Is BOK required for apoptosis induced by endoplasmic reticulum stress?

The B-cell lymphoma 2 (BCL-2)-related ovarian killer (BOK) shares sequence homology with the proapoptotic BCL-2 family members BAX and BAK. However, Bok cells are not protected from classic apoptotic triggers and evidence for a proapoptotic role of BOK is derivedmostly from overexpression studies (1). BOK localizes preferentially to the endoplasmic reticulum (ER) membrane, where it interacts with IP3-receptors (2, 3). Using cells from their newly generated Bok mouse strain, Carpio et al. propose that BOK is a critical inducer of BAX/BAK-dependent apoptosis in response to ER stress (4). This proposal is in contrast to our earlier report, in which we showed that loss of BOK did not confer resistance toward ER stress in several cell types (2). Underlying reasons for this discrepancy may lie in the initial Sv129:C57BL/6 mixed genetic background of the strain used by Carpio et al. (4) [which may influence the phenotype despite backcrossing (5)] and their targeting strategy of the Bok locus. By targeting exons 2 (containing the START codon) and 3, alternative splicing of exon 1–4 is enabled and is indeed readily detectable based on the RT-PCR analysis in figure 1C of Carpio et al. (4). A resulting ∼1-kb transcript (exon1/4/5), which is not occurring naturally, contains several predicted ORFs and may influence the phenotype of these mice. In contrast, our Bok strain was generated in a pure C57BL/6 genetic background, with no detectable transcript because of targeting of the exon 1 splice donor site along with exon 2 (1). Based on figure 6B of Carpio et al. (4), it is also important to discuss that the SV40-immortalized Bok mouse embryonic fibroblasts (MEFs), which they almost exclusively used for their studies, show a >50% reduction in the baseline levels of Bcl-2-interacting mediator of cell death (Bim) compared with WT controls (despite comparable Chop levels). Given the critical role of BIM in ER stress-induced apoptosis, this reduction of Bim may fully account for the reported resistance to ER stress. It is unclear whether this reduction of Bim is particular to these SV40 MEFs, which are prone to line-toline variations within the one genotype, or whether this is also seen in primary cells (e.g., primary MEFs, which were used for some experiments) from these mice. Importantly, we did not observe significant changes in Bim levels in SV40MEFs or tissues from ourBokmice (Fig. 1A). Overall, our analysis of SV40 MEFs, primary MEFs, myeloid progenitors, mast cells, and primary neutrophils did not support a proapoptotic role of BOK downstream of ER stress (2) (Fig. 1B andC). Furthermore, the rescue experiments in figure 5 of Carpio et al. (4) are in our view inconclusive, because BOK was overexpressed by transient transfection, a stress condition that induces BIM and PUMA (2). Carpio et al. (4) conclude with in vivo data on Bok mice being protected from thapsigargin-induced liver damage. Regretfully, however, no quantitative data on liver damage with statistical analysis were provided. Of note, we did not find any protection from ER stressors in Bok primary hepatocytes or immortalized human hepatocytes rendered BOK-deficient (Fig. 1 D and E). Taking these data together, we reason that BOK may not be essential in promoting ER stressinduced apoptosis.