Down-regulation of EPHX2 gene transcription by Sp1 under high-glucose conditions.
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.
Research Center for Environmental Bioscience and Department of Bioscience, School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda 669-1337, Japan.
Ami
A
Oguro
Research Center for Environmental Bioscience and Department of Bioscience, School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda 669-1337, Japan.
Shoko
S
Oida
Research Center for Environmental Bioscience and Department of Bioscience, School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda 669-1337, Japan imaoka@kwansei.ac.jp.
Susumu
S
Imaoka
D051698:Activating Transcription Factor 2 / Q000378:metabolism; D021581:Active Transport, Cell Nucleus; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive; D002460:Cell Line; D015536:Down-Regulation; D004851:Epoxide Hydrolases / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic; D055785:Gene Knockdown Techniques; D005947:Glucose / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D018384:Oxidative Stress; D008926:Plicamycin / Q000031:analogs & derivatives / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D016329:Sp1 Transcription Factor / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects
C497105:ATF2 protein, human; D051698:Activating Transcription Factor 2; D016329:Sp1 Transcription Factor; C108209:SP1 protein, human; C066851:mithramycin A; D004851:Epoxide Hydrolases; C506428:EPHX2 protein, human; D005947:Glucose; D008926:Plicamycin
activating protein 2α (AP2α); high glucose; oxidative stress; soluble epoxide hydrolase gene (EPHX2); specificity protein 1 (Sp1)