期刊
BIOCHEMISTRY
卷 46, 期 35, 页码 10170-10185出版社
AMER CHEMICAL SOC
DOI: 10.1021/bi700017z
关键词
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资金
- NCRR NIH HHS [P41 RR000954, P41-RR00954] Funding Source: Medline
- NHLBI NIH HHS [P01 HL057278, P01-HL57278] Funding Source: Medline
- NIDDK NIH HHS [P30 DK056341-06, R37-DK34388, P60 DK020579, P30 DK056341-07, P30 DK056341, P30-DK56341, R01 DK069455, P60-DK20579, R01 DK069455-04, R37 DK034388] Funding Source: Medline
- PHS HHS [R01-69455] Funding Source: Medline
beta-Cell mass is regulated by a balance between beta-cell growth and beta-cell death, due to apoptosis. We previously reported that apoptosis of INS-1 insulinoma cells due to thapsigargin-induced ER stress was suppressed by inhibition of the group VIA Ca2+-independent phospholipase A(2) (iPLA(2)beta), associated with an increased level of ceramide generation, and that the effects of ER stress were amplified in INS-1 cells in which iPLA(2)beta was overexpressed (OE INS-1 cells). These findings suggested that iPLA(2)beta and ceramides participate in ER stress-induced INS-1 cell apoptosis. Here, we address this possibility and also the source of the ceramides by examining the effects of ER stress in empty vector (V)-transfected and iPLA(2)beta-OE INS-1 cells using apoptosis assays and immunoblotting, quantitative PCR, and mass spectrometry analyses. ER stress induced expression of ER stress factors GRP78 and CHOP, cleavage of apoptotic factor PARP, and apoptosis in V and OE INS-1 cells. Accumulation of ceramide during ER stress was not associated with changes in mRNA levels of serine palmitoyltransferase (SPT), the rate-limiting enzyme in de novo synthesis of ceramides, but both message and protein levels of neutral sphingomyelinase (NSMase), which hydrolyzes sphingomyelins to generate ceramides, were temporally increased in the INS-1 cells. The increases in the level of NSMase expression in the ER-stressed INS-1 cells were associated with corresponding temporal elevations in ER-associated iPLA(2)beta protein and catalytic activity. Pretreatment with BEL inactivated iPLA(2)beta and prevented induction of NSMase message and protein in ER-stressed INS-1 cells. Relative to that in V INS-1 cells, the effects of ER stress were accelerated and/or amplified in the OE INS-1 cells. However, inhibition of iPLA(2)beta or NSMase (chemically or with siRNA) suppressed induction of NSMase message, ceramide generation, sphingomyelin hydrolysis, and apoptosis in both V and OE INS-1 cells during ER stress. In contrast, inhibition of SPT did not suppress ceramide generation or apoptosis in either V or OE INS-1 cells. These findings indicate that iPLA(2)beta activation participates in ER stress-induced INS-1 cell apoptosis by promoting ceramide generation via NSMase-catalyzed hydrolysis of sphingomyelins, raising the possibility that this pathway contributes to beta-cell apoptosis due to ER stress.
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