In this study, we provide evidence that WFS1 plays a crucial role in regulating ATF6α transcriptional activity through HRD1-mediated ubiquitination and proteasome-mediated degradation of ATF6α protein. Based upon the data provided, we propose a pathway for the negative-feedback regulation of the ER stress signaling network by WFS1 (Figure ). In healthy cells, WFS1 prevents dysregulated ER stress signaling by recruiting ATF6α to HRD1 and the proteasome for ubiquitin-mediated degradation under non–ER stress conditions. When stress is applied to the ER, such as through the chemical ER stress inducer DTT, ATF6α is released from WFS1. It is then released from the ER membrane and translocates to the nucleus, where it upregulates stress signaling targets. At later time points, WFS1 is induced by ER stress, causing eventual degradation of ATF6α when ER homeostasis is established. In patients with Wolfram syndrome or Wfs1–/– mice, ATF6α escapes from this proteasome-dependent degradation, leading to dysregulated ATF6α signaling. This ATF6α hyperactivation caused by the lack of WFS1 is probably involved in β cell apoptosis.
WFS1 controls steady-state levels of ATF6α protein and activation.
It has previously been shown that WFS1-deficient β cells are susceptible to ER stress–mediated apoptosis (11
). We confirmed that knockdown of WFS1 made β cells sensitive to ER stress–mediated cell death (Supplemental Figure 14). In addition, we found that ectopic expression of an active form of ATF6α in β cells caused apoptosis (Supplemental Figure 15). Although this fits to our model that hyperactivation of ATF6α has a harmful effect on β cells and leads to apoptosis, it may be contrary to previous studies showing beneficial effects of ATF6α upregulation. For example, it has been shown that activation of the ATF6α pathway by a chemical compound protects neuronal cells from ER stress–mediated apoptosis (30
). This effect is mediated by BiP upregulation. The induction of ATF6α has also been shown to protect cardiomyocytes from ischemia/reperfusion-mediated apoptosis (31
). This effect is also mediated by BiP and GRP94 upregulation. Conversely, a recent report has shown that ATF6α upregulation attenuates diet-induced obesity and insulin resistance (32
). More importantly, it has been shown that WT mice are better protected from ER stress in vivo than are ATF6α knockout mice. ATF6α knockout hepatocytes have been shown to be more sensitive to ER stress–mediated cell death compared with control hepatocytes (7
). It is not surprising that previous studies found induction of ATF6α to have beneficial effects on cell function and cell survival, because ATF6α is a major regulator for BiP, a central molecular chaperone in the ER (7
). The main conclusion of the present study is that chronic dysregulation of the UPR, more specifically hyperactivation of ATF6α signaling, has a negative effect on β cell survival. It has been suggested that the UPR regulates both adaptive and apoptotic effectors (2
). The balance between these effectors depends on the nature of the ER stress, whether it is tolerable or unresolvable. Thus, the UPR acts as a binary switch between life and death. Our results demonstrated that, in patients with Wolfram syndrome and Wfs1–/–
mice, unresolvable ER stress occurs in β cells and neurons, leading to a switch toward apoptosis.
ER stress is caused by both physiological and pathological stimuli that can lead to the accumulation of unfolded and misfolded proteins in the ER. Physiological ER stress can be caused by a large biosynthetic load placed on the ER, for example, during postprandial stimulation of proinsulin biosynthesis in pancreatic β cells. This stimulation leads to the activation of ER stress signaling and enhancement of insulin synthesis (34
). Under physiological ER stress conditions, activation of ER stress signaling must be tightly regulated because hyperactivation or chronic activation of this signaling pathway can cause cell death. For example, when eukaryotic translation initiation factor 2α, a downstream component of ER stress signaling, is hyperphosphorylated by the compound salubrinal in pancreatic β cells, apoptosis is induced in these cells (35
). Our results showed that WFS1 has an important function in the tight regulation of ER stress signaling through its interaction with a key transcription factor, ATF6α, thereby protecting cells from the damaging effects of hyperactivation of this signaling pathway.
On the basis of our present results, we believe WFS1 plays a similar role in mammals as HRD3 does in yeast: stabilizing and enhancing the activity of a key ER-resident E3 ligase, HRD1 (28
). Thus, a loss of functional WFS1 may affect ER stress levels in 2 ways: (a) enhancing ATF6α signaling by increasing the pool of ATF6α, and (b) destabilizing HRD1 protein and thus its activity. The latter would independently contribute to ER stress by promoting the buildup of unfolded and misfolded proteins in the ER. In support of this is our present finding that silencing of HRD1 in β cell lines indeed led to mild ER stress (Supplemental Figure 16). It has previously been shown that HRD1 is regulated by the IRE1–XBP-1 pathway (26
) and is activated at a later time point during ER stress. The ATF6α pathway, however, is activated at an earlier phase (36
). Thus, WFS1 may also function as a switch from the ATF6α pathway to the IRE1–XBP-1 pathway, through the stabilization of HRD1 and consequent destruction of ATF6α protein. A previous publication has reported that WFS1 deficiency could lead to increased HRD1 expression (17
), contrary to our findings. This discrepancy could be attributed to the fact that WFS1 deficiency can cause dysregulated ER stress signaling and can lead to hyperactivation of the IRE1–XBP-1 pathway under some circumstances.
It has been established that WFS1 is induced under ER stress (11
). However, WFS1 increased steadily over a 24-hour time period (data not shown). ATF6α upregulation, on the other hand, occurred much more rapidly. Thus, the initial pool of WFS1 protein induced under stress may not have an inhibitory effect on ATF6α protein. In addition, we have shown that the ER-resident chaperone BiP also bound to ATF6α. The release of BiP from ATF6α when unfolded/misfolded proteins accumulate in the ER may be a key step in how ATF6α escapes WFS1-mediated proteolysis. BiP binding may be essential for the interaction of ATF6α and WFS1, and, upon release, cause a conformational change in ATF6α, leading to its consequent release from WFS1.
WFS1 is highly expressed in pancreatic β cells that are specialized for the production and regulated secretion of insulin to control blood glucose levels. In β cells, ER stress signaling needs to be tightly regulated to adapt to the frequent fluctuations of blood glucose levels and to produce the proper amount of insulin in response to the need for it (34
). To achieve tight regulation, mammals may have developed WFS1 as a regulator of HRD1 function in addition to SEL1. Higher expression of WFS1 in β cells, therefore, prevents hyperactivation of ER stress signaling in these cells that are particularly sensitive to disruption of ER homeostasis and dysregulation of the UPR. Therefore, WFS1 has a role in protecting β cells from death by acting as an ER stress signaling suppressor.
Mutations in the gene encoding WFS1 cause Wolfram syndrome, a genetic form of diabetes and neurodegeneration. It has been proposed that a high level of ER stress causes β cell death and neurodegeneration in this disorder. Collectively, our results suggest that a loss-of-function mutation of WFS1 causes the instability of an E3 ligase, HRD1, leading to the upregulation of ATF6α protein and hyperactivation of ATF6α signaling. Therefore, we predict that a loss-of-function or hypomorphic mutation of the WFS1, HRD1, or ATF6α genes can cause ER stress–related disorders, such as diabetes, neurodegeneration, and bipolar disorder. Indeed, it has been shown recently that common variants in WFS1 confer risk of type 2 diabetes (14
), and there is a link between WFS1 mutations and type 1A diabetes (38
). It has also been shown that ATF6α polymorphisms and haplotypes are associated with impaired glucose homeostasis and type 2 diabetes (40
). Excessive β cell loss is a component of both type 1 and type 2 diabetes (41
); therefore, WFS1 may have a key role in the protection of these cells from apoptosis through the tight regulation of ER stress signaling, thereby suppressing the diabetes phenotype. In addition, about 60% of patients with Wolfram syndrome have some mental disturbance such as severe depression, psychosis, or organic brain syndrome, as well as impulsive verbal and physical aggression (42
). The heterozygotes who do not have Wolfram syndrome are 26-fold more likely than noncarriers to have a psychiatric hospitalization (43
). The relative risk of psychiatric hospitalization for depression was estimated to be 7.1 in these heterozygotes (44
). Therefore, it is possible that dysregulation of a negative feedback loop of ER stress signaling may have a pathological role in psychiatric illness.
In this study, we focused on determining the physiological function of WFS1 in ER stress signaling because of its implication in diabetes, neurodegeneration, and bipolar disorder. We propose that WFS1 has a critical function in the regulation of ER stress signaling and prevents secretory cells, such as pancreatic β cells, from dysfunction and premature death caused by hyperactivation of ER stress signaling through its interaction with the transcription factor ATF6α. WFS1 could therefore be a key target for prevention and/or therapy of ER stress–mediated diseases such as diabetes, neurodegenerative diseases, and bipolar disorder.