The Group VIA Calcium-Independent Phospholipase A₂ Participates in ER Stress-Induced INS-1 Insulinoma Cell Apoptosis by Promoting Ceramide Generation via Hydrolysis of Sphingomyelins by Neutral Sphingomyelinase

Abstract

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 A2 (iPLA2beta), associated with an increased level of ceramide generation, and that the effects of ER stress were amplified in INS-1 cells in which iPLA2beta was overexpressed (OE INS-1 cells). These findings suggested that iPLA2beta 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 iPLA2beta-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 iPLA2beta protein and catalytic activity. Pretreatment with BEL inactivated iPLA2beta 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 iPLA2beta 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 iPLA2beta 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.

Activation of the unfolded protein response pathway causes ceramide accumulation in yeast and INS-1E insulinoma cells

“…The high specifi c activity of CerS6 coupled with feedback inhibition could help to explain the transient increases in ceramides that are often seen under stress conditions or other stimulation where it has been proposed to play important roles in activating direct targets in signaling ( 47,48 ). In contrast to our fi ndings in INS-1E cells, thapsigargin-induced ER stress was previously associated with stimulated ceramide production due to increased neutral sphingomyelinase activity in INS832/13 cells ( 49 ). It may be speculated that the latter cells are more prone to ER stress-related ceramide production, as they, in contrast to INS-1E cells, express human insulin in addition to the two allelic rat insulins.…”

Unique sphingomyelin patches are targets of a beta-cell-specific antibody

“…These patches are only present on the surface of ␤ cells, making them suitable for ligand binding. We believe that our fi ndings have implications for identifying future ligands for the proposed antigen for in vivo imaging purposes as well as for potential therapy, as SM-derived ceramide has been shown to have a role in the regulation of insulin synthesis ( 13,14 ) and contributes to ␤ -cell apoptosis (15)(16)(17)(18).…”

Calcium-independent phospholipases A2 and their roles in biological processes and diseases

“…1 . iPLA 2 ␤ and sphingolipids. Stress stimuli induce the intrinsic (mitochondrial) apoptotic pathway, which is mitigated by suppressing iPLA 2 ␤ expression/activity ( 103,104,136,138,246 ). Intriguingly, the primary lipid signals promoting mitochondrial decompensation were ceramides, derived through hydrolysis of sphingomyelins by neutral sphingomyelinase-2 (NSMase-2) ( 103,104 ).…”