Tuberc Respir Dis.  2018 Apr;81(2):138-147. 10.4046/trd.2017.0115.

The Phosphodiesterase 4 Inhibitor Roflumilast Protects against Cigarette Smoke Extract-Induced Mitophagy-Dependent Cell Death in Epithelial Cells

• Kyung SY
• Kim YJ
• Son ES
• Jeong SH
• Park JW

• KMID: 2420602
• DOI: http://doi.org/10.4046/trd.2017.0115

Abstract

BACKGROUND
Recent studies show that mitophagy, the autophagy-dependent turnover of mitochondria, mediates pulmonary epithelial cell death in response to cigarette smoke extract (CSE) exposure and contributes to the development of emphysema in vivo during chronic cigarette smoke (CS) exposure, although the underlying mechanisms remain unclear.
METHODS
In this study, we investigated the role of mitophagy in the regulation of CSE-exposed lung bronchial epithelial cell (Beas-2B) death. We also investigated the role of a phosphodiesterase 4 inhibitor, roflumilast, in CSE-induced mitophagy-dependent cell death.
RESULTS
Our results demonstrated that CSE induces mitophagy in Beas-2B cells through mitochondrial dysfunction and increased the expression levels of the mitophagy regulator protein, PTEN-induced putative kinase-1 (PINK1), and the mitochondrial fission protein, dynamin-1-like protein (DRP1). CSE-induced epithelial cell death was significantly increased in Beas-2B cells exposed to CSE but was decreased by small interfering RNA-dependent knockdown of DRP1. Treatment with roflumilast in Beas-2B cells inhibited CSE-induced mitochondrial dysfunction and mitophagy by inhibiting the expression of phospho-DRP1 and -PINK1. Roflumilast protected against cell death and increased cell viability, as determined by the lactate dehydrogenase release test and the MTT assay, respectively, in Beas-2B cells exposed to CSE.
CONCLUSION
These findings suggest that roflumilast plays a protective role in CS-induced mitophagy-dependent cell death.

Keyword

Mitophagy; Roflumilast; Tobacco Use; Pulmonary Disease, Chronic Obstructive

MeSH Terms

Cell Death*
Cell Survival
Cyclic Nucleotide Phosphodiesterases, Type 4*
Emphysema
Epithelial Cells*
L-Lactate Dehydrogenase
Lung
Mitochondria
Mitochondrial Degradation
Mitochondrial Dynamics
Pulmonary Disease, Chronic Obstructive
Smoke*
Tobacco Products*
Tobacco Use
Cyclic Nucleotide Phosphodiesterases, Type 4
L-Lactate Dehydrogenase
Smoke

Figure

• 

  Figure 1 Effects of cigarette smoke extract (CSE) on mitochondrial dysfunction and mitophagy in Beas-2B epithelial cells. Human bronchial epithelial (Beas-2B) cells were treated with the indicated percent of CSE for 24 hours. (A) Flow cytometric analysis of CSE-treated Beas-2B cells left unstained or labeled with tetramethylrhodamine, ethyl ester (TMRE) and MitoSOX Red. Cells were induced with 10% CSE for 18 hours. (B, C) The expression of mitophagy-related proteins was determined by Western blot analysis. DRP1: dynamin-1-like protein; PINK1: PTEN-induced putative kinase-1.

• 

  Figure 2 Effects of dynamin-1-like protein (DRP1) and PTEN-induced putative kinase-1 (PINK1) silencing on cigarette smoke extract (CSE)–induced cell death in Beas-2B epithelial cells. Beas-2B cells were exposed to different concentrations of CSE for 18 hours. Cell viability was evaluated using the MTT assay and cytotoxicity was measured by the lactate dehydrogenase (LDH) release test. Beas-2B cells were pre-treated with control, DRP1, or PINK1 siRNAs for 48 hours prior to treatment with 10% CSE for 18 hours. Cell cytotoxicity (C) and viability (D) were estimated by the LDH and MTT assays, respectively. The data shown represent the mean±SD derived from three determinations. *p<0.05 and ***p<0.001, compared with the control siRNA-transfected group.

• 

  Figure 3 Effects of roflumilast on mitochondrial dysfunction and mitophagy in cigarette smoke extract (CSE)–induced Beas-2B epithelial cells. Beas-2B cells were pre-treated for 2 hours with 5 µM roflumilast and exposed to 10% CSE for 4 hours. (A) Detection of mitochondrial dysfunction (×400). Cells labeled with tetramethylrhodamine, ethyl ester (TMRE) or MitoSOX Red were incubated with control or 5 µM roflumilast and treated with 10% CSE for 4 hours. (B) The expression of mitophagy-related proteins was determined by Western blot analysis. (C) The expression of apoptosis-related proteins was determined by Western blot analysis. DRP1: dynamin-1-like protein; PINK1: PTEN-induced putative kinase-1; R5: roflumilast 5 µM.

• 

  Figure 4 Effects of roflumilast on apoptotic cell death in cigarette smoke extract (CSE)–induced Beas-2B epithelial cells. Cells were pre-treated for 2 hours with 5 µM roflumilast followed by treatment with 10% CSE for 18 hours. Cell viability (A) and cytotoxicity (B) were estimated by MTT and lactate dehydrogenase (LDH) assay, respectively. (C) Beas-2B cells were pre-treated with control, dynamin-1-like protein (DRP1), or PTEN-induced putative kinase-1 (PINK1) siRNAs for 48 hours prior to treatment with 10% CSE for 18 hours. R5: roflumilast 5 µM. The data shown represent the mean±SD derived from three determinations. *p<0.05, **p<0.01, and ***p<0.001, compared with the CSE-treated group.

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