Elsevier

Toxicology

Volume 266, Issues 1–3, 21 December 2009, Pages 30-37
Toxicology

Aromatic hydrocarbons upregulate glyceraldehyde-3-phosphate dehydrogenase and induce changes in actin cytoskeleton. Role of the aryl hydrocarbon receptor (AhR)

https://doi.org/10.1016/j.tox.2009.10.009Get rights and content

Abstract

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional enzyme involved in several cellular functions including glycolysis, membrane transport, microtubule assembly, DNA replication and repair, nuclear RNA export, apoptosis, and the detection of nitric oxide stress. Therefore, modifications in the regulatory ability and function of GAPDH may alter cellular homeostasis. We report here that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and β-naphthoflavone, which are well-known ligands for the aryl hydrocarbon receptor (AhR), increase GAPDH mRNA levels in vivo and in vitro, respectively. These compounds fail to induce GAPDH transcription in an AhR-null mouse model, suggesting that the increase in GAPDH level is dependent upon AhR activation. To analyse the consequences of AhR ligands on GAPDH function, mice were treated with TCDD and the level of liver activity of GAPDH was determined. The results showed that TCDD treatment increased GAPDH activity. On the other hand, treatment of Hepa-1 cells with β-naphthoflavone leads to an increase in microfilament density when compared to untreated cultures. Collectively, these results suggest that AhR ligands, such as polycyclic hydrocarbons, can modify GAPDH expression and, therefore, have the potential to alter the multiple functions of this enzyme.

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.

References (41)

  • E. Roche et al.

    Induction by glucose of genes coding for glycolytic enzymes in a pancreatic beta-cell line (INS-1)

    J. Biol. Chem.

    (1997)
  • M. Rodriguez-Sosa et al.

    Over-production of IFN-gamma and IL-12 in AhR-null mice

    FEBS Lett.

    (2005)
  • S. Sato et al.

    Low-dose dioxins alter gene expression related to cholesterol biosynthesis, lipogenesis, and glucose metabolism through the aryl hydrocarbon receptor-mediated pathway in mouse liver

    Toxicol. Appl. Pharmacol.

    (2008)
  • H.D. Schmitz et al.

    Glyceraldehyde-3-phosphate dehydrogenase associates with actin filaments in serum deprived NIH 3T3 cells only

    Cell Biol. Int.

    (2002)
  • M.A. Sirover

    New insights into an old protein: the functional diversity of mammalian glyceraldehyde-3-phosphate dehydrogenase

    Biochim. Biophys. Acta

    (1999)
  • T.H. Thatcher et al.

    Aryl hydrocarbon receptor-deficient mice develop heightened inflammatory responses to cigarette smoke and endotoxin associated with rapid loss of the nuclear factor-kappaB component RelB

    Am. J. Pathol.

    (2007)
  • K.E. Thompson et al.

    Differences between rats and mice in the involvement of the aryl hydrocarbon receptor in 4-vinylcyclohexene diepoxide-induced ovarian follicle loss

    Toxicol. Appl. Pharmacol.

    (2005)
  • E.J. Tisdale

    Glyceraldehyde-3-phosphate dehydrogenase is phosphorylated by protein kinase Ciota/lambda and plays a role in microtubule dynamics in the early secretory pathway

    J. Biol. Chem.

    (2002)
  • L. Xu et al.

    2,3,7,8 Tetrachlorodibenzo-p-dioxin induction of cytochrome P4501A in cultured rat and human hepatocytes

    Chem. Biol. Int.

    (2000)
  • M. Brownlee

    The pathobiology of diabetic complications: a unifying mechanism

    Diabetes

    (2005)
  • Cited by (14)

    • The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on the proteome of porcine granulosa cells

      2018, Chemosphere
      Citation Excerpt :

      We demonstrated that TCDD increased granulosal abundance of GAPDH after 12 h of treatment. The TCDD-induced increase in GAPDH gene expression and protein abundance was also reported in rat hepatocytes (Reyes-Hernandez et al., 2009) and placenta (Ishimura et al., 2002), respectively. GAPDH is a multifunctional enzyme involved in several cellular processes including glycolysis, membrane transport, microtubule assembly as well as DNA replication and repair.

    • Activation of aryl hydrocarbon receptor regulates the LPS/IFNγ-induced inflammatory response by inducing ubiquitin-proteosomal and lysosomal degradation of RelA/p65

      2018, Biochemical Pharmacology
      Citation Excerpt :

      Upon binding to its ligands, AhR translocates from the cytoplasm to the nucleus where it binds to xenobiotic response elements (XREs) located in the promoter of its target genes, such as cytochrome P450s (CYP1A1, CYP1A2, CYP1B1), NAD(P)H quinone oxidoreductase, and UDP-glucuronosyl-transferase 6 [1]. In addition to its role in xenobiotic metabolism, AhR has been implicated in several cellular processes, including liver development [2], neurogenesis [3], cholesterol and glucose metabolism [4,5], cell proliferation and apoptosis [6], and in the homeostasis of the immune system [7–9]. It was shown that AhR modulates the adaptive and innate immune responses and has been implicated in the differentiation of IL-17-producing helper T cells and Treg cells [10].

    • Activation of AHR mediates the ubiquitination and proteasome degradation of c-Fos through the induction of Ubcm4 gene expression

      2015, Toxicology
      Citation Excerpt :

      The result is an up-regulation of the expression of a battery of genes encoding xenobiotic-metabolizing enzymes, such as cytochrome P450s (CYP1A1, CYP1A2, CYP1B1), NAD(P)H quinone oxidoreductase, and UDP-glucuronosyl-transferase 6 (Gonzalez and Fernandez-Salguero 1998). AHR also modulates the expression of such genes as those involved in heart and liver development (Fernandez-Salguero et al., 1995; Fernandez-Salguero et al., 1997), homeostasis of the immune system (Rodriguez-Sosa et al., 2005), neurogenesis (Latchney et al., 2013), cholesterol and glucose metabolism (Reyes-Hernandez et al., 2009; Sato et al., 2008), and cell proliferation and apoptosis (Elizondo et al., 2000). Data from a recent study in which the liver gene profile between AhR-null and wild-type (WT) mice were compared suggest that AHR might control the expression of several genes coding for the ubiquitin-proteasome system (UPS) (Reyes-Hernandez et al., 2010).

    • Inhibition of glutathione biosynthesis alters compartmental redox status and the thiol proteome in organogenesis-stage rat conceptuses

      2013, Free Radical Biology and Medicine
      Citation Excerpt :

      Highly responsive to cellular conditions, including hypoxia, the mouse 5′ GAPDH flaking region contains several important response elements that are relevant to developmental signaling and are redox regulated. These include the AP1, SP1, HIF-1, XRE, MARE, GRE, and RARE elements that also regulate the expression of other developmentally relevant genes such as VEGF [64,65]. GAPDH has also recently been shown to have a direct role in cellular function as a mediator of RNA stability.

    • Ube2l3 gene expression is modulated by activation of the aryl hydrocarbon receptor: Implications for p53 ubiquitination

      2010, Biochemical Pharmacology
      Citation Excerpt :

      Upon binding TCDD, the AhR translocates to the nucleus, dimerizes with the AhR nuclear translocator protein (ARNT), binds xenobiotic responsive elements (XREs), and up-regulates the expression of a battery of genes encoding xenobiotic-metabolizing enzymes, such as the cytochrome P450s (CYP1A1, CYP1A2, CYP1B1), NAD(P)H quinone oxydoreductase, and UDP-glucuronosyl-transferase 6 [2]. Although AhR may function as part of an adaptive chemical response, several studies suggest that this transcription factor could have important functions in liver and cardiac development [3,4], cell proliferation [5], homeostasis of the immune system [6], circadian rhythmicity, and cholesterol and glucose metabolism [7,8]. In order to understand the physiologic role of AhR and identify genes under its control, we compared the basal liver gene expression between Ahr-null and wild-type (WT) mice by DNA microarray analysis.

    View all citing articles on Scopus
    View full text