Endoplasmic reticulum stress induces UPR, an adaptive mechanism that controls cell fate between survival and death in an intensity time-dependent manner. It involves three signal transduction pathways initiated by three ER transmembrane proteins: PERK, inositol requiring protein 1α (IRE1α, also known as ERN1), and activating transcription factor 6α (ATF6α) (27, 28). Each of the three maintains an inactive state in combination with the ER chaperone BiP (also named GRP78) in resting cells. Under chronic or overwhelming ER stress, all three mammalian UPR pathways uniquely lead to the initiation of CHOP transcription through binding sites within CHOP’s promoter. The cis-acting AARE1 and AARE2 as well as the composite CARE (29) are bound by ATF4, while ATF6α and X box-binding protein 1 (XBP1) bind to ERSE1 and ERSE2.

The PERK pathway is predominant in CHOP activation. Upon the luminal binding of misfolded proteins, PERK is activated through dimerization and trans-autophosphorylation (30). It phosphorylates eukaryotic translation initiator factor 2α (eIF2α), which then attenuates global protein synthesis. Upon activation, ATF4 translocates into the nucleus and transcriptionally upregulates CHOP as well as many UPR genes that are vital for amino acid metabolism and redox processes (31).

The activation of IRE1α is similar to PERK in that its luminal domains are first dimerized and then are trans-autophosphorylated. Activated IRE1α creates spliced XBP1(s) by cleaving a 26-nucleotide intron from the mRNA of unspliced XBP1 using a cytoplasmic RNase domain. IRE1α also mediates regulated IRE1-dependent decay of selective mRNAs (32). XBP1(s) enters the nucleus and induces the transcription of genes correlated with protein-folding capacity and ER-associated degradation. Hence, CHOP expression is upregulated by XBP1(s) (33, 34).

In the ATF6α branch, the type II ER located protein ATF6α is transported to the Golgi apparatus where it is processed by Site-1 and Site-2 proteases (SP1 and SP2, respectively). As a consequence of this processing, a cytosolic fragment of ATF6α is produced and enters the nucleus to regulate the expression of target genes, including BiP and CHOP (35, 36). Along with XBP1(s), ATF6f contributes to the augmentation of ER size and ER protein-folding capacity through target genes.

Integrated stress response serves as another cytoprotective mechanism against various stressors, such as ER stress, nutrition stress, oxidative stress, proteasome inhibition, hyperoxia, or viral infection (5, 37, 38). This common adaptive response initiates with four kinases consisting of general control nonderepressible 2, RNA-dependent protein kinase (39), heme regulated inhibitor, and PEAK, all four of which then converge on a core event, the phospho-eIF2α/ATF4 pathway, which in turn increases the transcription of CHOP (4, 40).

Reactive oxygen species disturb redox status and ER homeostasis, thus inducing ER stress responses. ROS have been reported to activate CHOP through the AP-1 element in the CHOP promoter (41). The MAPKs consisting of JNK, p38 MAPK, and ERK are canonical downstream mediators of ROS (42). There are a number of studies establishing the signaling axis of ROS-induced CHOP upregulation via MAPKs signaling pathways in different cells. The ROS–MAPKs–CHOP pathway has been reported to suppress migration of hepatocellular carcinoma (HCC) cells (43) and mediate the downstream death receptor pathway in a number of cancer cells (44–47). Moreover, a scavenger of ROS diminished the PERK/eIF2α/CHOP pathway (48). IRE1 can recruit TRAF2 to activate ASK1, which can, through different pathways, induce expression of JNK and p38 MAPK (49). Phosphorylation of CHOP by p38 MAPK is required for its activation. Thus, the way by which ROS activates MAPKs and CHOP may be the IRE1α or PERK pathway of UPR (50). The ATF4/ATF3 axis was also reported to induce CHOP expression under ROS-dependent ER stress (51, 52).

Several members of the CREB/ATF transcription factor family are capable of regulating CHOP expression. ATF3 can interact with the CARE elements within the CHOP promoter (29), while ATF5 activates the AARE1 site (53) under arsenite exposure. ATF2 binds the AARE sequence to regulate CHOP transcription in response to amino acid starvation (54). Conversely, some factors can inhibit the expression of CHOP, thereby reducing its detrimental effects. At early stage of ER stress, miR-211 expression, induced by PERK activation, can suppress CHOP transcription through histone methylation at its promoter (55). CHOP expression can also be repressed through toll-like receptor (TLR)–TRIF-dependent pathway which increases the activity of eIF2B to counteract the effect of p-eIF2α under the treatment of LPS, a TLR4 ligand in vitro and in vivo. Furthermore, the activation of eIF2B by TLR–TRIF signaling is attributed to serine dephosphorylation of eIF2Bε by protein phosphatase 2A. When TRIF was deficient in mice, CHOP induction, apoptosis, and organ dysfunction ensued (56, 57).

During a stress situation, UPR attempts to increase protein-folding capacity and remove misfolded and unfolded proteins. If the remedy is inadequate to restore homeostasis under chronic ER stress, terminal UPR will trigger apoptosis through abundant signaling mechanisms, mainly mediated by CHOP, JNK, and caspase-12, with CHOP as the most widely studied (60). It is notable that CHOP expression itself is not sufficient to induce apoptosis unless exposed to a stress signal. Studies in both cellular and animal models with CHOP gene deficiency have shed light on the pro-apoptotic role of CHOP during cellular stress (61, 62). Synoptically, CHOP-dependent apoptosis is mainly mediated by altering the expression of pro-apoptotic or anti-apoptotic genes, either directly or indirectly (1, 63). Both the intrinsic, mitochondrial pathway and extrinsic, death receptor pathway of classic apoptosis can be activated by CHOP and proceed with a set of initiator caspases and common executioner caspases (64). Overall, the apoptotic pathways mediated by downstream targets of CHOP form networks (Figure 3), wherein the molecular interaction mechanisms remain to be further understood.

To cope with an inadequate protein-folding environment in ER, cells activate autophagy, an early stress-adaptive self-eating process, responsible for lysosome-dependent degradation of protein aggregates and other cellular material through UPR signaling (90). Many cellular stresses can trigger autophagy or apoptosis depending on specific circumstances and autophagy usually precedes apoptosis. CHOP has been implicated in autophagy induced by amino acid starvation, ER stress, virus infection, and hypoxia.

During amino acid starvation and ER stress, CHOP binds to the promoters of a set of autophagy genes (91). The time course analysis provides a further understanding that its upregulation of autophagy genes is within a short period of leucine starvation in cells. However, as time goes on, CHOP turns to inhibit the activation of these genes. It also inhibits autophagic flux and the conversion of microtubule-associated protein 1A/1B-light chain 3B (referred to as LC3), which is a key step for autophagy formation. The UPR-controlled balance of cell fate is therefore inclined toward cell death (92). Moreover, CHOP modulates the induction of autophagosomes during ER stress, as evidenced by the inhibition of LC3-II expression and GFP-LC3B dots (93). The results also showed that CHOP upregulated IRE1α, which contributed to autophagy induction, but their specific role and relation in this autophagy pathway remain unknown. Similarly, CHOP-mediated Licochalcone A-induced autophagy in non-small cell lung cancer cells and HeLa cells, while knockdown of CHOP reversed autophagy by reducing LC3-II and GFP-LC3 expression (94). Another study implied that UPR-activated CHOP elicited complete autolysosome maturation in hepatitis C virus-induced autophagy via LC3B-II-dependent mechanism (95). In the context of hypoxia, CHOP induces the expression of the autophagy gene Atg5 by directly binding to its promoters (96).

C/EBP homologous protein is involved in the block of differentiation in mesenchymal lineages. It is a fundamental regulator of adipogenesis, a role that has been supported by numerous experiments. The terminal differentiation of adipocytes is necessary for efficient lipid storage. CHOP was initially found to negatively regulate adipocyte differentiation in vitro, in response to metabolic stress (97), hypoxia (98), and when phosphorylated by stress-induced p38 MAPK (11). In subsequent studies, both in vivo and in vitro adipocyte differentiation is inhibited by CHOP expression after PERK–eIF2α activation during ER stress (99). As for the specific mechanisms, under polyamine depletion, CHOP exerts the inhibitory effect through interaction with C/EBPβ, thus impairing its role in the execution of mitotic clonal expansion process and in the transcriptional activation of peroxisome proliferator-activated receptor γ (PPAR-γ) and C/EBPα; the predominant regulators of adipogenesis (100). This is consistent with the molecular mechanism whereby CHOP impairs the differentiation of preadipocyte in response to aging (101). Higher levels of CHOP have led to hyperplasia of adipose tissue with less differentiated adipocytes in mouse models and downregulation of CHOP mRNA is required for complete adipocyte differentiation of MEFs (102).

Besides adipocytes, CHOP was also identified as a negative modulator for osteoblast differentiation. In terms of the mechanism, CHOP inhibits the binding activity of C/EBPβ against Runx2, thus suppressing their induction of the osteocalcin (Ocn) gene and phosphatase activity (103). This negative modulation is also supported by recent experiments. Osteoblast proliferation and differentiation can be regulated by ADP-ribosylation-like factor 6 interacting protein 5 (Arl6ip5), whereas CHOP is required for reduction of the above two events and induces apoptosis in Arl6ip5-knockdown osteoblasts (104). The promotion of osteoblast differentiation by the transcription factor EB is coupled with reduced expression of ATF4 and CHOP. However, their expression was upregulated by TFEB overexpression in the stimulation of bone morphogenic protein 2 (BMP2), a potent inducer of osteoblast differentiation (105). It was previously proposed that CHOP may indirectly promote BMP2-induced osteogenesis (103) and enhance osteoblast differentiation of mesenchymal progenitor cells especially in the presence of BMP2 (106). Consistent with the finding, the PERK–eIF2α–ATF4 pathway promotes BMP2-induced osteoblast differentiation (107). Taken together, expression of CHOP exerts a dual role in osteoblast differentiation. It may be that the inhibitory action of CHOP is not sufficient to affect BMP2-induced differentiation processes. The specific association between CHOP and BMP2 in osteoblasts remains a topic of further exploration. Moreover, myoblast differentiation is inhibited by CHOP, which suppresses the transcription of myogenic regulatory factor in myoblasts by binding to its transcription regulatory sequences and affecting histone acetylation (108). Recent evidence has also unveiled that CHOP blocks the progression of myeloid lineage in granulo-monocytic progenitors (109).

Fibrosis progresses as an eventual pathological outcome of three stages of general wound-healing responses; injury, inflammation, and repair to persistent organ injury. A pathological hallmark of fibrosis is the excessive deposition of extracellular matrix (ECM) in the tissues. This increased protein synthesis may disturb ER homeostasis and induce the expression of CHOP (110). An emerging role of CHOP in promoting fibrotic response of internal organs is supported by alleviation of fibrosis in CHOP-knockout mice (111). Given that apoptosis is a common cellular event that leads to organ remodeling and fibrosis after insult, CHOP-initiated pro-apoptotic activity may be partly the underlying mechanism. Moreover, alternatively activated phenotype (M2) macrophages are considered to participate in promoting collagen deposition and fibrogenesis. In recent years, research advances have indicated that CHOP likely regulates the activation of M2 macrophages to trigger tissue fibrosis (112). These M2 macrophages would release various cytokines to create a microenvironment that favors fibrogenesis, including high levels of TGF-β1, which is a key factor that promotes the activation of ECM-producing myofibroblasts (113). Modulation of CHOP expression may be a potential treatment for organ fibrosis. Here, we show the vital role of CHOP in the formation of specific fibrotic disorders.

In states of uncontrolled proliferation and insufficient vascularization (e.g., in cancer), conditions of low nutrient supply, such as hypoxia and oxidative stress, may trigger ER stress and subsequent UPR activation that have been documented in various human cancers (140). Studies have shown evidence of CHOP activation in various types of cancer cells (125, 141). Thus, CHOP-induced apoptosis in ER stress has significant implications for cancers. Furthermore, CHOP mutations are found in some human tumors, although whether or not they contribute to tumorigenesis remains unknown (142). Accumulating data suggest that CHOP impinges upon several aspects of cancer including tumor formation as well as progression of tumors once formed. Nonetheless, how CHOP activation exerts tumor-supporting or tumor-suppressive roles remains to be elucidated.

In diabetes mellitus, the glucostatic cycle to maintain normoglycemia is dysregulated due to an insufficient mass of functioning pancreatic β-cells to synthesize the needed amounts of insulin for metabolism (162). For type 2 diabetes (T2D), insulin resistance under stress of excess nutrients, including hyperglycemia and hyperlipemia, causes progressive β-cell failure (163). Dissimilarly, in type 1 diabetes (T1D), β-cells are attacked by autoimmune activity and the workload of the remaining β-cells increases. Collectively, high demand of insulin synthesis and secretion overwhelms the capacity of β-cell ER and thereby activates UPR to compensate. As the process continues, terminal UPR leads to apoptosis of β-cells and the onset of diabetes (164).

Apoptosis has been the main focus of studies on β-cell dysfunction during diabetes, among which CHOP-induced apoptosis is the most studied, as it is a key event in the pathogenesis of diabetes (165). Indeed, numerous studies have found that islet cells from mice and patients with T1D or T2D manifests elevated levels of CHOP (7, 166). In 2002, studies conducted with Akita mice proved that genetic removal of CHOP alleviated β-cell loss and hereditary diabetes, which vividly links CHOP to β-cell apoptosis for the first time (167). CHOP deficiency also prevents oxidative damage with reduced ROS and thereby improves ER function in β-cells (168), while oxidative stress is proven as an important factor that gives rise to β-cell dysfunction in diabetes (169). Thus, past studies have identified drugs, such as vildagliptin (170), that promote β-cell survival by decreasing CHOP expression in diabetic mouse models, along with downregulation of ATF4 and TRIB3 in T2D db/db mice (171).

For T2D, it has been evidenced that CHOP is responsible for β-cell apoptosis and dysfunction both in genetic and diet-induced mouse models of T2D, as well as in vitro (168, 172). The PERK/eIF2α/CHOP signaling mediated β-cell sensitization to lipotoxicity and apoptosis under the challenge of palmitate (173, 174) and guanabenz (175). Ubiquitination and degradation of CHOP by cellular inhibition of apoptosis protein-1 prevented palmitate-induced lipotoxicity (173). Consistent with this notion, the inhibition effect of CHOP on adipocyte differentiation interferes with effective fatty acid storage, which may cause lipotoxicity. Furthermore, the human islet amyloid polypeptide (h-IAPP), also characteristic of T2D, induced dysfunction of autophagy and apoptosis through CHOP, but inhibition of CHOP alone may not be a durable therapeutic strategy for the β-cell toxicity of h-IAPP, considering multiple stress pathways are activated during this process (176). To unveil the specific mechanisms underlying β-cell apoptosis in T2D, researchers found that CHOP regulation of puma is essential for the apoptotic pathway during glucotoxicity T2D (177). Another critical event is that CHOP downregulates p21 to trigger β-cell apoptosis due to glucotoxicity, thus promoting the onset of T2D (85, 178). As such, the chemicals that inhibit CHOP expression protect β cells from apoptosis and dysfunction, such as 1,2,3-triazole derivatives (179).

For insulinopenic T1D, autoimmunity triggers an inflammatory response along with cytokine release which induces ER stress in β-cells. CHOP contributes to cytokine-induced apoptosis of β-cells via mitochondrial apoptotic pathways and indirect pro-inflammatory responses, indicating the role of CHOP in T1D. Mechanistic studies have shown that CHOP knockdown in insulinoma cell lines protected against the downregulation of anti-apoptotic BCL-2-like proteins, Bcl-2 and Mcl-1, while decreasing NF-κB activity and expression of its target genes, including inducible NO synthase (iNOS) and TNF receptor superfamily member 6 (FAS) (180). CHOP blocking by siRNA partially protected human beta cells against cytokine-induced apoptosis independent of NO, whereas CHOP induction was NO dependent and could be inhibited by iNOS blocker in rat insulin-producing cells (181). Moreover, CHOP acted as a mediator of β-cell apoptosis in islets deficient for Gata4, which belongs to a group of β-cell survival factors that contribute to T1D risk when they undergo mutations (182).

Overall, cumulating evidence suggests that CHOP is involved in the pathogenesis of diabetes, predominantly T2D, in response to glucotoxicity, lipotoxicity, as well as oxidative stress and islet amyloid derived from IAPP. With regard to diabetic complications, there is also some relevance to CHOP. For example, during murine DN, ATF6-dependent CHOP activation was induced by defective insulin signaling due to impaired nuclear translocation of sXBP1 in podocytes (34), whereas CHOP-null mice gained protection from DN (183). Tubules of diabetic mice and patients showed increased levels of CHOP protein, and other than apoptosis, CHOP-induced expression of fibronectin in tubule cells (123). As for diabetic cardiovascular complications, including diabetic cardiomyopathy, IL-1β-induced myocyte apoptosis was mediated by the IRAK-2/CHOP pathway (184). CHOP-induced apoptosis was partly targeted by Ginsenoside Rg1 to ameliorate diabetic myocardial damage (185). Correspondingly, myocyte apoptosis and cardiac dysfunction induced by methylglyoxal were attenuated in CHOP-null mice (62). Furthermore, streptozotocin-induced diabetic CHOP^−/− mice manifested not only reduced hyperglycemia but also lessened severity of oxidative-nitrative stress in their sciatic nerve and in their eventual diabetic peripheral neuropathy (186).

Neurodegenerative diseases are hallmarked by progressive loss of neuronal function. Many risk factors including aging, oxidative stress, and gene mutations of neurodegenerative process can cause toxic accumulation of misfolded proteins, which ultimately leads to neuronal cells undergoing ER stress-induced apoptosis. As a key player in ER stress and oxidative stress, CHOP expression is found to be elevated in many disorders related to neurodegeneration, such as the Parkinson disease (PD) (187), and CHOP induces neuronal apoptosis, which has been proposed as a target of treatments for some neurodegenerative diseases (188). In some cases, disruption of CHOP exerts a neuroprotective role through yet unknown mechanisms (189). We list here important studies on CHOP involvement in neurodegenerative diseases in recent years.

A prominent clinical hallmark of Alzheimer’s disease (AD) is progressive cognitive impairment. AD is attributed to pathological deposits of neurofibrillary tangles formed by hyperphosphorylated tau aggregates and abnormal aggregation of amyloid-β (Aβ) plaques. In the AD brain, ATF4 synthesis in axons locally exposed to Aβ_1–42 triggered retrograde cell loss through CHOP, and conversely, CHOP deletion hindered Aβ_1–42-mediated neurodegeneration (190). In the mouse model of AD and neuroblastoma cells, researchers analyzed the effect of palmitate and noticed that CHOP indispensably mediated increased β-site APP-cleaving enzyme 1 (BACE1) activity and ensuing Aβ production, but only partially (191). In agreement with this, silencing CHOP expression attenuated NF-κB activation and its binding to the BACE1 promoter, thus reducing Aβ production induced by 27-hydroxycholesterol (192). CHOP knockdown also alleviated the negative regulation of C/EBPα binding to the leptin promoter and subsequent leptin expression, which is able to decrease Aβ genesis and tau phosphorylation (193). As for PD, CHOP and ATF4 play a key role in regulating Trib3 and apoptosis in cellular models of PD, as evidenced by the protective role of CHOP and ATF4 knockdown in 6-OHDA and MPP(+) models (194). Inhibition of CHOP-mediated crocin-induced neuroprotection in the PD model through Wnt pathway in vitro (195).

In S63-deletion mice of Charcot–Marie–Tooth (CMT) disease type 1B, CHOP deletion decreased demyelination and rescued their motor deficit (196). Mechanistic studies subsequently revealed that CHOP targeted GADD34 to reactivate translation in the nerves of this model (197). Surprisingly, CHOP ablation did not rescue the abnormalities of Schwann cell development in R98C mouse model of type 1B CMT (198). Prion-related diseases are another type of neurodegenerative disorder, and accumulation of prion protein (PrP) defines the pathobiology. Upregulation of CHOP through the PERK pathway is a pathogenic factor of neurodegeneration induced by the membrane-tethered flexible tail of PrP (199). A role of CHOP in mild spinal cord injury is indicated by enhanced neuronal functional recovery in CHOP-deficient mice, partly due to decreased oligodendrocyte apoptosis (200). Moreover, CHOP and caspase12 induced neuron apoptosis at later stages of chemical hypoxia (201). In the context of neurodegeneration in retinas, a sustained upregulation of CHOP can result from optic nerve injury and CHOP deficiency increased the survival of retinal ganglion cells (202). Apoptotic cell death of photoreceptors was also mediated by CHOP in retinas deficient in autophagosomes (203). Likewise, in brain astrocytes, MA mediated CHOP upregulation downstream of the activation of all three ER stress pathways, which together lead to apoptosis via intrinsic caspase cascade (204). This arises as the mechanism of MA-mediated neurodegenerative effects. The PERK–eIF2α–ATF4–CHOP pathway mediated sevoflurane-induced neuroapoptosis in neonatal brains (205). Moreover, CHOP expression in the brain plays a pivotal role in the negative regulation of two neurotrophic cytokines, leptin and insulin-like growth factor-1 by palmitate (206).