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Is BOK Required for Apoptosis Induced by Endoplasmic Reticulum Stress?

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Is BOK Required for Apoptosis Induced by Endoplasmic Reticulum Stress? LETTER Is BOK required for apoptosis induced by endoplasmic reticulum stress? LETTER Yuniel Fernandez-Marreroa, Francine Keb,c, Nohemy Echeverrya,1, Philippe Bouilletb,c, Daniel Bachmanna, Andreas Strasserb,c, and Thomas Kaufmanna,2 The B-cell lymphoma 2 (BCL-2)-related ovarian killer comparable Chop levels). Given the critical role of BIM (BOK) shares sequence homology with the proapo- in ER stress-induced apoptosis, this reduction of Bim ptotic BCL-2 family members BAX and BAK. However, may fully account for the reported resistance to ER −/− Bok cells are not protected from classic apoptotic stress. It is unclear whether this reduction of Bim is par- triggers and evidence for a proapoptotic role of BOK ticular to these SV40 MEFs, which are prone to line-to- is derived mostly from overexpression studies (1). BOK line variations within the one genotype, or whether this localizes preferentially to the endoplasmic reticulum is also seen in primary cells (e.g., primary MEFs, which (ER) membrane, where it interacts with IP3-receptors were used for some experiments) from these mice. Im- −/− (2, 3). Using cells from their newly generated Bok portantly, we did not observe significant changes in Bim −/− mouse strain, Carpio et al. propose that BOK is a crit- levels in SV40 MEFs or tissues from our Bok mice (Fig. ical inducer of BAX/BAK-dependent apoptosis in re- 1A). Overall, our analysis of SV40 MEFs, primary MEFs, sponse to ER stress (4). This proposal is in contrast to myeloid progenitors, mast cells, and primary neutrophils our earlier report, in which we showed that loss of BOK did not support a proapoptotic role of BOK downstream did not confer resistance toward ER stress in several of ER stress (2) (Fig. 1 B and C). Furthermore, the rescue cell types (2). Underlying reasons for this discrepancy experiments in figure 5 of Carpio et al. (4) are in our may lie in the initial Sv129:C57BL/6 mixed genetic view inconclusive, because BOK was overexpressed background of the strain used by Carpio et al. (4) by transient transfection, a stress condition that in- [which may influence the phenotype despite back- duces BIM and PUMA (2). −/− crossing (5)] and their targeting strategy of the Bok Carpio et al. (4) conclude with in vivo data on Bok locus. By targeting exons 2 (containing the START co- mice being protected from thapsigargin-induced liver don) and 3, alternative splicing of exon 1–4 is enabled damage. Regretfully, however, no quantitative data on and is indeed readily detectable based on the liver damage with statistical analysis were provided. Of RT-PCR analysis in figure 1C of Carpio et al. (4). A result- note, we did not find any protection from ER stressors −/− ing ∼1-kb transcript (exon1/4/5), which is not occur- in Bok primary hepatocytes or immortalized human ring naturally, contains several predicted ORFs and hepatocytes rendered BOK-deficient (Fig. 1 D and E). may influence the phenotype of these mice. In con- Taking these data together, we reason that −/− trast, our Bok strain was generated in a pure BOK may not be essential in promoting ER stress- C57BL/6 genetic background, with no detectable induced apoptosis. transcript because of targeting of the exon 1 splice donor site along with exon 2 (1). Acknowledgments Basedonfigure6BofCarpio et al. (4), it is also im- Immortalized human hepatocytes were a kind gift −/− portant to discuss that the SV40-immortalized Bok from Dr. T. Brunner. This work was supported by mouse embryonic fibroblasts (MEFs), which they almost the Swiss National Science Foundation (SNF), Grant exclusively used for their studies, show a >50% reduc- 310030E150805, part of the D-A-CH initiative from the tion in the baseline levels of Bcl-2-interacting mediator SNF and the Deutsche Forschungsgemeinschaft of cell death (Bim) compared with WT controls (despite (DFG), FOR-2036. aInstitute of Pharmacology, Medical Faculty, University of Bern, 3010 Bern, Switzerland; bMolecular Genetics of Cancer Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia; and cDepartment of Medical Biology, University of Melbourne, Melbourne, VIC 3010, Australia Author contributions: Y.F.-M., A.S., and T.K. designed research; Y.F.-M., F.K., N.E., and D.B. performed research; Y.F.-M., F.K., N.E., P.B., D.B., and T.K. analyzed data; and A.S. and T.K. wrote the paper. The authors declare no conflict of interest. 1Present address: Laboratory of Molecular Oncology, Clinic of Oncology, University Hospital Zurich, 8044 Zurich, Switzerland. 2To whom correspondence should be addressed. Email: [email protected]. www.pnas.org/cgi/doi/10.1073/pnas.1516347113 PNAS Early Edition | 1of2 Downloaded by guest on September 24, 2021 A Bim mRNA, SV40 MEF Bim mRNA, mouse tissues D WT WT -/- 100 -/- 0.8 p=0.106 Bok 0.050 p=0.984 Bok 75 Hprt) 0.6 p=0.718 p=0.980 Hmbs/brain) 50 0.4 0.025 562nm OD 25 (%reduced MTT) Brefeldin A 20 g/mL (72 M) Tunicamycin 10 g/mL (12 M) (relative to 0.2 0 Ct (relative to - 0162448 0162448 Ct 2 0.000 0.0 -/- - WT Bok 2 Liver Kidney Spleen Time (h) B 100 WT E Bok-/- 23 kDa ) - 75 BOK / PI - 50 42 kDa -actin 25 1 Bortezomib Etoposide 2D4 1B2 1H10 2H1 0 empty hBOK 0.0 0.5 1.0 5.0 10.0 0.0 0.5 1.0 5.0 10.0 subclones from gRNA line 100 gRNA 75 Viability (% AnnexinV (% Viability IHH empty virus 100 50 IHH gRNA hBOK 25 75 Thapsigargin Brefeldin A 50 0 0.0 0.5 1.0 5.0 10.0 0.0 0.1 0.5 1.0 2.0 25 C Concentration ( M) Bortezomib Etoposide 0 ) - 100 100 Thapsigargin 50 nM Tunicamycin 500 ng/mL 562nm OD / PI - 80 (600 nM) WT (%reduced MTT) 75 60 Bok-/- 50 40 25 20 Thapsigargin Tunicamycin 0 Viability (% AnV (% Viability 0 3 0 5 0 0 182842 0182842 0.00 0.16 0.31 0.6 1.25 2.50 5.00 10.0 0.0 0.16 0.31 0.63 1.2 2.50 5.00 10. Time (h) Concentration ( M) Fig. 1. (A)LossofBok does not affect Bim expression. Quantitative PCR analysis of Bim mRNA levels in SV40-transformed MEFs (mean ± SEM −/− from three independent lines per genotype; Hprt was used as reference gene) or primary tissues isolated from WT and Bok mice (mean ± SEM, four mice per genotype per tissue, Hmbs was used as reference gene). (B) Early passage primary MEFs (t = 24 h; mean ± SD from four + −/− independent lines per genotype) and (C) primary bone marrow-derived Gr-1 neutrophils from Bok mice (mean ± SD, three mice per genotype) are not protected from ER stress-induced apoptosis. Note that primary MEFs were relatively resistant to etoposide-induced cell death. −/− Cell viability was determined by flow cytometry using FITC-Annexin V/propidium iodide staining. (D) Bok primary hepatocytes (mean ± SD, three mice per genotype) and (E) immortalized human hepatocytes (IHH) rendered BOK-deficient using CRISPR/Cas9 technology (t = 24 h; mean ± SD from three independent experiments; lentiCRISPR v2 was a gift from Feng Zhang, Broad Institute of MIT and Harvard, Cambridge, MA; Addgene plasmid #52961) are not protected from ER stress-induced apoptosis. Immunoblot (E) shows loss of BOK in IHH cells before subcloning (gRNA hBOK; BOK protein levels were reduced by 80%, as assessed by quantitative Western blot analysis with near-infrared fluorescence) and examples of derived subclones. Cell viability was determined by MTT assay performed in sextuplicates. Statistical analyses were performed using the unpaired t test. 1 Ke F, et al. (2012) BCL-2 family member BOK is widely expressed but its loss has only minimal impact in mice. Cell Death Differ 19(6):915–925. 2 Echeverry N, et al. (2013) Intracellular localization of the BCL-2 family member BOK and functional implications. Cell Death Differ 20(6):785–799. 3 Schulman JJ, Wright FA, Kaufmann T, Wojcikiewicz RJ (2013) The Bcl-2 protein family member Bok binds to the coupling domain of inositol 1,4,5-trisphosphate receptors and protects them from proteolytic cleavage. J Biol Chem 288(35):25340–25349. 4 Carpio MA, et al. (2015) BCL-2 family member BOK promotes apoptosis in response to endoplasmic reticulum stress. Proc Natl Acad Sci USA 112(23):7201–7206. 5 Vanden Berghe T, et al. (2015) Passenger mutations confound interpretation of all genetically modified congenic mice. Immunity 43(1):200–209. 2of2 | www.pnas.org/cgi/doi/10.1073/pnas.1516347113 Fernandez-Marrero et al. Downloaded by guest on September 24, 2021.
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