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

Kang, Shin Myung; Yoon, Jin Young; Kim, Yu Jin; Lee, Sang Pyo; Jeong, Sung Hwan; Park, Jeong Woong

doi:2011.71.2.88

Tuberc Respir Dis > Volume 71(2); 2011 > Article

Original Article

Tuberculosis and Respiratory Diseases 2011;71(2):88-96.
DOI: https://doi.org/10.4046/trd.2011.71.2.88 Published online August 1, 2011.

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

Shin Myung Kang, Jin Young Yoon, Yu Jin Kim, Sang Pyo Lee, Sung Hwan Jeong, Jeong Woong Park

Department of Pulmonary and Critical Care Medicine, Gachon University Gil Hospital, Gachon University of Medicine and Science, Incheon, Korea. jwpark@gilhospital.com

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.

Key Words: Apoptosis, Cigarette Smoking, Protein Kinase C-epsilon