G-protein signaling abnormalities mediated by CD95 in salivary epithelial cells

Salivary epithelial cells from patients with primary Sjogren's syndrome (SS ) undergo Fas-mediated apoptosis. Bcl-2 and Bcl-xL are apoptosis suppressin g oncogenes, Very little is known about the role of these oncogene molecule s in salivary epithelial cells. To investigate the possible prevention of s alivary glandular destruction in SS by Bcl-2 and Bcl-xL, stable transfectan ts expressing these molecules were made from HSY cells, a human salivary ep ithelial cell line. HSY cells were transfected with an expression vector fo r human Bcl-2 or Bcl-xL. Stable transfectants were selected and apoptosis w as induced by anti-fas antibody. Apoptosis was quantified by propidium iodi de staining followed by flow cytometry, Caspase activity was detected by im munohistochemical analysis and enzyme cleavage of DEVD-AMC, a fluorescent s ubstrate. Response to carbachol, a muscarinic receptor agonist, and EGF was measured by Ca2+ mobilization and influx. Fas-mediated apoptosis was signi ficantly inhibited in Bcl-2 and Bcl-xL transfectants compared to wild-type and control transfectants (empty vector). Surprisingly, caspase activity wa s not inhibited in Bcl-2 and Bcl-xL transfectants. Activation of the Fas pa thway in the Bcl-2 and Bcl-xL transfectants by antibody also inhibited carb achol and EGF responsiveness (i.e., Ca2+ mobilization and/or influx) by 50- 60%, This Fas-mediated inhibition of cell activation was partially or compl etely restored by specific peptide interference of caspase enzyme activity. The prevention of Fas-mediated apoptosis by the overexpression of Bcl-2 an d Bcl-xL in salivary gland epithelial cells results in injured cells expres sing caspase activity and unable to respond normally to receptor agonists, Such damaged cells may exist in SS patients and could explain the severe dr yness out of proportion to the actual number of apoptotic cells seen on sal ivary gland biopsy.