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. 2015 Apr 24;290(17):11021-31.
doi: 10.1074/jbc.M115.648956. Epub 2015 Mar 11.

Group VIA Phospholipase A2 (iPLA2β) Modulates Bcl-x 5'-Splice Site Selection and Suppresses Anti-apoptotic Bcl-x(L) in β-Cells

Suzanne E Barbour  1 , Phuong T Nguyen  1 , Margaret Park  1 , Bhargavi Emani  1 , Xiaoyong Lei  2 , Mamatha Kambalapalli  1 , Jacqueline C Shultz  1 , Dayanjan Wijesinghe  3 , Charles E Chalfant  4 , Sasanka Ramanadham  5
Affiliations

Group VIA Phospholipase A2 (iPLA2β) Modulates Bcl-x 5'-Splice Site Selection and Suppresses Anti-apoptotic Bcl-x(L) in β-Cells

Suzanne E Barbour et al. J Biol Chem. .

Abstract

Diabetes is a consequence of reduced β-cell function and mass, due to β-cell apoptosis. Endoplasmic reticulum (ER) stress is induced during β-cell apoptosis due to various stimuli, and our work indicates that group VIA phospholipase A2β (iPLA2β) participates in this process. Delineation of underlying mechanism(s) reveals that ER stress reduces the anti-apoptotic Bcl-x(L) protein in INS-1 cells. The Bcl-x pre-mRNA undergoes alternative pre-mRNA splicing to generate Bcl-x(L) or Bcl-x(S) mature mRNA. We show that both thapsigargin-induced and spontaneous ER stress are associated with reductions in the ratio of Bcl-x(L)/Bcl-x(S) mRNA in INS-1 and islet β-cells. However, chemical inactivation or knockdown of iPLA2β augments the Bcl-x(L)/Bcl-x(S) ratio. Furthermore, the ratio is lower in islets from islet-specific RIP-iPLA2β transgenic mice, whereas islets from global iPLA2β(-/-) mice exhibit the opposite phenotype. In view of our earlier reports that iPLA2β induces ceramide accumulation through neutral sphingomyelinase 2 and that ceramides shift the Bcl-x 5'-splice site (5'SS) selection in favor of Bcl-x(S), we investigated the potential link between Bcl-x splicing and the iPLA2β/ceramide axis. Exogenous C6-ceramide did not alter Bcl-x 5'SS selection in INS-1 cells, and neutral sphingomyelinase 2 inactivation only partially prevented the ER stress-induced shift in Bcl-x splicing. In contrast, 5(S)-hydroxytetraenoic acid augmented the ratio of Bcl-x(L)/Bcl-x(S) by 15.5-fold. Taken together, these data indicate that β-cell apoptosis is, in part, attributable to the modulation of 5'SS selection in the Bcl-x pre-mRNA by bioactive lipids modulated by iPLA2β.

Keywords: Alternative Splicing; Apoptosis; Bcl-x; Diabetes; Phospholipase A; Signal Transduction; β-Cell.

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Figures

FIGURE 1.
FIGURE 1.
Chemically induced ER stress correlates with reduced expression of anti-apoptotic Bcl-x(L) in β-cells. A, INS-1 cells were treated with 1 μm thapsigargin (T) or DMSO (c), and protein was extracted and used for immunoblot analysis of iPLA2β protein. A representative experiment is shown. B and C, INS-1 cells were cultured for 13 h in the presence of DMSO (c) or thapsigargin (T, 1 μm) and then RNA and protein were extracted. B, representative immunoblot analysis of Bcl-x(L) protein in c- and Tg-treated cells and quantification of three independent immunoblots. Each replicate was derived from an independent experiment that started with freshly plated cells. C, analysis of Bcl-x splice variants in a representative RT-PCR experiment (left panel inset), quantification of Bcl-x(L)/Bcl-x(S) ratio in four independent RT-PCR experiments, and quantification of Bcl-x(L)/Bcl-x(S) ratio in three independent qPCR experiments (right panel). (*, Tg group is significantly different from the c group, p < 0.05.).
FIGURE 2.
FIGURE 2.
Spontaneous ER stress correlates with reduced expression of anti-apoptotic Bcl-x(L) in β-cells. A, representative immunoblot comparing iPLA2β in wild-type and Akita (AK) β-cells. B, WT and Akita β-cell lines were cultured for 8 h, and RNA was then extracted and RT-PCR used to amplify Bcl-x splice variants. Shown are a representative experiment (inset) and quantification of Bcl-x(L)/Bcl-x(S) ratio in three independent experiments. C, islets were harvested from wild-type (WT) and Akita (AK) mice. RNA was extracted and RT-PCR used to amplify murine Bcl-x splice variants. Two independent experiments are shown. Each quantification is presented as mean ± S.E. (*, Akita (AK) group is significantly different form the WT group, p < 0.05.)
FIGURE 3.
FIGURE 3.
Chemical ablation of iPLA2β promotes and iPLA2β overexpression suppresses selection of downstream 5′SS in human Bcl-x minigene. A, representative immunoblot showing iPLA2β protein levels in INS-1 cells transfected with empty vector (V) or iPLA2β cDNA (OE). B, empty vector and OE INS-1 cells were transfected with plasmid expressing a functional human Bcl-x minigene. Cells were cultured for 13–16 h in the presence of DMSO (c) or (S)-BEL (10 μm). RNA was harvested, and RT-PCR performed to amplify minigene splice variants. Shown are a representative experiment (left panel) and the quantification of four independent experiments (right panel). (*, significantly different from c-treated INS-1 vector cells, p < 0.05; #, significantly different from c-treated INS-1 cells, p < 0.05.) Each quantification is presented as mean ± S.E.
FIGURE 4.
FIGURE 4.
Genetic ablation of iPLA2β promotes selection of the downstream Bcl-x 5′ SS in human Bcl-x minigene. INS-1 cells were transfected with control (c) or iPLA2β (iPLA2β) siRNA. A, representative immunoblot analysis of iPLA2β protein in transfected cells. B, INS-1 cells were transfected with control or iPLA2β siRNA and then treated with DMSO (c) or 1 μm thapsigargin (T). Cell death was quantified through trypan blue exclusion assays. Shown are mean ± S.E. from four independent experiments. (*, T group significantly different from control-c or iPLA2β-c, p < 0.0001; #, iPLA2β-T group significantly different from control T group, p < 0.0001.) C, INS-1 cells were co-transfected with Bcl-x minigene and c- or iPLA2β-siRNA. Cells were cultured for 13 h, and then RNA was harvested and RT-PCR performed to amplify minigene splice variants. Shown are a representative experiment (left panel) and quantification (right panel) of four independent experiments (mean ± S.E.). (*, significantly different from control siRNA treatment group, p < 0.05.) D, wild-type (WT) and Akita β-cells were transfected with control (left)- or iPLA2β (right)-siRNA and then treated with 1 μm thapsigargin for 4–16 h. RNA was extracted and RT-PCR performed to amplify Bcl-x splice variants. A representative experiment is shown. Each representative experiment was performed at least twice.
FIGURE 5.
FIGURE 5.
iPLA2β promotes selection of the upstream alternative 5′SS in endogenous islet Bcl-x. Islets were harvested from wild-type (WT), RIP-iPLA2β-Tg (Tg), and iPLA2β−/− (KO) mice. A, immunohistochemistry analysis of iPLA2β (red) and insulin (green). Islets were counterstained with DAPI (blue) to mark the nuclei of individual cells. Merged images are presented, where arrowheads indicate co-expression of iPLA2β and insulin. B, islets were isolated and then cultured in the presence of DMSO (−) or 2 μm thapsigargin (+). RNA was harvested and used for RT-PCR to amplify Bcl-x splice variants. Two mice were studied in each group, and results from both mice are shown. The average ratio of Bcl-x(L)/Bcl-x(S) is shown for each treatment group.
FIGURE 6.
FIGURE 6.
iPLA2β regulates Bcl-x 5′SS selection through both ceramide-dependent and -independent mechanisms. A, INS-1 cells were treated with DMSO (c) or 50 μm C6-ceramide (Cer) for 24 h. RNA was harvested and RT-PCR performed to amplify Bcl-x splice variants. Shown is the ratio of Bcl-x(L)/Bcl-x(S) in four independent experiments. B, INS-1 cells were treated with DMSO (c), 10 μm (S)-BEL (BEL), or 1 μm thapsigargin (T) alone or pretreated with (S)-BEL prior to treatment with thapsigargin ± 50 μm C6-ceramide (Cer) for 13 h. RNA was isolated and RT-PCR performed to amplify Bcl-x splice variants. A representative experiment is shown. C, INS-1 cells were treated with DMSO (c) or thapsigargin alone or pretreated with (S)-BEL (BEL) or 10 μm GW4869 (GW) prior to treatment with thapsigargin for 13 h, and Bcl-x RNAs were amplified. A representative experiment is shown. Each representative experiment was performed at least twice. Each quantification is presented as mean ± S.E.
FIGURE 7.
FIGURE 7.
iPLA2β modulates lipid mediators involved in Bcl-x 5′SS selection. A and B, INS-1 cells were treated with DMSO (c), 1 μm thapsigargin (T), or 10 μm (S)-BEL for 13 h. Culture supernatants were harvested, and ESI-MS/MS was performed to quantify polyunsaturated fatty acids and their derivatives. The lipids were normalized to RNA retrieved from cells that conditioned the media. Shown are means ± S.E. of EPA (A) or 5-HETE (B) in three replicates. C, Bcl-x(L) and Bcl-x(S) mRNAs were quantified in samples from A and B, and the Bcl-x(L)/Bcl-x(S) ratio is plotted against the ratio of 5-HETE/EPA. Linear regression analysis indicated a significant correlation (p = 0.003). D, iPLA2β mRNA was quantified in samples from A and B and is plotted against the ratio of 5-HETE/EPA. The 5-HETE/EPA ratio was significantly higher in cells with low iPLA2β expression (p < 0.005, Wilcoxon rank sum test). E, INS-1 cells were treated with vehicle (DMSO), 10 μm 5-HETE, or 10 μm EPA for up to 13 h. RNA was isolated and qPCR performed to quantify Bcl-x splice variants. The data are derived from three independent treatments. (*, significantly different from vehicle control treatment group, p < 0.05.).
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