Dioxin-Mediated Up-Regulation of Aryl Hydrocarbon Receptor Target Genes Is Dependent on the Calcium/Calmodulin/CaMKI Pathway

Regulation of genes targeted by the ligand-activated aryl hydrocarbon receptor (AhR) has been shown to be controlled by calcium (Ca ) changes induced by AhR agonists such as the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The present study was designed to characterize this link between Ca and the AhR pathway. We report that fast elevation of intracellular Ca in TCDD-exposed mammary MCF-7 cells was associated with transient enhanced activity of the Ca / calmodulin (CaM)-dependent protein kinase (CaMK) pathway. Chemical inhibition of this pathway using the CaM antagonist W7 or the CaMK inhibitor KN-93 strongly reduced TCDD-mediated induction of the AhR target gene CYP1A1. Small interfering RNA (siRNA)-mediated knockdown expression of CaMKI , one of the CaMK isoforms, similarly prevented CYP1A1 up-regulation. Both KN-93 and siRNA targeting CaMKI were found to abolish TCDDmediated activation of CYP1A1 promoter and TCDD-triggered nuclear import of AhR, a crucial step of the AhR signaling pathway. TCDD-mediated inductions of various AhR targets, such as the drug metabolizing CYP1B1, the cytokine interleukin-1 , the chemokines interleukin-8 and CCL1, the adhesion molecule 7 integrin, and the AhR repressor, were also prevented by KN-93 in human macrophages. Taken together, these data identified the Ca /CaM/CaMKI pathway as an important contributing factor to AhR-mediated genomic response. The aryl hydrocarbon receptor (AhR) is a helix-loop-helix transcription factor activated by endogenous ligands and xenobiotics such as the environmental contaminant 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD) (Barouki et al., 2007). In the absence of ligand, AhR, associated with 90-kDa heat shock protein, p23, and X-associated protein 2 (XAP2), is primarily located in the cytosol (Hankinson, 1995). After ligand binding, AhR moves to the nucleus, dissociates from the chaperone complex, and forms a heterodimer with the AhR nuclear translocator. This heterodimer binds to specific xenobiotic responsive elements (XRE) [core sequence: CACGCN(A/T)] found in the promoter of target genes and subsequently regulates their transcription (Swanson, 2002). In this way, TCDD and other AhR agonists [such as the carcinogenic environmental polycyclic aromatic hydrocarbons (PAHs)] markedly induce expression of the drug-metabolizing enzyme CYP1A1, known to detoxify but also to bioactivate carcinogens, including PAHs, and commonly considered a paradigm of AhR gene targets (Barouki et al., 2007). In addition to activation of AhR, TCDD and PAHs have been shown to increase intracellular concentration of Ca ([Ca ]i) (Burchiel et al., 1991; Archuleta et al., 1993; Davila et al., 1995; Tannheimer et al., 1997; Le Ferrec et al., 2002). It is noteworthy that blocking these [Ca ]i changes through the use of intracellular Ca chelator or store-operated channel blockers prevents induction of CYP1A1 (N Diaye et al., 2006), thus suggesting cross-talk between Ca variations This work was supported by grants from the Ligue Nationale contre le Cancer, the University of Rennes I, and the Agence Francaise de Sécurité Sanitaire de l’Environnement et du Travail (AFSSET). P.M. received a fellowship from the Ligue Nationale contre le Cancer Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.107.043125. ABBREVIATIONS: AhR, aryl hydrocarbon receptor; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; XAP2, X-associated protein 2; XRE, enobiotic responsive elements; PAH, polycyclic aromatic hydrocarbon; CaM, calmodulin; CaMK, Ca /CaM-dependent protein kinase; 2-APB, 2-Aminoethoxydiphenylborate; BAPTA-AM, 1,2-bis(O-aminophenoxy)ethane-N,N,N ,N -tetraacetic acid tetra(acetoxymethyl) ester; KN-93, 2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzenesulfonyl)amino-N-(4-chlorocinnamyl)-N-methylbenzylamine; KN-92, 2-[N-(4-methoxybenzenesulfonyl)]amino-N-(4chlorocinnamyl)-N-methylbenzylamine; W7, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide; AM, acetoxymethyl ester; Ab, antibody; EROD, ethoxyresorufin O-deethylase; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; siRNA, small interfering RNA. 0026-895X/08/7303-769–777$20.00 MOLECULAR PHARMACOLOGY Vol. 73, No. 3 Copyright © 2008 The American Society for Pharmacology and Experimental Therapeutics 43125/3311269 Mol Pharmacol 73:769–777, 2008 Printed in U.S.A. 769 at A PE T Jornals on Sptem er 6, 2017 m oharm .aspeurnals.org D ow nladed from and AhR-dependent regulatory pathways. Such interactions remain however to be characterized. For this purpose, it may be suitable to focus on key mediators of signaling pathways activated by Ca . Among these, the CaMKs represent a major one (Braun and Schulman, 1995). CaMKs correspond to a family of structurally related serine/threonine protein kinases that play important roles in proliferation (RodriguezMora et al., 2005) and differentiation (Zayzafoon, 2006). It is noteworthy that such cellular processes are also known to be affected upon AhR activation (Barouki et al., 2007). This has led us in the present study to analyze the putative contribution of CaMKs to AhR-dependent genomic response. Using mainly mammary MCF-7 cells, we report that activity of CaMKI , one of the CaMK isoforms, is required for TCDDtriggered nuclear translocation of AhR and subsequent upregulation of AhR target genes, especially of CYP1A1. Such data therefore highlight the Ca /CaM/CaMKI pathway as an important contributing factor to AhR-mediated genomic response. Materials and Methods Chemicals and Reagents. 2-Aminoethoxydiphenylborate (2-APB), ethoxyresorufin, and salicylamide were purchased from Sigma-Aldrich (St Louis, MO). TCDD was obtained from Cambridge Isotope Laboratories (Cambridge, MA), whereas 1,2-bis(O-aminophenoxy)ethaneN,N,N ,N -tetraacetic acid tetra(acetoxymethyl) ester (BAPTA-AM), the CaMK inhibitor KN-93 (2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzenesulfonyl)amino-N-(4-chlorocinnamyl)-N-methylbenzylamine), and its inactive structural analog KN-92 (2-[N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine), W7 (N(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide), and ionomycin were obtained from Calbiochem (La Jolla, CA). Pluronic acid and Fura-2-AM were provided from Invitrogen (Carlsbad, CA). Ficoll and TRIzol reagents were obtained from Invitrogen. Granulocyte macrophage–colony-stimulating factor (specific activity, 1.2 10 U/mg) was purchased from Schering-Plough (Lyon, France). Polyclonal goat antihuman CYP1A1/2 antibody (Ab) was obtained from Daiichi Pure Chemicals Co. Ltd. (Tokyo, Japan), polyclonal rabbit anti-AhR Ab from BIOMOL Research Laboratories (Plymouth Meeting, PA) and rabbit nonspecific IgG from GE Healthcare (Chalfont St. Giles, Buckinghamshire, UK). Monoclonal mouse anti-actin and polyclonal rabbit anti-p38 mitogen-activated protein kinase, antiglyceraldehyde-3-phosphate dehydrogenase, anti-phospho-CaMKI (Thr), and goat anti-lamin A/C Abs were purchased from Santa Cruz Biotechnology (La Perray en Yvelines, France). [ -P] ATP was from Amersham Biosciences. FITC-labeled anti-rabbit IgG Ab was purchased from Jackson Immunoresearch (Suffolk, UK). Chemicals were commonly used as stock solution in dimethyl sulfoxide. Final concentration of solvent did not exceed 0.2% (v/v); control cultures received the same volume of solvent as for treated counterparts. Cell Culture. Human mammary MCF-7 cells were cultured in Dulbecco’s modified Eagle’s medium with 4500 mg/l D-glucose, 110 mg/l sodium pyruvate, and nonessential amino acids, supplemented with 100 U/ml penicillin, 100 U/ml streptomycin, and 10% fetal calf serum. Primary human macrophages were obtained from granulocyte macrophage-colony-stimulating factor–exposed blood monocytes and cultured as described previously (van Grevenynghe et al., 2003). Intracellular Ca Measurements. Variations in [Ca ]i were analyzed by spectrofluorometry using the Ca -sensitive probe Fura2-AM, as reported previously (Le Ferrec et al., 2002). In brief, MCF-7 cells were cultured in 24-well plates and incubated with the acetoxy cell-permeant form of Fura-2 (Fura-2 AM; 1.5 M) for 30 min at 37°C in HEPES-buffered medium (10 mM HEPES, 134.8 mM NaCl, 4.7 mM KCl, 1 mM MgCl2, 1.2 mM KH2PO4, 1 mM CaCl2, and 10 mM glucose, pH 7.4, at 37°C), supplemented with 0.006% Pluronic acid. After removing Fura-2-AM, cells were placed in the spectrofluorometer (SpectraMax Gemini XS; Molecular Devices, Toronto, ON, Canada) and a well scan was performed at indicated times. Cells were irradiated alternately with light at wavelengths of 340 and 380 nm, and fluorescence from the trapped dye was measured at 510 nm. The ratio of fluorescence intensities recorded after excitation at 340 nm and 380 nm, defined as the F340/F380 ratio, was used to estimate [Ca ]i changes, knowing that [Ca 2 ]i increase results in an enhanced F340/F380 ratio (Le Ferrec et al., 2002; N Diaye et al., 2006). Effects of chemical treatment on [Ca ]i were expressed as F340/ F380 ratio (i.e., the F340/F380 ratio after the addition of chemicals minus the F340/F380 ratio measured before treatment). Ethoxyresorufin O-deethylase Activity Assay. EROD activity, corresponding to the O-deethylation of ethoxyresorufin, and mainly supported by CYP1A1 enzyme in living MCF-7 cells, was measured as described previously (Sparfel et al., 2006). In brief, MCF-7 cells were incubated in phosphate-buffered saline, pH 7.4, containing 50 M ethoxyresorufin and 1.5 mM salicylamide, and kinetic reading was performed at 37°C with a SpectraMax Gemini SX spectrofluorometer over a 30 min-period. RNA Isolation and Analysis. Total RNAs, extracted using the TRIzol method (Invitrogen), were subjected to reverse transcriptionreal-time quantitative polymerase chain reaction (RT-qPCR) analyses as described previously (Monteiro et al., 2007). Relative quantification of mRNA levels was performed after normalization of the total amount of cDNA tested to an 18 S RNA endogenous reference. The sequences of the primers used for RT-qPCR analysis are given in Table 1. Reverse Transfection of siRNA. For inhibiting expression of CaMKs, we first used “The Human siArray Reverse Transfection Format siRNA library,” targeting the various isoforms of the CaMK family (Dharmacon RNA Technologies, Lafayette, CO). This siRNA library corresponds to pools of four siRNA directed against different regions of targeted mRNA (Smart pool) for each CaMK isoform and spotted in 96 multiwell plates. In each well, 6.25 pmol of desiccated siRNAs were rehydrated using 0.125 l of Dharmafect I reagent diluted in 25 l of transfection medium (Opti-MEM; Invitrogen) for 40 min at room temperature. Next, 35,000 MCF-7 cells diluted in complete culture medium were added to each well. Twenty-four hours later, transfection medium was replaced by fresh medium for an