Showing papers on "Transcription Factor CHOP published in 2001"

Feedback Inhibition of the Unfolded Protein Response by GADD34-Mediated Dephosphorylation of eIF2α

Abstract: Phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2α) on serine 51 integrates general translation repression with activation of stress-inducible genes such as ATF4, CHOP, and BiP in the unfolded protein response. We sought to identify new genes active in this phospho-eIF2α–dependent signaling pathway by screening a library of recombinant retroviruses for clones that inhibit the expression of a CHOP::GFP reporter. A retrovirus encoding the COOH terminus of growth arrest and DNA damage gene (GADD)34, also known as MYD116 (Fornace, A.J., D.W. Neibert, M.C. Hollander, J.D. Luethy, M. Papathanasiou, J. Fragoli, and N.J. Holbrook. 1989. Mol. Cell. Biol. 9:4196–4203; Lord K.A., B. Hoffman-Lieberman, and D.A. Lieberman. 1990. Nucleic Acid Res. 18:2823), was isolated and found to attenuate CHOP (also known as GADD153) activation by both protein malfolding in the endoplasmic reticulum, and amino acid deprivation. Despite normal activity of the cognate stress-inducible eIF2α kinases PERK (also known as PEK) and GCN2, phospho-eIF2α levels were markedly diminished in GADD34-overexpressing cells. GADD34 formed a complex with the catalytic subunit of protein phosphatase 1 (PP1c) that specifically promoted the dephosphorylation of eIF2α in vitro. Mutations that interfered with the interaction with PP1c prevented the dephosphorylation of eIF2α and blocked attenuation of CHOP by GADD34. Expression of GADD34 is stress dependent, and was absent in PERK−/− and GCN2−/− cells. These findings implicate GADD34-mediated dephosphorylation of eIF2α in a negative feedback loop that inhibits stress-induced gene expression, and that might promote recovery from translational inhibition in the unfolded protein response.

Nitric oxide-induced apoptosis in pancreatic β cells is mediated by the endoplasmic reticulum stress pathway

Abstract: Excessive nitric oxide (NO) production in cytokine-activated β cells has been implicated in β cell disruption in type 1 diabetes. β cells are very vulnerable to NO-induced apoptosis. However, the mechanism underlying this phenomenon is unclear. Low concentrations of NO that lead to apoptosis apparently do not cause severe DNA damage in mouse MIN6 β cells. CHOP, a C/EBP homologous protein that is induced by endoplasmic reticulum (ER) stress and plays a role in growth arrest and cell death, was induced by a NO donor, S-nitroso-N-acetyl-d,l-penicillamine (SNAP). SNAP increased cytosolic Ca2+, and only agents depleting ER Ca2+ induced CHOP expression and led to apoptosis, suggesting that NO depletes ER Ca2+. Overexpression of calreticulin increased the Ca2+ content of ER and afforded protection to cells against NO-mediated apoptosis. Furthermore, pancreatic islets from CHOP knockout mice showed resistance to NO. We conclude that NO depletes ER Ca2+, causes ER stress, and leads to apoptosis. Thus, ER Ca2+ stores are a new target of NO, and the ER stress pathway is a major mechanism of NO-mediated β cell apoptosis.

Inhibition of CHOP translation by a peptide encoded by an open reading frame localized in the chop 5′UTR

Abstract: Chop is a ubiquitously expressed mammalian gene encoding a small nuclear protein related to the CCAAT/enhancer-binding protein (C/EBP) family of transcription factors. CHOP protein plays an important role in various cellular processes such as growth, differentiation and programmed cell death. CHOP expression is strongly increased in response to a large variety of stresses including perturbation of the endoplasmic reticulum function, DNA damage and nutrient deprivation. Multiple mechanisms including transcriptional and post-transcriptional controls are involved in the regulation of CHOP expression. We show here that the 5′UTR of the Chop transcript plays an important role in controlling the synthesis of CHOP protein. In particular, the 5′UTR contains a conserved uORF which encodes a 31 amino acid peptide that inhibits the expression of the downstream ORF. Mutational analysis of the 5′ leader region and peptide coding sequences suggests that the peptide itself inhibits expression of the downstream ORF. Such results suggest a role for uORF in limiting ribosomal access to downstream initiation sites. With respect to the importance of CHOP protein in the regulation of cellular functions, the mechanisms that regulate its basal level are of considerable interest.

Induction of the C/EBP homologous protein (CHOP) by amino acid deprivation requires insulin-like growth factor I, phosphatidylinositol 3-kinase, and mammalian target of rapamycin signaling.

Abstract: In mammalian cells, gene regulation by amino acid deprivation is poorly understood. Here, we examined the signaling pathways involved in the induction of the C/EBP homologous protein (CHOP) by amino acid starvation. CHOP is a transcription factor that heterodimerizes with other C/EBP family members and may inhibit or activate the transcription of target genes depending on their sequence-specific elements. Amino acid deficiency, when accompanied by insulin-like growth factor I signaling, results in the accumulation of CHOP messenger RNA and protein in AKR-2B and NIH-3T3 cells. The phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 are able to block CHOP induction in response to amino acid deprivation. Rapamycin is also able to abrogate CHOP expression, suggesting that the mammalian target of rapamycin is involved in CHOP induction by amino acid deficiency. LY294002 and rapamycin are also able to block CHOP induction by hydrogen peroxide, but do not affect expression induced by sodium arsenite or A23187. This is the first evidence that the insulin-like growth factor I/phosphatidylinositol 3-kinase/mammalian target of rapamycin pathway is required for gene regulation by amino acid deprivation and that this pathway is involved in the induction of CHOP by both amino acid deficiency and oxidative stress by hydrogen peroxide. (Endocrinology 142: 221‐228, 2001)

Nuclear Factor 1 Is a Negative Regulator of gadd153 Gene Expression in Vascular Smooth Muscle Cells

Abstract: Growth arrest and DNA damage inducible gene 153 (gadd153) is expressed at very low levels in growing cells but is markedly induced in response to cellular stresses, including glucose deprivation, exposure to genotoxic agents, and other growth-arresting situations. Forced expression of GADD153 can induce cell cycle arrest and/or apoptosis in many types of cells. Recently, we reported that GADD153 was induced in vascular smooth muscle cells (VSMCs) in neointimal lesions of balloon-injured carotid arteries. To investigate the underlying molecular mechanisms of gadd153 gene expression in VSMCs, we isolated and characterized a promoter region of the rat gadd153 gene. Sequence alignments of this region revealed 1 TATA-like sequence and several well-known cis elements. The 5'-deletion analysis for this region showed that a domain spanning -447 through -368 drastically reduced the promoter activity to almost equal levels of promoterless control. Because this domain contained a consensus sequence for the nuclear factor 1 family of proteins (NF1), DNA-binding studies were performed by use of 2 types of NF1 consensus probes. Both probes were specifically shifted by nuclear extracts from proliferating VSMCs and were supershifted by antiserum against CCAAT transcription factor/NF1. In addition, promoter activity of a mutant luciferase vector, which was generated by a point mutation at the NF1 binding motif of the gadd153 gene, was 14-fold higher than that of a wild-type one. These results suggest that gadd153 gene expression in VSMCs is negatively regulated by an NF1-binding motif, and NF1 may act as an antiapoptotic factor by continuously suppressing gadd153 gene expression in growing VSMCs.

Peptides derived from pro-growth hormone-releasing hormone activate p38 mitogen-activated protein kinase in GH3 pituitary cells.

Abstract: Posttranslational processing of the pro-growth hormone-releasing hormone (proGHRH) peptide can result in the formation of at least two peptide products: GHRH and the C-terminal peptide, GHRH-related peptide (GHRH-Rp). While cyclic adenosine monophosphate transduces many of the actions of GHRH, other pathways also have been implicated in its actions. The aims of this study were to examine and characterize the activation of mitogen-activated protein kinase (MAPK) pathways by GHRH, and GHRH-RP in pituitary-derived GH3 cells, as well as the activation of the transcription factors that serve as substrates for these kinases. GHRH rapidly increased p44/p42 MAPK activity in GH3 cells in a protein kinase A-dependent and a protein kinase C-independent manner and stimulated the activation of the transcription factor Elk-1. By contrast, GHRH-RP, and p75-92NH2 had no effect on p44/p42 MAPK phosphorylation in these cells. Additionally, we determined that all three peptides, GHRH, GHRH-RP, and p75-92NH2, rapidly and specifically increase phosphorylation of p38 MAPK and stimulate the activation of the nuclear factor CHOP. These are the first studies to demonstrate the activation of Elk-1 by GHRH and the activation of p38 MAPK and CHOP by GHRH, GHRH-RP, and p75-92NH2. We conclude, that members of the GHRH family of peptides differentially activate multiple intracellular signaling pathways and suggest that the biologic actions of GHRH may be far more diverse than previously thought.