Aromatic hydrocarbons upregulate glyceraldehyde-3-phosphate dehydrogenase and induce changes in actin cytoskeleton. Role of the aryl hydrocarbon receptor (AhR)
Introduction
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a halogenated aromatic hydrocarbon and an environmental contaminant. Exposure to TCDD, the most toxic dioxin, results in several deleterious effects, including wasting syndrome, immunotoxicity, hepatotoxicity, teratogenicity, and cancer (Huff et al., 1994). These effects are mediated by the aryl hydrocarbon receptor (AhR), a ligand-activated receptor that is a member of the basic helix–loop–helix–PAS (bHLH-Per-Arnt-Sim) family of transcription factors. Upon binding TCDD, the AhR translocates to the nucleus, dimerises with the AhR nuclear-translocator protein (ARNT), and binds dioxin responsive elements (DRE, also known as xenobiotic responsive elements or XREs) found at the regulatory sequences of responsive genes. This chain of events results in upregulation of the expression of a battery of genes encoding xenobiotic-metabolizing enzymes, such as cytochrome P450s (CYP1A1, CYP1A2, CYP1B1), NAD(P)H quinone oxydoreductase, and UDP-glucoronosyl-transferase-6 (Gonzalez and Fernandez-Salguero, 1998). Although the AhR function as part of an adaptive chemical response, several studies suggest that this transcription factor may have important functions in liver and cardiac development (Fernandez-Salguero et al., 1995, Fernandez-Salguero et al., 1997), cell proliferation (Elizondo et al., 2000), immune homeostasis (Rodriguez-Sosa et al., 2005), circadian rhythmicity, and cholesterol and glucose metabolism (Sato et al., 2008).
Evidence has shown that TCDD can disrupt glucose metabolism at multiple levels. In mice, TCDD alters gluconeogenesis by decreasing pyruvate carboxylase levels in an AhR-dependent manner (Ryu et al., 1995). More recently, it was reported that TCDD-treated rats present an increase in glycogen content and glucose transporter 3 (GLUT3) mRNA levels in the placenta (Ishimura et al., 2002). In contrast, TCDD downregulates the expression levels of GLUT1 and 3 in pluripotent P19 mouse embryonic carcinoma cells (Tonack et al., 2007). Moreover, several epidemiological studies indicate that exposure to TCDD increases the risk of diabetes mellitus by, among other mechanisms, decreasing glucose uptake (Longnecker and Michalek, 2000).
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays an important role in glucose metabolism and is involved in some complications related to diabetes. GAPDH is a glycolytic enzyme, with a high degree of conservation across species, which catalyses the conversion of glyceraldehyde-3-phosphate to 1,3-diphosphoglycerate. Although GAPDH is considered a constitutive housekeeping gene because its transcription levels remain constant under most experimental conditions, its expression is regulated by several factors and circumstances, such as glucose (Roche et al., 1997), 1α,25-dihydroxyvitamin D3 (Desprez et al., 1992), hypoxia (Graven et al., 2003), insulin (Nasrin et al., 1990), calcium (Chao et al., 1990), and cell proliferation (Mansur et al., 1993).
Recently, several investigations have revealed that GAPDH is involved in different cellular processes besides glycolysis. For instance, GAPDH participates in membrane transport, microtubule assembly, DNA replication and repair, and nuclear RNA export (Sirover, 1999). It also acts as a pro-apoptotic molecule in age-related neuronal disorders and as a sensor of nitric oxide stress (Chuang et al., 2005). Therefore, alterations in GAPDH levels may disturb multiple cellular pathways.
Xenobiotics such as TCDD also have the potential to modify cellular levels of GAPDH as described in human epidermal keratinocytes (McNulty and Toscano, 1995). The goal of the present study was to determine the effect of TCDD and β-naphthoflavone on GAPDH expression and function, as well as to evaluate the role of the AhR on the regulation of GAPDH transcription. Our results suggest that TCDD increase GAPDH mRNA and protein levels via an AhR-dependent mechanism, resulting in an increase in GAPDH activity and in β-actin polymerisation.
Section snippets
Materials
Mouse hepatoma cells (Hepa-1) were purchased from ATCC (Manassas, VA, USA). TCDD was purchased from AccuStandart, Inc. (New Haven, CT). β-naphthoflavone, Triton X-100, sodium fluoride, sodium arsenate and NAD were purchased from Sigma (St. Louis, MO). TCDD and β-naphthoflavone were dissolved in corn oil and DMSO, respectively.
Animals
AhR-null mice were provided by Frank J. Gonzalez (NIH, Bethesda, MD). The generation of AhR-null mice has been previously described (Fernandez-Salguero et al., 1995).
Results
Although GAPDH is commonly thought of as a constitutive housekeeping gene, its induction by several compounds or physiological conditions may make this gene an inappropriate control for RNA quantification under certain circumstances. Moreover, induction of GAPDH by xenobiotics, such as AhR ligands, may alter cellular homeostasis, since this enzyme is involved in several biological processes. In this study, we first determined the effect of TCDD, an AhR ligand, on GAPDH mRNA levels. Fig. 1 shows
Discussion
In the present study, we show that TCDD induce GAPDH mRNA and protein levels as well as enzymatic activity. These results are in agreement with a previous report where GAPDH activity was increased in cultured human keratinocytes after treatment with TCDD (McNulty and Toscano, 1995). However, more recently in vivo studies suggested that GAPDH expression is not altered by TCDD treatment (Pohjanvirta et al., 2006). This discrepancy may be explained by several factors. For instance, the latter
Conflict of interest
None.
Acknowledgment
This work was supported by CONACYT grant 48786.
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