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

Cytokines induce apoptosis in pancreatic beta cells, but the exact mechanis ms and sequence of events are not clear. Here, we investigate a role for tu mor necrosis factor alpha (TNF-alpha) in the apoptosis of beta cells. Using the ribonuclease (RNase) protection assay and the reverse transcriptase-po lymerase chain reaction (RT-PCR) method, we confirmed that TNF receptor 1 ( TNFR1). TNFR1-associated death domain protein (TRADD), Fas receptor-associa ted intracellular protein with death domain (FADD), and FADD-like interleuk in-1 beta-converting enzyme (FLICE) were expressed in the pancreatic beta c ell line, MIN6 cells. Fluorescent microscopic examination using Hoechst 333 42 dye (Sigma. St Louis, MO) demonstrated that TNF-alpha induced time- and dose-dependent apoptotic nuclear changes in these beta cells. In situ end-l abeling (ISEL) DNA analysis revealed that 10 nmol/L TNF-alpha generated new 3'-OH DNA strand breaks. Moreover, qualitative assessment of the induced D NA damage on agarose gels showed that 10 nmol/L TNF-alpha produced characte ristic apoptotic patterns of DNA fragments formed by internucleosomal hydro lysis of static chromatin. In addition, CS-ceramides and natural ceramides dispersed in a solvent mixture of ethanol and dodecane induced characterist ic features of apoptosis in MIN6 cells, mimicking TNF-induced DNA damage. W e also determined endosomal ceramide production after TNF-alpha (10 nmol/L) treatment in MIN6 cells using the diacylglycerol kinase assay. These resul ts suggest that TNF-alpha can cause apoptosis in pancreatic beta cells thro ugh TNFR1-linked apoptotic factors, TRADD, FADD, and FLICE, and TNF-induced ceramide production may be involved in the pathways. Copyright(C) 1999 by W.B. Saunders Company.