Benzo[a]pyrene vs. Tapinarof: unraveling ligand-specific AHR transcriptional responses in immortalized keratinocytes.

The aryl hydrocarbon receptor (AHR) integrates structurally diverse ligands to control xenobiotic metabolism, immune responses, and cell fate decisions. Ligand-specific AHR effects in human skin are increasingly recognized, but underlying mechanisms remain incompletely understood. Here, we compared transcriptomic responses of immortalized human keratinocytes to the environmental AHR ligand benzo[a]pyrene (BaP) and the therapeutic AHR agonist Tapinarof. Using AHR-proficient and AHR-mutant HaCaT keratinocytes, we performed bulk RNA-sequencing, pathway enrichment analysis, and targeted validation by qPCR and immunoblot analyses. Both ligands elicited largely AHR-dependent yet distinct gene expression programs. As expected, both ligands increased the expression of canonical AHR/ARNT pathway target genes, including cytochrome P450 (CYP) 1A1, CYP1B1, and artemin. However, BaP uniquely activated a non-canonical AHR signaling pathway involving epidermal growth factor receptor (EGFR) and downstream signaling that stimulates NRF2 transcriptional activity. Tapinarof, while initiating similar initial events (e.g., c-Src phosphorylation), interfered with this pathway by inhibiting phosphoinositide 3-kinase/AKT signaling. Another gene set related to antiviral responses was also solely modulated by BaP and sensitive to CYP1A inhibition. This gene set also responded to etoposide, which induces DNA double-strand breaks independently of AHR. Indeed, inhibiting CYP1A-catalyzed metabolic activation of BaP reduced the level of phosphorylated histone H2AX, a marker for DNA double-strand breaks, and respective gene expression. Collectively, our data show that BaP and Tapinarof engage overlapping but mechanistically distinct AHR-dependent networks in immortalized keratinocytes, with BaP uniquely combining non-canonical AHR-EGFR-NRF2 signaling and CYP1A1-dependent genotoxic stress responses. Our results further suggest an off-target effect of Tapinarof, i.e., AKT inhibition.