Dear Editor,
The unfolded protein response (UPR) constitutes the main cellular response to perturbed protein homeostasis. It also exerts a critical function during the differentiation of activated B lymphocytes into antibody-secreting plasma cells. During the UPR, IRE1α activation causes processing of the Xbp-1 mRNA, which encodes a master regulator of plasma cell differentiation.1
A recent paper in the EMBO J by Rodriguez et al. described a role for the pro-apoptotic BH3-only BCL-2 family members PUMA and BIM in the processing of Xbp-1 and thus the upstream modulation of the UPR response.2 This conclusion was based on the finding that PUMA and BIM could bind to IRE1α in a BH3 domain-dependent manner and that pro-survival BCL-2 was required for this interaction. Finally, the authors reported that BIM modulates the function of IRE1α in regulating Xbp-1 splicing, resulting in a substantial reduction in IgM secretion by mitogen (LPS)-activated B cells from Bim−/− mice.
These findings are counterintuitive for several reasons. First, the interaction between IRE1α with BIM or PUMA was stated to depend on their BH3 domains and also pro-survival BCL-2 (Figures 4, 5 and 7).2 We originally discovered BIM through λ phage expression library screening using BCL-2 as the bait and revealed in co-immunoprecipitation studies that this interaction requires the BH3 domain of BIM.3 Subsequent studies extended this finding to demonstrate that not only BIM but in fact all BH3-only proteins (including PUMA) bind to the pro-survival BCL-2 family members via their BH3 domain.4 Thus, it is inconceivable that the BH3 domain of BIM or PUMA could engage IRE1α and BCL-2 simultaneously. We were unable to reproduce the interaction between the cytoplasmic domain of IRE1αwith a VSV-tag) and BIM or PUMA in co-immunoprecipitation studies, whereas interaction of BIM with MCL-1 or LC8 could be readily detected in the same samples (positive controls; Figure 1a and data not shown).
Second, Rodriguez et al. reported that LPS-stimulated Bim−/− B cells were greatly impaired in their ability to secrete IgM antibodies owing to a defect in Xbp-1 splicing (Figure 7).2 This is inconsistent with the well-established fact that Bim−/− mice have abnormally increased serum levels of IgM and IgG,5 probably due to the protection of plasma cells from ER stress-induced apoptosis, which in lymphoid and certain other cell types requires BIM for initiation.6 Similarly, they reported that loss of PUMA reduced Xbp-1 splicing in LPS-treated B cells and greatly impaired IgM secretion (on its own). Contrary to the findings by Rodriguez et al., we found that LPS-treated B cells from wt, Bim−/−, Puma−/− and Bim−/−Puma−/− mice showed no differences in Xbp-1 splicing (Figure 1b). In addition, we conducted digital droplet PCR analyses for the quantitation of spliced Xbp-1 mRNA and expression of its target ERdj4, but were unable to find any differences between cells of the different genotypes (Figure 1b graph and Figure 1c). If at all, Bim−/− MEFs from two different mice showed increased ERdj4 expression upon tunicamycin treatment. Most importantly, we found no significant differences in IgM secretion between cultures of B cells from the knock-out strains compared with wt B cells (Figure 1d).
In conclusion, we were unable to reproduce the findings by Rodriguez et al. using identical experimental conditions. In light of our observations and based on the well-established essential roles of BIM and PUMA in the initiation of apoptosis triggered by ER stress6, 7 and several other apoptotic stimuli,5, 8 we conclude that these BH3-only proteins function exclusively downstream but not upstream in the UPR pathway.
References
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Rodriguez DA et al. EMBO J 2012; 31: 2322–2335.
O'Connor L et al. EMBO J 1998; 17: 384–395.
Chen L et al. Mol Cell 2005; 17: 393–403.
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Puthalakath H et al. Cell 2007; 129: 1337–1349.
Reimertz C et al. J Cell Biol 2003; 162: 587–597.
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Herold, M., O'Reilly, L., Lin, A. et al. Evidence against upstream regulation of the unfolded protein response (UPR) by pro-apoptotic BIM and PUMA. Cell Death Dis 5, e1354 (2014). https://doi.org/10.1038/cddis.2014.290
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DOI: https://doi.org/10.1038/cddis.2014.290
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