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The Aryl Hydrocarbon Receptor Regulates Focal Adhesion Sites Through a Non-Genomic FAK/Src Pathway

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The Aryl Hydrocarbon Receptor Regulates Focal Adhesion Sites Through a Non-Genomic FAK/Src Pathway Oncogene (2013) 32, 1811–1820 & 2013 Macmillan Publishers Limited All rights reserved 0950-9232/13 www.nature.com/onc ORIGINAL ARTICLE The aryl hydrocarbon receptor regulates focal adhesion sites through a non-genomic FAK/Src pathway C Tomkiewicz1,2, L Herry1,2,3, L-C Bui1,2,CMe´ tayer1,2, M Bourdeloux1,2, R Barouki1,2,3 and X Coumoul1,2 The aryl hydrocarbon receptor (AhR) is commonly described as a transcription factor, which regulates xenobiotic-metabolizing enzymes. Recent studies have suggested that the binding of ligands to the AhR also activates the Src kinase. In this manuscript, we show that the AhR, through the activation of Src, activates focal adhesion kinase (FAK) and promotes integrin clustering. These effects contribute to cell migration. Further, we show that the activation of the AhR increases the interaction of FAK with the metastatic marker, HEF1/NEDD9/CAS-L, and the expression of several integrins. Xenobiotic exposure, thus, may contribute to novel cell-migratory programs. Oncogene (2013) 32, 1811–1820; doi:10.1038/onc.2012.197; published online 4 June 2012 Keywords: AhR; cell plasticity; FAK; integrin; Src INTRODUCTION Ultimately, the phosphorylation of FAK leads to the dissociation of The aryl hydrocarbon receptor (AhR) is a xenobiotic-activated the FAS and cell migration. transcription factor involved in the detoxication pathways.1 It TCDD (2,3,7,8-p-Tetrachlorodibenzo-p-dioxin) is one of the most belongs to the basic helix loop helix/Per AhR nuclear translocator potent ligands of the AhR. Genome-wide transcriptome analyses that Sim family. Pollutant ligands of the AhR include dioxins, explore the effects of TCDD on gene expression have shown that furans, polychlorinated biphenlys and polycyclic aromatic several other biological pathways are regulated in addition to 23–25 hydrocarbons.2,3 Ubiquitous in mammals, the AhR forms a xenobiotic metabolism. We have shown that TCDD induces the 13,15,26,27 cytoplasmic complex with heat shock proteins. Upon ligand expression of the Hef1/Cas-L/Nedd9, Agr2 and Sos1 genes, binding, the AhR translocates into the nucleus where it interacts genes that are involved in the ‘epithelial mesenchymal transition’ with AhR nuclear translocator. The AhR/AhR nuclear translocator pathway and survival and metastasis. The increase in the cellular heterodimer recognizes xenobiotic-responsive elements in the mobility and morphological changes observed during the epithelial promoters of target genes and controls their expression.4 The mesenchymal transition elicited following 24–72 h of treatment with AhR signaling pathway is best known for the transcriptional TCDD and were found to depend upon the induction of the Hef1 13,28 regulation of xenobiotic-metabolizing enzymes, which are gene. However, in the present study, we found that subtle involved in the metabolism of drugs and pollutants. This elegant morphological changes in the FASs were observed after only 1–4 h of adaptive pathway allows the coordinate detection and elimination treatment with TCDD, well before any significant increase in the HEF1 of pollutants and, thus, protects organisms against foreign protein occurred. We, therefore, hypothesized that those rapid chemicals.4 effects, which require neither transcription nor translation, could be Recently, however, other genes and alternative pathways have at the origin of the initial changes in the cell morphology. Further, we been identified as AhR targets.1,5 Studies with gene-knockout suspected that these effects could be synergistic with the gene- models (in both vertebrates and invertebrates) have suggested regulatory effects of late onset to yield, together, the full spectrum of that the AhR has other functions including the regulation of cell morphological and migratory changes triggered by TCDD. migration during development.6–12 Several groups, including our Circumstantial support for this hypothesis comes from the own, have shown that, upon xenobiotic binding, the AhR observations (including a recent elegant study using fluorescence stimulates the migration and the invasion of several types of resonance energy transfer (FRET) technology) that the AhR, in the cells.6,10,13–17 cytoplasm, is bound to several chaperone proteins and to the Src 29–31 Cellular migration is a consequence, in part, of a redistribu- kinase. The binding of xenobiotics to the AhR not only initiates tion of the focal adhesion sites (FASs), which are composed of theeffectsoftheAhRongeneexpressionbutalso,perhapsas multiple transmembrane and cytoplasmic proteins (integrins, importantly, leads to the release of Src. This rapid liberation of Src, kinasesy).18,19 One component of the FASs, which has a crucial which is a non-transcriptional event, may lead to its activation and to role in cellular migration, is the focal adhesion kinase (FAK). FAK is the subsequent phosphorylation of Src partners, for example, those activated by autophosphorylation (tyrosine 397) following the at the FASs. In the present study, we show that the binding of clustering of integrins. This activation leads to the recruitment of xenobiotic to the AhR activates the redistribution of FAS and leads to SH2-domain-containing regulators, such as Src and other cell plasticity through non-transcriptional events and that these members of the SFK family (Src family of tyrosine kinases). events act in synergy to the transcriptional regulation of the major These kinases phosphorylate multiple other sites on FAK.20–22 genes involved in cell migration. 1INSERM UMR-S 747, Toxicologie Pharmacologie et Signalisation Cellulaire, Paris, France; 2Universite´ Paris Descartes, Sorbonne Paris Cite´, Centre Universitaire des Saints-Pe`res, Paris, France and 3AP-HP, Hoˆpital Necker Enfants Malades, Service de Biochimie Me´tabolique, Paris, France. Correspondence: Professor X Coumoul, Universite´ Paris Descartes, Sorbonne Paris Cite´, 45 rue des Saints-Pe`res, Paris 75006, France. E-mail: [email protected] Received 26 September 2011; revised 6 March 2012; accepted 30 March 2012; published online 4 June 2012 The AhR/Src pathway regulates integrin function C Tomkiewicz et al 1812 RESULTS AhR regulates early FAK and Src activation AhR activates early integrin clustering The FAK regulates FAS reorganization and integrin clustering.32 The reorganization of FAS in HepG2 cells treated with TCDD was We, therefore, hypothesized that FAK is an early target of TCDD. observed by immunostaining of paxillin (a component of FAS) and Examination of the activation of FAK and Src, as measured by their staining of actin. Morphological changes, which consisted of an profiles of phosphorylation, showed (Figure 2a) that the phos- increase in cell spreading, the formation of stress fibers, an phorylation of FAK on tyrosine 397 (Y397) is significantly increased extension of the FAS and the loss of cell contacts in the HepG2 after only 1 h of exposure of the cells to TCDD. This is an cells, occurred rapidly (4-8 h) following treatment with TCDD autophosphorylation, which creates a binding site for Src. Src, also, (Figure 1a). All of these changes depend upon the activation of is activated significantly after only 15 min of exposure of cells to the AhR because they were inhibited by the AhR antagonist, TCDD (Figure 2b), in agreement with previous studies.30,33–38 The a-naphthoflavone (aNF) (Figure 1b). Because the formation of new total amounts of FAK and Src remain constant during the course FAS during cell migration depends upon integrin clustering, we of those experiments. looked for changes in the distribution of integrins with specific Increased phosphorylation of tyrosines 861 and 925 in FAK also staining. After only 2–4 h of treatment with TCDD, clusters of was observed following treatment of the cells with TCDD. integrins associated with actin fibers were present at the cell However, the increase in the phosphorylation of Y925, which membrane (Figure 1c). This clustering was also blocked by aNF was significant after only 2 h of exposure of cells to TCDD, was still (Figure 1d). slower than that of tyrosine 397 and 861 (Figure 2a), as well as Figure 1. TCDD/AhR activates early morphological changes, redistribution of FASs and triggers integrin b1 clustering in HepG2 cells. (a) HepG2 cells were treated for 4, 8 or 48 h with TCDD (25 nM) and stained with DAPI (blue, nucleus), phalloidin (green, actin) and immunostained to detect paxillin (red, FASs). Scale bar, 10 mm. (b) HepG2 cells were treated with TCDD (25 nM) and/or aNF (5 mM) for 24 h and immunostained to detect paxillin (white, FASs, upper part of the panel) stained with phalloidin (white, actin, middle part of the panel). The merged images are shown in the lower panel. Scale bar, 10 mm. (c) HepG2 cells were treated with TCDD (25 nM) for 30, 120 or 240 min and immunostained to detect integrin b1 (green, upper part of the panel) and stained with phalloidin (magenta, middle part of the panel). The merged images are shown in the lower panel. White arrows show integrin clusters linked by sub-membrane actin fibers. Scale bar, 10 mm. (d) HepG2 cells were treated with TCDD (25 nM) and/or aNF (5 mM) for 24 h and immunostained to detect integrin b1 (green, upper part of the panel) and stained with phalloidin (magenta, actin, middle part of the panel). The merged images are shown in the lower panel. White arrows show integrin clusters linked by sub-membrane actin fibers. Scale bar, 10 mm. A full colour version of this figure is available at the Oncogene journal online. Oncogene (2013) 1811 – 1820 & 2013 Macmillan Publishers Limited The AhR/Src pathway regulates integrin function C Tomkiewicz et al 1813 Figure 2. TCDD triggers the early phosphorylation and activation of FAK/Src through an AhR-dependant pathway. (a and b) HepG2 cells were treated for increasing lengths of time (0,1 and 2 h) with TCDD (25 nM). Western blots were performed with cells extracts using (a) antibodies to: FAK total, FAK pY397, FAK pY861 or FAK pY925; (b) antibodies to: pan-Src or Src-pY416.
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