The aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor that is widely expressed across human cells and tissues. Once activated, it translocates to the nucleus and associates with the ARNT protein to form a complex that recognizes a specific DNA sequence, the xenobiotic response element (XRE), located in the promoter regions of its target genes, among which are members of the cytochrome P450 1 family such as CYP1A1. AHR is involved in xenobiotic metabolism, immune response, and cell differentiation, and regulates the response to numerous environmental contaminants such as dioxin and polycyclic aromatic hydrocarbons. Despite the importance of this pathway, studies have been hampered by the lack of a robust method to rapidly assess AHR transcriptional activity: conventional luciferase assays indeed require cell lysis.
To overcome this limitation, the authors constructed two novel reporter genes placed under the control of XRE elements: a fluorescent reporter combining the GFP protein with histone H2B (XRE-H2B-eGFP) and a secreted nanoluciferase (XRE-pNL1.3[secNluc]). These tools were validated in several cell types: primary human villous cytotrophoblasts, as well as NIH/3T3 mouse embryonic fibroblast lines and BeWo human placental cell lines. Cells were transfected and then treated with a strong AHR ligand, benzo[a]pyrene (B[a]P), or with an inhibitor, CH223191.
Compared with control cells, a significant increase in AHR activity was observed after treatment with 0.5 and/or 2 µM B[a]P, and a significant decrease after exposure to 3 µM CH223191. These responses, measured both by nuclear fluorescence and by secreted nanoluciferase, proved to be concentration-dependent. CYP1A1 protein expression, monitored in parallel by immunoblot, confirmed this profile, with dose-dependent induction under B[a]P. The three cell types did not, however, display the same degree of activation under 2 µM B[a]P, with induction reaching approximately 4-fold in cytotrophoblasts, 2-fold in NIH/3T3 cells, and only 1.2-fold in BeWo cells, suggesting a degree of cell-type specificity. The authors further note a nuclear localization of AHR and a basal activation, possibly related to endogenous ligands such as kynurenine or to compounds present in the culture medium.
These two systems complement existing tools while offering notable advantages: compatibility with different cell types and species, and the absence of cell lysis. The secreted nanoluciferase stands out for its sensitivity, requiring only a small volume of supernatant, whereas the fluorescent reporter allows live-cell imaging and real-time kinetic analysis. These plasmids pave the way for various applications, in particular the screening of endogenous or exogenous AHR ligands.