Effect of arsenic on transcription factor AP-1 and NF-κB DNA binding activity and related gene expression
Introduction
Extensive studies have shown that arsenic is a major environmental contaminant and is associated with the occurrence of adverse health effects such as skin alterations, peripheral vascular disease and cancer, including skin, lung and bladder cancer (Sommers and McManus, 1953, Hu et al., 1988, Chiang et al., 1993, Chiou et al., 1995, Tsuda et al., 1995, Tseng et al., 1996, Smith et al., 1998, Chiou et al., 2001). On the other hand, arsenic compounds have been used as medicinals for many years. Recently, arsenic trioxide has been used as an effective therapeutic for acute promyelocytic leukemia (Chen et al., 2001a, Chen et al., 2001b, Chen et al., 2001c). However, the mechanism for carcinogenesis induced by arsenic is still unclear. Interference with signal transduction pathways is one of several modes of action that have been proposed for arsenic as a genotoxicant and carcinogen (Porter et al., 1999, Trouba et al., 2000, Kitchin, 2001, Vega et al., 2001).
Alterations in intracellular oxidation/reduction (redox) reactions have been shown to activate signal cascades that regulate early response genes. These genes are believed to function in a protective or reparative capacity. Stress-induced activation of these early response genes appears to rely, at least in part, on changes in intracellular redox status. Transcription factors, such as activating protein-1 (AP-1) and nuclear factor kappa B (NF-κB), play a very important role in these responses. Both AP-1 and NF-κB are considered stress response transcription factors, which regulate the expression of a variety downstream target genes, such as pro-inflammatory genes that are known to be involved with cellular anti-oxidant defence mechanisms (Kapahi et al., 2000). NF-κB is maintained in the cytoplasm of non-stimulated cells through interaction with specific inhibitors, IκBs (Delhase et al., 1999). In response to pro-inflammatory stimuli, the IκBs are rapidly phosphorylated and degraded by ubiquitin-dependent proteolysis, resulting in the release of free NF-κB dimers, which translocate to the nucleus to induce transcription of target genes (Karin and Delhase, 2000). In contrast, the AP-1 heterodimers are constitutively localised within the nucleus and transactivation of AP-1 is achieved through phosphorylation of its activation domain by c-Jun N-terminal kinase (JNK) (Hsu et al., 2000).
There are two levels of modulation of these transcription factor activities by redox status: induction and translocation of the transcription factor and transcription factor DNA binding activity (Galter et al., 1994, Schenk et al., 1994, Sen and Packer, 1996, Hirota et al., 1997, Jin et al., 1997, Nakamura et al., 1997). Both of these processes can be modulated by reactive oxygen species (ROS) and redox proteins such as thioredoxin (Trx) and Redox activating factor-1 (Ref-1). Trx is a small multi-functional protein that has a conserved redox-active disulfide at the active site. It has been reported that Trx functions both intra-cellularly and extra-cellularly as one of the key regulators of signalling in the cellular responses against various stresses (Nakamura et al., 1997, Kumar and Holmgren, 1999, Arner and Holmgren, 2000). Trx translocates from the cytosol into the nucleus in response to a variety of cellular stresses and regulates the activity of DNA binding proteins, including Jun/Fos and NF-κB (Tanaka et al., 1997, Hirota et al., 1999, Tanaka et al., 2000). Trx also interacts with intracellular Ref-1 (also known as APE/Ref-1) which is a bifunctional protein with both DNA repair activity and redox activity. Ref-1 also enhances the activity of Jun/Fos, and consequently regulates the expression of various genes (Hirota et al., 1997, Diamond et al., 1999).
Arsenic has been shown previously to affect transcription and expression of proto-oncogenes in mammalian cells in culture (Barchowsky et al., 1996, Germolec et al., 1996, Parrish et al., 1999, Porter et al., 1999, Kapahi et al., 2000). However, many previous studies used only short-term exposures and significantly higher (more toxic) doses of arsenic. There is limited evidence showing how long term exposure with low dose (sub-micro-molar) arsenic affects signal transduction pathways. Chronic exposure to low doses is more representative of human exposure to arsenic.
We investigated the effect of long-term and short-term exposure to arsenic on the DNA binding activity of transcription factors AP-1 and NF-κB and the expression of related genes such as Trx, APE/Ref-1, c-Jun, c-fos. We also explored the significance of interactions between arsenic and other pro-oxidant treatments, such as phorbol 12-myristate 13-acetate (TPA) and hydrogen peroxide (H2O2).
Section snippets
Chemicals
Sodium arsenite (NaAsO2) and c-fos, c-jun, thioredoxin, and APE/Ref-1 PCR primers were purchased from Sigma Chemical Co. (St. Louis, MO). Oligonucleotide probes having the consensus-DNA binding sequences for AP-1 and NF-κB were purchased from Promega (Madison, WI). Anti-c-Jun, c-Fos, and APE/Ref-1 antibodies were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA). All other chemicals were purchased from Sigma Chemical Co.
Cell culture and chemical treatment
Human GM847 fibroblast cells (from the Murdoch Institute,
Cellular toxicity
We used the neutral red dye uptake assay to measure cytotoxicity in GM847 cells after 24 h treatment with As(III). The initial results indicated that the dose required to reduce viability by 50% (IC50) after a 24 h exposure to As(III) in this cell line is 50 μM. We then used sub-toxic concentrations of 0.1 and 0.5 μM As(III) for long-term treatment with arsenic, as described in the methods. We found that after 50–120 days treatment with these low concentrations of arsenic, human fibroblast
Discussion
Arsenic is a well-known co-mutagen and a human carcinogen. Epidemiological studies indicate that long-term arsenic exposure results in increased risk for human cancer of the skin, respiratory tract, and urinary bladder via inhalation and ingestion (Sommers and McManus, 1953, Chiang et al., 1993, Chiou et al., 1995, Tsuda et al., 1995, Chiou et al., 2001).
Several hypotheses have been proposed to describe the mechanism of arsenite-induced carcinogenesis (Simeonova and Luster, 2000, Kitchin, 2001)
Acknowledgements
This work was supported in part by the US Environmental Protection Agency's Science Achieve Results (STAR) program; the Electric Power Research Institute, Contract No. EP-P4898/C2396; the New York University School of Medicine's NIEHS Center (ES00260); the Kaplan Comprehensive Cancer Center of NYU Medical Center; and the Centre for Cellular and Molecular Biology, School of Biological and Chemical Sciences, Deakin University, Australia.
References (57)
- et al.
Arsenic induces oxidant stress and NF-kappa B activation in cultured aortic endothelial cells
Free Radic. Biol. Med.
(1996) - et al.
Stimulation of reactive oxygen, but not reactive nitrogen species, in vascular endothelial cells exposed to low levels of arsenite
Free Radic. Biol. Med.
(1999) - et al.
Low levels of arsenic trioxide stimulate proliferative signals in primary vascular cells without activating stress effector pathways
Toxicol. Appl. Pharmacol.
(1999) - et al.
Opposite effect of NF-kappa B and c-Jun N-terminal kinase on p53-independent GADD45 induction by arsenite
J. Biol. Chem.
(2001) c-Jun NH2-terminal kinase-mediated redox-dependent degradation of IkappaB: role of thioredoxin in NF-kappaB activation
J. Biol. Chem.
(2001)- et al.
Redox factor-1 (Ref-1) mediates the activation of AP-1 in HeLa and NIH 3T3 cells in response to heat shock
J. Biol. Chem.
(1999) - et al.
Redox regulation of NF-kappa B activation
Free Radic. Biol. Med.
(1997) - et al.
Arsenic induces overexpression of growth factors in human keratinocytes
Toxicol. Appl. Pharmacol.
(1996) - et al.
Distinct roles of thioredoxin in the cytoplasm and in the nucleus. A two-step mechanism of redox regulation of transcription factor NF-kappaB
J. Biol. Chem.
(1999) - et al.
An endogenous redox molecule, thioredoxin, regulates transactivation of epidermal growth factor receptor and activation of NF-kappaB by lysophosphatidic acid
FEBS Lett.
(2001)