title: 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.
authors.affiliation: Department of Internal Medicine II, Chiba University School of Medicine, Japan.
authors.forename: N
authors.initials: N
authors.lastname: Ishizuka
authors.forename: K
authors.initials: K
authors.lastname: Yagui
authors.forename: Y
authors.initials: Y
authors.lastname: Tokuyama
authors.forename: K
authors.initials: K
authors.lastname: Yamada
authors.forename: Y
authors.initials: Y
authors.lastname: Suzuki
authors.forename: J
authors.initials: J
authors.lastname: Miyazaki
authors.forename: N
authors.initials: N
authors.lastname: Hashimoto
authors.forename: H
authors.initials: H
authors.lastname: Makino
authors.forename: Y
authors.initials: Y
authors.lastname: Saito
authors.forename: A
authors.initials: A
authors.lastname: Kanatsuka
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D002352:Carrier Proteins / Q000378:metabolism; D002518:Ceramides / Q000096:biosynthesis / Q000494:pharmacology; D053938:DNA Fragmentation; D053200:Fas-Associated Death Domain Protein; D006801:Humans; D007515:Islets of Langerhans / Q000378:metabolism / Q000502:physiology*; D051379:Mice; D011506:Proteins / Q000378:metabolism; D018124:Receptors, Tumor Necrosis Factor / Q000378:metabolism; D047888:Receptors, Tumor Necrosis Factor, Type I; D015398:Signal Transduction / Q000502:physiology*; D053199:TNF Receptor-Associated Death Domain Protein; D047990:TNF Receptor-Associated Factor 1; D014407:Tumor Cells, Cultured; D047988:Tumor Necrosis Factor Receptor-Associated Peptides and Proteins; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology / Q000502:physiology*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D015703:Antigens, CD; D002352:Carrier Proteins; D002518:Ceramides; C505533:FADD protein, human; C505534:Fadd protein, mouse; D053200:Fas-Associated Death Domain Protein; D011506:Proteins; D018124:Receptors, Tumor Necrosis Factor; D047888:Receptors, Tumor Necrosis Factor, Type I; D053199:TNF Receptor-Associated Death Domain Protein; D047990:TNF Receptor-Associated Factor 1; C481226:Tradd protein, mouse; D047988:Tumor Necrosis Factor Receptor-Associated Peptides and Proteins; D014409:Tumor Necrosis Factor-alpha