H Nakamura1, A Kawakami, H Ida, T Koji, K Eguchi. 1. First Department of Internal Medicine, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan. nhideki@nagasaki-u.ac.jp
Abstract
OBJECTIVES: Cell death is detected in the ducts of labial salivary glands (LSG) of patients with primary Sjögren's syndrome (pSS). However, the counter-mechanism to inhibit the apoptotic process remains unclear. In this study, we studied the ability of epidermal growth factor (EGF) to activate the PI3K-Akt pathway and NF-kB in primary cultured salivary gland epithelial cells (SGEC) of pSS patients. METHODS: SGEC, obtained from 2 female pSS patients, were cultured and used for Hoechst staining and deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL) assay. The frequency of apoptosis, detected by Hoechst staining, was quantified, and statistical significance was determined through unpaired student's t-test. RESULTS: Following twelve hours of stimulation, both PI3K inhibitors and anti-Fas antibody failed to induce apoptosis in primary cultured SGEC. However, the combination of anti-Fas antibody, along with LY294002 or Bay 11-7082, induced apoptosis which was statistically more significant than apoptosis found in the control cells (p < 0.01). Interestingly, the apoptosis induced by anti-Fas antibody along with LY294002 was clearly inhibited by the addition of 10 ng/ml EGF. Furthermore, the results of the TUNEL assay clearly indicated apoptosis through stimulation with anti-Fas antibody and LY294002 or Bay 11-7082. Furthermore, the apoptosis was completely blocked by the addition of EGF. CONCLUSION: Our results suggest that salivary epithelial cells are protected from Fas mediated apoptosis, through cell survival factors including either the PI3K-Akt pathway or NF-kB.
OBJECTIVES: Cell death is detected in the ducts of labial salivary glands (LSG) of patients with primary Sjögren's syndrome (pSS). However, the counter-mechanism to inhibit the apoptotic process remains unclear. In this study, we studied the ability of epidermal growth factor (EGF) to activate the PI3K-Akt pathway and NF-kB in primary cultured salivary gland epithelial cells (SGEC) of pSSpatients. METHODS: SGEC, obtained from 2 female pSSpatients, were cultured and used for Hoechst staining and deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL) assay. The frequency of apoptosis, detected by Hoechst staining, was quantified, and statistical significance was determined through unpaired student's t-test. RESULTS: Following twelve hours of stimulation, both PI3K inhibitors and anti-Fas antibody failed to induce apoptosis in primary cultured SGEC. However, the combination of anti-Fas antibody, along with LY294002 or Bay 11-7082, induced apoptosis which was statistically more significant than apoptosis found in the control cells (p < 0.01). Interestingly, the apoptosis induced by anti-Fas antibody along with LY294002 was clearly inhibited by the addition of 10 ng/ml EGF. Furthermore, the results of the TUNEL assay clearly indicated apoptosis through stimulation with anti-Fas antibody and LY294002 or Bay 11-7082. Furthermore, the apoptosis was completely blocked by the addition of EGF. CONCLUSION: Our results suggest that salivary epithelial cells are protected from Fas mediated apoptosis, through cell survival factors including either the PI3K-Akt pathway or NF-kB.