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SHORT COMMUNICATION

CERAMIDE, AN APOPTOTIC RHEOSTAT, INHIBITS CCAAT/ENHANCER BINDING PROTEIN-ß AND NF-E2-RELATED FACTOR-2 ACTIVATION: THE ROLE IN GLUTATHIONE S-TRANSFERASE A2 GENE REPRESSION

National Research Laboratory, College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul, South Korea

(Received January 5, 2004; accepted May 25, 2004)

	Abstract

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Ceramide is a sphingolipid that acts as a second messenger in signaling systems. Sphingomyelinase generates ceramide in response to cytotoxic stimuli. CCAAT/enhancer binding protein-ß (C/EBPß) and NF-E2-related factor-2 (Nrf2) are both involved in the regulation of the genes encoding phase II detoxification enzymes including glutathione S-transferase (GST). In the present study, we examined the effects of ceramide on C/EBPß or Nrf2 activation and on the inducible GSTA2 gene transactivation. C2-ceramide (C2), a cell-permeable analog, inhibited GSTA2 induction by oltipraz or tert-butylhydroquinone (t-BHQ) in H4IIE cells, whereas dihydro-C2-ceramide (dihydro-C2), an inactive analog, had no effect. Immunoblot analysis revealed that C2 prevented increase in the level of nuclear C/EBPß by oltipraz, whereas the level of C/EBPß in total cell lysates was not changed. Increase in nuclear Nrf2 by t-BHQ was also prevented by C2 treatment. Decreases in nuclear C/EBPß and Nrf2 by C2 were reversed by treatment of cells with N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132), a proteasome inhibitor, verifying the previous observations that the transcription factors were degraded by the proteasome system. In another study, we found that ceramide decreased nuclear hepatic nuclear factor-1 (HNF1), whose binding to the HNF1-response element in the GSTA2 gene was responsible for the constitutive and inducible gene expression. To define the role of C/EBPß or Nrf2 repression in GST expression under the condition excluding the negative regulation by C2-mediated HNF1 suppression, luciferase activity was determined in the cells transfected with HNF-pGL-1651 plasmid lacking the HNF1-response element. In the cells transfected with HNF-pGL-1651, C2 decreased the luciferase induction by oltipraz or t-BHQ. Thus, ceramide inhibits C/EBPß or Nrf2 activation, which contributes to repression of GSTA2 gene transactivation.

Glutathione S-transferases (GSTs) are an important family of detoxifying and cytoprotective enzymes in the liver. Glutathione conjugates of xenobiotics can be excreted through bile or urine. In our previous studies and others, analyses of the 5'-flanking region of the GSTA2 gene revealed the specific sequences defining the location of the antioxidant response element (ARE) and the CCAAT/enhancer binding protein-ß (C/EBPß) binding site (Huang et al., 2000; Kang et al., 2001, 2003). The PI3-kinase pathway controls nuclear translocation of NF-E2-related factor-2 (Nrf2) and regulates the ARE in the promoter of GSTA2 (Kang et al., 2001). Also, the pathway regulates nuclear translocation of C/EBPß and activation of the C/EBP response element in the promoter of the GSTA2 gene (Kang et al., 2001, 2003).

Despite the previous extensive studies on the ceramide regulation of intracellular effectors such as kinases, phosphatases, and proteases (Scheid and Duronio, 1998; Chalfant et al., 1999; Cuvillier, 2002), the effects of ceramide on major transcription factors have not been completely studied. In particular, no information is available on the effect of ceramide on Nrf2 activation. Although ceramide has been shown to repress C/EBP and ß forms in adipocytes (Reginato et al., 1998; Sprott et al., 2002), the role of C/EBPß repression by ceramide in the expression of phase II detoxification enzyme has never been explored.

We previously found that the hepatic nuclear factor-1 (HNF1) response element (HRE) in the GSTA2 promoter region was involved in the constitutive and inducible gene expression in H4IIE hepatocytes and that decrease in nuclear HNF1 by ceramide was associated with the repression of GST gene transactivation (Park et al., 2004b). As part of the comprehensive studies on the role of ceramide in activation of major transcription factors and alterations of GST gene expression, we were tempted to examine the inhibitory effects of ceramide on Nrf2 and C/EBPß in association with GSTA2 gene repression.

	Materials and Methods

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Materials. Anti-C/EBPß and anti-Nrf2 antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). These antibodies specifically recognized their respective transcription factors without any cross-reactivity as determined in our previous reports (Kang et al., 2001, 2002, 2003). C2 and dihydro-C2 were purchased from Calbiochem (San Diego, CA). Oltipraz was kindly provided as a gift from CJ Co. (Seoul, South Korea). tert-Butylhydroquinone (t-BHQ) (97%) was obtained from Sigma-Aldrich (St. Louis, MO). MG132 was purchased from BIOMOL Research Laboratories (Plymouth Meeting, PA). Horseradish peroxidase-conjugated goat anti-rabbit IgG was supplied from Zymed Laboratories (South San Francisco, CA). The plasmid pGTB-1.65 construct containing the rat GSTA2-promoter region (-1651 bp to +66 bp) was kindly provided by Dr. C. B. Pickett (Schering Plough, Kenilworth, NJ).

Cell Culture. H4IIE, a rat hepatocyte-derived cell line, was obtained from the American Type Culture Collection (Manassas, VA). Cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, 50 U/ml penicillin, and 50 µg/ml streptomycin at 37°C in a humidified atmosphere with 5% CO₂. C2 (20 µM) or dihydro-C2 (20 µM) was dissolved in dimethyl sulfoxide. For some experiments, H4IIE cells (10⁵ cells/well, 9.6 cm²/well in a six-well plate) were incubated with 10 µM oltipraz or 10 µM t-BHQ in the presence or absence of C2 or dihydro-C2 at 37°C for 24 h. MG132 (10 µM) was used to inhibit proteasomal degradation. Cells were washed twice with ice-cold PBS before sample preparation. We used H4IIE cells because pharmacological inhibition and transcription factor activation have been extensively studied in the cells (Kang et al., 2001, 2002, 2003). In H4IIE cells, major cytochromes P450 including CYP1A1/2, CYP3A, CYP2B, and CYP2C11 were expressed, as was observed in human hepatocyte cell lines (e.g., HepG2 cells).

Preparation of Nuclear Extracts and Total Cell Lysates. Nuclear extracts were prepared according to previously published methods (Kang et al., 2003). Briefly, H4IIE cells in dishes were washed twice with ice-cold PBS, scraped from the dishes with PBS, and transferred to microtubes. Cells were then centrifuged at 3000g for 3 min and allowed to swell after the addition of hypotonic buffer containing 10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.5% Nonidet P-40, 1 mM dithiothreitol, and 0.5 mM phenylmethylsulfonyl fluoride (PMSF). The lysates were incubated for 10 min on ice and then centrifuged at 7200g for 6 min at 4°C. Pellets containing crude nuclei were resuspended in 50 µl of extraction buffer containing 20 mM HEPES (pH 7.9), 400 mM NaCl, 1 mM EDTA, 10 mM dithiothreitol, and 1 mM PMSF, and then incubated for 30 min on ice. The samples were centrifuged at 15,000g for 10 min to obtain supernatants containing nuclear fractions.

To obtain total cell lysates, cells were lysed in buffer containing 20 mM Tris-Cl (pH 7.5), 1% Triton X-100, 137 mM sodium chloride, 10% glycerol, 2 mM EDTA, 1 mM sodium orthovanadate, 25 mM ß-glycerophosphate, 2 mM sodium pyrophosphate, 1 mM PMSF, and 1 µg/ml leupeptin. Cell lysates were boiled for 5 min and then centrifuged at 15,000g for 15 min at 4°C to remove debris.

Immunoblot Analysis. SDS-polyacrylamide gel electrophoresis and immunoblot analysis were performed according to the previously published procedure (Kang et al., 2003). Briefly, protein samples were separated by 7.5% or 12% gel electrophoresis and electrophoretically transferred to nitrocellulose paper. The nitrocellulose paper was incubated with each of the antibodies directed against GST (Detroit R&D, Inc., Detroit, MI), C/EBPß, and Nrf2 (Santa Cruz Biotechnology, Inc.), followed by incubation with a horseradish peroxidase-conjugated secondary antibody. Immunoreactive protein was visualized by an ECL chemiluminescence detection kit (Amersham Biosciences UK, Ltd., Little Chalfont,, Buckinghamshire, UK) (Kang et al., 2002). Equal loading of proteins was verified by actin immunoblotting with goat anti-actin antibody (Santa Cruz Biotechnology, Inc.). At least three separate experiments were performed with different samples to confirm changes in the protein levels. Scanning densitometry of the immunoblots was performed as described previously with Image Scan and Analysis System (Alpha Innotech, San Leandro, CA) (Kang et al., 2003).

Construction of GSTA2 Promoter-Luciferase Constructs and Luciferase Assay. The pGL-1651 reporter gene construct was generated by ligating the 1.65-kilobase upstream region from the transcription start site of the GSTA2 gene to the firefly luciferase reporter gene coding sequence (Kang et al., 2003). To specifically delete the HRE from pGL-1651, the DNA fragments containing the nucleotides from -1651 bp to -873 bp and from -844 bp to +66 bp were polymerase chain reaction-amplified using specific primers. The DNA fragments were ligated and cloned into the KpnI/XhoI and XhoI/BglII sites of pGL3 (Promega, Madison, WI) luciferase reporter plasmid, which was indicated as HNF-pGL-1651. The DNA sequences of the constructs were verified by sequence analysis using an ABI7700 DNA cycle sequencer. We used the dual-luciferase reporter assay system to determine the promoter activity (Promega), as described previously (Kang et al., 2003).

Statistical Analysis. One-way analysis of variance was used to assess statistical significance of differences among treatment groups. For each statistically significant effect of treatment, the Newman-Keuls test was used for comparisons between multiple group means. The data were expressed as means ± S.E.

	Results and Discussion

Top Abstract Materials and Methods Results and Discussion References

 
In our previous study, changes in the constitutive expression of GSTA2 were assessed in response to various concentrations of C2. The levels of GSTA2 protein were 52% to 80% suppressed by 20 and 25 µM C2 in H4IIE cells, whereas dihydro-C2 did not change the level of constitutive GSTA2 (Park et al., 2004b). In the present study, we verified the inhibitory effects of ceramide on GSTA2 induction by oltipraz or t-BHQ. C2 (20 µM) inhibited oltipraz (10 µM)-inducible GSTA2 induction in H4IIE cells, whereas dihydro-C2, an inactive analog, did not change the expression of GSTA2 (Fig. 1A). We next confirmed inhibition of t-BHQ-inducible GSTA2 expression by C2. C2 (20 µM), but not dihydro-C2 (20 µM), notably inhibited GSTA2 induction by t-BHQ (10 µM) (Fig. 1B), which indicated that GST repression was indeed mediated with ceramide.

View larger version (18K): [in this window] [in a new window]   FIG. 1. Repression of GSTA2 induction by C2. A, representative immunoblot of GSTA2 in H4IIE cells incubated with oltipraz (Olt, 10 µM), oltipraz + C2 (20 µM), or oltipraz + dihydro-C2 (20 µM) for 24 h. B, immunoblot analysis of GSTA2 in H4IIE cells incubated with t-BHQ (10 µM), t-BHQ + C2 (20 µM), or t-BHQ + dihydro-C2 (20 µM) for 24 h. Each lane was loaded with 10 µg of cytosolic proteins. Equal loading of proteins was verified by probing the replicate blot for actin.

Oltipraz promotes nuclear translocation of C/EBPß, which is dependent on PI3-kinase, and induces the GSTA2 gene via C/EBPß binding to the C/EBP binding site in the promoter region of the gene (Kang et al., 2003). N-terminal transactivation domains of C/EBPß that binds to the DNA binding element interacts with p300/CBP and enhances gene transactivation (Mink et al., 1997). t-BHQ, a representative prooxidant, primarily induces Nrf2 translocation into the nucleus and causes the binding of Nrf2 to the ARE in the promoter region of the target gene (GSTA2) for transactivation (Kang et al., 2001). Given the previous observations, we determined whether C2 inhibited activation of C/EBPß or Nrf2 in the cells treated with oltipraz or t-BHQ. Immunoblot analyses showed that C2 blocked increases in the levels of nuclear C/EBPß and Nrf2 activated by oltipraz or t-BHQ (Fig. 2). The levels of C/EBPß and Nrf2 in total cell lysates, however, were not notably changed by C2 treatment (Fig. 2, A and B). Therefore, decreases in the transcription factors in nuclear fractions may have resulted from inhibition of C/EBPß and Nrf2 activation, which possibly involves inhibition of phosphorylation and/or translocation of the proteins.

View larger version (21K): [in this window] [in a new window]   FIG. 2. The effects of C2 on C/EBPß and Nrf2 activation. Immunoblot analyses were performed of the levels of C/EBPß (A) and Nrf2 (B) in the nuclear fractions of H4IIE cells treated with oltipraz (Olt; 10 µM) or t-BHQ (10 µM) with or without 20 µM C2 for 6 h. The levels of C/EBPß and Nrf2 were also determined in total cell lysates, which verified equal loading of proteins. Each lane contains 15 µg of nuclear proteins or 10 µg of total cell lysates. Results were confirmed by three separate experiments and representative immunoblots are shown. Data represent the mean ± S.E. with three separate experiments [**, significant as compared with control, p < 0.01; ##, significant as compared with oltipraz alone, p < 0.01 (A); ##, significant as compared with t-BHQ alone, p < 0.01 (B)]. (The level of C/EBPß or Nrf2 in control cells = 100%.)

Studies have shown that both C/EBPs and Nrf2 are multi-ubiquitinated and subsequently degraded by the proteasomes (Pulford and Hayes, 1996; Hattori et al., 2003; Itoh et al., 2003; Stewart et al., 2003). To confirm that C/EBPß and Nrf2 were subjected to ubiquitin-mediated proteasomal degradation, immunoblot analyses were performed with the nuclear fractions prepared from the cells treated with C2, C2 + MG132, or MG132 (Fig. 3, A and B). The levels of nuclear C/EBPß and Nrf2 were both decreased by treatment of H4IIE cells with C2 for 6 h, which was reversed by concomitant treatment with MG132, a proteasome inhibitor. Treatment of H4IIE cells with MG132 alone caused accumulation of C/EBPß or Nrf2 in nuclear fractions. Thus, MG132 may inhibit proteasomal degradation in the nucleus. The lesser extent of nuclear accumulation of C/EBPß or Nrf2 by MG132 + C2 than that by MG132 alone indicates that the step inhibited by C2 precedes that affected by MG132.

View larger version (17K): [in this window] [in a new window]   FIG. 3. Immunoblot analyses of nuclear C/EBPß and Nrf2. The levels of C/EBPß (A) and Nrf2 (B) were determined in the nuclear fractions obtained from H4IIE cells treated with C2 (20 µM), C2 + MG132 (10 µM), or MG132. Data represent the mean ± S.E. with three separate experiments (**, significant as compared with control, p < 0.01; ##, significant as compared with C2 treatment, p < 0.01). (The nuclear level of C/EBPß or Nrf2 in control cells = 100%.) Olt, oltipraz.

We found that ceramide decreased the level of nuclear HNF1 and HNF1-mediated GST gene expression (Park et al., 2004b). We also determined the effects of C2 on luciferase expression of pGL-1651 in the cells treated with oltipraz or t-BHQ (Park et al., 2004b). Deletion of the C/EBP and ARE binding sites in the gene cannot allow us to define the role of these binding sites because the C/EBP and ARE binding sites are required for full gene transactivation (Park et al., 2004a). To define the effect of C2-mediated inhibition of nuclear C/EBPß on transactivation of the GSTA2 gene under the condition excluding the negative transcriptional regulation by C2-mediated HNF suppression, luciferase expression was determined in the cells transfected with the HNF-pGL-1651 plasmid (Fig. 4A). In HNF-pGL-1651-transfected cells, C2 significantly decreased the luciferase inducibility by oltipraz. Treatment of the cells transfected with HNF-pGL-1651 with t-BHQ + C2 resulted in a 62% decrease compared with t-BHQ alone (Fig. 4B). This finding supports the role of C/EBPß and Nrf2 inhibition by ceramide in GSTA2 gene repression.

View larger version (23K): [in this window] [in a new window]   FIG. 4. The effects of C2 on the luciferase reporter activity in H4IIE cells transfected with HNF-pGL-1651. A, the C/EBP, HNF1, and ARE binding sites are located in the 1.65-kilobase upstream region of the GSTA2 gene. The HNF-pGL-1651 plasmid lacks HRE. B, dual luciferase reporter assays were performed in the cells treated with oltipraz (Olt) or t-BHQ in the presence or absence of C2 after transient transfection with the HNF-pGL-1651 plasmid. The luciferase reporter gene assay was performed, as described under Materials and Methods. The experimental value for luciferase activity represents the mean ± S.E. with three separate experiments (significant as compared with oltipraz or t-BHQ alone, *, p < 0.05; **, p < 0.01).

Previous studies from this laboratory have shown that oltipraz induces GSTA2 through activation of C/EBPß and its binding to the C/EBP response element (Kang et al., 2003). PI3-kinase regulated nuclear translocation of C/EBPß by oltipraz for GST induction (Kang et al., 2003). In the present study, we found for the first time that ceramide decreased the level of nuclear C/EBPß in H4IIE cells, which accompanied GST repression. The pleiotropic effects of C/EBPs result from tissue-specific expression and post-transcriptional modifications. Some forms of C/EBPs, including C/EBP and C/EBP, are constitutively multi-ubiquitinated and subsequently degraded by the proteasomes (Hattori et al., 2003). Nonfunctional C/EBPß is degraded via the ubiquitin conjugating system involving the ubiquitin ligase or modification enzymes (Hochstrasser, 1995; Hattori et al., 2003). The ubiquitin ligase or the enzymes in catalyzing ubiquitination of C/EBPß seem to specifically recognize monomer forms of C/EBPs, indicating that the cellular levels of C/EBPß are controlled by post-translational modification involving degradation via the ubiquitin/proteasome pathway. Whether decrease in nuclear C/EBPß by ceramide results from an increase in ubiquitin-mediated degradation remains to be determined.

Transcription factor Nrf2 belongs to the cap-n-collar transcription factor family characterized by the presence of a 45-amino acid homology region referred to as the CNC domain. Nrf2 binds to the ARE in the target genes (McMahon et al., 2001). Oxidative stress by decreased glutathione or prooxidant (e.g., t-BHQ) activates the PI3-kinase pathway, which plays an essential role in nuclear translocation of Nrf2 for GST induction (Huang et al., 2000; Kang et al., 2001, 2002). Nrf2 undergoes rapid degradation by the ubiquitin-dependent pathway, which is mediated by the 26S proteasomes (Nguyen et al., 2003; Stewart et al., 2003), whereas electrophiles cause nuclear translocation of Nrf2 with concomitant stabilization (Itoh et al., 2003). In the present study, we provide evidence that ceramide as an apoptotic signal inhibits Nrf2 activation by t-BHQ and thus leads, at least in part, to GST repression.

Sphingolipid is involved in the down-regulation of the liver-specific genes. The expression of GST and many other genes requires the liver-specific transcription factors for the maximal transcription activity (Pimental et al., 1993; Enomoto et al., 2001). The present study provides the evidence that ceramide inhibits activation of the transcription factors C/EBPß and Nrf2 and that the ability of ceramide in suppressing the transcription factors is accompanied by the well characterized repression of the GST gene. Our experiments revealed that ceramide inhibited the GSTA2 gene transactivation in the HNF-pGL1651-transfected cells exposed to either oltipraz or t-BHQ, which resulted from inhibition of nuclear C/EBPß and Nrf2, but not that of HNF1. Thus, it is highly likely that cooperative assembly of C/EBPß and Nrf2 with constitutively active HNF1 is required for the formation of a large transactivation complex that promotes the maximal transcription of the target gene.

Ceramide was shown to increase the activities of serine/threonine protein phosphatase PP1 and PP2A (Chalfant et al., 1999), which may be responsible for specific dephosphorylation of certain proteins (e.g., Bcl-2). Ceramide may decrease C/EBPß, Nrf2, or HNF1 binding to specific DNA sequences as a result of increases in their dephosphorylation. Therefore, suppression of C/EBPß, Nrf2, and HNF1 transcription factors by ceramide may be mediated with inactivation of the proteins (i.e., decreases in phosphorylation and nuclear translocation). It has also been shown that ceramide-induced cell death accompanies direct inhibition of mitochondrial respiratory chain complex III (Gudz et al., 1997). Formation of long-chain ceramide species resulted in proteasomal activation and subsequent activation of effector caspases (Kroesen et al., 2003). The link of GST repression and decrease in cell viability may also stem from suppression of these transcription factors.

C/EBPß, Nrf2, and HNF1 are required as the transcription factors for other liver-specific gene expression. The promoter regions of the GSTA3 (GenBank accession number AF067442 [GenBank] ) and GSTA5 (GenBank accession number AH005038 [GenBank] ) genes contain the consensus DNA binding site(s) for C/EBP and/or Nrf2 (Pulford and Hayes, 1996). We found that ceramide was capable of repressing the induction of other GST forms (i.e., GSTA3/5) by chemical inducers (Park et al., 2004b). It is highly likely that both C/EBPß and Nrf2 serve transcriptional components of the transactivator complex for the constitutive and inducible GST gene expression. Inhibition by ceramide of these transcription factors may also lead to repression of other genes (e.g., CYP2C11) (Chen et al., 1995; Merril et al., 1999). In summary, the present study demonstrated that ceramide inhibits C/EBPß and Nrf2, which are necessary for cooperative assembly of an activated transcription complex at the target gene promoter, and that ceramide inhibition of these transcription factors contributes to repression of the GSTA2 gene transactivation.

	Acknowledgments

 
The kind donation of pGTB-1.65 containing the GSTA2-promoter region from Dr. Cecil B. Pickett is gratefully acknowledged.

	Footnotes

 
This work was supported by the National Research Laboratory Program (2001), Korea Institute of Science and Engineering Evaluation and Planning, Ministry of Science and Technology, The Republic of Korea.

ABBREVIATIONS: PI3-kinase, phosphatidylinositol 3-kinase; ARE, antioxidant response element; C2, C2-ceramide; C/EBP, CCAAT/enhancer binding protein; dihydro-C2, dihydro-C2-ceramide; GST, glutathione S-transferase; HNF1, hepatic nuclear factor-1; HRE, HNF1 response element; Nrf2, NF-E2-related factor-2; t-BHQ, tert-butylhydroquinone; MG132, N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal; bp, base pair(s); PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride.

Address correspondence to: Dr. Sang Geon Kim, College of Pharmacy, Seoul National University, Sillim-dong, Kwanak-gu, Seoul 151-742, South Korea. E-mail: sgk{at}snu.ac.kr

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