Tuberc Respir Dis.  2011 Aug;71(2):88-96. 10.4046/trd.2011.71.2.88.

Inhibition of PKC Epsilon Attenuates Cigarette Smoke Extract-Induced Apoptosis in Human Lung Fibroblasts (MRC-5 Cells)

• Kang SM
• Yoon JY
• Kim YJ
• Lee SP
• Jeong SH
• Park JW

• KMID: 1846415
• DOI: http://doi.org/10.4046/trd.2011.71.2.88

Abstract

BACKGROUND
It is known that cigarette smoke (CS) causes cell death. Apoptotic cell death is involved in the pathogenesis of CS-related lung diseases. Some members of the protein kinase C (PKC) family have roles in cigarette smoke extract (CSE)-induced apoptosis. This study was conducted to investigate the role of PKC epsilon in CSE-induced apoptosis in human lung fibroblast cell line, MRC-5.
METHODS
Lactate dehydrogenase release was measured using a cytotoxicity detection kit. The MTT assay was used to measure cell viability. Western immunoblot, Hoechst 33342 staining and flow cytometry were used to demonstrate the effect of PKCepsilon. Caspase-3 and caspase-8 activities were determined using a colorimetric assay. To examine PKCepsilon activation, Western blotting was performed using both fractions of membrane and cytosol.
RESULTS
We showed that CSE activated PKCepsilon by demonstrating increased expression of PKCepsilon in the plasma membrane fraction. Pre-treatment of PKCepsilon peptide inhibitor attenuated CSE-induced apoptotic cell death, as demonstrated by the MTT assay (13.03% of control, 85.66% of CSE-treatment, and 53.73% of PKCepsilon peptide inhibitor-pre-treatment, respectively), Hoechst 33342 staining, and flow cytometry (85.64% of CSE-treatment, 53.73% of PKCepsilon peptide inhibitor-pre-treatment). Pre-treatment of PKCepsilon peptide inhibitor reduced caspase-3 expression and attenuated caspase-3, caspase-8 activity compared with CSE treatment alone.
CONCLUSION
PKCepsilon seem to have pro-apoptotic function and exerts its function through the extrinsic apoptotic pathway in CSE-exposed MRC-5 cells. This study suggests that PKCepsilon inhibition may be a therapeutic strategy in CS-related lung disease such as chronic obstructive pulmonary disease.

Keyword

Apoptosis; Cigarette Smoking; Protein Kinase C-epsilon

MeSH Terms

Apoptosis
Benzimidazoles
Blotting, Western
Caspase 3
Caspase 8
Cell Death
Cell Line
Cell Membrane
Cell Survival
Fibroblasts
Flow Cytometry
Humans
L-Lactate Dehydrogenase
Lung
Lung Diseases
Membranes
Protein Kinase C
Protein Kinase C-epsilon
Pulmonary Disease, Chronic Obstructive
Smoke
Smoking
Tobacco Products
Benzimidazoles
Caspase 3
Caspase 8
L-Lactate Dehydrogenase
Protein Kinase C
Protein Kinase C-epsilon
Smoke

Figure

• 

  Figure 1 CSE induces MRC-5 cell death. (A) MRC-5 cells were exposed to 20% CSE at the indicated times, and cell viability was assessed using the MTT assay. (B) MRC-5 cells were exposed to 20% CSE at the indicated times, and cell cytotoxicity was assessed LDH assay. Data from each time point were compared with untreated cells at each time points. (C) MRC-5 cells were treated with 20% CSE for the indicated time periods. Activation of caspase-3 was assessed by Western blotting in total cell lysates. Data are expressed as the mean±SE. Significance was determined using t-test. *p<0.05, †p<0.005. CSE: cigarette smoke extract; LDH: lactate dehydrogenase; SE: standard error.

• 

  Figure 2 PKCε inhibition increases cell viability. (A) Translocation of PKCε from cytosol to the membrane fraction was analyzed in MRC-5 treated with 20% CSE for 12 hours. Expression of PKCε was detected by Western blot analysis as described. Cytosol and membrane fractions were isolated as described in Materials and Methods. All results are representative of three independent experiments. (B) Cell viability was evaluated after 12 hours incubation at the different concentration of PKCε peptide inhibitor using the MTT assay as described in Materials and Methods. Data are expressed as the mean±SE. Significance was determined using t-test. *p<0.05, †p<0.005. PKCε: protein kinase C epsilon; CSE: cigarette smoke extract; SE: standard error.

• 

  Figure 3 Flow cytometry analysis using annexin V and PI staining and Hoechst 33342 staining. (A) The apoptosis level of MRC-5 cells pretreated with PKCε peptide inhibitor, 20% CSE exposure alone, or control was compared using flow cytometry. Results are representative of three independent experiments. Annexin V (+) PI (-) cells (right lower) are considered apoptotic. (B) Histogram represents data from flow cytometry. Data are expressed as the mean±SE. Significance was determined using t-test. (C) Nuclei of MRC-5 cells were stained with Hoechst 33342 to detect apoptosis morphologically. White arrows point to the fragmented condensed nuclei. Photographs are representative results from three independent experiments. Images from phase contrast microscopy were also displayed (×200). *p<0.05. CSE: cigarette smoke extract; PI: propidium iodide; PKCε: protein kinase C epsilon; SE: standard error.

• 

  Figure 4 Inhibition of PKCε decreases caspase-3 and caspase-8 activities in MRC-5 cells after CSE exposure. (A) MRC-5 cells were pretreated with PKCε peptide inhibitor and Western blotted with cleaved caspase-3. (B) Histogram represents results from Western blotting. (C, D) The caspase-3 and caspase-8 activities were assessed by colorimetric assay. Data are expressed as the mean±SE. Significance was determined using t-test. *p<0.05. CSE: cigarette smoke extract; PKCε: protein kinase C epsilon; SE: standard error.

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