sEH (soluble epoxide hydrolase), which is encoded by the EPHX2 gene, regulates the actions of bioactive lipids, EETs (epoxyeicosatrienoic acids). Previously, we found that high-glucose-induced oxidative stress suppressed sEH levels in a hepatocarcinoma cell line (Hep3B) and sEH was decreased in streptozotocin-induced diabetic mice in vivo. In the present study, we investigated the regulatory mechanisms underlying EPHX2 transcriptional suppression under high-glucose conditions. The decrease in sEH was prevented by an Sp1 (specificity protein 1) inhibitor, mithramycin A, and overexpression or knockdown of Sp1 revealed that Sp1 suppressively regulated sEH expression, in contrast with the general role of Sp1 on transcriptional activation. In addition, we found that AP2α (activating protein 2α) promoted EPHX2 transcription. The nuclear transport of Sp1, but not that of AP2α, was increased under high glucose concomitantly with the decrease in sEH. Within the EPHX2 promoter −56/+32, five Sp1-binding sites were identified, and the mutation of each of these sites showed that the first one (SP1_1) was important in both suppression by Sp1 and activation by AP2α. Furthermore, overexpression of Sp1 diminished the binding of AP2α by DNA-affinity precipitation assay and ChIP, suggesting competition between Sp1 and AP2α on the EPHX2 promoter. These findings provide novel insights into the role of Sp1 in transcriptional suppression, which may be applicable to the transcriptional regulation of other genes.

INTRODUCTION

sEH (soluble epoxide hydrolase) catalyses the conversion of EETs (endogenous epoxyeicosatrienoic acids) into DHETs (dihydroxyeicosatrienoic acids), and regulates the multiple actions of EETs. EETs contribute to vascular homoeostasis through their actions in vasodilation, anti-inflammation, anti-aggregation, anti-apoptosis and angiogenesis [1,2]. Because DHETs lose the functions that EETs have, or have different biological actions from EETs, the expression level of sEH is critical for the functions of EETs. On the other hand, sEH also has phosphatase activity, and its functions in cholesterol synthesis in hepatic cells and the growth of cells were recently investigated [3,4]. Previously we found that sEH had high activity towards LPAs (lysophosphatidic acids), which are lipid signalling molecules that control cell proliferation and motility [5]. Therefore sEH levels are also critical for the function of the phosphatase domain.

Alterations of sEH levels in the tissues of several pathological conditions were found, including increased sEH levels in the renal tissues of SHRs (spontaneously hypertensive rats) [6], suppression of sEH levels in renal and hepatic malignant neoplasms [7], and our previous finding that sEH levels were decreased in liver and kidney in streptozotocin-induced diabetic mice [8]. The decrease in sEH in diabetic mice was restored by treatment with insulin. In addition, in human hepatocarcinoma Hep3B cells, sEH was decreased under high-glucose conditions, and the decrease in sEH was due to increased production of ROS (reactive oxygen species) by the activation of NADPH oxidase. It has been shown that EETs stimulate insulin secretion in rat pancreatic islets [9] and that sEH inhibition abrogated endothelial dysfunction in db/db mice [10]. Hence the decrease in sEH under high glucose results in increased levels and physiological actions of EETs in diabetes. However, the mechanism by which high-glucose levels regulate sEH gene expression has not been clarified.

The sEH gene is encoded by EPHX2, and the transcription of EPHX2 is shown to be up-regulated by angiotensin II through the activation of the AP1 (activating protein 1) transcription factor [11]. Homocysteine also up regulates EPHX2 transcription via the binding of ATF6 (activating transcription factor 6) to the EPHX2 promoter [12]. Tanaka et al. [13] showed that the EPHX2 promoter lacks a TATA box or CAAT box motif, but is characterized by several GC-rich regions within the promoter [13]. The methylation of the EPHX2 promoter was also demonstrated [14]. The binding of a transcriptional factor, Sp1 (specificity protein 1), to the GC-rich motif within the EPHX2 promoter has been shown, but the regulation of its transcription by Sp1 remains unknown.

Sp1 is a member of the family of zinc-finger transcription factors, which binds to the GC-rich motif in the promoters of numerous genes. Sp1 is a ubiquitous nuclear factor that plays a role in maintaining the basal transcription of numerous housekeeping genes with TATA-less promoters, but it can also activate the transcription of several genes in response to physiological stimuli such as oxidative stress or serum stimulation [15]. The level or activity of Sp1 is also increased in many type of tumours, such as breast cancer [16] and hepatic cancer [17].

In the present study, we found that Sp1 negatively regulated EPHX2 promoter activity under high-glucose conditions, and the Sp1-binding site within the promoter was also important in positive regulation by AP2α (activating protein 2α). These findings reveal a new aspect of Sp1 as a negative regulator of genes in addition to its general function of transcriptional activation.

EXPERIMENTAL

Materials

FBS, horseradish peroxidase conjugated to goat anti-mouse IgG and catalase were from Sigma. Nitrocellulose membrane and horseradish peroxidase conjugated to goat anti-rabbit IgG were obtained from Bio-Rad Laboratories. DMEM (Dulbecco's modified Eagle's medium, high glucose) and anti-Myc antibody were purchased from Wako Pure Chemical Industries. Mithramycin A was purchased from Santa Cruz Biotechnology. Anti-Sp1 antibody was from Santa Cruz Biotechnology or Gene Tex. Anti-AP2α antibody was from Cell Signaling Technology.

Cell culture

The human hepatoma cell line Hep3B was obtained from the Cell Resource Center for Biomedical Research at the Institute of Development, Aging and Cancer of Tohoku University (Tohoku, Japan). HEK (human embryonic kidney)-293T cells were a gift from Professor Shintaro Suzuki of Kwansei Gakuin University (Sanda, Japan). Cells were cultured in DMEM containing 10% (v/v) FBS, 100 units/ml penicillin and 100 μg/ml streptomycin, and maintained at 37°C in 5% CO2 and 95% air. Cells were cultured in medium containing normal glucose (25 mM) or high glucose (75 mM) for 72 h, and the medium was renewed at 48 h, in the presence or absence of catalase (600 units/ml) or mithramycin A (10 or 100 nM). For catalase treatment, the concentration of FBS in the medium was decreased to 1%.

Isolation of upstream sequence of human EPHX2 from Hep3B and HEK-293T genomes

The transcriptional start site of EPHX2 has been identified by Tanaka et al. [13]. Genomic DNA was isolated from Hep3B cells, and the sequence upstream of the transcriptional start site of human EPHX2, −1091 to +32, was amplified using primer set 1 and 2 as shown in Table 1. The sequence isolated upstream of EPHX2 was compared with GenBank® sequence EU584434, revealing differences of G to A at −274, G to C at −389, and G to A at −466. C was inserted at −505, and the region from −444 to −417 was deleted. The fragment was inserted into a pGL3-promoter vector (Promega) whose SV40 promoter was removed by self-ligation after digestion with BglII and HindIII. Each 5′-endo-deleted upstream region of EPHX2 was amplified with reverse primer 2 and each forward primer 3, 4, 5, 6, 7, 8, 9, or 10 using–1091/+32 as a template, and each fragment was inserted into the pGL3 vector. A series of mutants of the EPHX2 promoter was constructed based on promoter −56/+32 in the pGL3 construct. The upstream region of the human APOA1 (apolipoprotein A-I) gene was isolated from the HEK-293T genome with primer set 11 and 12, then inserted into the pGL3 vector with SacI and XhoI sites.

Table 1
Primers used in the present study

Fw, forward; Rv, reverse.

Primer number Sequence Comments 
5′-AGACGAGCTCAAACCCACGGCTCTGGTCAATCCTG-3′ Fw; upstream of EPHX2 −1091 to −1067; underline, SacI site 
5′-TAGCTCGAGCAGCTAACCTGGGAGATGCGCGAAG-3′ Rv; upstream of EPHX2 +8 to +32; underline, XhoI site 
5′-CTTGGAGCTCAAGAGCGTGCCTAGAGGAGTGGTCAGG-3′ Fw; upstream of EPHX2 −586 to −560; underline, SacI site 
5′-GATCGAGCTCTTCCCAGGCATTCCAAGTC-3′ Fw; upstream of EPHX2 −374 to −356; underline, SacI site 
5′-TAAGAGCTCCTTTCCCGGCCAGAGTCCAGC-3′ Fw; upstream of EPHX2 −124 to −104; underline, SacI site 
5′-TAATGAGCTCCAGAGGGCGGAGTCCCGTTAA-3′ Fw; upstream of EPHX2 −92 to −72; underline, SacI site 
5′-TAATGAGCTCAGGCGGGGCCAGGGCAGGGG-3′ Fw; upstream of EPHX2 −56 to −37; underline, SacI site 
5′-TAAGAGCTCGCAGGGGCGGGGCAGAGCCGGG-3′ Fw; upstream of EPHX2 −43 to −22; underline, SacI site 
5′-TAAGAGCTCGCGGGGCAGAGCCGGGCCAAGCT-3′ Fw; upstream of EPHX2 −37 to −15; underline, SacI site 
10 5′-TAAGAGCTCAGCTGGGCGGGTCATGCGCCCT-3′ Fw; upstream of EPHX2 −18 to +4; underline, SacI site 
11 5′-TAATGAGCTCCTTGGAGAGAGGCCTGGAGGACCTG-3′ Fw; upstream of APOA1 −500 to −476; underline, SacI site 
12 5′-ATTCTCGAGAGCAGGACGCACCTCCTTCTCGCAG-3′ Rv; upstream of APOA1 +6 to +30; underline, XhoI site 
13 5′-CTGAATTCCCATGAGCGACCAAGATCACTCCAT-3′ Fw; 1–23 of SP1 cDNA; underline, EcoRI site; bold, start codon 
14 5′-TAACTCGAGGTATGGCCCATATGTCTCTGGCC-3′ Rv; downstream of SP1 cDNA; underline, XhoI site; stop codon is upstream of this primer 
15 5′-TAGAATTCCTATGACCGCTCCCGAAAAGCC-3′ Fw; 1–20 of SP3 cDNA; underline, EcoRI site; bold, start codon 
16 5′-TGGCTCGAGACCACAATGAATAAGTATTTG-3′ Rv; downstream of SP3 cDNA; underline, XhoI site; stop codon is upstream of this primer 
17 5′-GTCAAGCTTACCATGGCATCAATGCAGAA-3′ Fw; 1–17 of Myc Tag sequence; underline, HindIII site; bold, start codon 
18 5′-AAGAATTCAAATGCTTTGGAAATTGACGGATAATA-3′ Fw; 1–25 of TFAP2A cDNA; underline, EcoRI site; bold, start codon 
19 5′-ATTCTCGAGTCACTTTCTGTGCTTCTCCTCTTTG-3′ Rv; 1290–1314 of TFAP2A cDNA; underline, XhoI; bold, stop codon 
20 5′-CCCTTTCCTGTGCCCCTCCC-3′ Fw; upstream of EPHX2 −151 to −132 for ChIP 
21 5′-CAGCTAACCTGGGAGATGCG-3′ Rv; upstream of EPHX2 +13 to +32 for ChIP 
22 5′-AGTGTGGAAATCGGTGATTA-3′ Fw; upstream of EPHX2 −791 to −772 for ChIP 
23 5′-CTTTGCCAAGGCAAATGTGT-3′ Rv; upstream of EPHX2 −606 to −587 for ChIP 
Primer number Sequence Comments 
5′-AGACGAGCTCAAACCCACGGCTCTGGTCAATCCTG-3′ Fw; upstream of EPHX2 −1091 to −1067; underline, SacI site 
5′-TAGCTCGAGCAGCTAACCTGGGAGATGCGCGAAG-3′ Rv; upstream of EPHX2 +8 to +32; underline, XhoI site 
5′-CTTGGAGCTCAAGAGCGTGCCTAGAGGAGTGGTCAGG-3′ Fw; upstream of EPHX2 −586 to −560; underline, SacI site 
5′-GATCGAGCTCTTCCCAGGCATTCCAAGTC-3′ Fw; upstream of EPHX2 −374 to −356; underline, SacI site 
5′-TAAGAGCTCCTTTCCCGGCCAGAGTCCAGC-3′ Fw; upstream of EPHX2 −124 to −104; underline, SacI site 
5′-TAATGAGCTCCAGAGGGCGGAGTCCCGTTAA-3′ Fw; upstream of EPHX2 −92 to −72; underline, SacI site 
5′-TAATGAGCTCAGGCGGGGCCAGGGCAGGGG-3′ Fw; upstream of EPHX2 −56 to −37; underline, SacI site 
5′-TAAGAGCTCGCAGGGGCGGGGCAGAGCCGGG-3′ Fw; upstream of EPHX2 −43 to −22; underline, SacI site 
5′-TAAGAGCTCGCGGGGCAGAGCCGGGCCAAGCT-3′ Fw; upstream of EPHX2 −37 to −15; underline, SacI site 
10 5′-TAAGAGCTCAGCTGGGCGGGTCATGCGCCCT-3′ Fw; upstream of EPHX2 −18 to +4; underline, SacI site 
11 5′-TAATGAGCTCCTTGGAGAGAGGCCTGGAGGACCTG-3′ Fw; upstream of APOA1 −500 to −476; underline, SacI site 
12 5′-ATTCTCGAGAGCAGGACGCACCTCCTTCTCGCAG-3′ Rv; upstream of APOA1 +6 to +30; underline, XhoI site 
13 5′-CTGAATTCCCATGAGCGACCAAGATCACTCCAT-3′ Fw; 1–23 of SP1 cDNA; underline, EcoRI site; bold, start codon 
14 5′-TAACTCGAGGTATGGCCCATATGTCTCTGGCC-3′ Rv; downstream of SP1 cDNA; underline, XhoI site; stop codon is upstream of this primer 
15 5′-TAGAATTCCTATGACCGCTCCCGAAAAGCC-3′ Fw; 1–20 of SP3 cDNA; underline, EcoRI site; bold, start codon 
16 5′-TGGCTCGAGACCACAATGAATAAGTATTTG-3′ Rv; downstream of SP3 cDNA; underline, XhoI site; stop codon is upstream of this primer 
17 5′-GTCAAGCTTACCATGGCATCAATGCAGAA-3′ Fw; 1–17 of Myc Tag sequence; underline, HindIII site; bold, start codon 
18 5′-AAGAATTCAAATGCTTTGGAAATTGACGGATAATA-3′ Fw; 1–25 of TFAP2A cDNA; underline, EcoRI site; bold, start codon 
19 5′-ATTCTCGAGTCACTTTCTGTGCTTCTCCTCTTTG-3′ Rv; 1290–1314 of TFAP2A cDNA; underline, XhoI; bold, stop codon 
20 5′-CCCTTTCCTGTGCCCCTCCC-3′ Fw; upstream of EPHX2 −151 to −132 for ChIP 
21 5′-CAGCTAACCTGGGAGATGCG-3′ Rv; upstream of EPHX2 +13 to +32 for ChIP 
22 5′-AGTGTGGAAATCGGTGATTA-3′ Fw; upstream of EPHX2 −791 to −772 for ChIP 
23 5′-CTTTGCCAAGGCAAATGTGT-3′ Rv; upstream of EPHX2 −606 to −587 for ChIP 

Preparation of constructs for overexpression of Sp1, Sp3 and AP2α

The entire coding region of human SP1 isoform A (GenBank® accession number NM_138473.2) and that of human SP3 isoform 1 (GenBank® accession number NM_003111.4) were amplified by PCR with primer sets 13 and 14, and 15 and 16 respectively, as shown in Table 1. The amplified fragment was inserted into a pCMV-Myc vector (Clontech Laboratories) with EcoRI and XhoI sites. The Myc tag sequence-fused SP1 or SP3 cDNA was amplified with primer set 17 and 14, and 17 and 16 respectively, and inserted into pcDNA3.1(+) (Invitrogen) with HindIII and XhoI sites. For the knockdown of Sp1 with shRNA, a specific region of the SP1 sequence was designated (967–987 of the SP1 cDNA sequence). The sense, antisense and hairpin sequences were inserted into the pBAsi-hU6 Neo vector (TaKaRa Bio) stepwise according to the manufacturer's instructions. The sequence of inserted nucleotides for Sp1 knockdown is as follows: 5′-AATGCCAATAGCTACTCAACTCTGTAAGCCACAGAT-GGGAGTTGAGTAGCTATTGGCATTTTTTTT-3′ (underlined are the 967th–987th nucleotides in SP1 and complementary sequences in each other; in bold is the hairpin region). The nucleotide sequence for control shRNA against GFP is as follows: 5′-CTCGAGTACAACTATAACTCACTGTAAGCCA-CAGATGGGTGAGTTATAGTTGTACTCGAGTTTTTT-3′. The entire coding region of human TFAP2A isoform A (GenBank® accession number NM_003220.2) was isolated from Hep3B cells with primer set 18 and 19, then inserted into the pCMV-HA vector (Clontech Laboratories) with EcoRI and XhoI sites.

Nuclear extraction and immunoblotting

Nuclear extracts were prepared from Hep3B cells using the method of Chang and Huang [18] with minor modifications. Cells were washed with PBS and lysed with buffer A (10 mM Hepes, pH 7.9, containing 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT and 0.1% Triton X-100) with a protease inhibitor cocktail (Sigma) on ice for 10 min. Nuclei were precipitated by centrifugation at 12000 g for 2 min. Pellets were resuspended in buffer C (20 mM Hepes, pH 7.9, containing 25% glycerol, 1.5 mM MgCl2, 0.2 mM EDTA, 420 mM NaCl and 0.5 mM DTT) with a protease inhibitor cocktail and were allowed to stand on ice for 20 min. The suspension was centrifuged at 12000 g for 15 min, and the supernatants obtained were collected as the nuclear fraction. For immunoblotting, anti-sEH or anti-β-actin antibodies were prepared as described previously [4].

Luciferase reporter gene assay

The EPHX2 promoter in pGL3 (0.5 μg) and pRL-TK (25 ng) were transfected into HEK-293T cells (3×103) in a 24-well plate with GenePORTER TM2 transfection reagent (Gene Therapy Systems). After incubation for 24 h, the culture medium was replaced with fresh medium containing normal (25 mM) or high (75 mM) glucose and cultured for 72 h. Luciferase activity was assayed with a luminometer (Lumat LB9507; Berthold) using the Dual-Luciferase Reporter assay system (Promega). SP1, SP3 or TFAP2A in pCMV (0.25 μg) was co-transfected with EPHX2 promoter in pGL3 (0.25 μg) and pRL-null (25 ng) into HEK-293T cells. After incubation for 24 h, the culture medium was replaced with fresh medium, and luciferase activity was measured 48 h after transfection.

DNA-affinity precipitation assay

The SP1 in pCMV-Myc was transfected into HEK-293T cells at various concentrations, and the nuclear extract was pre-cleared with streptavidin–agarose resin and poly(dI-dC) at 4°C for 1 h. After centrifugation, the supernatant (80 μg of protein) was incubated with 3 μg of biotin-labelled double-stranded oligonucleotide containing EPHX2 upstream region −58/−26 (5′-biotin-GGAGGCGGGGCCAGGGCAGGGGCGGGGCAG-AGC-3′) (Hokkaido System Science) in binding buffer (20 mM Hepes, pH 7.9, 0.1 mM KCl, 2 mM MgCl2, 20 mM NaCl, 0.2 mM EDTA, 1 mM DTT and 10% glycerol containing protease inhibitor cocktail) at 4°C overnight. DNA complex was precipitated with 20 μl of streptavidin–agarose (Thermo Fisher) at 4°C for an additional 1 h. The complex was washed three times with the binding buffer containing 0.5% NP40, and analysed by Western blotting with anti-Sp1 or anti-AP2α antibody.

ChIP assay

HEK-293T cells overexpressing Sp1 or AP2α were incubated with 1.5% (w/v) formaldehyde for 10 min and quenched by adding glycine to a final concentration of 125 mM. Cells were washed with PBS three times and resuspended in ChIP buffer (150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.5% NP40, 50 mM Tris/HCl, pH 7.5, 0.5 mM PMSF, 10 mM NaF and 0.5 mM DTT), and disrupted on ice with a sonicator followed by centrifugation at 14000 g for 10 min. The supernatant was incubated with control IgG, anti-Sp1 or anti-AP2α antibody for 30 min at room temperature. Then Protein A–Sepharose (GE Healthcare) was added and incubated for 45 min at 4°C. The beads were rinsed five times sequentially with ChIP buffer, and elution buffer (1% SDS and 0.1 M NaHCO3) was added and incubated for 15 min at room temperature. To reverse the cross-linking, 0.4 M NaCl was added and incubated at 65°C overnight. The mixture was treated with proteinase K for 30 min at 50°C, and the DNA was purified. The upstream regions of EPHX2, −151 to +32, and −791 to −587, were amplified by PCR with primer sets 20 and 21, and 22 and 23 respectively as shown in Table 1.

Statistical analysis

Statistical analysis for single comparisons between means was carried out with the Student's t test, and P<0.05 was considered statistically significant. For multiple comparisons, one-way ANOVA followed by a Bonferroni/Dunn post-hoc test was used.

RESULTS

Down-regulation of EPHX2 promoter activity by high glucose

Previously we found that sEH mRNA levels were suppressed in cells treated with high glucose for 72 h. In the present study, we investigated the EPHX2 promoter activity under normal- or high-glucose conditions by luciferase reporter assay in HEK-293T cells. High glucose suppressed the promoter activity of the construct containing the upstream region −1091/+32 of the EPHX2 gene (Figure 1A). The basal promoter activity was gradually decreased by the consecutive 5′-end-deletion of the upstream region of EPHX2. The suppressive effect of high glucose on the promoter activity remained in the region −56/+32, although the decrease in the activity of the construct containing −124/+32 under high glucose was restored, in part, by the 5′-deletion from −124 to −92. A motif-search analysis by Transfac indicated that the region within −124 and −92 contained several Sp1-binding sites. The additional deletion from −56 to −43 almost abolished the suppression of the activity under high glucose, although the basal activity was decreased by the deletion (Figure 1B).

Luciferase reporter activity of EPHX2 promoter under high-glucose conditions

Figure 1
Luciferase reporter activity of EPHX2 promoter under high-glucose conditions

(A and B) The upstream region −1091/+32 (A) or −56/+32 (B) of EPHX2 in pGL3 or its 5′-endo deletion construct was co-transfected with pRL-TK into HEK-293T cells. After incubation with medium containing normal or high glucose for 72 h, luciferase activity was measured using the Dual-Luciferase Reporter Assay system. The ratio of the activity of EPHX2 promoter −1091/+32 (A) or −56/+32 (B) to pRL-TK under normal glucose was set at 1. Results are means±S.D. for three separate experiments. *P<0.05, **P<0.01.

Figure 1
Luciferase reporter activity of EPHX2 promoter under high-glucose conditions

(A and B) The upstream region −1091/+32 (A) or −56/+32 (B) of EPHX2 in pGL3 or its 5′-endo deletion construct was co-transfected with pRL-TK into HEK-293T cells. After incubation with medium containing normal or high glucose for 72 h, luciferase activity was measured using the Dual-Luciferase Reporter Assay system. The ratio of the activity of EPHX2 promoter −1091/+32 (A) or −56/+32 (B) to pRL-TK under normal glucose was set at 1. Results are means±S.D. for three separate experiments. *P<0.05, **P<0.01.

Increased nuclear Sp1 levels under high-glucose conditions

To investigate Sp1's involvement in the suppression of sEH by high glucose, sEH levels were analysed under high glucose in the presence of an Sp1 inhibitor, mithramycin A, in Hep3B cells. The decrease in sEH levels under high glucose was abolished in the presence of 10 nM mithramycin A (Figure 2A). Next, the Sp1 levels under high glucose were investigated. Although the levels of SP1 mRNA and Sp1 protein were not altered under high-glucose conditions (Figures 2B and 2C), Sp1 was detected in both cytosol and nucleus, and the nuclear Sp1 levels were significantly increased by treatment with high glucose for 72 h concomitantly with the decrease in sEH levels (Figure 2D).

Participation of Sp1 in the suppression of sEH, and increased Sp1 levels under high-glucose conditions

Figure 2
Participation of Sp1 in the suppression of sEH, and increased Sp1 levels under high-glucose conditions

(A) Hep3B cells were cultured under normal- or high-glucose conditions for 72 h in the absence or presence of mithramycin A, and sEH expression was analysed by Western blotting. (B and C) SP1 mRNA levels (B) or total Sp1 protein levels (C) under normal- or high-glucose conditions for 72 h were analysed by real-time PCR or Western blotting. A representative blot of three independent experiments is shown. (D) Hep3B cells were cultured under normal- or high-glucose conditions for 24, 48 or 72 h, and nuclear or cytosol Sp1 levels were analysed. Results are means±S.D. for three separate experiments. **P<0.01.

Figure 2
Participation of Sp1 in the suppression of sEH, and increased Sp1 levels under high-glucose conditions

(A) Hep3B cells were cultured under normal- or high-glucose conditions for 72 h in the absence or presence of mithramycin A, and sEH expression was analysed by Western blotting. (B and C) SP1 mRNA levels (B) or total Sp1 protein levels (C) under normal- or high-glucose conditions for 72 h were analysed by real-time PCR or Western blotting. A representative blot of three independent experiments is shown. (D) Hep3B cells were cultured under normal- or high-glucose conditions for 24, 48 or 72 h, and nuclear or cytosol Sp1 levels were analysed. Results are means±S.D. for three separate experiments. **P<0.01.

Increased nuclear Sp1 levels by high-glucose-induced oxidative stress

Previously, we found that suppression of sEH under prolonged high glucose for 72 h was mediated by increased cellular ROS levels [8]. The decrease in sEH under high glucose was abolished by the addition of catalase, which metabolizes hydrogen peroxide (Figure 3A), whereas the nuclear transport of Sp1 under high glucose was also inhibited by catalase (Figure 3B). These results suggest that increased nuclear Sp1 levels by high-glucose-induced ROS contribute to the decrease in sEH expression.

Increased Sp1 levels by high-glucose-induced ROS

Figure 3
Increased Sp1 levels by high-glucose-induced ROS

(A and B) Cells were cultured under normal- or high-glucose conditions for 72 h in the presence or absence of 600 units/ml catalase, and sEH protein levels in whole-cell lysates (A) and nuclear or cytosol Sp1 protein levels (B) were analysed by Western blotting. Results are means±S.D. for three separate experiments. *P<0.05, **P<0.01.

Figure 3
Increased Sp1 levels by high-glucose-induced ROS

(A and B) Cells were cultured under normal- or high-glucose conditions for 72 h in the presence or absence of 600 units/ml catalase, and sEH protein levels in whole-cell lysates (A) and nuclear or cytosol Sp1 protein levels (B) were analysed by Western blotting. Results are means±S.D. for three separate experiments. *P<0.05, **P<0.01.

Sp1 negatively regulates sEH expression

To investigate Sp1's suppressive effect on sEH expression, Sp1 was overexpressed in Hep3B cells and sEH levels were analysed. Overexpression of Myc-tag-fused Sp1 was detected by Western blotting with anti-Myc antibody. Overexpression of Sp1 decreased sEH expression under both normal- and high-glucose conditions for 72 h (Figure 4A). Sp3 and Egr1 are known to bind the same motif as Sp1 and display a parallel or opposing transcription effect with Sp1, depending on the promoter. Overexpression of Sp3 had a similar effect to that of Sp1 on sEH expression (Figure 4A), whereas overexpression of Egr1 did not alter sEH expression (results not shown). Next, the effect of Sp1 knockdown on sEH expression was investigated. Decreases in Sp1 levels by the shRNA with the Sp1-targeting sequence were detected by Western blotting with anti-Sp1 antibody. Knockdown of Sp1 increased sEH levels under both normal- and high-glucose conditions (Figures 4B and C). These results indicate that Sp1 negatively regulates sEH expression. The correlation between sEH and Sp1 levels was investigated in different cell types, including Hep3B, HEK-293T and HeLa cells. A decrease in sEH under high glucose was observed in all these cells (results not shown). HeLa cells, which expressed high levels of Sp1, showed low levels of sEH, whereas high levels of sEH were observed in Hep3B cells, which expressed low levels of Sp1 (Figure 4D). These results indicate that sEH levels depend on Sp1 levels.

sEH levels in Sp1-overexpressing or Sp1-knockdown cells

Figure 4
sEH levels in Sp1-overexpressing or Sp1-knockdown cells

(A) Sp1 or Sp3 in pcDNA-Myc was transfected in Hep3B cells and the overexpression was confirmed by Western blotting with anti-Myc antibody (arrowheads or arrow indicate overexpressed Sp1 or Sp3 respectively). These cells were cultured under normal- or high-glucose conditions for 72 h, and sEH protein levels were analysed by Western blotting. (B and C) Sp1-knockdown cells were generated using shRNA in Hep3B (B) or HeLa (C) cells, and knockdown of Sp1 was confirmed by Western blotting with anti-Sp1 antibody (upper panel). sEH protein levels in these cells under normal- or high-glucose conditions for 72 h (B), or normal-glucose conditions (C) were analysed. (D) Sp1 and sEH proteins in Hep3B, HEK-293T, and HeLa cells were analysed by Western blotting. A representative blot of two independent experiments is shown. Results are means±S.D. for three separate experiments.*P<0.05, **P<0.01. OE, overexpression.

Figure 4
sEH levels in Sp1-overexpressing or Sp1-knockdown cells

(A) Sp1 or Sp3 in pcDNA-Myc was transfected in Hep3B cells and the overexpression was confirmed by Western blotting with anti-Myc antibody (arrowheads or arrow indicate overexpressed Sp1 or Sp3 respectively). These cells were cultured under normal- or high-glucose conditions for 72 h, and sEH protein levels were analysed by Western blotting. (B and C) Sp1-knockdown cells were generated using shRNA in Hep3B (B) or HeLa (C) cells, and knockdown of Sp1 was confirmed by Western blotting with anti-Sp1 antibody (upper panel). sEH protein levels in these cells under normal- or high-glucose conditions for 72 h (B), or normal-glucose conditions (C) were analysed. (D) Sp1 and sEH proteins in Hep3B, HEK-293T, and HeLa cells were analysed by Western blotting. A representative blot of two independent experiments is shown. Results are means±S.D. for three separate experiments.*P<0.05, **P<0.01. OE, overexpression.

Transcriptional regulation of EPHX2 promoter by Sp1

To investigate the transcriptional regulation of EPHX2 gene by Sp1, Sp1 in pCMV-Myc was co-transfected with the EPHX2 promoter −1091/+32 in pGL3, and luciferase activity was measured. Overexpression of Sp1 significantly suppressed EPHX2 promoter activity, and overexpression of Sp3 showed similar effects (Figure 5A). In general, Sp1 positively regulates the transcription of a large number of genes. Therefore, to confirm that overexpressed Sp1 in the present study had proper transcriptional activity, the luciferase activity of the APOA1 gene promoter, which is typically activated by Sp1 [19,20], was measured in Sp1-overexpressing cells. As a result, the activity of APOA1 promoter −500/+30 was significantly increased by overexpression of Sp1, as opposed to the suppression of EHPX2 promoter activity by Sp1 (Figure 5B). These results indicate that the regulation of EPHX2 transcription is unique in that it is negatively regulated by Sp1. Overexpression of Sp1 also decreased the promoter activity of the construct containing the EPHX2 upstream region −56/+32, and the suppressive effect of Sp1 was not observed in the region −43/+32, suggesting that the region from −56 to −43 is important for the negative regulation by Sp1 (Figure 5D). These results are consistent with the suppression of EPHX2 promoter activity in response to high glucose as shown in Figure 1B. Within the region from −56 to +32, five Sp1-binding sites, SP1_1 to SP1_5, were identified by Transfac analysis, and SP1_1 and SP1_2 sites were located in the region from −56 to −43 (Figure 5C). To determine the contribution of the Sp1-binding sites to EPHX2 promoter activity, each Sp1 site was individually mutated in EPHX2 promoter −56/+32 in the pGL3 vector. The mutation of the SP1_1 site blunted the suppression of promoter activity by overexpression of Sp1, but the mutation also decreased basal promoter activity (Figure 5E). This result suggests that the SP1_1 site is important for the negative regulation by Sp1, but this site may also be necessary for the binding of the other transcriptional factors that positively regulate the EPHX2 promoter activity. On the other hand, the mutation of the SP1_3 site significantly decreased basal promoter activity, and this activity was slightly increased by overexpression of Sp1. Interestingly, when Sp1 was overexpressed, the activity of wild-type and that of any mutants of the promoter were almost the same. The mutation of the SP1_1 site also prevented the response of EPHX2 promoter activity to high glucose (Figure 5F).

Suppression of EPHX2 promoter activity by Sp1

Figure 5
Suppression of EPHX2 promoter activity by Sp1

(A) EPHX2 promoter −1091/+32 in pGL3 and pRL-null were co-transfected with Sp1 or Sp3 in pCMV-Myc into HEK-293T cells. Cells were cultured under normal- or high-glucose conditions for 72 h, and luciferase activity was measured. Overexpression of Sp1 and Sp3 was confirmed by Western blotting with anti-Myc antibody. (B) Empty pGL3, APOA1 promoter −500/+30 in pGL3, or EPHX2 promoter −1091/+32 in pGL3 and pRL-null were co-transfected with Sp1 in pCMV into cells, and luciferase activity was measured at 48 h after transfection. (C) Sp1-binding sites SP1_1–SP1_5 within the EPHX2 promoter −56/+32. (D) EPHX2 promoter −56/+32 or its 5′-endo deletion construct, and pRL-null were co-transfected with Sp1 in pCMV into cells, and luciferase activity was measured. (E) Wild-type EPHX2 promoter −56/+32 or mutant construct in which each Sp1-binding site was mutated, and pRL-null were co-transfected with Sp1 in pCMV into cells, and luciferase activity was measured. (F) Wild-type of EPHX2 promoter −56/+32 or the mutant construct, and pRL-null were transfected into cells. These cells were incubated under normal- or high-glucose conditions for 72 h, and luciferase activity was measured. Results are means±S.D. for three separate experiments. *P<0.05, **P<0.01. OE, overexpression.

Figure 5
Suppression of EPHX2 promoter activity by Sp1

(A) EPHX2 promoter −1091/+32 in pGL3 and pRL-null were co-transfected with Sp1 or Sp3 in pCMV-Myc into HEK-293T cells. Cells were cultured under normal- or high-glucose conditions for 72 h, and luciferase activity was measured. Overexpression of Sp1 and Sp3 was confirmed by Western blotting with anti-Myc antibody. (B) Empty pGL3, APOA1 promoter −500/+30 in pGL3, or EPHX2 promoter −1091/+32 in pGL3 and pRL-null were co-transfected with Sp1 in pCMV into cells, and luciferase activity was measured at 48 h after transfection. (C) Sp1-binding sites SP1_1–SP1_5 within the EPHX2 promoter −56/+32. (D) EPHX2 promoter −56/+32 or its 5′-endo deletion construct, and pRL-null were co-transfected with Sp1 in pCMV into cells, and luciferase activity was measured. (E) Wild-type EPHX2 promoter −56/+32 or mutant construct in which each Sp1-binding site was mutated, and pRL-null were co-transfected with Sp1 in pCMV into cells, and luciferase activity was measured. (F) Wild-type of EPHX2 promoter −56/+32 or the mutant construct, and pRL-null were transfected into cells. These cells were incubated under normal- or high-glucose conditions for 72 h, and luciferase activity was measured. Results are means±S.D. for three separate experiments. *P<0.05, **P<0.01. OE, overexpression.

Activation of EPHX2 transcription by AP2α with overlapping binding sites for Sp1

Next we searched for a transcriptional factor that binds upstream of EPHX2 overlapping with the SP1_1 site and activates its promoter. A motif analysis by Transfac revealed that the region from −56 to −43 contained sequences matching the consensus sequence of the binding site of AP2α, and that this overlapped with the SP1_1 site. Overexpression of AP2α increased EPHX2 promoter activity within the region −1091/+32 (Figure 6A). Especially in the region −56/+32, the activation of the promoter by AP2α was high. This effect was diminished by the deletion of the 5′-end sequence from −56 to −43 and almost abolished by the additional deletion from −56 to −37 (Figure 6A), indicating that the region between −56 and −37 was important for the activation by AP2α. Within the region −56/+32, the mutation of the SP1_1 site diminished the increase in activity by AP2α (Figure 6B), suggesting that the SP1_1 site was important for both suppression by Sp1 and activation by AP2α. In addition, the mutation of SP1_3 at the position of −37 completely abolished the effect of AP2α, indicating that the SP1_3 site was also essential for the effect of AP2α. Overexpression of AP2α also increased sEH protein levels in Hep3B cells (Figure 6C). Furthermore, endogenous AP2α was detected in the nuclei, but not the cytosol of Hep3B cells, and its expression levels in nuclei were not altered under high glucose, whereas Sp1 expression was increased in nuclei (Figure 6D).

Transcriptional activation of EPHX2 promoter by AP2α

Figure 6
Transcriptional activation of EPHX2 promoter by AP2α

(A) EPHX2 promoter −1091/+32 or its 5′-endo deletion construct, and pRL-null were co-transfected with AP2α in pCMV-HA into HEK-293T cells, and luciferase activity was measured. (B) Wild-type EPHX2 promoter −56/+32 or mutant construct in which each Sp1-binding site was mutated, and pRL-null were co-transfected with AP2α in pCMV-HA into cells, and luciferase activity was measured. (C) AP2α in pCMV-HA was transfected into Hep3B cells, and sEH protein levels were analysed. (D) Hep3B cells were cultured under normal- or high-glucose conditions for 72 h, and nuclear and cytosol fractions were isolated. Sp1 and AP2α protein levels were analysed by Western blotting. A representative blot of three independent experiments is shown. Results are means±S.D. for three separate experiments. **P<0.01. OE, overexpression.

Figure 6
Transcriptional activation of EPHX2 promoter by AP2α

(A) EPHX2 promoter −1091/+32 or its 5′-endo deletion construct, and pRL-null were co-transfected with AP2α in pCMV-HA into HEK-293T cells, and luciferase activity was measured. (B) Wild-type EPHX2 promoter −56/+32 or mutant construct in which each Sp1-binding site was mutated, and pRL-null were co-transfected with AP2α in pCMV-HA into cells, and luciferase activity was measured. (C) AP2α in pCMV-HA was transfected into Hep3B cells, and sEH protein levels were analysed. (D) Hep3B cells were cultured under normal- or high-glucose conditions for 72 h, and nuclear and cytosol fractions were isolated. Sp1 and AP2α protein levels were analysed by Western blotting. A representative blot of three independent experiments is shown. Results are means±S.D. for three separate experiments. **P<0.01. OE, overexpression.

Binding of Sp1 and AP2α to the EPHX2 promoter

To elucidate the binding of Sp1 and AP2α to the EPHX2 promoter, DNA-affinity precipitation assay was carried out using biotin-labelled oligonucleotides containing EPHX2 promoter −58/−26 with SP1_1–SP1_3 sites (Figure 7A). The binding of Sp1 and AP2α was detected, and the binding of AP2α to the promoter was diminished by overexpression of Sp1, although the levels of AP2α were not altered by overexpression of Sp1. In addition, ChIP analysis showed that Sp1 and AP2α bound to the EPHX2 upstream region −151/+32 containing a lot of Sp1-binding sites, but not −791/−587 with few Sp1-binding sites (Figure 7B). The binding of AP2α to the promoter was diminished concomitantly with the increase in Sp1 binding by overexpression of Sp1. These results suggest competition between Sp1 and AP2α within the upstream region of EPHX2.

Binding of Sp1 and AP2α to EPHX2 promoter

Figure 7
Binding of Sp1 and AP2α to EPHX2 promoter

(A) DNA-affinity precipitation assay: HEK-293T cells were transfected with various amounts of Sp1 in pCMV-Myc or empty vector, and their nuclear extract was precipitated with biotin-labelled oligonucleotide containing the EPHX2 upstream region −58/−26. DNA–protein complex was isolated with streptavidin–agarose beads, and analysed by Western blotting with anti-Sp1 or anti-AP2α antibody. A representative blot of two independent experiments is shown. (B) ChIP assay: AP2α was co-overexpressed with different doses of Sp1 in HEK-293T cells, and their expression levels were detected by Western blotting (upper panel). These cells were incubated with formaldehyde, and sonicated DNA fragments were precipitated with antibody against Sp1 or AP2α. After reversing the cross-linking, the DNA was amplified using primers specific for the EPHX2 promoter region −151 to +32, or −791 to −587. The indicated Sp1-binding sites within the EPHX2 promoter were searched by Transfac analysis. Ab, antibody.

Figure 7
Binding of Sp1 and AP2α to EPHX2 promoter

(A) DNA-affinity precipitation assay: HEK-293T cells were transfected with various amounts of Sp1 in pCMV-Myc or empty vector, and their nuclear extract was precipitated with biotin-labelled oligonucleotide containing the EPHX2 upstream region −58/−26. DNA–protein complex was isolated with streptavidin–agarose beads, and analysed by Western blotting with anti-Sp1 or anti-AP2α antibody. A representative blot of two independent experiments is shown. (B) ChIP assay: AP2α was co-overexpressed with different doses of Sp1 in HEK-293T cells, and their expression levels were detected by Western blotting (upper panel). These cells were incubated with formaldehyde, and sonicated DNA fragments were precipitated with antibody against Sp1 or AP2α. After reversing the cross-linking, the DNA was amplified using primers specific for the EPHX2 promoter region −151 to +32, or −791 to −587. The indicated Sp1-binding sites within the EPHX2 promoter were searched by Transfac analysis. Ab, antibody.

DISCUSSION

The present study revealed that Sp1 down-regulated EPHX2 transcription via its binding to the EPHX2 promoter under high-glucose conditions. The EPHX2 promoter has been identified as a TATA-less promoter and contains multiple GC-rich regions for Sp1 binding [13]. Although two AP1-binding sites within EPHX2 upstream of −586 and −92 and an ATF6-binding site within −374 and −124 have been identified as regulation sites for its transcription [11,12], promoter analysis with the consecutive 5′-end deletion indicated that neither AP1 nor ATF6 explained its down-regulation by high glucose. Overexpression of Sp1 significantly suppressed sEH protein levels and the promoter activity of EPHX2 from −1091 to +32, despite the fact that the proper activity of Sp1 in the present study was confirmed by its transcriptional activation with the APOA1 promoter. These results could not exclude the participation of other factors that co-operate with Sp1 and are masked by overexpression of Sp1. However, knockdown of Sp1 increased sEH levels, indicating that endogenous Sp1 also negatively regulates sEH expression. Indeed, sEH levels were dependent on Sp1 levels, and an inverse correlation of sEH and Sp1 levels was observed in different cell types (Figure 4D). Previously, we found that sEH expression was also decreased under prolonged hypoxia for 72 h in Hep3B cells and in the liver of mice with hypoxic exposure [4]. Increased Sp1 levels have been found in mice exposed to hypoxia for 72 h [21], and we confirmed the increase in nuclear Sp1 under hypoxia for 72 h in Hep3B cells (results not shown). Together, these results suggest that Sp1 might down-regulate sEH expression under several conditions in which Sp1 was induced. Indeed, decreased sEH levels are found in renal and hepatic malignant neoplasms [7], whereas Sp1 is increased in many type of tumours, including hepatic cancer [17].

Our results indicated that nuclear Sp1 levels and the binding of Sp1 to the EPHX2 promoter were increased by high-glucose-induced oxidative stress. High glucose was known to increase the production of ROS via several mechanisms, including autoxidation of glucose, the polyol pathway, facilitation of the mitochondrial electron transport chain and activation of NADPH oxidase [22]. We have also confirmed the previous finding that increased ROS levels by high glucose were inhibited by DPIC (diphenyleneiodonium chloride), an NADPH oxidase inhibitor [8]. ROS can activate numerous intracellular kinase pathways [23]. We found that an increase in Sp1 was suppressed by an inhibitor of p38, SB203580, which also inhibited the decrease in sEH under high glucose (results not shown), implying the involvement of the p38 MAPK (mitogen-activated protein kinase) pathway in the increased nuclear Sp1. The facilitation of Sp1 transfer to the nucleus via phosphorylation of Sp1 by p38 has been suggested [24,25], and it has been shown that oxidative stress stimulated the binding of Sp1 to GC-rich regions of the caveolin-1 promoter through p38 MAPK signalling [26].

In the present study, we found that Sp1 bound to the SP1_1 site within −56 and +32, which was essential for the suppressive effect of Sp1. On the other hand, the SP1_1 site was also important for the activation of the promoter by AP2α. AP2α plays important roles in embryogenesis, notably cranial closure and craniofacial development [27]. Although the expression levels of AP2α are known to be low in endoderm-derived tissue such as liver [28], nuclear AP2α was detected in Hep3B cells in the present study, and was also expressed in mouse hepatocytes [29]. AP2α binds to not only its consensus sequences, GCCN3/4GGC and GCCN3/4GGG [30], but also to GC-rich sequences widely as well as Sp1. Although the opposite roles of Sp1 as an activator and AP2α as a repressor were found on the dystrophin DP71 promoter [31] and the SOD2 promoter [32], to the best of our knowledge down-regulation of transcription by Sp1 with a regulatory site overlapping with activation by AP2α was observed for the first time in this study. In addition, the binding of AP2α to the EPHX2 promoter was diminished by increased Sp1 binding by DNA-affinity precipitation and ChIP assays, suggesting that Sp1 bound to the Sp1-binding sites via competition with the binding of AP2α.

In the present study, we focused on the minimal promoter region within −56 and +32, which responded to high glucose or Sp1 overexpression. However, not only the SP1_1 site, but also the multiple Sp1-binding sites upstream of this region might be involved in the regulation by Sp1 and AP2α. In addition, Sp3, which binds to the same motif as Sp1, seems to have a similar suppressive effect on the EPHX2 promoter as that of Sp1, although the contribution of Sp3 to the high-glucose-induced decrease in sEH levels was not clear.

The findings of the present study provide novel insights into the role of Sp1 in transcriptional suppression, which may be applicable to the transcriptional regulation of other genes besides EPHX2.

AUTHOR CONTRIBUTION

Ami Oguro mainly carried out the experiments and drafted the paper. Shoko Oida participated in the transcriptional analysis. Susumu Imaoka conceived the study, and participated in its design and co-ordination and helped to draft the paper.

FUNDING

This study was supported in part by the Project to Assist Private Universities in Developing Bases for Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (S1001051). It was also supported in part by a Grant-in-aid from Kwansei Gakuin University.

Abbreviations

     
  • AP1

    activating protein 1

  •  
  • AP2α

    activating protein 2α

  •  
  • ATF

    activating transcription factor

  •  
  • DHET

    dihydroxyeicosatrienoic acid

  •  
  • DMEM

    Dulbecco’s modified Eagle’s medium

  •  
  • EET

    endogenous epoxyeicosatrienoic acid

  •  
  • HEK

    human embryonic kidney

  •  
  • MAPK

    mitogen-activated protein kinase

  •  
  • ROS

    reactive oxygen species

  •  
  • sEH

    soluble epoxide hydrolase

  •  
  • Sp1

    specificity protein 1

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