Tumor necrosis factor alpha signaling pathway and apoptosis in pancreatic β cells
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- 作者：Nobuko Ishizuka ; Kazuo Yagui ; Yoshiharu Tokuyama ; Katsumi Yamada ; Yoshifumi Suzuki ; Jun-ichi Miyazaki ; Naotake Hashimoto ; Hideichi Makino ; Yasushi Saito ; Azuma Kanatsuka
- 刊名：Metabolism
- 出版年：1999
- 出版时间：December 1999
- 年：1999
- 卷：48
- 期：12
- 页码：1485-1492
- 全文大小：4777 K
文摘
Cytokines induce apoptosis in pancreatic β cells, but the exact mechanisms and sequence of events are not clear. Here, we investigate a role for tumor necrosis factor alpha (TNF-α) in the apoptosis of β 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-1β—converting enzyme (FLICE) were expressed in the pancreatic β cell line, MIN6 cells. Fluorescent microscopic examination using Hoechst 33342 dye (Sigma, St Louis, MO) demonstrated that TNF-α induced time- and dose-dependent apoptotic nuclear changes in these β cells. In situ end-labeling (ISEL) DNA analysis revealed that 10 nmol/L TNF-α generated new 3′-OH DNA strand breaks. Moreover, qualitative assessment of the induced DNA damage on agarose gels showed that 10 nmol/L TNF-α 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-α (10 nmol/L) treatment in MIN6 cells using the diacylglycerol kinase assay. These results suggest that TNF-α can cause apoptosis in pancreatic β cells through TNFR1-linked apoptotic factors, TRADD, FADD, and FLICE, and TNF-induced ceramide production may be involved in the pathways.