title: Activation of p38 Mitogen-Activated Protein Kinase by Clotrimazole Induces Multidrug Resistance-Associated Protein 3 Activation through a Novel Transcriptional Element.
abstract: Multidrug resistance-associated protein 3 (MRP3) is a basolaterally localized transporter in the liver and contributes to the transport of various metabolites such as conjugates of endogenous compounds and drugs from hepatocytes. MRP3 expression in the human liver is low under normal physiologic conditions but is induced by drug treatment. Although several studies have identified a region necessary for the basal transcription of MRP3, no region that responds to drugs has been reported. To identify the xenobiotic-responsive elements of MRP3, we constructed a luciferase reporter plasmid containing the MRP3 5'-flanking region up to -10 kb upstream from the transcription start site. Among typical nuclear receptor ligands, clotrimazole dramatically enhanced MRP3 reporter activity in HepG2 cells, whereas rifampicin had no effect. We then conducted MRP3 reporter assays with deletion or mutation constructs to identify a clotrimazole-responsive element. The element was located approximately -6.8 kb upstream from the MRP3 transcription start site. Overexpression of the pregnane X receptor did not enhance clotrimazole-mediated transcription. We found that clotrimazole was toxic to HepG2 cells and we therefore investigated whether mitogen-activated protein kinase (MAPK) activation is involved in the transactivation of MRP3 by clotrimazole. p38 MAPK inhibitor SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole] suppressed MRP3 mRNA expression induced by clotrimazole, whereas c-Jun N-terminal kinase inhibitor SP600125 (1,9-pyrazoloanthrone) and extracellular signal-regulated kinase inhibitor PD98059 [2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one] did not. Phosphorylated p38 MAPK was detected in HepG2 cells treated with clotrimazole. These results suggest that activation of the p38 MAPK pathway induces the transcriptional activation of MRP3.
authors: Department of Environmental and Health Science (T.S., K.I., Y.N., K.F., M.Y., T.K., K.N.) and Clinical Pharmacotherapeutics (S.K.), Faculty of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University, Aoba-ku, Sendai, Miyagi, Japan; Department of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizuoka, Suruga-ku, Shizuoka, Japan (T.S., K.Y.); and Division of Drug Metabolism and Molecular Toxicology, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba-ku, Sendai, Miyagi, Japan (S.M., T.T., Y.Y.).
authors: Takamitsu
authors: T
authors: Sasaki
authors: Department of Environmental and Health Science (T.S., K.I., Y.N., K.F., M.Y., T.K., K.N.) and Clinical Pharmacotherapeutics (S.K.), Faculty of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University, Aoba-ku, Sendai, Miyagi, Japan; Department of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizuoka, Suruga-ku, Shizuoka, Japan (T.S., K.Y.); and Division of Drug Metabolism and Molecular Toxicology, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba-ku, Sendai, Miyagi, Japan (S.M., T.T., Y.Y.).
authors: Keita
authors: K
authors: Inami
authors: Department of Environmental and Health Science (T.S., K.I., Y.N., K.F., M.Y., T.K., K.N.) and Clinical Pharmacotherapeutics (S.K.), Faculty of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University, Aoba-ku, Sendai, Miyagi, Japan; Department of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizuoka, Suruga-ku, Shizuoka, Japan (T.S., K.Y.); and Division of Drug Metabolism and Molecular Toxicology, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba-ku, Sendai, Miyagi, Japan (S.M., T.T., Y.Y.).
authors: Yoshihiro
authors: Y
authors: Numata
authors: Department of Environmental and Health Science (T.S., K.I., Y.N., K.F., M.Y., T.K., K.N.) and Clinical Pharmacotherapeutics (S.K.), Faculty of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University, Aoba-ku, Sendai, Miyagi, Japan; Department of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizuoka, Suruga-ku, Shizuoka, Japan (T.S., K.Y.); and Division of Drug Metabolism and Molecular Toxicology, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba-ku, Sendai, Miyagi, Japan (S.M., T.T., Y.Y.).
authors: Kodai
authors: K
authors: Funakoshi
authors: Department of Environmental and Health Science (T.S., K.I., Y.N., K.F., M.Y., T.K., K.N.) and Clinical Pharmacotherapeutics (S.K.), Faculty of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University, Aoba-ku, Sendai, Miyagi, Japan; Department of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizuoka, Suruga-ku, Shizuoka, Japan (T.S., K.Y.); and Division of Drug Metabolism and Molecular Toxicology, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba-ku, Sendai, Miyagi, Japan (S.M., T.T., Y.Y.).
authors: Midori
authors: M
authors: Yoshida
authors: Department of Environmental and Health Science (T.S., K.I., Y.N., K.F., M.Y., T.K., K.N.) and Clinical Pharmacotherapeutics (S.K.), Faculty of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University, Aoba-ku, Sendai, Miyagi, Japan; Department of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizuoka, Suruga-ku, Shizuoka, Japan (T.S., K.Y.); and Division of Drug Metabolism and Molecular Toxicology, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba-ku, Sendai, Miyagi, Japan (S.M., T.T., Y.Y.).
authors: Takeshi
authors: T
authors: Kumagai
authors: Department of Environmental and Health Science (T.S., K.I., Y.N., K.F., M.Y., T.K., K.N.) and Clinical Pharmacotherapeutics (S.K.), Faculty of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University, Aoba-ku, Sendai, Miyagi, Japan; Department of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizuoka, Suruga-ku, Shizuoka, Japan (T.S., K.Y.); and Division of Drug Metabolism and Molecular Toxicology, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba-ku, Sendai, Miyagi, Japan (S.M., T.T., Y.Y.).
authors: Shuichi
authors: S
authors: Kanno
authors: Department of Environmental and Health Science (T.S., K.I., Y.N., K.F., M.Y., T.K., K.N.) and Clinical Pharmacotherapeutics (S.K.), Faculty of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University, Aoba-ku, Sendai, Miyagi, Japan; Department of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizuoka, Suruga-ku, Shizuoka, Japan (T.S., K.Y.); and Division of Drug Metabolism and Molecular Toxicology, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba-ku, Sendai, Miyagi, Japan (S.M., T.T., Y.Y.).
authors: Satomi
authors: S
authors: Matsui
authors: Department of Environmental and Health Science (T.S., K.I., Y.N., K.F., M.Y., T.K., K.N.) and Clinical Pharmacotherapeutics (S.K.), Faculty of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University, Aoba-ku, Sendai, Miyagi, Japan; Department of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizuoka, Suruga-ku, Shizuoka, Japan (T.S., K.Y.); and Division of Drug Metabolism and Molecular Toxicology, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba-ku, Sendai, Miyagi, Japan (S.M., T.T., Y.Y.).
authors: Takayoshi
authors: T
authors: Toriyabe
authors: Department of Environmental and Health Science (T.S., K.I., Y.N., K.F., M.Y., T.K., K.N.) and Clinical Pharmacotherapeutics (S.K.), Faculty of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University, Aoba-ku, Sendai, Miyagi, Japan; Department of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizuoka, Suruga-ku, Shizuoka, Japan (T.S., K.Y.); and Division of Drug Metabolism and Molecular Toxicology, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba-ku, Sendai, Miyagi, Japan (S.M., T.T., Y.Y.).
authors: Yasushi
authors: Y
authors: Yamazoe
authors: Department of Environmental and Health Science (T.S., K.I., Y.N., K.F., M.Y., T.K., K.N.) and Clinical Pharmacotherapeutics (S.K.), Faculty of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University, Aoba-ku, Sendai, Miyagi, Japan; Department of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizuoka, Suruga-ku, Shizuoka, Japan (T.S., K.Y.); and Division of Drug Metabolism and Molecular Toxicology, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba-ku, Sendai, Miyagi, Japan (S.M., T.T., Y.Y.).
authors: Kouichi
authors: K
authors: Yoshinari
authors: Department of Environmental and Health Science (T.S., K.I., Y.N., K.F., M.Y., T.K., K.N.) and Clinical Pharmacotherapeutics (S.K.), Faculty of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University, Aoba-ku, Sendai, Miyagi, Japan; Department of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizuoka, Suruga-ku, Shizuoka, Japan (T.S., K.Y.); and Division of Drug Metabolism and Molecular Toxicology, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba-ku, Sendai, Miyagi, Japan (S.M., T.T., Y.Y.) nagataki@tohoku-mpu.ac.jp.
authors: Kiyoshi
authors: K
authors: Nagata
mesh_terms: D003022:Clotrimazole / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D056945:Hep G2 Cells; D006801:Humans; D008024:Ligands; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics*; D009154:Mutation; D000077297:Pregnane X Receptor; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011987:Receptors, Steroid / Q000378:metabolism; D015533:Transcriptional Activation / Q000187:drug effects*; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D003022:Clotrimazole; D008024:Ligands; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D000077297:Pregnane X Receptor; D047428:Protein Kinase Inhibitors; D012333:RNA, Messenger; D011987:Receptors, Steroid; D048051:p38 Mitogen-Activated Protein Kinases; C114462:multidrug resistance-associated protein 3