Tuberc Respir Dis.  2017 Jan;80(1):83-89. 10.4046/trd.2017.80.1.83.

The Impact of Autophagy on the Cigarette Smoke Extract-Induced Apoptosis of Bronchial Epithelial Cells

• Lee CH
• Lee KH
• Jang AH
• Yoo CG

• KMID: 2396345
• DOI: http://doi.org/10.4046/trd.2017.80.1.83

Abstract

BACKGROUND
Previous studies report that apoptosis and autophagy are involved in the pathogenesis of emphysema, and macroautophagy is one of the processes regulating the apoptosis pathway. However, few studies have evaluated whether chaperone-mediated autophagy (CMA) contributes to the regulation of apoptosis. In this study, we investigated the impact of autophagy, including both macroautophagy and CMA, on the apoptosis in bronchial epithelial cells.
METHODS
Cigarette smoke extract (CSE) was injected intratracheally into C57BL/6 mice, and emphysema and apoptosis were evaluated in the lungs. After treatment with CSE, apoptosis, macroautophagy, and CMA were measured in BEAS2-B cells, and the impact of autophagy on the apoptosis was evaluated following knockdown of autophagy-related genes by short interfering RNAs (siRNAs).
RESULTS
Intratracheal CSE injection resulted in the development of emphysema and an increase in apoptosis in mice. CSE increased the apoptosis in BEAS2-B cells, and also elevated the expression of proteins related to both macroautophagy and CMA in BEAS2-B cells. The knockdown experiment with siRNAs showed that macroautophagy increases apoptosis in BEAS2-B cells, while CMA suppresses apoptosis.
CONCLUSION
The intratracheal injection of CSE induces pulmonary emphysema and an increase in apoptosis in mice. CSE also induces apoptosis, macroautophagy, and CMA of bronchial epithelial cells. Macroautophagy and CMA regulate apoptosis in opposite directions.

Keyword

Tobacco Products; Smoke; Apoptosis; Autophagy; Epithelial Cells

MeSH Terms

Animals
Apoptosis*
Autophagy*
Emphysema
Epithelial Cells*
Lung
Mice
Pulmonary Emphysema
RNA, Small Interfering
Smoke*
Tobacco Products*
RNA, Small Interfering
Smoke

Figure

• 

  Figure 1 Intratracheal CSE injection led to the development of emphysema and an increase of apoptosis in mice. (A) Control group (H&E stain, ×100). (B) CSE group (H&E stain, ×100). (C) Control group (TUNEL, ×400). (D) CSE group (TUNEL, ×400). The arrows indicate TUNEL positive cells. CSE: cigarette smoke extract; TUNEL: terminal deoxynucleotidyl transferase dUTP nick-end labeling.

• 

  Figure 2 CSE decreased BEAS2-B cell viability. (A) MTT assay. (B) LDH release. *p<0.05 by ANOVA. CSE: cigarette smoke extract; MTT: 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide; LDH: lactose dehydrogenase.

• 

  Figure 3 CSE increased apoptosis of BEAS2-B cells. (A) Annexin/PI statin. (B) Western blot. CSE: cigarette smoke extract; PI: propidium iodide; PARP-1: poly(ADP-ribose) polymerase-1; GAPDH: glyceraldehyde 3-phosphate dehydrogenase.

• 

  Figure 4 CSE increased macroautophagy and chaperone-mediated autophagy in BEAS2-B cells. CSE: cigarette smoke extract; LC3B: microtubule-associated protein 1 light chain 3B; GAPDH: glyceraldehyde 3-phosphate dehydrogenase.

• 

  Figure 5 Macroautophagy and chaperone-mediated autophagy have opposite effects on apoptosis of BEAS2-B cells. (A) LC3B was successfully inhibited by siRNA. (B) The inhibition of LC3B decreased PARP-1 cleavage (see full length PARP-1 in S.E. and N-terminal cleavage product in L.E.). (C) LAMP2a was successfully inhibited by siRNA. (D) The inhibition of LAMP2a increased PARP-1 cleavage (see full length PARP-1 in S.E. and N-terminal cleavage product in L.E.). LC3B: microtubule-associated protein 1 light chain 3B; siRNA: short interfering RNA; PARP-1: poly(ADP-ribose) polymerase-1; S.E.: short exposure; L.E.: long exposure; LAMP2a: lysosome-associated membrane protein 2a; CSE: cigarette smoke extract; GAPDH: glyceraldehyde 3-phosphate dehydrogenase.

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