Unraveling the differential impact of PAHs and dioxin-like compounds on AKR1C3 reveals the EGFR extracellular domain as a critical determinant of the AHR response

Abstract

Polycyclic aromatic hydrocarbons (PAHs), dioxin-like compounds (DLCs) and structurally-related environmental pollutants may contribute to the pathogenesis of various diseases and disorders, primarily by activating the aryl hydrocarbon receptor (AHR) and modulating downstream cellular responses. Accordingly, AHR is considered an attractive molecular target for preventive and therapeutic measures. However, toxicological risk assessment of AHR-modulating compounds as well as drug development is complicated by the fact that different ligands elicit remarkably different AHR responses. By elucidating the differential effects of PAHs and DLCs on aldo-keto reductase 1C3 expression and associated prostaglandin D₂ metabolism, we here provide evidence that the epidermal growth factor receptor (EGFR) substantially shapes AHR ligand-induced responses in human epithelial cells, i.e. primary and immortalized keratinocytes and breast cancer cells. Exposure to benzo[a]pyrene (B[a]P) and dioxin-like polychlorinated biphenyl (PCB) 126 resulted in a rapid c-Src-mediated phosphorylation of EGFR. Moreover, both AHR agonists stimulated protein kinase C activity and enhanced the ectodomain shedding of cell surface-bound EGFR ligands. However, only upon B[a]P treatment, this process resulted in an auto-/paracrine activation of EGFR and a subsequent induction of aldo-keto reductase 1C3 and 11-ketoreduction of prostaglandin D₂. Receptor binding and internalization assays, docking analyses and mutational amino acid exchange confirmed that DLCs, but not B[a]P, bind to the EGFR extracellular domain, thereby blocking EGFR activation by growth factors. Finally, nanopore long-read RNA-seq revealed hundreds of genes, whose expression is regulated by B[a]P, but not by PCB126, and sensitive towards pharmacological EGFR inhibition. Our data provide novel mechanistic insights into the ligand response of AHR signaling and identify EGFR as an effector of environmental chemicals.

Keywords: Aldo-keto reductase 1C3; Aryl hydrocarbon receptor; Dioxin-like compounds; Environmental pollutants; Epidermal growth factor receptor; Polycyclic aromatic hydrocarbons.

Fig. 1.. B[a]P induces AKR1C3 expression in an AHR-dependent manner.

a qRT-PCR analyses of CYP1A1 and AKR1C3 in NHEKs stimulated with B[a]P (2.5 μM), PCB126 (1 μM) or solvent (0.1 % DMSO) for 24 h. n = 9. *, p ≤ 0.05 compared to DMSO; #, p ≤ 0.05 compared to B[a]P. b qRT-PCR analysis of AKR1C3 in HaCaT keratinocytes stimulated as indicated for 24 h. n = 6. *, p ≤ 0.05 compared to DMSO control, #, p ≤ 0.05, compared to B[a]P of the same concentration. c qRT-PCR analyses of CYP1A1 and AKR1C3 in HaCaT-shAHR and HaCaT-EV keratinocytes exposed to 2.5 μM B[a]P, 1 μM PCB126 or 0.1 % DMSO for 24 h. n = 4. *, p ≤ 0.05 compared to EV DMSO, #, p ≤0.05 compared to EV B[a]P. d Western blot analyses of AKR1C3 protein content in HaCaT-shAHR and HaCaT-EV keratinocytes stimulated as described in c. GAPDH level served as loading control. n = 3, representative picture. e LC-MS analyses of supernatants derived from HaCaT-shAHR and HaCaT-EV cells stimulated with 0.1 % DMSO, 1 μM PCB126 or 2.5 μM B[a]P for 24 h. Afterwards, cells were treated with 1 μM PGD₂ in conditioned medium and supernatants were collected at indicated time points. n = 3. *, p ≤ 0.05 compared to DMSO. f LC-MS analyses of the supernatants of HaCaT cells treated with 0.1 % DMSO and 2.5 μM B[a]P for 24 h. In addition, HaCaT cells pretreated for 23 h with 2.5 μM B[a]P were co-exposed for 1 h to 50 μM flufenamic acid (FFA) and B[a]P. Subsequently, cells were treated with 1 μM PGD2 in conditioned medium and the supernatant was collected at indicated time points. Supernatants were collected at indicated time points. n = 3. *, p ≤ 0.05 compared to DMSO.

Fig. 2.. B[a]P stimulates AKR1C3 expression in an EGFR-dependent manner.

a HaCaT keratinocytes were transiently transfected with ARNT-targeted or non-silencing siRNA for 24 h. Next, cells were treated with 2.5 μM B[a]P or solvent for another 24 h. The protein level of CYP1A1, ARNT and AKR1C3 was detected by western blot analyses, α-tubulin was used as loading control. n = 2, representative pictures. b qRT-PCR analyses of CYP1A1 and AKR1C3 in HaCaT-shAHR and HaCaT-EV cells treated with 10 μM PP2 or 0.1 % DMSO for 24 h. n = 4. *, p ≤ 0.05 compared to EV DMSO, #, p ≤ 0.05 compared to EV B[a]P. c qRT-PCR analyses of CYP1A1 and AKR1C3 in HaCaT-shAHR and HaCaT-EV keratinocytes treated as indicated for 24 h. n = 4–7. *, p ≤ 0.05 compared to EV DMSO, #, p ≤ 0.05 compared to EV B[a]P. d NHEKs were treated with 20 ng/ml AREG for 24 h and AKR1C3 transcript level were analyzed by qRT-PCR. e Western blot analyses of SRC and its phosphorylated form (Y416). HaCaT keratinocytes were treated with B[a]P (2.5 μM), PCB126 (1 μM) or 0.1 % DMSO for the indicated time. n = 3, representative pictures. f Phosphorylation of EGFR in HaCaT keratinocytes treated with B[a]P (2.5 μM) or PCB126 (1 μM) was examined by western blot analysis. Cells were treated for 15 min and 2 h. DMSO (0.1 %) served as solvent control, EGF (10 ng/ml) as positive control. n = 3, representative picture. g Levels of activated PKC were quantified using a non-radioactive protein kinase activity assay. HaCaT keratinocytes were pre-treated with Bosutinib (1 μM), MNF (20 μM), CH223191 (10 μM) or 0.1 % DMSO for 1 h. Afterwards, cells were stimulated with B[a]P (2.5 μM) or PCB126 (1 μM) and 0.1 % DMSO. After 2 h, cells were lysed and PKC activity was determined. ELISA-based quantification of h AREG and i TGFα in the cell culture supernatants. Cells were treated as described in g. Supernatants were collected 2 h after treatment. n = 4. *, p ≤ 0.05 compared to DMSO, #, p ≤ 0.05 compared to either B[a]P or PCB126 treated cells respectively.

Fig. 3.. B[a]P stimulates AKR1C3 expression through a non-canonical signaling pathway.

Western blot analyses of ERK1/2 phosphorylation upon exposure to a B[a]P (2.5 μM) and b PCB126 (1 μM) for the indicated time points. In the upper panel the densitometric quantification, in the lower panel representative blots are shown. n = 3 – 5. *, p ≤ 0.05 compared to DMSO. Western blot analysis of HaCaT cells treated with c B[a]P (2.5 μM) and d PCB126 (1 μM) for 15 min or e B[a]P (2.5 μM) for 2 h. In parallel, cells were co-treated with bosutinib (1 μM), marimastat (1 μM), PD153035 (1 μM), RO-31–8220 (1 μM), MNF (20 μM), CH223191 (10 μM) or DMSO (0.1 %). In c and d EGFR phosphorylation at residue Y845 and in e EGFR phosphorylation at residue Y1068 was examined. All results were normalized to total EGFR, GAPDH was used as loading control. n = 3, representative pictures. f qRT-PCR analyses of AKR1C3 in HaCaT keratinocytes. The cells were treated with B[a]P (2.5 μM) in the absence and presence of either Bosutinib (1 μM), PD153035 (1 μM), BP1-QII (1 μM), Cobimetinib (1 μM), EGFR-blocking antibody (4 μg/ml), Marimastat (1 μM) or DMSO (0.1 %) for 24 h. n = 3 – 6. *, p ≤ 0.05 compared to DMSO, #, p ≤ 0.05 compared to B[a]P. g Western blot analysis of AKR1C3 protein level in HaCaT keratinocytes. Cells were treated as indicated at the concentrations depicted in f for 24 h. In the lower panel a representative blot, in the upper panel the densitometric quantification is shown. n = 3. *, p ≤ 0.05 compared to DMSO, #, p ≤ 0.05 compared to B[a]P. h qRT-PCR analyses of AKR1C3 in NHEKs treated for 24 h with B[a]P (2.5 μM) in the absence and presence of either Bosutinib (1 μM), PD153035 (1 μM), Marimastat (1 μM), MNF (20 μM), CH223191 (10 μM) or DMSO (0.1 %). Additional cells were treated with 20 ng/ml AREG. n = 7. *, p ≤ 0.05 compared to DMSO, #, p ≤ 0.05 compared to B[a]P.

Fig. 4.. Induction of AKR1C3 depends on NRF2 in HaCaT keratinocytes.

a Western blot analyses of NRF2 protein stabilization. Cells were treated as indicated, the co-treatment with D3T (70 μM) and MG-132 (10 μM) served as positive control. Whole cell lysates were prepared 6 h post stimulation. GAPDH was used as loading control. On the left a representative western blot, on the right the densitometric quantification is shown. n = 4. *, p ≤ 0.05 compared to DMSO, #, p ≤ 0.05 compared to B[a]P. b qRT-PCR analyses of CYP1A1 and AKR1C3 in HaCaT and HaCaT-NRF2-KO (DU34) keratinocytes. Cells were treated with B[a]P (2.5 μM), PCB126 (1 μM) or DMSO (0.1 %) DMSO for 24 h. n = 3. *, p ≤ 0.05 compared to DMSO HaCaT control, #, p ≤ 0.05 compared to the respective proficient HaCaT control. c ROS formation was analyzed using the DCF-DA assay. HaCaT cells were treated as indicated for 6 h. As a positive control cells were treated with H₂O₂ 30 min prior to staining. n = 3. *, p ≤ 0.05 compared to DMSO. d mRNA level of AKR1C3 in HaCaT and HaCaT-NRF2-KO (DU34) cells treated for 24 h as indicated. Test compounds were used at the following concentrations: DMSO (0.1 %), B[a]P (2.5 μM), D3T (70 μM) and GSH (100 μM). n = 3. *, p ≤ 0.05 compared to DMSO, #, p ≤ 0.05 compared to the respective HaCaT control sample. e qRT-PCR analyses of AKR1C3 in HaCaT and HaCaT-NRF2-KO (ΔNRF2) keratinocytes treated as indicated for 24 h. n = 4. *, p ≤ 0.05 compared to DMSO, #, p ≤ 0.05 compared to the respective HaCaT control sample.

Fig. 5.. TCDD and PCB126 interfere with the activation and internalization of EGFR.

a The effect of the DLCs on the interaction of EGF with EGFR was analyzed by using a cell-free EGF/EGFR AlphaLISA binding kit. n = 3. *, p ≤ 0.05 compared to DMSO. b Effect of B[a]P and PCB126 on EGFR activation upon TGFα stimulation was analyzed via western blot analyses. HaCaT keratinocytes were starved for 3 h and next stimulated with TGFα (20 ng/ml) for 2.5 min on ice. Afterwards, either DMSO (0.1 %), PCB126 (1 μM) or B[a]P (2.5 μM) was added and cells were incubated at 37 °C and 5 % CO₂ for 30 min. Levels of total and phosphorylated EGFR (Y1068, Y1137) were determined; β-Actin was used as loading control. n = 3. representative pictures. c Influence of PCB126 (1 μM), TCDD (0.1 μM) and B[a]P (2.5 μM) on EGFR internalization was investigated by EGFR internalization assay and subsequent high content screening microscopy. On the left representative pictures, on the right results from the automated quantification are shown. n = 4–7. *, p ≤ 0.05 compared to DMSO. #, p ≤ 0.05 compared to B[a]P. d Colorimetric BrdU incorporation assay to assess the influence of PCB126, TCDD and PD153035 on AREG-induced DNA synthesis. HaCaT keratinocytes were treated as indicated for 4 h. Absorption was measured at a wavelength of 370 nm (reference wavelength 492 nm). n = 3. *, p ≤ 0.05 compared to DMSO. #, p ≤ 0.05 compared to AREG/DMSO. e Colorimetric BrdU incorporation assay in HaCaT-AHR-KO (DU26) keratinocytes. Cells were treated as indicated and the experiment was performed as described in e. n = 7. *, p ≤ 0.05 compared to DMSO. #, p ≤ 0.05 compared to AREG/DMSO.

Fig. 6.. PCB126 and TCDD bind to EGFR and inhibit its growth factor-induced activation.

a In silico docking analyses predicting the binding of PCB126 (blue) and TCDD (orange) to the ECD of EGFR (grey). EGF (magenta; taken from PDB ID: 1ivo) is superimposed. Interacting amino acid residues of EGFR ECD for b PCB126 and c TCDD shown as stick models. d Potentially interacting residues were converted to alanine by site directed mutagenesis. HepG2 cells were either transfected with an empty vector (pCMV3), pCMV3-EGFR plasmid or a plasmid bearing one of the following point mutations: EGFR^Q8A, EGFR^S11A or EGFR^Q408A. 24 h post transfection, cells were starved for 3 h and treated with EGF (10 ng/ml) alone and in combination with 1 μM PC126 for 15 min. Phosphorylation of EGFR residue Y1068 was assessed by western blotting and the results were normalized to total EGFR. GAPDH was used as loading control. Bar graph shows the densitometric quantification. Signals were compared with the respective DMSO control. n = 3. *, p ≤ 0.05 compared to DMSO, #, ≤ 0.05 compared to EGF. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 7.. RNA-Seq analysis reveals distinct expression patterns upon B[a]P treatment.

RNA-Seq analyses of HaCaT keratinocytes using oxford nanopore long-read RNA sequencing technology. Cells were treated with DMSO (0.1 %), PCB126 (1 μM), B[a]P (2.5 μM) or B[a]P (2.5 μM) + PD153035 (1 μM) for 24 h. n = 3. a Differentially expressed genes compared to DMSO with a |log₂ fold change| ≥ 1.5 depicted in a venn diagram. b Gene set enrichment analysis of biological processes of a subset of filtered genes, which were solely regulated by B[a]P and where EGFR inhibition of EGFR signaling with PD153035 counteracted this regulation. Following filter strategy was applied: PCB126 vs. DMSO |log₂ fold change| ≤ 1 → B[a]P vs PCB126 |log₂ fold change| ≥ 2 → B[a]P vs B[a]P + PD153035 |log₂ fold change| ≥ 1. Data set of differentially expression analysis of PCB126 vs. B[a]P was filtered for the remaining genes. Regulation of biological processes was depicted in a dot plot. c Genes filtered for b were employed on a KEGG pathway analysis. Regulated pathways are shown in a dot plot. d Variance heatmap of predefined expression patterns. Shown are the top 10 genes with the highest variance of each gene expression type. Type A: PCB126 vs. DMSO log₂ fold change > 1.5 → B[a]P vs. DMSO log₂ fold change > 1.5 → B[a]P + PD153035 vs. B[a]P log₂ fold change > 0. Type B: PCB126 vs. DMSO log₂ fold change < 1 → B[a]P vs. DMSO log₂ fold change > 1.5 → B[a]P vs. B[a]P + PD153035 log₂ fold change > 0. Type C: PCB126 vs. DMSO log₂ fold change > 0 → B[a]P vs. DMSO log₂ fold change < −1 → B[a]P vs. B[a]P + PD153035 log₂ fold change < 0. e qRT-PCR analysis of genes representing the different types of gene expression patterns. n = 4. *, p ≤ 0.05 compared to DMSO, #, ≤ 0.05 compared to B[a]P.