Tuberc Respir Dis.  2015 Jan;78(1):8-17. 10.4046/trd.2015.78.1.8.

Role of AMP-Activated Protein Kinase (AMPK) in Smoking-Induced Lung Inflammation and Emphysema

• Lee JS
• Park SJ
• Cho YS
• Huh JW
• Oh YM
• Lee SD

• KMID: 2114643
• DOI: http://doi.org/10.4046/trd.2015.78.1.8

Abstract

BACKGROUND
AMP-activated protein kinase (AMPK) not only functions as an intracellular energy sensor and regulator, but is also a general sensor of oxidative stress. Furthermore, there is recent evidence that it participates in limiting acute inflammatory reactions, apoptosis and cellular senescence. Thus, it may oppose the development of chronic obstructive pulmonary disease.
METHODS
To investigate the role of AMPK in cigarette smoke-induced lung inflammation and emphysema we first compared cigarette smoking and polyinosinic-polycytidylic acid [poly(I:C)]-induced lung inflammation and emphysema in AMPKalpha1-deficient (AMPKalpha1-HT) mice and wild-type mice of the same genetic background. We then investigated the role of AMPK in the induction of interleukin-8 (IL-8) by cigarette smoke extract (CSE) in A549 cells.
RESULTS
Cigarette smoking and poly(I:C)-induced lung inflammation and emphysema were elevated in AMPKalpha1-HT compared to wild-type mice. CSE increased AMPK activation in a CSE concentration- and time-dependent manner. 5-Aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR), an AMPK activator, decreased CSE-induced IL-8 production while Compound C, an AMPK inhibitor, increased it, as did pretreatment with an AMPKalpha1-specific small interfering RNA.
CONCLUSION
AMPKalpha1-deficient mice have increased susceptibility to lung inflammation and emphysema when exposed to cigarette smoke, and AMPK appears to reduce lung inflammation and emphysema by lowering IL-8 production.

Keyword

AMP-Activated Protein Kinases; Pulmonary Disease, Chronic Obstructive; Emphysema; Inflammation; Interleukin-8

MeSH Terms

AMP-Activated Protein Kinases*
Animals
Apoptosis
Cell Aging
Emphysema*
Inflammation
Interleukin-8
Mice
Oxidative Stress
Pneumonia*
Poly I-C
Pulmonary Disease, Chronic Obstructive
RNA, Small Interfering
Smoke
Smoking
Tobacco Products
AMP-Activated Protein Kinases
Interleukin-8
Poly I-C
RNA, Small Interfering
Smoke

Figure

• 

  Figure 1 Generation of heterozygous AMPKα1-deficient (AMPKα1-HT) C57BL/6 mice. (A) Genotyping of mice by multiplex polymerase chain reaction; this yielded amplification products of 350 bp (knockout) and 450 bp (wild-type). (B) Western blot analysis of AMPKα expression in lung tissue. AMPK: AMP-activated protein kinase; WT: wild-type littermate; HT: heterozygous AMPKα1-deficient mouse; M: marker. Data represent mean±SEM (n=3). Each data point is based on three independent Western blots. *p<0.05, vs. WT mice.

• 

  Figure 2 Increased cigarette smoking- and poly(I:C)-induced lung inflammation and emphysema in AMPKα1-deficient (AMPKα1-HT) mice. (A) Histological assessment at 2 months of lung sections stained with hematoxylin and eosin (×100). (B) Total bronchoalveolar lavage cell counts and differential cell counts. (C) Morphometric analysis of mean linear intercepts. AMPK: AMP-activated protein kinase; WC: wild-type no smoke (n=4); HC: AMPKα1-HT no smoke (n=5); WS: wild-type exposed to smoke (n=4); HS: AMPKα1-HT exposed to smoke (n=5). Data represent mean±SEM. * and † denotes significant differences (p<0.05) between the WC and WS group, and between the WS and HS group, respectively.

• 

  Figure 3 CSE increases the production of IL-8 in A549 cells. (A, C) Cells were exposed to 0%-4.5% CSE for 24 hours. (B, D) Cells were incubated with medium alone (time 0) or exposed to 3% CSE for the times indicated. Protein levels in cell lysates (A, B) were analyzed by Western blot, and protein levels in culture media (C, D) were analyzed by enzyme-linked immunosorbent assay. CSE: cigarette smoke extract; IL-8: interleukin 8. Data in each group are means±SEM of three independent experiments. *p<0.05, vs. 0% CSE (A, C) or time 0 (B, D).

• 

  Figure 4 CSE activates AMPK in A549 cells. (A) Cells were incubated with medium alone or exposed to 3% CSE for the times indicated. (B) Cells were exposed to 0-10 mM AICAR (an AMPK activator) for 5 hours. (C) Cells were exposed to 0-100 µM Compound C (an AMPK inhibitor) for 5 hours. (D) Cells were transfected with various concentrations of AMPK siRNA for 48 hours. CSE: cigarette smoke extract; AMPK: AMP-activated protein kinase; AICAR: 5-aminoimidazole-4-carboxamide-1-β-4-ribofuranoside.

• 

  Figure 5 AMPK plays a vital role in the induction of IL-8 by CSE in A549 cells. (A, C) Cells were incubated with medium alone or exposed to 3% CSE for 3 hours after pretreatment with AICAR (1 mM) and Compound C (5 µM). (B, D) Cells were incubated with medium alone or exposed to 3% CSE for 24 hours after pretreatment with 10 nM AMPK siRNA or negative siRNA. Protein levels in cell lysates (A, B) were analyzed by Western blot, and protein levels in culture media (C, D) by enzyme-linked immunosorbent assay. The immunoblot results are representative of three independent experiments. AMPK: AMP-activated protein kinase; CSE: cigarette smoke extract; AICAR: 5-aminoimidazole-4-carboxamide-1-β-4-ribofuranoside; IL-8: interleukin 8; DMSO: dimethyl sulfoxide. Data in each group are means±SEM (n=3). *p<0.05, vs. medium only. †p<0.05 vs CSE without drug or siRNA treatment.

• 

  Figure 6 Inhibition of AMPK activation by Compound C increases IL-1β-induced IL-8 secretion in A549 cells. Cells were incubated with medium alone or exposed to 5 ng/mL IL-1β for 24 hours after pretreatment with AICAR (1 mM) or Compound C (10 µM) for 5 hours. (A) Protein levels in cell lysates were analyzed by Western blot. (B) Protein levels in culture media were analyzed by enzyme-linked immunosorbent assay. The immunoblot results are representative of three independent experiments. AMPK: AMP-activated protein kinase; IL-1β, interleukin-1β; AICAR: 5-aminoimidazole-4-carboxamide-1-β-4-ribofuranoside. Data in each group are means±SEM (n=3). *p<0.05, vs. medium only. †p<0.05, vs. CSE without AICAR or Compound C.

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