Wolfram syndrome 1 gene negatively regulates ER stress signaling in rodent and human cells (original) (raw)
WFS1 forms an ER stress–mediated complex with ATF6α and suppresses its activity. In order to further define the role of WFS1 in the UPR, we assessed whether WFS1 expression affects the function of UPR components. Transcriptional activity of a transmembrane transcription factor and master regulator of the UPR, ATF6α, is attenuated by WFS1 expression. Under ER stress, the N-terminal DNA binding domain of ATF6α is cleaved and released from the ER to upregulate UPR target genes in the nucleus (3–5). As expected, when full-length ATF6α was transfected with the ATF6α binding site reporter gene ATF6GL3, this reporter was induced 12-fold by ATF6α (20), an induction reduced to 3-fold by cotransfection with WFS1 (Figure 1A). ATF6α has also been shown to strongly activate the BiP/GRP78 promoter (4). To confirm that WFS1 regulates ATF6α transcriptional activity on the BiP/GRP78 promoter, full-length ATF6α or cleaved ATF6α (ΔATF6α) was cotransfected with WFS1 and a rat GRP78 promoter reporter gene containing the ER stress response element (ERSE). This reporter was induced by both full-length ATF6α and ΔATF6α; however, only full-length ATF6α activity was suppressed by WFS1 expression (Figure 1A). In addition, full-length ATF6α protein expression decreased when it was coexpressed with WFS1 (Figure 1B). BiP has previously been shown to anchor full-length ATF6α to the ER membrane and prevent ATF6α activation (6, 21). To compare the ability of WFS1 to suppress ATF6α with that of BiP, the GRP78 promoter reporter was cotransfected with full-length ATF6α and BiP, with full-length ATF6α and WFS1, or with full-length ATF6α, BiP, and WFS1. Suppression of ATF6α activity by WFS1 was stronger than that by BiP (Figure 1C). Collectively, these results indicate that WFS1 suppresses ATF6α transcriptional activity before its translocation to the nucleus.
WFS1 interacts with ATF6α in an ER stress–dependent manner and suppresses ATF6α transcriptional activation. (A) COS7 cells were transfected with a full-length ATF6α expression plasmid or ΔATF6α with a WFS1 plasmid together with the following luciferase reporter genes: ATF6α binding site reporter gene ATF6GL3, ATF6α mutant site reporter ATF6m1GL3, and GRP78 promoter reporter gene ERSE. Relative intensity of luciferase was then measured (n = 3). (B) Protein lysates from the luciferase assay were analyzed by IB using anti-HA (ATF6α), anti-Flag (WFS1), and anti-actin antibodies. ATF6α and ΔATF6α are denoted by single and double asterisks, respectively. (C) COS7 cells were transfected with a full-length ATF6α expression plasmid with a BiP expression plasmid, WFS1 expression plasmid, or WFS1 and BiP expression plasmid together with the GRP78 reporter gene (n = 3). (D) An anti-WFS1 antibody was used to IP WFS1 protein from INS1 832/13 cells untreated (UT) or treated with the ER stress inducer DTT (1 mM) for 0.5, 1.5, or 3 hours. IPs were then subject to IB analysis using anti-ATF6α, anti-WFS1, and anti-actin antibodies (n = 3). (E) INS1 832/13 cells were treated with DTT (1 mM) for 2 hours and then chased in normal media for 0, 1, or 2 hours. WFS1 was subjected to IP from cell lysates, and IPs were analyzed by IB using anti-ATF6α, anti-WFS1, and anti-actin antibodies (n = 3).
Both WFS1 and ATF6α are transmembrane proteins localized to the ER (3, 10), raising the possibility that the suppression of the ATF6α reporter by WFS1 might be mediated by direct interaction between the WFS1 and ATF6α proteins. To confirm this, the association of WFS1 with ATF6α was examined in the pancreatic β cell line INS-1 832/13. WFS1 associated with ATF6α under nonstress conditions (Figure 1D). To examine whether this interaction was maintained during ER stress conditions, the cells were treated with the ER stress inducer dithiothreitol (DTT), which caused a dissociation of ATF6α from WFS1 in a time-dependent manner, with almost complete dissociation 3 hours after treatment (Figure 1D). This ER stress–dependent interaction was also observed in cells treated with another ER stress inducer, thapsigargin (Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI39678DS1). To confirm that this interaction was recovered after stress, cells were treated for 2 hours with DTT and then chased in normal media. As expected, the interaction of ATF6 and WFS1 began to recover after a 1-hour chase (Figure 1E). This interaction was also seen in the neuronal cell line Neuro2A (Supplemental Figure 2). Together, these data suggest that ATF6 is released from WFS1 under stress in order to activate its target UPR genes.
WFS1 has a function in the degradation of ATF6α through the ubiquitin-proteasome pathway. Suppression of ATF6α transcriptional activity by WFS1 and the formation of an ATF6α-WFS1 complex led to the prediction that WFS1 regulates ATF6α function at the posttranslational level. To test this prediction, we derived a pancreatic β cell line, MIN6 cells, stably expressing a shRNA directed against WFS1. Full-length as well as nuclear ATF6α protein levels increased approximately 2-fold compared with control cells (Figure 2A). To confirm that upregulation of ATF6α protein is directly regulated by WFS1, we reintroduced a lentivirus expressing WFS1 into the cells expressing shRNA directed against WFS1; ATF6 protein expression levels were again reduced when WFS1 was reintroduced (Figure 2A).
WFS1 regulates ATF6α protein levels. (A) IB analysis measured ATF6α and WFS1 levels in MIN6 cells expressing shGFP (control) or shWFS1, as well as in MIN6 cells expressing shWFS1 or expressing shWFS1 and rescued with WFS1 (n = 3). (B) IB analysis measuring ATF6α, WFS1, IRE1α, and PERK levels in INS1 832/13 cells (treated with 2 mM DTT for 3 hours) overexpressing GFP (control) or WFS1 (n = 3). (C) Quantitative real-time PCR analysis of BiP, total Xbp-1, Chop, Ero1-α, Glut2, and Ins2 mRNA levels in INS1 832/13 cells overexpressing GFP (control) or WFS1 (n = 3). (D) IB analysis of ATF6α and WFS1 in COS7 cells transfected with ATF6α-HA or ATF6α-HA and WFS1-FLAG at 2 different ratios, and in INS1 832/13 cells expressing inducible WFS1 and treated with or without MG132. (E) IB analysis of ATF6α and WFS1 in MIN6 cells expressing shWFS1 and transfected with WT WFS1-FLAG or mutant P724L WFS1-FLAG and G695V WFS1-FLAG (n = 3). (F) IB analysis measuring ATF6α and WFS1 levels in INS1 832/13 cells expressing WT WFS1 or P724L WFS1 (n = 3). (G) WFS1 was subjected to IP from COS7 cells expressing ATF6α-HA or ATF6α-HA with WT, P724L, or G695V WFS1-Flag using an anti-Flag antibody. IPs and input proteins were analyzed using anti-HA and anti-Flag antibodies. **P < 0.01.
ATF6α mRNA was unchanged in the WFS1-knockdown INS-1 832/13 cells, but ATF6α target genes, such as p58IPK and BiP (7, 8), were upregulated as predicted (Supplemental Figure 3). To further confirm that this upregulation is directly regulated by ATF6α, we suppressed ATF6α expression by siRNA in WFS1 knockdown INS-1 832/13 cells and then measured expression levels of its major target, BiP. Upregulation of BiP by WFS1 inhibition was cancelled out by ATF6α inhibition (Supplemental Figure 4).
ATF6α protein levels were also measured in INS-1 832/13 cells overexpressing WFS1. Full-length and nuclear ATF6α protein levels were suppressed in these cells, whereas there was no significant change in protein levels of the other 2 master regulators of the UPR, IRE1 and PERK (Figure 2B). IRE1 and PERK protein expression levels were not decreased even with higher levels of WFS1 expression (Supplemental Figure 5). Suppression of ATF6α protein expression was also seen in a neuronal cell line (Supplemental Figure 6). ATF6 target gene mRNA levels were also suppressed in β cells overexpressing WFS1 (Figure 2C). The relationship of WFS1 and ATF6 protein expression was found to be dose-dependent: increased expression of WFS1 leads to a decrease in ATF6 protein expression (Supplemental Figure 7). We asked whether this relationship was proteasome dependent. Treatment of 2 WFS1-overexpressing cell lines with the proteasome inhibitor MG132 rescued ATF6α protein levels (Figure 2D). We cloned 2 missense mutants, WFS1 P724L and WFS1 G695V, and 1 inactivating mutant, WFS1 ins483fs/ter544, from patient samples (13). Mutant variants of WFS1 did not affect ATF6α protein levels in MIN6 cells expressing shRNA directed against WFS1 (Figure 2E and Supplemental Figure 8). This was also confirmed in INS-1 832/13 cells expressing the missense mutant WFS1 P724L (Figure 2F) and in neuronal cells expressing the missense mutant WFS1 G695V (Supplemental Figure 9). Although ATF6α weakly interacted WFS1 P724L and WFS1 G695V, there was no significant decrease in ATF6α protein levels in these cells (Figure 2G).
To assess the impact of WFS1 on ATF6α protein degradation, cycloheximide experiments were performed. In MIN6 cells expressing shRNA directed against WFS1, there was a block in ATF6α protein degradation, whereas in cells overexpressing WFS1, there was minimal ATF6α protein expression (Figure 3, A and B). WFS1 could not enhance the degradation of 2 other ER proteins susceptible to misfolding, TCRα and mutant alpha-1-antitrypsin NHK3 (refs. 22–24 and Supplemental Figure 10), which indicates that WFS1 specifically degrades ATF6α protein. WFS1 also enhanced the ubiquitination of ATF6α. In cells expressing shRNA directed against WFS1, there was a decrease in ATF6α ubiquitination after blocking proteasome activity (Figure 3C), whereas in cells overexpressing WFS1, there was an enhancement of ATF6α ubiquitination (Figure 3D). In Wfs1–/– mouse pancreata, ATF6α protein expression was strikingly high compared with control littermate pancreata (Figure 3E), indicating that WFS1 functions in ATF6α protein expression in vivo. In samples from patients with WFS1 mutations, there was a higher expression of ATF6α protein compared with control samples (Supplemental Figure 11). Together, these results indicate that WFS1 is important for regulating ATF6α protein expression. When WFS1 was not present, there was increased expression of ATF6α protein and hyperactivation of its downstream effectors. This suggests that in response to ER stress, ATF6α escapes from WFS1-dependent degradation, is cleaved in the Golgi to its active form, and then translocates to the nucleus to upregulate its UPR target genes.
WFS1 enhances ATF6α ubiquitination and degradation. (A) IB analysis measuring ATF6α, WFS1, and actin levels in MIN6 cells stably expressing shGFP (control) or shWFS1 treated with 40 μM cycloheximide (CX) for 0, 2, and 4 hours (n = 3). (B) IB analysis measuring ATF6α, WFS1, and actin levels in INS1 832/13 cells expressing GFP (control) or WFS1 treated with 40 μM cycloheximide for 0, 2, and 6 hours (n = 3). (C) ATF6α was subjected to IP using an anti-ATF6α antibody from an INS1 832/13 cells inducibly expressing shWFS1 (treated for 48 hours with 2 μM doxycycline) and treated with MG132 (20 μM) for 3 hours. IPs were then subjected to IB with anti-ubiquitin and anti-ATF6α antibodies, and input lysates were blotted with anti-ATF6α, anti-WFS1, and anti-actin antibodies (n = 3). (D) ATF6α was subjected to IP using an anti-ATF6α antibody, from INS1 832/13 cells overexpressing GFP (control) or WFS1, then treated with MG132 (0.1 μM) overnight. IPs were subjected to IB with anti-ubiquitin and anti-ATF6α antibodies. Input lysates were subjected to IB with anti-ATF6α, anti-WFS1, and anti-actin antibodies (n = 3). (E) Wfs1–/– and WT littermate mouse pancreata were analyzed by immunohistochemistry using anti-ATF6α and anti-insulin antibodies. Scale bars: 50 μm.
These data raised the possibility that WFS1 recruits ATF6α to the proteasome for its degradation. As we predicted, WFS1 formed a complex with the proteasome (Figure 4A). When glycerol-gradient fractionation was performed on ER-isolated lysates, the proteasome ATF6α and WFS1 comigrated in the same high–molecular weight fractions, and a complex between them was formed (Figure 4, B and C).
WFS1 forms a complex with the proteasome and ATF6α. (A) WFS1 was subjected to IP from INS1 832/13 cells using an anti-WFS1 specific antibody. IPs were subjected to IB with anti–alpha 5 20S proteasome and anti-WFS1 antibodies. (B) IB analysis measuring CREB, actin, and PDI levels using whole cell lysates or ER-isolated lysates of INS1 832/13 cells. ER-isolated lysates of INS1 832/13 cells were also subjected to fractionation using a 10%–40% glycerol gradient. Fractions were analyzed by IB using anti–alpha 5 20s proteosome, anti-ATF6α, and anti-WFS1 antibodies. Lanes were run on separate gels and were not contiguous. (C) WFS1 was subjected to IP from a mixture of fractions 10 and 11 using an anti-WFS1 antibody, and IP products were subjected to IB analysis using anti-alpha 5 20s proteosome, anti-ATF6α, and anti-WFS1 antibodies. ATF6 was subjected to IP from a mixture of fractions 9 and 12, and IP products were analyzed by IB with anti–alpha 5 20s proteosome and anti-ATF6α (n = 3).
WFS1 stabilizes HRD1, which functions as an E3 ligase for ATF6α. Because WFS1 is localized to the ER membrane and recruits ATF6α to the proteasome, but is not itself an E3 ligase, we searched for ER-localized E3 ligases with which WFS1 could interact. A top candidate was the ER-resident E3 ligase HRD1, which has a known role in ER stress signaling (25, 26). SEL1/HRD3, which has an important function in hydroxy-3-methylglutaryl-CoA reductase (HMG-R) degradation (27), has been shown to interact with and stabilize HRD1 (28), raising the possibility that WFS1 may also have a similar function and could interact with HRD1. Indeed, WFS1 and HRD1 formed a complex (Figure 5A). We next asked whether WFS1 also plays a role in HRD1 protein expression. Inducible suppression of WFS1 in INS-1 832/13 cells expressing shRNA directed against WFS1 suppressed HRD1 protein expression (Figures 5B). To test the effect of WFS1 on HRD1 protein stability, we performed cycloheximide experiments using MIN6 cells stably expressing shRNA directed against WFS1. HRD1 protein expression was significantly decreased in WFS1 knockdown cells compared with control cells, and it was difficult to measure the stability of HRD1 (Figure 5C).
WFS1 interacts with and stabilizes the E3 ligase HRD1. (A) Hrd1 was subjected to IP from INS1 832/13 cells, and IPs were subjected to IB analysis using anti-WFS1 and anti-Hrd1 antibodies (n = 3). (B) Total lysates from INS1 832/13 cells inducibly expressing shWFS1 (treated with 2 μM doxycycline for 48 hours) were analyzed by IB using anti-WFS1, anti-Hrd1, and anti-actin antibodies (n = 3). (C) IB analysis measuring HRD1 levels in MIN6 cells stably expressing shGFP (control) or shWFS1 treated with 40 μM cycloheximide for 0, 0.5, 1, and 2 hours (n = 3). (D) Wfs1–/– and WT littermate mouse pancreata were analyzed by immunohistochemistry using anti-HRD1 and anti-insulin antibodies (n = 3). Scale bars: 100 μm. (E) COS7 cells were transfected with pcDNA3, HRD1–c-Myc, HRD1–c-Myc and WT WFS1, or HRD1–c-Myc and WFS1 mutants (P724L, G695V, and ins483fs/ter544) expression plasmids. Expression levels of HRD1–c-Myc, WFS1, and actin were measured by IB using anti–c-Myc, anti-WFS1, and anti-actin antibodies, respectively. WT and mutant WFS1 are denoted by single and double asterisks, respectively. (F) COS7 cells were transfected with pcDNA3, HRD1–c-Myc, HRD1–c-Myc and WT WFS1-Flag, HRD1–c-Myc and WFS P724L-Flag, and HRD1–c-Myc and WFS1 G695V-Flag expression plasmids. The lysates were subjected to IP with anti-Flag antibody and IB with anti–c-Myc antibody to study the interaction between HRD1 and WFS1.
We further confirmed the effects of WFS1 on HRD1 protein expression in vivo using Wfs1–/– mice. As expected from the results using β cell lines, HRD1 expression was undetectable in islets of Wfs1–/– mice (Figure 5D). In addition, in samples from patients with Wolfram syndrome, there was less HRD1 protein expression compared with control samples (Supplemental Figure 12A). HRD1 expression did not affect WFS1 protein expression (Supplemental Figure 12B).
We next sought to compare the effects of WT WFS1 and WFS1 mutants on HRD1 protein expression. Ectopic expression of WT WFS1 increased HRD1 protein expression, whereas ectopic expression of missense and inactivating WFS1 mutants did not increase or decrease HRD1 expression (Figure 5E). To determine whether WFS1 mutants interact with HRD1, comparable amounts of WT and missense mutant WFS1 proteins were expressed together with HRD1 in COS7 cells, and the interaction was monitored by co-IP. HRD1 interacted with WT WFS1, but not with WFS1 mutants (Figure 5F). Collectively, these results demonstrated that WFS1 stabilizes and enhances the function of the E3 ligase HRD1 through direct binding.
Based on the ability of WFS1 to regulate ATF6α protein, as well as its function in stabilizing HRD1, it followed that WFS1 may be recruiting ATF6α to HRD1 and that ATF6α is a substrate of HRD1. Indeed, HRD1 interacted with ATF6α (Figure 6A). In glycerol-gradient fractionation experiments of ER-isolated lysates, HRD1, ATF6α, and WFS1 were found to form a complex (Supplemental Figure 13). We next analyzed the interaction between ATF6α and HRD1 under ER stress conditions. ATF6α was released from HRD1 by DTT and thapsigargin treatments (Figure 6B), which indicates that the interaction between these proteins is disrupted by ER stress. To study the relationship between HRD1 and ATF6α protein expression levels, the stability of ATF6α protein was measured in MIN6 cells stably expressing shRNA directed against HRD1 and control cells. HRD1 suppression in cells enhanced ATF6α protein stability (Figure 6C). In contrast, overexpression of HRD1 enhanced ATF6α protein degradation (Figure 6D). HRD1 also enhanced ATF6α ubiquitination, and lack of HRD1 decreased ATF6α ubiquitination (Figure 6, E and F). Collectively, these results indicate that the WFS1-HRD1 complex enhances ATF6α ubiquitination and degradation.
HRD1 is an E3 ligase for ATF6α. (A) HRD1 was subjected to IP from INS1 832/13 cells treated for 3 hours with 30 μM MG132. IPs and input proteins were analyzed by IB using anti-ATF6α and anti-HRD1 antibodies. Lanes were run on the same gel but were noncontiguous (white line). (B) An anti-HRD1 antibody was used to IP HRD1 protein from INS1 832/13 cells untreated or treated with DTT (1 mM) and thapsigargin (Tg; 1 μM) for 3 hours. IPs were then subjected to IB analysis using anti-ATF6α, anti-HRD1, and anti-actin antibodies (n = 3). (C) IB analysis measuring ATF6α levels in MIN6 cells stably expressing shGFP (control) or shRNA shHRD1 treated with 40 μM cycloheximide for 0, 4, and 6 hours (n = 3). (D) COS7 cells transfected with ATF6α-HA expression plasmid (control) or ATF6α-HA together with Hrd1-myc expression plasmids (Hrd1) were treated with 40 μM cycloheximide for 0, 4, and 6 hours. Whole cell lysates were subjected to IB with an anti-HA antibody (n = 3). (E) ATF6α was subjected to IP using an anti-ATF6α antibody from INS1 832/13 cells either mock transfected (control) or transfected with a Hrd1-Myc expression plasmid and treated with MG132 (20 μM) for 3 hours. IPs were then subjected to IB with anti-ubiquitin and anti-ATF6α antibodies, and input lysates were blotted with anti-ATF6α, anti–c-Myc, and anti-actin antibodies (n = 3). (F) ATF6 was subjected to IP using an anti-ATF6α antibody from MIN6 cells stably expressing shGFP (control) or shHRD1 and treated with MG132 (20 μM) for 3 hours. IPs were then subjected to IB with anti-ubiquitin and anti-ATF6α antibodies, and input lysates were blotted with anti-HRD1, anti-ATF6α, and anti-actin antibodies (n = 3).