p38 Mitogen-activated Protein Kinase and Nuclear Factor-κB Facilitate CD40-mediated Salivary Epithelial Cell Death

Abstract

Objective.Our previous studies indicated that CD40-mediated Fas-dependent apoptosis is important for the glandular destruction of Sjögren’s syndrome (SS), although other immune and nonimmune mechanisms are also involved in exocrine dysfunction. We investigated the roles of p38 mitogen-activated protein kinase (p38MAPK) and nuclear factor-κB (NF-κB) in salivary epithelial cell death in SS.Methods.Expression of p38, phosphorylated p38 (pp38), and IκB-α was examined by Western blotting upon CD40 ligation. Activity of NF-κB induced by anti-CD40 monoclonal antibody (mAb) was examined by electrophoretic mobility shift assay (EMSA) and Western blotting. Expression of Fas was analyzed by flow cytometry and Western blotting with or without the p38-specific inhibitor SB203580 or the NF-κB-specific inhibitor caffeic acid phenethyl ester (CAPE). Induction of apoptosis in salivary epithelial cells was examined by DNA fragmentation and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay. Expression of phosphorylated p38MAPK and NF-κB was measured by immunohistochemistry.Results.pp38MAPK and NF-κB p65 were predominantly expressed in the ductal and acinar epithelium adjacent to lymphoid infiltrates of SS salivary gland by immunohistochemistry. CD40 ligation strongly enhanced p38MAPK and NF-κB activity by EMSA and Western blotting in cultured salivary epithelial cells. Treatment of cells with anti-CD40 mAb resulted in significantly upregulated Fas expression and induction of Fas-dependent apoptosis. Inhibition of p38MAPK and NF-κB activity by SB203580 and/or CAPE reduced Fas expression and apoptosis in salivary epithelial cells, establishing p38MAPK and NF-κB as proapoptotic factors in this context.Conclusion.CD40 ligation plays an important role in activation of p38MAPK, NF-κB, and Fas molecules to initiate proapoptotic signaling. p38MAPK and NF-κB collaborate in regulation of proapoptotic signaling in CD40-mediated Fas-dependent apoptosis in salivary epithelial cells.