Tumor necrosis factor alpha signaling pathway and apoptosis in pancreatic beta cells.

Abstract

Cytokines induce apoptosis in pancreatic beta cells, but the exact mechanisms and sequence of events are not clear. Here, we investigate a role for tumor necrosis factor alpha (TNF-alpha) in the apoptosis of beta cells. Using the ribonuclease (RNase) protection assay and the reverse transcriptase-polymerase chain reaction (RT-PCR) method, we confirmed that TNF receptor 1 (TNFR1), TNFR1-associated death domain protein (TRADD), Fas receptor-associated intracellular protein with death domain (FADD), and FADD-like interleukin-1beta-converting enzyme (FLICE) were expressed in the pancreatic beta cell line, MIN6 cells. Fluorescent microscopic examination using Hoechst 33342 dye (Sigma, St Louis, MO) demonstrated that TNF-alpha induced time- and dose-dependent apoptotic nuclear changes in these beta cells. In situ end-labeling (ISEL) DNA analysis revealed that 10 nmol/L TNF-alpha generated new 3'-OH DNA strand breaks. Moreover, qualitative assessment of the induced DNA damage on agarose gels showed that 10 nmol/L TNF-alpha produced characteristic apoptotic patterns of DNA fragments formed by internucleosomal hydrolysis of static chromatin. In addition, C2-ceramides and natural ceramides dispersed in a solvent mixture of ethanol and dodecane induced characteristic features of apoptosis in MIN6 cells, mimicking TNF-induced DNA damage. We also determined endosomal ceramide production after TNF-alpha (10 nmol/L) treatment in MIN6 cells using the diacylglycerol kinase assay. These results suggest that TNF-alpha can cause apoptosis in pancreatic beta cells through TNFR1-linked apoptotic factors, TRADD, FADD, and FLICE, and TNF-induced ceramide production may be involved in the pathways.