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KMID : 0383820110710020088
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Tuberculosis and Respiratory Diseases
2011 Volume.71 No. 2 p.88 ~ p.96
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Inhibition of PKC Epsilon Attenuates Cigarette Smoke Extract- Induced Apoptosis in Human Lung Fibroblasts (MRC-5 Cells)
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Kang Shin-Myung
Yoon Jin-Young
Kim Jin-Yu
Lee Sang-Pyo
Jeong Sung-Hwan
Park Jeong-Woong
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Abstract
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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.
Materials and 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 PKCε. Caspase-3 and caspase-8 activities were determined using a colorimetric assay. To examine PKCε activation, Western blotting was performed using both fractions of membrane and cytosol.
Results: We showed that CSE activated PKCε by demonstrating increased expression of PKCε in the plasma membrane fraction. Pre-treatment of PKCε 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 PKCε peptide inhibitor-pre-treatment, respectively), Hoechst 33342 staining, and flow cytometry (85.64% of CSE-treatment, 53.73% of PKCε peptide inhibitor-pre-treatment). Pre-treatment of PKCε peptide inhibitor reduced caspase-3 expression and attenuated caspase-3, caspase-8 activity compared with CSE treatment alone.
Conclusion: PKCε 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 PKCε inhibition may be a therapeutic strategy in CS-related lung disease such as chronic obstructive pulmonary disease.
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KEYWORD
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Apoptosis, Cigarette Smoking, Protein Kinase C-epsilon
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