Epithelial cell mitochondrial dysfunction and PINK1 are induced by transforming growth factor-beta1 in pulmonary fibrosis - PubMed (original) (raw)

. 2015 Mar 18;10(3):e0121246.

doi: 10.1371/journal.pone.0121246. eCollection 2015.

Jin Woo Song 2, Sarah G Chu 1, Kenji Mizumura 3, Juan C Osorio 1, Ying Shi 1, Souheil El-Chemaly 1, Chun Geun Lee 4, Ivan O Rosas 5, Jack A Elias 6, Augustine M K Choi 3, Danielle Morse 1

Affiliations

Epithelial cell mitochondrial dysfunction and PINK1 are induced by transforming growth factor-beta1 in pulmonary fibrosis

Avignat S Patel et al. PLoS One. 2015.

Abstract

Background: Epithelial cell death is a major contributor to fibrogenesis in the lung. In this study, we sought to determine the function of mitochondria and their clearance (mitophagy) in alveolar epithelial cell death and fibrosis.

Methods: We studied markers of mitochondrial injury and the mitophagy marker, PTEN-induced putative kinase 1 (PINK1), in IPF lung tissues by Western blotting, transmission electron microscopy (TEM), and immunofluorescence. In vitro experiments were carried out in lung epithelial cells stimulated with transforming growth factor-β1 (TGF-β1). Changes in cell function were measured by Western blotting, flow cytometry and immunofluorescence. In vivo experiments were performed using the murine bleomycin model of lung fibrosis.

Results: Evaluation of IPF lung tissue demonstrated increased PINK1 expression by Western blotting and immunofluorescence and increased numbers of damaged mitochondria by TEM. In lung epithelial cells, TGF-β1 induced mitochondrial depolarization, mitochondrial ROS, and PINK1 expression; all were abrogated by mitochondrial ROS scavenging. Finally, Pink1-/- mice were more susceptible than control mice to bleomycin induced lung fibrosis.

Conclusion: TGF-β1 induces lung epithelial cell mitochondrial ROS and depolarization and stabilizes the key mitophagy initiating protein, PINK1. PINK1 ameliorates epithelial cell death and may be necessary to limit fibrogenesis.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1

Fig 1. PINK1 Expression and Mitochondrial Dysfunction in IPF Lung Tissue.

A) Western blot of mitochondrial fraction of PINK1 in human lung tissue from control and IPF patients showing increased PINK1 levels in IPF samples. B) Densitometry of blot in A. C) mRNA expression of Pink1 in human lung tissue from control (n = 5) and IPF (n = 5) samples. D) Representative transmission electron micrographs from control and IPF lung tissue showing mitochondria (*). Magnif = 18500x; scale bars = 200nm. E) Quantification of total and abnormal mitochondria. Total mitochondria per μm2 in control and IPF was 0.52 ± 0.39 vs. 0.46 ± 0.34. Abnormal mitochondria per μm2 in control and IPF was 0.12 ± 0.19 vs. 0.32 ± 0.32, p = 0.003. F) Confocal immunofluorescence against PINK1(red) and LC3 (green) in control and IPF lung (magnification 10x). G) Magnified view (60x) of confocal immunofluoresence with DAPI (blue) and PINK1 (red) in control and IPF lung.

Fig 2

Fig 2. TGF-β1 increases PINK1 expression and induces mitochondrial fission in vitro.

A) TGF-β1 induced PINK1 expression of Beas-2B cells in a time- and dose-dependent manner. B) Quantification by densitometry of PINK1 expression in Beas-2B cells stimulated with TGF-β1 for 6 hours. C) Confocal microscopy of Beas-2B cells stimulated with TGF-β1 (6h) showed that TGF-β1 induces formation of PINK1 puncta (red) (magnification 120x). D) Quantification of colocalization of LC3 and PINK1 punctae (* p = 0.035, ** p = 0.001). E) Confocal microscopy of Beas-2B cells transfected with vectors staining mitochondria (green) and lysosomes (red) and with TGF-β1 stimulation for 6 hours (magnification 60x). F) Western blot of Beas-2B cells stimulated with TGF-β1 (6hrs) showed increased expression of pDRP1 (ser616). G) Confocal microscopy of Beas-2B cells stimulated with TGF-β1 (5 ng/mL, 24 hrs) showed more fragmentation (fission) of mitochondria stained with MitoTracker Green (magnification 120x).

Fig 3

Fig 3. TGF-β1 induces mitochondrial depolarization.

A) Beas-2B cells stimulated with TGF-β1 and CCCP (positive controls) were stained with JC-1 for 15 min and analyzed by flow cytometric analysis. TGF-β1 treated cells showed decreased red fluorescence (mitochondrial depolarization). B) Quantification of relative MFI for experiment in A (p = 0.06). C) Beas-2B cells stimulated with TGF-β1 were stained with MitoSOX for 10 min and analyzed by flow cytometry. TGF-β1 treated cells showed increased red fluorescence (mitochondrial ROS production) and mitochondria-specific antioxidant (MitoTEMPO) reversed the effect of TGF-β1. D) Western blot of the mitochondrial fraction of Beas-2B cells stimulated with TGF-β1 (5ng/mL) +/- MitoTEMPO (200μM) showing decreased PINK1 expression in presence of MitoTEMPO.

Fig 4

Fig 4. Loss of PINK1 augments TGF-β1 induced cell death.

A) Measurement of mitochondrial ROS in murine type II alveolar epithelial cells from Pink1 -/- and Pink1 WT mice by flow cytometry and MitoSOX staining. B) Western blot against PINK1 demonstrated knockdown of expression with PINK1 siRNA. C) Beas-2B cells were treated with siRNA (50 nM, 24hrs). Then, they were stimulated with TGF-β1 (5 ng/mL, 24hrs) and analyzed by Annexin V/PI flow cytometry. D) Quantification of cell death from experiment in C (*p = 0.0005, **p = 0.0119, ***p = 0.032). Loss of PINK1 exaggerated cell death in cells treated with TGF-β1 relative to transfection with control siRNA.

Fig 5

Fig 5. Loss of PINK1 aggravates bleomycin induced lung fibrosis.

Pink1 -/- mice and their littermate controls (male, 8–10 weeks old) were treated with 3U/kg of intratracheal bleomycin sulfate or saline on day 1 and sacrificed on Day 21. Pink1 -/- mice in the bleomycin group showed higher levels of hydroxyproline compared to controls (85.4 μg/mL vs. 97.0 μg/mL; *p = .05, **p<0.001).

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