not known. Therefore, we studied the effects of nintedanib on fibroblasts isolated from the lungs of patients with IPF. Protein and gene expression of profibrotic markers were assessed by Western immunoblotting and real-time PCR. Autophagy markers and signaling events were monitored by biochemical assays, Western immunoblotting, microscopy, and immunofluorescence staining. Silencing of autophagy effector proteins was achieved with small interfering RNAs. Nintedanib down-regulated protein and mRNA expression of extracellular matrix (ECM) proteins, fibronectin, and collagen 1a1 while inhibiting transforming growth factor (TGF)-β1-induced myofibroblast differentiation. Nintedanib also induced beclin-1-dependent, ATG7-independent autophagy. Nintedanib's ECM-suppressive actions were not mediated by canonical autophagy. Nintedanib inhibited early events in TGF-β signaling, specifically tyrosine phosphorylation of the type II TGF-β receptor, activation of SMAD3, and p38 mitogen-activated protein kinase. Nintedanib down-regulates ECM production and induces noncanonical autophagy in IPF fibroblasts while inhibiting TGF-β signaling. These mechanisms appear to be uncoupled and function independently to mediate its putative antifibrotic effects.

Keywords: autophagy; fibroblasts; fibrosis; nintedanib; transforming growth factor-β.

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Figures

Figure 1.
Figure 1.
Nintedanib decreases constitutive expression of extracellular matrix proteins fibronectin and collagen 1a1 in idiopathic pulmonary fibrosis (IPF) fibroblasts. (A and B) IPF fibroblasts were treated with increasing doses of nintedanib (0.5, 1, or 2 μM) (A) or nintedanib (2 μM) for increasing durations (24, 48, or 72 h) (B). Expression of fibronectin and collagen 1a1 was evaluated by Western immunoblotting. (C) Densitometric analysis of independent experiments on different IPF fibroblast cell lines treated with nintedanib (2 μM) for 72 hours. Error bars represent mean ± SEM (n = 3). **P < 0.01. (D) IPF fibroblasts were treated with nintedanib (2 μM) for 72 hours, cells were lysed, and RNA was extracted. RT-PCR was performed to assess steady-state mRNA levels of fibronectin and collagen 1a1, compared with β-actin (control). Results are depicted graphically. Error bars represent mean ± SEM (n = 3). **P < 0.01. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
Figure 2.
Figure 2.
Nintedanib induces vacuolar changes in fibroblasts. (A and B) IPF fibroblasts were treated with vehicle (A) or nintedanib (2 μM) (B) for 6 hours, and light microscopy was performed. Scale bars, 50 μm.
Figure 3.
Figure 3.
Nintedanib enhances autophagic flux in fibroblasts. (A) IPF fibroblasts were treated with increasing doses of nintedanib for 72 hours. LC3 expression was evaluated by Western blotting. (B) Densitometric analysis of independent experiments performed as in A showing light chain 3B-II (LC3-II)/LC3-I/GAPDH. Error bars represent mean ± SEM (n = 3). **P < 0.01. (C) IPF fibroblasts were treated with nintedanib (2 μM) for increasing durations. (D) Densitometric analysis of independent experiments performed as in C showing LC3-II/LC3-I/GAPDH. Error bars represent mean ± SEM (n = 3). **P < 0.01. (E and F) IPF fibroblasts were treated with vehicle (E) or nintedanib (2 μM) (F) for 6 hours, and laser confocal microscopy was performed to detect LC3 by indirect immunofluorescence. Scale bars, 10 μm. (G) IPF fibroblasts were treated with nintedanib (2 μM) for 6 hours, and chloroquine (20 μM) was added 2 hours before harvest. LC3 expression was evaluated by Western blotting. (H) Densitometric analysis of independent experiments performed as in G showing LC3-II/LC3-I/GAPDH. Error bars represent mean ± SEM (n = 3). *P < 0.05. DAPI, 4′,6-diamidino-2-phenylindole; ns, not significant.
Figure 4.
Figure 4.
Autophagy induced by nintedanib is beclin-1 dependent and ATG7 independent. (A) IPF fibroblasts were subjected to small interfering RNA (siRNA)-mediated knockdown of ATG7 and treated with nintedanib (2 μM) for 48 hours. ATG7 and LC3 expression were evaluated by Western blotting. (B) Densitometric analysis of independent experiments performed as in A showing LC3-II/LC3-I/GAPDH. Error bars represent mean ± SEM (n = 3). (C) IPF fibroblasts were subjected to siRNA-mediated knockdown of beclin-1 and treated with nintedanib (2 μM) for 48 hours. Beclin-1 and LC3 expression were evaluated by Western blotting. (D) Densitometric analysis of independent experiments performed as in C showing LC3-II/LC3-I/GAPDH. Error bars represent mean ± SEM (n = 5). *P < 0.05. NT, nontargeting siRNA. ATG7, autophagy-related protein 7.
Figure 5.
Figure 5.
Autophagy inhibition does not abrogate the effects of nintedanib on ECM proteins. (A) IPF fibroblasts were subjected to siRNA-mediated knockdown of beclin-1 and treated with nintedanib (2 μM) for 48 hours. Expression of ECM proteins (fibronectin and collagen 1a1) was evaluated by Western blotting. (B and C) Densitometric analysis of independent experiments performed as in A showing fibronectin (B) and collagen 1 (C) expression relative to GAPDH. Error bars represent mean ± SEM (n = 4). *P < 0.05; **P < 0.01. (D) IPF fibroblasts were subjected to siRNA-mediated knockdown of LC3 and treated with nintedanib (2 μM) for 48 hours. Expression of ECM proteins was evaluated by Western blotting. (E and F) Densitometric analysis of independent experiments performed as in D showing fibronectin (E) and collagen 1 (F) expression relative to GAPDH. Error bars represent mean ± SEM (n = 4). *P < 0.05; **P < 0.01.
Figure 6.
Figure 6.
Nintedanib has divergent effects on constitutive and transforming growth factor (TGF)-β1–induced α-smooth muscle actin (α-SMA) expression. (A and B) Expression of α-SMA by Western blotting was assessed in IPF fibroblasts treated with increasing doses of nintedanib (0.5, 1, or 2 μM) (A) or nintedanib (2 μM) for increasing duration (24, 48, or 72 h) (B). (C) Densitometric analysis of independent experiments on different IPF fibroblast cell lines treated with nintedanib (2 μM) for 72 hours showing α-SMA expression relative to GAPDH. Error bars represent mean ± SEM (n = 3). *P < 0.05. (D) IPF fibroblasts were treated with nintedanib (2 μM) for 72 hours, cells were lysed, and RNA was extracted. RT-PCR was performed to assess steady-state mRNA levels of α-SMA compared with β-actin (control). Results are depicted graphically. Error bars represent mean ± SEM (n = 3). **P < 0.01. (E) IPF fibroblasts serum starved overnight were cotreated with or without TGF-β1 (2.5 ng/ml) and increasing doses of nintedanib (0.5, 1, and 2 μM) for 48 hours. α-SMA expression was evaluated by Western blotting. (F) Expression of α-SMA by Western blotting was assessed in IPF fibroblasts serum starved overnight and cotreated with or without TGF-β1 (2.5 ng/ml) and nintedanib (2 μM) for 48 hours. (G) Densitometric analysis of independent experiments performed as in F showing α-SMA expression relative to GAPDH. Error bars represent mean ± SEM (n = 3). *P < 0.05.
Figure 7.
Figure 7.
Nintedanib inhibits early events in TGF-β1 signaling. (A) IMR-90 fibroblasts were serum starved overnight and cotreated with or without TGF-β1 (2.5 ng/ml) and increasing doses of nintedanib (0.5, 1, and 2 μM) for 1 hour. Expression of pS423/425-SMAD3, SMAD3, pT180/Y182-p38 mitogen-activated protein kinase (MAPK), and p38 MAPK was evaluated by Western blotting. (B) Expression of pS423/425-SMAD3, and SMAD3 by Western blotting was assessed in IMR-90 fibroblasts that were serum starved overnight and cotreated with or without TGF-β1 (2.5 ng/ml) and nintedanib (2 μM) for 20 minutes. (C) Densitometric analysis of independent experiments performed as in B showing pS423/425-SMAD3 relative to GAPDH. Error bars represent mean ± SEM (n = 3). *P < 0.05. (D) Levels of phospho-tyrosine residues were assessed by Western blotting in IMR-90 fibroblasts under experimental conditions similar to those in B and subjected to immunoprecipitation (IP) to enrich TβR-II. Input levels of TβR-II, along with GAPDH and IgG staining with Ponceau S in the immunoprecipitated samples, represent loading controls. p-Tyr, phospho-tyrosine; TβR, TGF β receptor, type II.
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References

    1. Richeldi L, Costabel U, Selman M, Kim DS, Hansell DM, Nicholson AG, Brown KK, Flaherty KR, Noble PW, Raghu G, et al. Efficacy of a tyrosine kinase inhibitor in idiopathic pulmonary fibrosis. N Engl J Med. 2011;365:1079–1087. - PubMed
    1. Richeldi L, du Bois RM, Raghu G, Azuma A, Brown KK, Costabel U, Cottin V, Flaherty KR, Hansell DM, Inoue Y, et al. Efficacy and safety of nintedanib in idiopathic pulmonary fibrosis. N Engl J Med. 2014;370:2071–2082. - PubMed
    1. Chaudhary NI, Roth GJ, Hilberg F, Muller-Quernheim J, Prasse A, Zissel G, Schnapp A, Park JE. Inhibition of PDGF, VEGF and FGF signalling attenuates fibrosis. Eur Respir J. 2007;29:976–985. - PubMed
    1. Hilberg F, Roth GJ, Krssak M, Kautschitsch S, Sommergruber W, Tontsch-Grunt U, Garin-Chesa P, Bader G, Zoephel A, Quant J, et al. BIBF 1120: triple angiokinase inhibitor with sustained receptor blockade and good antitumor efficacy. Cancer Res. 2008;68:4774–4782. - PubMed
    1. Wollin L, Maillet I, Quesniaux V, Holweg A, Ryffel B. Antifibrotic and anti-inflammatory activity of the tyrosine kinase inhibitor nintedanib in experimental models of lung fibrosis. J Pharmacol Exp Ther. 2014;349:209–220. - PubMed

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