Abstract
This study was designed to analyze the effects of the Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) inhibitor STO-609 (7-oxo-7H-benzimidazo[2,1-a]benz[de]isoquinoline-3-carboxylic acid) toward the aryl hydrocarbon receptor (AhR) pathway because Ca2+/calmodulin-dependent protein kinase (CaMK) Iα, known as a downstream CaMKK effector, has been recently shown to contribute to the AhR cascade. STO-609 failed to alter up-regulation of the AhR target CYP1A1 in response to the potent AhR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in MCF-7 cells. STO-609, used at a 25 μM concentration known to fully inhibit CaMKK activity, was surprisingly found to markedly induce CYP1A1 expression and activity by itself in MCF-7 cells; it similarly up-regulated various other AhR target genes in human macrophages. STO-609-related CYP1A1 induction was prevented by chemical inhibition or small interfering RNA-mediated knockdown expression of AhR. Moreover, STO-609 was demonstrated to physically interact with the ligand-binding domain of AhR, as assessed by TCDD binding competition assay, and to induce AhR translocation to the nucleus. As already reported for AhR agonists, STO-609 triggered the increase of [Ca2+]i and activation of CaMKIα, whose inhibition through the use of the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid-acetoxymethyl ester or the CaMK inhibitor KN-93 (2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine), respectively, prevented STO-609-mediated CYP1A1 activity induction. Taken together, these results demonstrate that the CaMKK inhibitor STO-609 can act as an AhR ligand and, in this way, fully activates the Ca2+/CaMKIα/AhR cascade. Such data, therefore, make unlikely any contribution of CaMKK activity to the AhR pathway and, moreover, suggest that caution may be required when using STO-609 as a specific inhibitor of CaMKKs.
Footnotes
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This work was supported by grants from the Ligue Nationale contre le Cancer and from the Agence Francaise de Sécurité Sanitaire de l'Environnement et du Travail. P.M. received a fellowship from the Ligue Nationale contre le Cancer.
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.108.023333.
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ABBREVIATIONS: AhR, arylhydrocarbon receptor; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; PAH, polycyclic aromatic hydrocarbon; XRE, xenobiotic-responsive element; MAPK, mitogen-activated protein kinase; CaMK, Ca2+/calmodulin-dependent protein kinase; CaMKK, Ca2+/calmodulin-dependent protein kinase kinase; STO-609, 7-oxo-7H-benzimidazo[2,1-a]benz[de]isoquinoline-3-carboxylic acid; U0126, 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophynyltio)butadiene; PD98059, 2′-amino-3′-methoxyflavone; 2-APB, 2-aminoethoxydiphenylborate; αNF, α-naphthoflavone; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; AM, acetoxymethyl ester; KN-93, 2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine; W7, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide; Ab, antibody; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; AMPK, AMP-activated protein kinase; FITC, fluorescein isothiocyanate; DMSO, dimethyl sulfoxide; EROD, ethoxyresorufin O-deethylase; RT, reverse transcription; qPCR, real-time quantitative polymerase chain reaction; IL, interleukin; ITG, integrin; siRNA, small interfering RNA; iAhR, small interfering RNA targeting AhR; iNT1, nontargeting siRNA; a.u., arbitrary unit(s); SP600125, 1,9-pyrazoloanthrone.
- Received July 11, 2008.
- Accepted August 26, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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