The ER is a vast membranous network responsible for the synthesis, maturation, and trafficking of a wide range of proteins. It is also a critical site for Ca2+
homeostasis. As a central regulator of protein folding, quality control, trafficking, and targeting, the ability of the ER to adapt its capacity to manage synthetic, metabolic, and other adverse conditions is of paramount importance for the cell. Under conditions that challenge ER function, particularly an increase in newly synthesized, unfolded proteins in the ER lumen, this organelle elicits an elaborate adaptive response known as the UPR (Ron and Walter, 2007
In eukaryotic cells, monitoring of the ER lumen and signaling through the canonical branches of the UPR are mediated by three ER membrane-associated proteins, PERK (PKR-like eukaryotic initiation factor 2α kinase), IRE1 (inositol requiring enzyme 1), and ATF6 (activating transcription factor-6). In a well-functioning and “stress-free” ER, these three transmembrane proteins are bound by a chaperone, BiP/GRP78, in their intralumenal domains (amino-terminal of IRE1 and PERK and carboxy-terminal of ATF6) and rendered inactive (Bertolotti et al., 2000
; Shen et al., 2002
). There may also be additional mechanisms controlling the activity of each UPR sensor and simple disruption of the interaction with BIP may not always result in constitutive activation (Oikawa et al., 2007
; Zhou et al., 2006
). Accumulation of improperly folded proteins and increased protein cargo in the ER results in the recruitment of BiP away from these UPR sensors. This and potentially other yet to be discovered lumenal events result in oligomerization and activation of the two kinases, PERK and IRE1, and engage a complex downstream signaling pathway (Ron and Walter, 2007
). Activation of the third branch of the UPR requires translocation of ATF6 to the Golgi apparatus where it is processed by the serine protease site-1 protease (S1P) and the metalloprotease site-2 protease (S2P) to produce an active transcription factor (Chen et al., 2002
). ATF6 is reduced in response to ER stress, and only the reduced monomeric ATF6 can reach the Golgi apparatus, indicating that redox status is also a potential determinant of ATF6 activation (Nadanaka et al., 2007
). Together these three arms mitigate ER stress by reducing protein synthesis, facilitating protein degradation, and increasing production of chaperones that help proteins in the ER lumen to fold (). The result is that the ER stress resolves, and if it does not then the cell is functionally compromised and may undergo apoptosis.
The oldest branch of the UPR, in an evolutionary sense, is mediated through the stress-regulated kinase and ribonuclease IRE1, which is conserved from yeast to humans (Calfon et al., 2002
; Patil and Walter, 2001
). The endoribonuclease activity of IRE1α cleaves a 26 base-pair segment from the mRNA of the X-box binding protein-1 (XBP1), creating an alternative message that is translated into the active (or spliced) form of the transcription factor (XBP1s) (Sidrauski and Walter, 1997
). XBP1s, alone or in conjunction with ATF6α, launches a transcriptional program to produce chaperones (such as Grp78) and proteins involved in ER biogenesis, phospholipid synthesis, ER-associated protein degradation (ERAD), and secretion (for example, EDEM, ERdj4, PDI). Thus, XBP1s activates one of the major pathways for enhancing the folding capacity of the ER and for dealing with ER stress (Lee et al., 2003
). XBP1 was the only known substrate of IRE1, but recently this endoribonuclease was reported to target other mRNAs during ER stress, although through a different mechanism. These mRNAs are degraded by IRE1, thereby preventing their translation, an additional measure to relieve ER stress (Hollien et al., 2009
; Hollien and Weissman, 2006
). This is an intriguing new aspect of signaling through the regulated IRE1-dependent decay (RIDD) pathway that is likely to produce additional insights into the biological outcomes of ER stress beyond the simple degradation of unwanted mRNAs.
When activated, ATF6 moves to the nucleus to stimulate the expression of genes containing ER stress elements (ERSE-I, -II), UPR elements (UPRE), and cAMP response elements (CRE) in their promoters. ERAD as well as production of the ER degradation-enhancing α-mannosidase-like protein (EDEM) are boosted by these events, facilitating clearance and degradation of misfolded proteins from the ER lumen (Kokame et al., 2001
; Yoshida et al., 1998
). Currently, ATF6 and XBP1—a target of ATF6, which is further processed and activated by the ribonuclease activity of the IRE1 branch—are viewed as the predominant regulators of the transcriptional response programs triggered during the UPR (Yoshida et al., 2000
). In addition to regulating XBP1
mRNA expression, ATF6 can also interact directly with the XBP1 protein to target UPR quality-control genes. Interestingly, however, mice lacking ATF6α or ATF6β do not have a major phenotype, whereas the dual deficiency is lethal (Wu et al., 2007
; Yamamoto et al., 2007
). This suggests either highly specialized adaptive functions carried out through this branch or the existence of further redundancies in the transcriptional responses of the UPR.
The recent identification of a number of ATF6α-related proteins suggests that the traditional model of a three-armed UPR may not be complete. To date, five proteins—Luman (CREB3), Oasis (CREB3L1), BBF2H7 (CREB3L2), CREBH (CREB3L3), and Tisp40 (CREB4, CREB3L4)—share a region of high sequence similarity with ATF6α: a transmembrane domain adjacent to a conserved bZIP region (Bailey and O'Hare, 2007
). Like ATF6α, these proteins are anchored to the ER and in response to activation by specific stimuli undergo regulated intramembrane proteolysis in the Golgi and subsequent translocation to the nucleus. These factors have all been implicated in the ER stress response due to their ability to respond to traditional ER stressors, activate known UPR targets, or show activity at UPR response elements (Bailey and O'Hare, 2007
). Despite the similarities among these transmembrane bZIPs and ATF6α, differences in activating stimuli, tissue distribution, and response element binding indicate unique roles for each of these factors in regulating the UPR. For example, CREBH may respond to inflammatory stimuli induced by lipopolysaccharide and cytokines in the liver and may help to integrate the UPR with the acute phase response by stimulating the production of serum amyloid, serum amyloid P component, and C-reactive protein (Zhang et al., 2006a
), whereas Oasis is highly expressed in the skeletal system with a potential role in bone formation and osteoblast activity (Murakami et al., 2009
). The existence of a family of ATF6-like proteins with distinct tissue distributions and activation profiles suggests the evolution of a more fine-tuned ER stress response in mammals that allows for a distinct response depending on the cell or tissue involved, the specific stressor encountered, and the duration of the stress. Of course, the transcriptional program of the UPR may have even greater complexity beyond this transcription factor family and may involve other as yet unknown molecules that carry out specialized functions, including those related to metabolism.
Activation of the third arm of the UPR through PERK results in phosphorylation of eIF2α (eukaryotic translational initiation factor 2α) at serine 51, which converts eIF2α to a competitor of eIF2B and reduces the rate of formation of the ternary complex, resulting in reduced global protein synthesis and a subsequent reduction in the workload of the ER (Harding et al., 1999
; Shi et al., 1998
). PERK is one of four protein kinases that can mediate eIF2α phosphorylation; the other three kinases are PKR (double-stranded RNA-activated protein kinase), GCN2 (general control non-derepressible kinase 2), and HRI (heme-regulated inhibitor kinase). The role of eIF2α kinases other than PERK in ER stress remains unclear, although recent studies show that PKR is activated during ER stress and influences the UPR and related inflammatory signaling events (Nakamura et al., 2010
) (). In addition to an overall reduction in protein synthesis, this branch of the UPR is also linked to broad transcriptional regulation through several distinct mechanisms, including the transcriptional regulation of ribosomal RNA (DuRose et al., 2009
). This results in activation of ATF4 (activating transcription factor-4), Nrf2 (nuclear erythroid 2 p45-related factor 2), and NF-κB (nuclear factor kappa β), a master transcription factor with numerous functions including regulation of the inflammatory response. ATF4 is produced through alternative translation and induces expression of genes involved in apoptosis (CHOP, C/EBP homologous protein), ER redox control (ERO1, endoplasmic reticulum oxidoreductin), the negative feedback release of eIF2α inhibition (Gadd34, growth arrest, and DNA damage-inducible protein), and glucose metabolism (fructose 1,6-bisphosphate; glucokinase, and phosphoenolpyruvate carboxykinase) (Harding et al., 2000b
; Ma et al., 2002
). PERK-dependent phosphorylation triggers dissociation of Nrf2/Keap1 complexes and allows subsequent Nrf2 nuclear import (Cullinan et al., 2003
). Recent studies have shown that NF-κB can be activated through this pathway via translational suppression of inhibitory kappa B (IκB), resulting in the regulation of mediators of inflammation (Deng et al., 2004
; Jiang et al., 2003
) such as IL-6 and TNF-α.
The selective increase in production or activity of a subset of proteins by PERK also aids in recovery from stress. For example, one consequence of ER stress is the accumulation of reactive oxygen species (ROS) that promotes a state of oxidative stress (Cullinan and Diehl, 2006
). In anticipation of generating these potentially hazardous by-products, the UPR has incorporated an antioxidant defense system as well. PERK signaling, via activation of the Nrf2 and ATF4 transcription factors, engages survival responses, coordinates the convergence of ER stress with oxidative stress signaling, and orchestrates the execution of the antioxidant response element-dependent gene transcription program. This includes expression of genes encoding heme oxygenase-1 (HO-1), thioredoxin reductase 1 (TXNRD1), and the glutathione S
-transferases GSTP1, GSTM1, and GSTm2 (Cullinan and Diehl, 2006
It is clear that the UPR does not always result in successful alleviation of ER stress and establishment of a functional equilibrium in the ER. Often driven by severe or prolonged stress signals, the UPR can induce cell death via apoptosis (Rao et al., 2004
). Like most other ER stress responses, the signaling pathways involved in this response are tightly regulated. CHOP is induced through the PERK pathway, caspase-12 and JNK signaling are activated, and proapoptotic Bcl-2 proteins BAX and BAK are switched on by the IRE1α pathway (Hetz et al., 2006
; Rao et al., 2004
). In addition, ER stress-induced IRE1α phosphorylation leads to the recruitment of TRAF2 (tumor necrosis factor receptor-associated factor 2) and ASK1 (apoptosis signal-regulating kinase 1) to the cytosolic leaflet of the ER membrane (Kaneko et al., 2003
). Activation of both the PERK and IRE1 pathways also leads to regulation of the NF-κB-IKK signaling pathway during ER stress through activation of IKK or degradation of the p65 subunit (Deng et al., 2004
; Hu et al., 2006
). The ATF6 branch can also regulate NF-κB activity (Yamazaki et al., 2009
). All of these signals contribute to the triggering of apoptotic responses when ER stress is excessive, prolonged, or insufficiently neutralized.
The mechanisms resulting in a commitment to cell death in response to ER stress remain a challenging aspect of ER biology that has yet to be decoded. Differential activation of the three UPR pathways may be a critical determinant of apoptosis (Ron and Walter, 2007
). For example, attenuation of IRE1 and ATF6 activities by persistent ER stress and prolonged signaling through PERK can create an appropriate condition for apoptosis (Lin et al., 2007
). Similarly, disproportionate engagement of the ribonuclease activity of IRE1 (which produces XBP1s as a protective measure) versus its ability to trigger JNK activity (or release proapoptotic mediators) (Hetz et al., 2006
) and divergent effects of IRE1-mediated XBP1 splicing and IRE1α-triggered degradation of mRNAs localized in the ER may be critical determinants of the life and death outcomes resulting from ER stress (Han et al., 2009
). Hence, it is likely that IRE1 is a critical lever in the UPR that controls commitment to cell death or promotes survival (Ron and Walter, 2007
). But there remain important gaps in our understanding of the ability of individual UPR initiators to recognize or respond to various forms of ER stress. Moreover, additional work is required to determine how and whether different branches of the UPR are specialized to respond to particular conditions and different cellular environments by engaging distinct survival responses. Although disruption of distal death mediators has yielded important insights, the proximal engagement of these pathways is also vital in linking ER stress to physiological functions and disease pathogenesis.
Although the function of the ER is predominantly viewed from a protein processing perspective, many conditions other than (or in addition to) increased protein synthesis and the presence of mutant or misfolded proteins can also trigger UPR activation. These conditions include an imbalance in ER calcium levels, glucose and energy deprivation, hypoxia, pathogens or pathogen-associated components, certain lipids, and toxins. In particular, studies to explore ER function under metabolic and inflammatory challenge may prove fruitful and are discussed in the following sections.