CHOP
Overview
Newly synthesized proteins translocated into the lumen of the endoplasmic reticulum (ER client proteins) are folded, post-translationally modified, assembled into oligomeric complexes, and ultimately exported. This load, which can be very heavy in secretory cells, imposes a physiological ER stress that is counteracted by a stereotyped set of adaptations collectively known as the unfolded protein response (UPR) (Patil and Walter 2001; Harding et al. 2002; Kaufman 2002). The UPR adapts the capacity of the secretory system to the load of client proteins by transcriptional up-regulation of genes that function in all aspects of ER client protein processing and metabolism. The UPR also transiently attenuates client protein synthesis, reducing the load on the organelle; but ultimately, the cell strives to defend and promote physiological levels of secretory activity. The importance of the UPR to secretory cell homeostasis is revealed by the phenotype of mutations affecting UPR signaling; these are associated with enhanced cell death and with defective secretory capacity (Harding et al. 2000b, 2001; Scheuner et al. 2001; Shen et al. 2001; Zhang et al. 2002). (1)
Death is also conspicuous in cells with a normally functioning UPR that have encountered insurmountable ER stress. Thus, exposure to the glycosylation inhibitor tunicamycin, to calcium ionophores that deplete ER calcium stores, or to reducing agents such as dithiothreitol that block disulfide bond formation all lead to ER stress and cell death (Kaufman 1999). Even more informative is the phenotype of certain toxic gain-of-function mutations that compromise the folding of abundantly expressed ER client proteins and lead to death of the producing cell. Examples include the C92 → Y mutation in insulin 2 in the Akita mouse model of early onset Diabetes Mellitus (Oyadomari et al. 2002b) and mutations affecting the folding of myelin constituents that lead to oligodendrocyte death (Gow et al. 1998). (2)
These pharmacological models and rare diseases point to the potential lethal consequences of ER stress. ER stress also accompanies common pathophysiological conditions, such as tissue ischemia (Paschen and Doutheil 1999; Kumar et al. 2001), viral infection (Jordan et al. 2002; Su et al. 2002), free cholesterol loading of macrophages in atherosclerotic lesions (Feng et al. 2003), and even β-cell exhaustion in Diabetes Mellitus (Oyadomari et al. 2002a). Various downstream effectors of cell death induced by ER stress have been identified (Nakagawa et al. 2000; Rao et al. 2001; Wei et al. 2001); however, the proximal steps that mobilize them during ER stress are poorly understood. (3)
CHOP
C/EBP homologous protein (CHOP) is a transcription factor that regulates apoptosis in response to cellular stress. CHOP also known as growth arrest and DNA damage 153 (GADD153) was first cloned because of its induction in response to genotoxic stress such as UV irradiation. CHOP has now been shown to be induced mainly by ER-stress (4). CHOP is normally expressed at low levels and localizes to the cytoplasm. Cellular stress triggers an upregulation of CHOP levels and accumulation in the nucleus where it can act as either a transcriptional repressor or activator (5). CHOP contains an N-terminal transcriptional activation domain and a C-terminal basic leucine zipper domain responsible for DNA binding (4). In the nucleus CHOP forms heterodimers with C/EBP family transcription factors to either enhance promoter binding or to inhibit their activity. In this manner CHOP regulates genes involved in cell survival and death (5). ER-stress induced apoptosis mediated through CHOP is implicated in various diseases including diabetes and neurodegeneration. In these cases inactivation of CHOP may provide a useful tool for blocking the enhanced apoptosis observed in these diseases (4).
Antibodies
CHOP/GADD153 antibodies have provided excellent tools to study ER-stress induced apoptosis. Lee et al. explored the use of autophagy and ER-stress related markers as prognostic indicators in hepatocellular carcinoma (2). In their study they performed immunohistochemisty using the CHOP/GADD153 antibody along with antibodies for LC3, Beclin 1, and GRP78 (6). While strong positive correlations with these markers were not observed, LC3 expression did correlate with good prognosis (5). Lin et al. used the CHOP/GADD153 antibody in their characterization of temozolomide, a chemotherapeutic drug used in the treatment of brain tumors . They monitored CHOP/GADD153 levels through western blotting to examine ER-stress induction in response to temozolomide . A group from the University of Washington sought to examine the role of hIAPP in the development of type II diabetes (4). Previous studies had shown induction of ER-stress by hIAPP expression. However, the authors of this study immunostained pancreatic islet cells with the CHOP/GADD153 antibody to show ER-stress is not induced at physiological levels of hIAPP (4). The Ron group at New York University identified carbonic anhydrase VI (CA-VI) as the first positively upregulated CHOP target gene (5). Using the CHOP/GADD153 antibody for western blotting the authors were able to show CHOP dependent induction of CA-VI (7). They then went on to show CHOP is present at the CA-VI promoter using in vitro footprinting assays.
Functions
It is likely that CHOP sensitizes cells to ER stress-mediated death by directly regulating target genes in the nucleus, as mutations affecting dimerization or DNA binding neutralize CHOP (Ubeda et al. 1996; McCullough et al. 2001). However, the identities of these target genes and the manner by which they might be related to death of ER stressed cells remain unknown. (8,9)
Relevance
Some studies have used expression profiling to identify potential CHOP target genes and have explored the leads provided by this analysis to better characterize the phenotype of CHOP knockout cells and animals. Some have reported on the surprising observation that CHOP target genes promote ER client protein load and an oxidative environment in the organelle. Thus, CHOP's role in the death of ER-stressed cells is better explained by its proximal effects on ER function than by distal interactions with the cell-death machinery. These findings have important implications for the function of the mammalian UPR.
Footnotes
- Papathanasiou MA, Kerr NC, Robbins JH, McBride OW, Alamo I Jr, Barrett SF, Hickson ID, Fornace AJ Jr (March 1991). "Induction by ionizing radiation of the gadd45 gene in cultured human cells: lack of mediation by protein kinase C". Mol Cell Biol 11 (2): 1009–16. PMC 359769. PMID 1990262.
- "Entrez Gene: DDIT3 DNA-damage-inducible transcript 3".
- Chen BP, Wolfgang CD, Hai T (March 1996). "Analysis of ATF3, a transcription factor induced by physiological stresses and modulated by gadd153/Chop10". Mol. Cell. Biol. 16 (3): 1157–68. PMC 231098. PMID 8622660.
- Ubeda M, Vallejo M, Habener JF (November 1999). "CHOP enhancement of gene transcription by interactions with Jun/Fos AP-1 complex proteins". Mol. Cell. Biol. 19 (11): 7589–99. PMC 84780. PMID 10523647.
- Hattori T, Ohoka N, Hayashi H, Onozaki K (April 2003). "C/EBP homologous protein (CHOP) up-regulates IL-6 transcription by trapping negative regulating NF-IL6 isoform". FEBS Lett. 541 (1-3): 33–9. doi:10.1016/s0014-5793(03)00283-7. PMID 12706815.
- Fawcett TW, Eastman HB, Martindale JL, Holbrook NJ (June 1996). "Physical and functional association between GADD153 and CCAAT/enhancer-binding protein beta during cellular stress". J. Biol. Chem. 271 (24): 14285–9. doi:10.1074/jbc.271.24.14285. PMID 8662954.
- Ubeda M, Habener JF (October 2003). "CHOP transcription factor phosphorylation by casein kinase 2 inhibits transcriptional activation". J. Biol. Chem. 278 (42): 40514–20. doi:10.1074/jbc.M306404200. PMID 12876286.
- Cui K, Coutts M, Stahl J, Sytkowski AJ (March 2000). "Novel interaction between the transcription factor CHOP (GADD153) and the ribosomal protein FTE/S3a modulates erythropoiesis". J. Biol. Chem. 275 (11): 7591–6. doi:10.1074/jbc.275.11.7591. PMID 10713066.
- CHOP genes in myxoid liposarcoma". Oncogene 12 (3): 489–94. PMID 8637704.
