Subsequently, the 1990s were marked by major discoveries for identification of the unfolded protein response (UPR) as a major ER stress responsive pathway. The accumulation of unfolded, misfolded proteins or otherwise damaged proteins pose a proteotoxic threat to the survival of the cell which leads to the activation of a well-documented ultrastructural response to ER stress (UPR). UPR effectors sense unfolded proteins in the ER and transmit this information via the activation of three key transcription factors (XBP1, ATF4, and ATF6) in the nucleus, where they regulate the expression of a variety of gene products collectively involved in managing ER stress and promoting cell survival (Garg et al. 2015). In case of sustained ER stress, proapoptotic signaling events begin to dominate shifting the balance toward cell death. CHOP, also known as GADD153 (growth arrest and DNA damage-inducible gene 153), was first reported as a central executor of this latter process (ER stress-induced apoptosis) (Fig. 1) (Garg et al. 2015).
Studies in the last two decades have led to the identification of key transcription factors belong to the network of CCAAT enhancer binding protein (C/EBP) family, with pathophysiological conditions associated with defective complement function. The first C/EBP protein was identified by Steve McKnight as a heat-stable factor that was interacting with the CCAAT box motif present in several areas of sequence homology with viral enhancers (Ramji and Foka 2002). CHOP protein was identified to be a member of the C/EBPs (Ramji and Foka 2002). Under normal conditions, CHOP is present at low constitutive levels in the cytosol but exhibited a markedly increased expression in response to ER stress. The mid-1990s to late 2000s period was hallmarked by findings providing evidence of CHOP signaling in a variety of pathologies (Fig. 1).
Structure Properties of C/EBP Homologous Protein (CHOP)
CHOP, also known as growth arrest and DNA damage-inducible gene 153 (GADD153), DNA–damage-inducible transcript 3 (DDIT3) and C/EBPζ, is a 29 kDa protein consisting of 169 (human) or 168 (rodents) amino acid residues. CHOP was first identified on the basis of DNA-damage-induced growth arrest. GADD genes are a group of genes that are linked with cell cycle arrest, apoptosis, growth arrest and certain forms of differentiation (Ron and Habener 1992). There are three distinct GADD genes, GADD34, GADD45 and GADD153, but there is no similarity among them (Luethy et al. 1990). C/EBPs define a family of transcription factors that all contain a highly conserved, basic leucine zipper domain at the C-terminus that is implicated in dimerization and DNA binding. C/EBPα, C/EBPβ, and C/EBPδ are activators of transcription allow to transactivate a variety of target genes (Oyadomari and Mori 2004). In contrast, C/EBPγ and C/EBPζ (CHOP) lack transactivation domains and instead act as transdominant repressors of transcription. C/EBPζ serves as a dominant negative inhibitor of C/EBPs. The C/EBPs are essential for phosphorylation-mediated changes in DNA-binding activity, gene transcription, translation, protein–protein interactions, and nuclear localization. Once bound to C/EBPs increase coactivator recruitment (e.g. CBP), that in turn can open up or recruit basal transcription factors (Sylvester et al. 1994).
CHOP promoter consists of a conserved C/EBP binding site, located between positions −339 and −320, through which other C/EBPs interact to transactivate CHOP expression, in liver hepatoma cells response, and in response to sodium arsenite treatment (metabolic stress) of rat pheochromocytoma PC12 cells. A closer examination between C/EBPβ and components of the ATF/CREB family of transcription factors provide insight in the regulation of Gadd153 during the mammalian stress response (Fawcett et al. 1999).
The expression of CHOP is transcriptional and posttranscriptional and is induced by amino acid starvation. Low levels of amino acids enhance CHOP expression independently of cellular stress via the PI3K-Akt-mTOR signaling pathways that converge on the amino-acid-responsive element located between positions −310 and −302. This amino-acid-responsive element related to the C/EBP–ATF-2 composite site and interacts with ATF-2 showing to be essential for the transcriptional activation of CHOP by leucine starvation (Fafaournoux et al. 2000). The −75 to −104 region of the CHOP promoter is required for both constitutive and ER-stress-inducible activation of the CHOP.
The ER stress responsive element consists of two overlapping regions that share conserved regulatory domains and protein-binding sites, present in the promoters of GRP78 (glucose-regulated protein 78), GRP94, PDI (protein disulphide-isomerase), and calreticulin. The transcription factor NF-Y interacts with the serine residues (79 and 82) and mediates constitutive activation of ER-stress gene transcription (Ubeda and Habener 2000).
Contribution of C/EBP Homologous Protein (CHOP) in the Signaling Pathways of ER Stress-Mediated Apoptosis
Expression Profile of CHOP
Endoplasmic Reticulum (ER) Stress and C/EBP Homologous Protein CHOP
UPR is orchestrated by three ER stress sensors: PERK (protein kinase RNA (PKR)-like endoplasmic reticulum kinase), IRE1 (inositol-requiring enzyme 1), and ATF6 (activating transcription factor 6). According to the most accepted model, in resting cells, these ER stress transducers are kept inactive through binding to the ER chaperone BiP. Accumulation of unfolded proteins in the ER lumen promote the dissociation of BiP from the ER stress signal transducers, which triggers activation of the UPR major branches promoting cell survival. However, during persistent or overwhelming ER stress, UPR fails to re-establish the normal function of the ER, and apoptosis is activated (Oyadomari and Mori 2004; Bertolotti et al. 2000).
During ER stress, ATF6, after dissociation of BiP, translocates to the Golgi apparatus, where it is cleaved sequentially by the serine proteases S1P and S2P (site 1 and site 2 proteases, respectively) to release its cytosolic domain and induces target ER genes. Active ATF6 induces ER chaperone proteins and the transcription factors CHOP and X box-binding protein 1 (XBP1) (Fig. 3) (Bertolotti et al. 2000).
Transcriptional Regulation of CHOP
The promoter site of CHOP contains at least two ERSE motifs (CHOP ERSE-1 and CHOP ERSE-2) and one AARE motif. The two ERSE motifs are positioned in opposite directions. These motifs present high homology with other ERSEs identified in the promoters of the GRP94, BiP, PDI, and calreticulin (CRT) genes. CHOP AARE motif is required for amino acid activation of the CHOP promoter. Upon ER stress, ATF4, binds to cis-acting elements (AARE1, AARE2) pATF6 and XBP-1 bind to the CACG part of cis-acting elements (ERSE1 and ERSE2), inducing the transcription of CHOP. The active ATF6 translocates to the nucleus and binds to the ER stress response element of the promoter of various genes to induce expression. ATF2 and ATF4 are shown to be directly involved in the amino acid regulation of CHOP expression. ATF4 binds to the amino acid response element (AARE1 and AARE2) in the promoter region of the target genes. ATF2 and ATF3 are able to bind to the CHOP AARE sequence. However, the role of ATF2 and ATF3 in the increased expression of CHOP upon ER stress requires further research to be clarified. (Fig. 4) NF-Y binds to the CCAAR part of ERSE1 and ERSE2 constitutively (Oyadomari and Mori 2004; Bertolotti et al. 2000).
Roles of CHOP in the Apoptotic Pathways
New Insights into the Role of CHOP/GADD153 in Disease
Recent evidence has shown that the stress of the ER is intrinsically related to the pathogenesis of Type 2 Diabetes (T2D) by a variety of mechanisms. Among them of particular significance are the increased demands for insulin production and secretion that induce ER stress in the pancreatic β cells that ultimately die of apoptosis (Oakes and Papa 2014). Under ER stress the UPR response is initially adaptive and aims to restore tissue homeostasis while at subsequent stages triggers apoptosis (Oakes and Papa 2014). In regulating gene expression, CHOP competes with the other c/EBPs and displaces them from DNA, operating as an inhibitor of transcription. During diabetes, CHOP-associated apoptosis compromises the overall functionality of the pancreatic β cells as implied by the resistance of CHOP-deficient mice to diabetes. This observation illustrates the contribution of CHOP and the relevance of ER stress-related apoptosis in the development of diabetes. Thus, it is conceivable that interference with ER function causes apoptosis in pancreatic β cells may be beneficial since it could counteract the progressive loss of β cell mass (Oakes and Papa 2014).
Diabetogenic UPR dysregulation is evident in PERK-knockout mice which develop progressive apoptosis in their β-cells and show progressive failure of pancreatic β-cells to secrete insulin. A rare human diabetic syndrome (Wolcott-Rallison) is caused by PERK null gene mutations, with severe infant diabetes caused by pancreatic hypoplasia and frequently a reduced number of β-cells (Oakes and Papa 2014). Recent studies indicate that Chop deletion in mouse models of T2D had improved glycemic control and expanded β cell mass, prevented glucose intolerance and induced cell survival. In addition, isolated islets from wild type mice displayed increased expression of UPR and oxidative stress response genes highlighting that CHOP is a fundamental factor that links ER and apoptosis in β-cells under conditions of increased insulin demand (Oakes and Papa 2014).
Recently, published observations showed that CHOP mediates the expression of the cell cycle regulator with antiapoptotic activity p21 (WAF1) (Mihailidou et al. 2010, 2014). Deficiency of p21 sensitizes pancreatic β-cells to glucotoxicity. Conversely, pharmacological stimulation of p21 expression, by an inhibitor of p53–MDM2 interaction (nutlin-3a), improves pancreatic function by increasing β-cell mass and by restoring the function of the pancreas. These findings indicate that stimulation of p21 activity can be beneficial for the diabetes management (Mihailidou et al. 2015).
Protein misfolding and accumulation is involved in the pathogenesis of neurodegenerative disorders. Neurodegeneration is a collective term describing progressive neuronal dysfunction and death (Alzheimer’s, Parkinson’s, and Huntington’s diseases). Many mutations in functional proteins are involved in the induction of CHOP. Mutations in presenilin-1, which increase the b-amyloid levels, were shown to induce CHOP protein and generate oxidative damage via ROS sensitizing neuronal cells to apoptosis using presenilin-1 mutant knock in mice. CHOP is involved in cell death induced by neurotoxins such as 6-hydroxydopamine and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Evidence suggested that mutations in Parkin gene result in loss of ubiquitin-protein ligase activity, which is one the most common causes of familial Parkinson’s disease (Oakes and Papa 2014).
Although the overexpression of CHOP promotes apoptosis in several cell lines, whether CHOP ultimately inhibits or induces tumor growth in patients remains an area of intense research. In a human glioma model, it has been demonstrated that genetic deletion of IRE1α resulted in smaller tumors and reduced angiogenesis. Low levels of CHOP contribute to the prosurvival effects of VEGF (Oakes and Papa 2014).
Mice expressing a transgene of Xbp1s (that is missing the 26-nt intron) in B lymphocytes resulted in myeloma. There is also strong evidence that proteasome inhibition with bortezomib (Velcade) leads to myeloma cell death in part by preventing disposal of misfolded proteins via the ERAD pathway and thus activating ER stress–induced apoptosis via CHOP. Although the above findings propose an oncogenic role for XBP1s in the development of myeloma, the recent findings have emerged that challenge this notion. It was recently reported that genetic ablation of IRE1α or XBP1 in human myeloma cell lines is well tolerated and leads to bortezomib resistance, challenging the rationale for using IRE1α inhibitors in this disease (Oakes and Papa 2014). Recent findings have shown that ATF4 and CHOP participate in the regulation of ER stress-mediated autophagy which is a self-cannibalization process whereby cytoplasmic materials and organelles are engulfed through lysosomal degradation is capable of inducing programmed cell death (Kania et al. 2015).
Since our understanding of UPR, knowledge of how ER is triggering specific cellular responses has accumulated. Numerous physiological and pathological conditions, as well as a variety of pharmacological agents, that disrupt ER function lead to accumulation of unfolded proteins and therefore pose the risk of proteotoxicity. The cell reacts to ER stress by triggering a set of transcriptional and translational events, which constitute the UPR response, an ER quality control system aimed at adaptation and safeguarding cellular survival. However, in cases of excessively severe stress, or malfunction of the ER quality control system, cells commit to self-destruction by apoptosis. Perturbation of the ER is a powerful inducer of CHOP. CHOP is the first identified protein which has been characterized as one of the most important mediators of ER stress-response machinery that induces apoptosis. CHOP is also considered as a key contributor to a growing list of several conformational disorders. Significant information has started to emerge into the critical downstream mediators of CHOP-induced apoptosis and implying the design of “smart” combinatorial therapies and new areas of potential clinical application. Although significant information has started to emerge for the abnormal expression of CHOP in ER stress, future work, resulting in a better understanding of CHOP pathways regulated, is clearly an avenue of research worth exploring.
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