Received for publication July 11, 2008.
Revised August 26, 2008.
Accepted for publication August 26, 2008.

Activation of the aryl hydrocarbon receptor by the calcium/calmodulin-dependent protein kinase kinase inhibitor STO-609

Abstract

This study was designed to analyse the effects of the Ca²⁺/calmodulin-dependent protein kinase kinase (CaMKK) inhibitor STO-609 towards the arylhydrocarbon receptor (AhR) pathway, since Ca²⁺/calmodulin-dependent protein kinase (CaMK) I, 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 siRNA-mediated knock-down expression of AhR. Moreover, STO-609 was demonstrated to physically interact with 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 increase of [Ca²⁺]_i and activation of CaMKI, whose inhibition through the use of the Ca²⁺ chelator BAPTA-AM or the CaMK inhibitor KN93, 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, by this way, fully activates the Ca²⁺/CaMKI/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.

Key words: Ah receptor, CYP induction, CYP1A, halogenated hydrocarbons