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Methyl Methacrylate Activates the Gsta1 Promoter
1 Department of Periodontology, Correspondence: * corresponding author, srp-psho{at}dpc.agu.ac.jp
Residual monomers in resin-based biomaterials cause cytotoxicity. We previously showed that methyl methacrylate (MMA) induced mRNA expression of the glutathione S-transferase alpha 1 gene (Gsta1) located downstream of the cis-acting anti-oxidant responsive element (ARE). Herein, we tested the hypothesis that MMA activated the Gsta1 promoter through the ARE. HepG2 cells were transfected with a luciferase reporter vector containing the ARE and the Gsta1 promoter (–990 to +46 bp) and cultured for 12 hrs with MMA (initial concentration, 10 mM). Analysis of the expressed luciferase activity indicated that MMA activated the promoter 2.6-fold. MMA (from 1 to 30 mM) dose-dependently increased the promoter activity, which reached a plateau between 6 and 12 hrs. In HepG2 cells transfected with a reporter vector containing 2 AREs and a TATA-like promoter, 10 mM MMA increased the reporter expression 2.8-fold. These results suggest that MMA increases Gsta1 transcription through ARE-mediated promoter activation.
Key Words: methyl methacrylate • glutathione S-transferase • anti-oxidant responsive element • reactive oxygen species • luciferase reporter assay Abbreviations: ANOVA, analysis of variance • ARE, anti-oxidant responsive element • Gsta1, mouse glutathione S-transferase alpha 1 gene • GSH, glutathione • GST, glutathione S-transferase • Keap1, Kelch ECH associating protein 1 • MMA, methyl methacrylate • NQO1, NAD(P)H:quinone oxidoreductase 1 • Nrf2, nuclear factor erythroid 2-related factor 2 • PCR, polymerase chain-reaction • ROS, reactive oxygen species • SEM, standard error of mean • t-BHQ, tert-butylhydroquinone.
Methyl methacrylate (MMA) polymerizes easily, and the MMA polymer has been widely used as a resin material in dentistry and medicine because of its excellent formability and operability. However, unpolymerized resin monomers are released from the resin and could cause cytotoxic effects, such as micronucleus formation (Schweikl et al., 2001), cell growth arrest, and reduced cell viability (Jinno et al., 2006). Using DNA microarray and real-time polymerase chain-reaction (PCR) analyses, we previously showed that MMA up-regulates gene expression of phase II enzymes such as glutathione S-transferases (GSTs) and NAD(P) H:quinone oxidoreductase 1 (NQO1) in L929 cells (Ishikawa et al., 2006). Since MMA is an electrophilic compound possessing a beta carbon with a positive partial charge (Schweikl et al., 2006), the reported results are consistent with the idea that phase II enzymes are induced by various electrophilic compounds (Talalay et al., 2003). GSTs catalyze conjugation of glutathione (GSH) to numerous electrophilic substances, most of which are derived from oxidative metabolism of xenobiotics combining covalently with DNA, RNA, or protein (Czosnek et al., 1984; Talalay et al., 1988; Rushmore and Pickett, 1993). Conjugated compounds are usually less toxic, more water-soluble, and easily excreted in the urine or bile (Ishikawa et al., 1997). Phase II enzymes are also induced by certain anti-oxidants such as tert-butylhydroquinone (t-BHQ) (Rushmore and Pickett, 1990). Anti-oxidant and electrophilic reagents have been shown to enhance transcription of phase II enzymes through the anti-oxidant responsive element (ARE) present in the 5'-flanking regions of their genes (Rushmore et al., 1991). The name ARE was introduced by Rushmore (Rushmore and Pickett, 1990). An element with sequence identity to the ARE was also found by Friling and called the ‘electrophile responsive element’ (Friling et al., 1990). The central transcription factor involved in ARE-mediated gene expression is nuclear factor erythroid 2-related factor 2 (Nrf2), which is retained under basal conditions by a cytosolic repressor protein, Kelch ECH associating protein 1 (Keap1) (Itoh et al., 1999, 2004; Talalay et al., 2003; Kensler et al., 2007). Nrf2 is released from Keap1 upon transcriptional activation, translocates into the nucleus, and binds to the ARE after forming heterodimeric complexes with other transcription factors, such as small Maf proteins. Keap1 has reactive sulfhydryl groups. Modification of these sulfhydryl groups induces conformational changes of Keap1, disrupting the Nrf2-Keap1 complex (Wakabayashi et al., 2004). It is possible that dental resin monomers lower the cellular-reducing capacity (see below) and oxidize cysteine residues on Keap1, leading to release of Nrf2 from Keap1, and activation of the ARE. GSH also functions as a major intracellular-reducing agent, maintaining the intracellular environment in a reduced state, and acting as a scavenger for reactive oxygen species (ROS) generated in various cellular processes (Dickinson and Forman, 2002). As described above, electrophilic xenobiotics are conjugated to GSH and decrease its cellular level. In fact, the dental resin co-monomer triethylene glycol dimethacrylate causes an almost complete depletion of intracellular GSH at sublethal concentrations (Geurtsen and Leyhausen, 2001). When cellular GSH is exhausted, unscavenged ROS accumulate in cells. Dental resin monomers thus could exert their toxic effects through ROS (Schweikl et al., 2006). In the present study, we focused on the mechanism through which MMA induced mRNA expression of mouse GST alpha 1 gene (Gsta1) (for nomenclature of GST genes, see Mannervik et al., 2005), and tested the hypothesis that MMA induced the Gsta1 transcription through ARE-mediated promoter activation.
General Recombinant DNA Techniques DNA fragments for reporter plasmid construction were amplified by PCR on a GeneAmp 2400 thermal cycler (Applied Biosystems, Foster City, CA, USA) with KOD -plus- DNA polymerase (Toyobo, Osaka, Japan). Amplified DNA fragments were purified by agarose gel electrophoresis, extracted with a QIAquick gel extraction kit (Qiagen, Chatsworth, CA, USA), and ligated into pTA2 cloning vectors (Toyobo) with a TArget CloneTM -Plus- cloning kit (Toyobo). Recombinant plasmids were transformed into competent E. coli DH5  cells (Nippon Gene, Tokyo, Japan), and the cells were cultured overnight at 37°C on an LB plate containing 50 µg/mL ampicillin. Ampicillin-resistant cells were picked and grown overnight at 37°C in LB medium containing 50 µg/mL ampicillin. Plasmids were collected and purified with the QIAprep Spin Miniprep Kit (Qiagen). Sequences of recombinant plasmids were determined by DNA sequencing with an ABI PRISM 3100 Avant Genetic analyzer, with the Big Dye terminator v3.1 cycle sequencing kit (Applied Biosystems). DNA fragments were ligated into luciferase reporter vectors with the Ligation-Convenience Kit (Nippon Gene). For transfection of plasmids into mammalian cells, they were purified with the EndoFree Plasmid Maxi Kit (Qiagen).
Reporter Plasmid Preparation A luciferase reporter vector containing a 5'-flanking region of Gsta1 (–990 to +46 bp; Gsta1pro990 in the Appendix Fig.) was prepared as follows. The 5'-flanking region was obtained from the lysate of a bacterial artificial chromosome E. coli clone containing Gsta1 (Invitrogen, RPCI23.C-302E8; Carlsbad, CA, USA) by PCR with the primers Gsta1-P1 (5'-GGTACCTGAAGAGAAATTAGCAGTGGACAT-3 ', KpnI site underlined) and Gsta1-P2 (5'-AAGCTTCTTCTCC ACTCAGCTCCCAGT-3', HindIII site underlined). The PCR was done for 30 cycles of 15 sec at 98°C, 15 sec at 60°C, and 1 min at 68°C. The PCR product, which had a KpnI site on one end and a HindIII site on the other, was gel-purified, ligated into the pTA2 cloning vector, and amplified in E. coli. The resultant plasmids were sequenced to ensure that no error had occurred during PCR. Subsequent digestion of the plasmid with KpnI and HindIII provided a fragment containing the region between –990 and +46 bp of Gsta1. This fragment was subcloned into a KpnI/ HindIII-digested pGL4.10 promoterless firefly luciferase vector (Promega, Madison, WI, USA) to create pGL4.10-Gsta1pro990 (see Appendix Fig.).
ARE-firefly luciferase reporter vectors were prepared as follows. The sense and antisense oligonucleotides of the Gsta1-derived ARE (–729 to –689 bp; sense oligonucleotide, 5'-TAGCTT GGAAATGACATTGCTAATGGTGACAAAGCAACTTT-3'; 2 ARE consensus sequences underlined) were phosphorylated by T4 polynucleotide kinase (TaKaRa Bio, Otsu, Japan), annealed, and inserted into the SmaI site upstream of a proximal promoter region of herpes simplex thymidine kinase in a luciferase reporter vector (pTAL-Luc; Clontech, Palo Alto, CA, USA) that was designed for analyzing enhancer sequences. The resultant vectors were amplified in E. coli. Cells containing vectors carrying one copy or two copies of the Gsta1-derived ARE were selected by DNA sequencing; vectors were collected from the selected cells and used for subsequent experiments (pTAL-1E-Luc and pTAL-2E-Luc in Fig. 3A
Cell Culture Human hepatoma HepG2 cells (RIKEN Cell Bank, RCB1648; Tsukuba, Japan) were maintained in minimum essential medium (Gibco BRL, Carlsbad, CA, USA) supplemented with 10% heat-inactivated fetal bovine serum (Equitech-Bio Inc., Kerrville, TX, USA) at 37°C in a 5% CO2 incubator. The medium was changed every other day.
Transient Transfection and Luciferase Assay
Statistical Analyses
MMA Increases the Gsta1 Promoter Activity When HepG2 cells were transfected with a firefly luciferase reporter vector containing a 5'-flanking region of Gsta1 (pGL4.10-Gsta1pro990, Fig. 1A  ) and incubated without or with MMA (initial concentration, 10 mM) for 12 hrs, MMA increased the Gsta1 promoter activity, represented by the reporter luciferase activity, 2.6-fold (Fig. 1B). We used -BHQt as a positive control for induction of the Gsta1 gene (Daniel et al., 1989; Friling et al., 1990, 1992; Paulson et al., 1990; Rushmore et al., 1991; Wasserman and Fahl, 1997; Li and Johnson, 2002) and found that MMA increased the Gsta1 promoter activity to similar extents as -BHQ (data not shown).t In HepG2 cells exposed to 10 mM MMA, an increase in the Gsta1 promoter activity was detectable within 3 hrs; the promoter activity reached a plateau between 6 and 12 hrs (Fig. 2A); MMA increased the promoter activity in a dose-dependent manner between 1 and 30 mM (Fig. 2B).
MMA Induces ARE-mediated Transcription When HepG2 cells were transfected with the ARE-luciferase reporter vector pTAL-2E-Luc, containing 2 tandem copies of the Gsta1-derived ARE upstream of a TATA-like promoter (Fig. 3A  ), and incubated for 12 hrs without additives or with MMA (initial concentration, 10 mM) or the ARE activator t-BHQ (90 µM), MMA and -BHQ enhanced the expression oft the reporter luciferase 2.8- and 2.4-fold, respectively (Fig. 3B). Neither of them enhanced the reporter expression in HepG2 cells transfected with the vector containing either a single or no copy of the ARE (Fig. 3B).
In this study, we showed that MMA increased Gsta1 promoter activity. As described above, the 5'-flanking region of Gsta1 carries an ARE. To examine whether MMA acted through the ARE, we constructed a luciferase reporter vector containing 2 copies of the Gsta1-derived ARE upstream of a TATA-like promoter. In HepG2 cells transfected with this vector, MMA induced the TATA-like promoter activation as effectively as t-BHQ, which acts through the ARE (Rushmore et al., 1991). These results suggest that MMA activates the Gsta1 promoter through the ARE. Neither of them activated the TATA-like promoter downstream of a single copy of the ARE, suggesting that the TATA-like promoter, used here as a model, required the presence of 2 AREs. The results thus suggest the following pathway on MMA-induced Gsta1 activation: (1) MMA is conjugated to GSH and depletes cellular GSH. (2) The cellular-reducing capacity consequently decreases with accumulation of ROS. (3) Cysteine sulfhydryl groups of Keap1 are oxidized; the resultant conformational change of Keap1 releases Nrf2 from the Keap1-Nrf2 complex in the cytosol. (4) Nrf2 translocates into the nucleus, associates with other transcription factors such as small Maf proteins, binds to the ARE, and increases Gsta1 transcription. MMA also increases mRNA expression of NQO1 (Ishikawa et al., 2006), which is located downstream of the ARE (Kensler et al., 2007). It is likely that MMA induces the transcription of NQO1 via the Keap1-Nrf2-ARE pathway as well. In preliminary studies, we transfected the reporter vector pGL4.10-Gsta1pro990 into mouse fibroblast L929 cells instead of HepG2 cells, and studied the effect of MMA on Gsta1 promoter activity. MMA-induced activity of the Gsta1 promoter, as well as the basal activity, was lower in L929 cells than in HepG2 cells. Similar results have been reported by others (Daniel et al., 1989; Paulson et al., 1990; Friling et al., 1990). These results are consistent with the facts that the liver is the major organ involved in detoxification of xenobiotics, and that GSTA1 exists in high amounts in hepatocytes and in lower amounts in other cells (Czosnek et al., 1984). We have been exploring a possibility that we could use the ARE-luciferase reporter assay as a means to detect ARE-related effects of resin monomers and other biomaterials quantitatively. Hepatic cells, like HepG2 cells, seem to be suitable for use in such an assay. It has been reported that Nrf2 is phosphorylated by protein kinase C, mitogen-activated protein kinases, and endoplasmic reticulum-resistant kinase (Schweikl et al., 2006; Kensler et al., 2007). Nrf2 phosphorylated at Ser-40 by protein kinase C is released from the Nrf2-Keap1 complex and enhances ARE-mediated transcription. In addition, ARE activation is reported to be dependent on phosphatidylinositol 3-kinase (Lee et al., 2001; Schweikl et al., 2006). Possible involvement of some of these enzymes also remains to be studied.
This work was supported by a grant-in-aid for the "AGU High-Tech Research Center" Project (2003–2007) for Private Universities from MEXT (Ministry of Education, Culture, Sports, Science and Technology, Japan) and by funds from the Aichi Gakuin University.
A supplemental appendix to this article is published electronically only at http://jdr.iadrjournals.org/cgi/content/full/87/12/1117/DC1. Received for publication February 6, 2008. Revision received May 16, 2008. Accepted for publication August 28, 2008.
Journal of Dental Research, Vol. 87, No. 12,
1117-1121 (2008)
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ABSTRACT
INTRODUCTION
30 mM) did not exert observable effects on cell number and viability. The normalized firefly luciferase activity was essentially independent of the cell number. 
