Toxic chemicals, extreme temperatures and other abnormal environmental conditions can cause the cells in our bodies to become stressed. Several kinds of stresses overwhelm a compartment in the cell called the endoplasmic reticulum, which is critical for processing new proteins so that they can work correctly. Endoplasmic reticulum stress has been linked to long-term diseases such as diabetes, cancer and neurodegenerative diseases.

Most of what is known about how cells sense and respond to endoplasmic reticulum stress comes from studies on isolated cells that were subjected to harsh conditions that cells cannot tolerate for longer than a day or two. By contrast, little is known about how cells within whole organisms respond to milder but longer-lasting endoplasmic reticulum stress, which is closer to what occurs during disease.

To investigate this issue, Gomez and Rutkowski treated mice repeatedly with a chemical that causes mild endoplasmic reticulum stress in the liver. The cells exposed to this persistent stress responded differently to those exposed to severe short-term stress. Whereas short-term stress causes liver cells to turn on genes that help the endoplasmic reticulum to process proteins more efficiently, long-term stress causes cells to turn off some of those genes.

Further investigation revealed that cells in the livers of obese mice show similar patterns of gene activity as cells exposed to long-term endoplasmic reticulum stress. The findings presented by Gomez and Rutkowski could therefore also help us to understand more about the liver problems that often occur during obesity and diabetes.

Further studies are now needed to examine exactly how long-lasting stress can shut off the cells’ protective mechanisms. Future experiments could also investigate whether other types of cells and organs respond to long-term endoplasmic reticulum stress in the same way as cells in the liver.

The western obesity epidemic has exposed one of the consequences of modernity: the increasing prevalence of non-infectious chronic diseases that progress down increasingly irreversible paths over the course of years to decades. Diabetes, atherosclerosis, hypertension, steatohepatitis, and the like join neurodegenerative disorders, cancers, lung disease and other chronic diseases in driving morbidity and mortality in the western world (Ezzati et al., 2002). As a class, these diseases necessarily entail a gradual deterioration of cellular and organ function, rather than an acute collapse. Thus, treating or reversing them requires understanding how persistent but otherwise modest stimuli alter the activity of key cellular pathways.

One cellular pathway increasingly implicated in the progression of a number of chronic diseases is the Unfolded Protein Response (UPR). The UPR is activated by disruption of the protein folding capacity of the endoplasmic reticulum, otherwise known as ‘ER stress’ (Walter and Ron, 2011). Unremitted ER stress and/or a dysregulated UPR appear to contribute to hepatic steatosis and steatohepatitis (Malhi and Kaufman, 2011), atherosclerosis (Zhou and Tabas, 2013), colitis and inflammatory bowel disease (Kaser et al., 2013), hypertension (Young and Davisson, 2015), and many others. As an organelle that carries out several essential cellular processes (protein processing, calcium storage, lipogenesis, certain metabolic steps, etc.) and that is physically and functionally intertwined with many other critical cellular pathways, the ER is sensitive to a range of diverse stimuli. For example, ER stress is observed in the livers of obese mice (Ozcan et al., 2004), and this response has been attributed to an excess load of nascent client proteins in the organelle due to nutrient-mediated stimulation of mTOR activity (Ozcan et al., 2008). Other physiological stimuli that elicit ER stress include nutritional status and the activity of metabolic pathways (Oyadomari et al., 2008; Tyra et al., 2012; Shao et al., 2014), differentiation cues (Iwakoshi et al., 2003; van Anken et al., 2003; Lee et al., 2005), inflammatory signals (Zhang et al., 2006; Hotamisligil, 2010), and many others. While the UPR is capable of responding to excessive ER stress by initiating cell death cascades (Sano and Reed, 2013), chronic stress is instead likely to result not in cell death (at least not for most cells, most of the time), but instead in a persistent burden on ER function that must be accommodated by the UPR. And yet, for many chronic diseases in which ER stress is implicated, it is not clear whether disease results from a UPR that becomes progressively dysregulated and thus responds inappropriately; or whether the UPR becomes progressively neutered and simply becomes increasingly unresponsive.

Experimental manipulation of the UPR has sketched the framework of a canonical UPR that is initiated by three ER-resident proteins and that culminates in transcriptional augmentation of the ER protein processing capacity and other non-transcriptional mechanisms to alleviate ER load. The inositol-requiring enzyme 1α (IRE1α) pathway results in both the production of the XBP1 transcription factor (Yoshida et al., 2001; Calfon et al., 2002; Lee et al., 2002) and the degradation of ER-associated mRNAs in a process known as Regulated IRE1-Dependent Decay via the IRE1 endonuclease activity (Hollien and Weissman, 2006; Hollien et al., 2009). The PKR-like endoplasmic reticulum kinase (PERK) stimulates production of the ATF4 transcription factor (Harding et al., 2000) and also transiently reduces ER protein load by phosphorylation of the translation initiation factor eIF2α (Shi et al., 1998; Harding et al., 1999). ATF6 (comprising both α and β paralogs) initiates the third pathway when ATF6 is itself cleaved by regulated intramembrane proteolysis in the Golgi, liberating a transcriptionally active N-terminal fragment (Ye et al., 2000). These pathways are all robustly activated by acute exposure of cells to high doses of ER stress-inducing drugs, which is an effective tool for understanding what the system is capable of but not as effective for understanding how the cellular response to chronic stress differs from these canonical pathways.

Evidence for the involvement of ER stress in chronic diseases in general comes from two lines of investigation: detecting markers of ER stress in the affected tissues of human patients or mouse models thereof, and observing disease-like phenotypes in mice with genetic ablations of UPR signaling pathways. Yet while studies of this sort provide prima facie evidence for UPR involvement, they do not address how the UPR becomes dysregulated during chronic disease, if in fact it even does. Further, animal models of chronic diseases such as those associated with obesity tend to influence innumerable cellular pathways and make it difficult to isolate the effects of ER stress per se from the many other confounding factors.

With these considerations in mind, we set out to address how the UPR responds to chronic stress in vivo. Following the example of our previous work in vitro (Rutkowski et al., 2006), here we describe how UPR signaling in the liver becomes altered during repeated exposure to stress, the mechanisms by which these alterations occur, and their potential relevance to liver dysfunction during obesity.

In this paper, we have described the first, to our knowledge, experimental reconstitution of chronic, tractable ER stress in a mammal in vivo. This work builds upon previous efforts to recreate chronic ER stress in fish larvae (Cinaroglu et al., 2011), and to elicit chronic ER stress in mammals by overexpression of misfolded ER client proteins or deletion of ER chaperones (e.g., [Ji et al., 2011; Zode et al., 2011; Kawasaki et al., 2015]). We have used this system as a tool to probe how UPR signaling becomes altered during persistent and/or repeated activation. Our investigation has several novel implications for the mechanisms of UPR signaling.

The data presented here lead us to propose a working model to account for the altered expression of Hspa5 (Bip) and other UPR transcriptional targets during chronic stress (Figure 7). In the basal (i.e., nominally ‘unstressed’) state, activation of ATF6α contributes to the expression of Hspa5 mRNA. We arrive at this conclusion because expression of Hspa5 was diminished in the absence of ATF6α even without an exogenous stress (Figure 3D). This finding implies that normal physiological function in the liver entails periods of UPR activation—perhaps not surprisingly, given that feeding itself activates the UPR (Figure 6A). BiP is one of the most abundant proteins in the ER lumen (Gething, 1999), and other non-stress-dependent mechanisms contribute to its basal regulation as well (Resendez et al., 1988). Upon exposure to a conventional ER stress, all three limbs of the UPR are activated (Rutkowski et al., 2006), leading to robust production of stress-specific target transcripts such as Ddit3 (Chop), as well as augmentation of transcripts encoding ER chaperones, such as Hspa5 and Hsp90b1 (Grp94). The attendant improvement of ER protein folding then presents a problem for the cell: how to avoid overproduction of UPR targets. As the UPR stress-sensing molecules become deactivated, naturally labile factors such as CHOP (both mRNA and protein) are readily lost (Rutkowski et al., 2006). However, ER chaperones such as BiP and GRP94 are long-lived at both the protein and mRNA levels (Rutkowski et al., 2006), and continued overproduction of these proteins would presumably greatly tax cellular resources and potentially constitute their own burden on the organelle. Thus, BiP overexpression appears to initiate a negative feedback loop, both suppressing UPR activity (including the basal activity of ATF6α) and also indirectly stimulating the degradation of its own mRNA. In the context of incidental exposure to stress, or of the low-level stresses that occur during normal physiological function, we presume that this depression of Hspa5 mRNA levels is transient. However, during repeated stress, we propose that it constitutes a resetting to a new quasi-stable setpoint for UPR transcriptional regulation.

Figure 7

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One of the founding observations of the UPR field was that activation of the ER stress sensing molecules required their dissociation from BiP in both yeast and mammals (Bertolotti et al., 2000; Shen et al., 2002). Originally, the purpose attributed to BiP binding was to hold the stress sensors in a quiescent state and so to play a role in UPR activation. However, the suggestion that at least yeast Ire1p can be activated by direct binding to unfolded proteins (Kimata et al., 2004; Gardner and Walter, 2011; Gardner et al., 2013) has cast doubt on this model. Our data raise the possibility that perhaps the role of BiP binding is to instead modulate UPR deactivation.

The objective of this work from the outset was to develop an experimental system for eliciting chronic ER stress specifically, in order to be able to determine how the regulation and output of the response changed under such conditions and so identify fingerprints of chronic ER stress associated with disease states. An important caveat is that most agents other than TM that specifically induce ER stress in vitro fail to do so easily or effectively in vivo. While relatively specific in inducing ER stress due to its blockage of N-linked glycosylation, TM also is capable of disrupting carbohydrate metabolism (Olden et al., 1979) and protein palmitoylation (Patterson and Skene, 1995), although whether it does so at the doses used here is unclear. In addition, it is conceivable that the chronic stress phenotype is caused not by the ER stress-inducing effect of TM, but by underglycosylation of some specific key protein. However, we do not favor this alternative explanation in part because the degree of protein underglycosylation under these conditions is relatively modest (e.g., Figure 1E), and also because of the ability of BiP overexpression to phenocopy Hspa5 downregulation (Figure 5).

In addition, the strikingly similar phenotype in Lep^ob/ob and Lepr^db/db mice vouches for this approach. One difference between the chronic condition on one hand and the steady-state in obese mice on the other is that the latter do not appear to express elevated BiP protein levels (not shown), which at face value seems at odds with the idea that elevated BiP protein is needed to both suppress ATF6α and activate or perpetuate RIDD. However, as we have demonstrated, the magnitude of ER stress elicited by feeding is considerably lower than that elicited by tunicamycin (Figure 6; also compare Figure 6A with Figure 4D), yet also much more repetitive than the once-daily TM challenge. It is plausible that the less robust but more constant stimulus of overfeeding results in modest alterations in expression of BiP protein that have little effect on Hspa5 mRNA over the course of meals or days but great effect over the course of months—essentially that small increases in BiP protein levels could produce small decreases in BiP mRNA expression that accumulate over time. It is possible that further moderating the dose of TM and lengthening its extent (from days to weeks/months) would more closely mimic the phenotype observed in obese animals, although this would be experimentally cumbersome. It is also worth noting that the suppression of Hspa5 and Hsp90b1 mRNAs might be achieved by other mechanisms that suppress ATF6α activation while perpetuating RIDD. For instance, it has been reported that ATF6α expression is lost in obese animals (Wang et al., 2009). Along these lines, we observed that Atf6 mRNA is also significantly downregulated by chronic stress, albeit not as robustly as Hspa5 (not shown), raising the possibility of a positive feedback loop for suppressing Hspa5 expression even as BiP protein levels subside. Alternatively, posttranslational modifications that alter BiP’s interactions with its binding partners (Chambers et al., 2012; Wang et al., 2014; Preissler et al., 2015) might also play a role. Ultimately, the clearest way to test whether the mechanism we describe here is at work in obese animals might be to intercross these animals with Atf6-/- animals; as when given repeated TM injections, these animals should have basally suppressed Hspa5 mRNA expression even at an early age, but then there should be no further suppression as the animals age and become obese. Even if the mechanism turns out to be entirely distinct, the description here of suppressed expression of Hspa5, and Hsp90b1 and potentially elevated RIDD activity in these animals has potential implications for their ability to maintain ongoing ER homeostasis. It is conceivable that an altered UPR ‘setpoint’ with reduced expression of Hspa5 and other UPR mRNA targets could render the animals more sensitive to ER stress-induced cell death and exacerbate the hepatic inflammation associated with obesity (Gregor and Hotamisligil, 2011).

A surprising conclusion from this work is that RIDD might remain active in the chronic condition, even as UPR-dependent transcriptional signaling is shut off. Admittedly, conclusively demonstrating RIDD activity in vivo is challenging; here we have relied on repression of the well-documented RIDD target Bloc1s1 as the primary readout for RIDD activity. Bloc1s1 expression was repressed in the chronic condition (Figure 4G), upon BiP overexpression (Figure 5D), and in Lepr^db/db animals (Figure 6E). Phos-tag immunoblotting showed substantial phosphorylation of IRE1α only in animals treated with a high acute dose of TM, and antibodies purported to be specific for phospho-IRE1 did not produce bands that were absent in cells lacking IRE1α in control experiments (not shown). These limitations made it impossible to directly test whether IRE1α remained phosphorylated in the chronic condition. In addition, there is as yet no functional assay for confirming that RIDD is active in vivo. Rather, substrates are generally identified as putative RIDD targets based on an in vitro assay using purified IRE1α (Hollien and Weissman, 2006; Hollien et al., 2009). Then, their RIDD dependence is confirmed by their suppression during ER stress in an IRE1α-dependent XBP1-independent manner. However, Hspa5 and Hsp90b1 are both also transcriptionally upregulated by XBP1 (Lee et al., 2003), which would likely confound analysis of their regulation in animals lacking IRE1α. In any case, our data hint at a previously unappreciated role for RIDD, which has generally been considered an anti-adaptive signaling mechanism to this point (Han et al., 2009; Lerner et al., 2012; Upton et al., 2012; Maurel et al., 2014). The purpose of the clearance of ER-resident proteins might not so much be to neuter the organelle and hasten its dysfunction during severe stress (Maurel et al., 2014) as to help turn the response off once stress has been overcome. If RIDD is indeed active under these conditions, such a role could help explain the previously puzzling finding that Hspa5 is a potential RIDD target (Han et al., 2009).

Assuming RIDD is active during chronic stress, our data provide further evidence that the Xbp1 splicing and RIDD activities of IRE1α can be dissociated (Lin et al., 2007; Han et al., 2008). They raise the possibility that this dissociation occurs not necessarily to separate adaptive from apoptotic outputs so much as to separate activation from deactivation. Given that most stresses encountered by most cells most of the time are likely to be mild and transient—such as that encountered by feeding—the death-accelerating function of RIDD might simply be a byproduct of a normal physiological role in restoring the UPR rheostat. Indeed, a parallel can be drawn with the UPR-regulated transcription factor CHOP which, though it clearly promotes cell death during severe stress (Zinszner et al., 1998) also might promote normal cellular function during milder stress both by potentiating the dephosphorylation of eIF2α (Marciniak et al., 2004) and by regulating lipid metabolism (Tyra et al., 2012; Chikka et al., 2013) toward the ultimate improvement of ER function.

The approach we took to experimentally reconstituting chronic ER stress in vivo was conceptually analogous to our earlier reconstitution of chronic stress in vitro in MEFs (Rutkowski et al., 2006). However, neither MEFs nor even cultured hepatocytes showed the same suppression of Hspa5 expression as seen in vivo. This discrepancy points to the existence of other as yet unappreciated factors that influence UPR output in the liver. Indeed, it is now becoming clear that, overlaid atop the structural framework of the canonical UPR lie modulatory pathways for altering UPR sensitivity and output, with potential implications for signaling during chronic stress. These include posttranslational modifications either of the stress sensors—such as S-nitrosylation of IRE1α (Yang et al., 2015)—or of ER chaperones, such as ADP ribosylation (Chambers et al., 2012) or AMPylation (Preissler et al., 2015) of BiP. It will be interesting to test whether any of these modifications is active during the chronic condition.

Finally, our results underscore the remarkable capacity of the UPR to adapt to chronic stress. While the severe experimental stresses that lead to massive cell death and organ dysfunction have proven useful in elucidating the signaling capabilities of the UPR when activated to its maximum, they yield little about the ebb and flow of the response at it is most likely to be activated in physiological scenarios. We hope that our approach will stimulate further efforts to more closely mimic physiological stresses in vivo, as these will be essential in linking chronic ER stress to human disease.

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Author details

1. Javier A Gomez

   Graduate Program in Molecular and Cellular Biology, University of Iowa Carver College of Medicine, Iowa City, United States

   Contribution

   JAG, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

2. D Thomas Rutkowski

   Departments of Anatomy and Cell Biology and Internal Medicine, University of Iowa Carver College of Medicine, Iowa City, United States

   Contribution

   DTR, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   thomas-rutkowski@uiowa.edu

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0001-6586-4449

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