Modulation of Ceramide-Induced Apoptosis in Enteric Neurons by Aryl Hydrocarbon Receptor Signaling: Unveiling a New Pathway beyond ER Stress

Abstract

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a persistent organic pollutant and a potent aryl hydrocarbon receptor (AHR) ligand, causes delayed intestinal motility and affects the survival of enteric neurons. In this study, we investigated the specific signaling pathways and molecular targets involved in TCDD-induced enteric neurotoxicity. Immortalized fetal enteric neuronal (IM-FEN) cells treated with 10 nM TCDD exhibited cytotoxicity and caspase 3/7 activation, indicating apoptosis. Increased cleaved caspase-3 expression with TCDD treatment, as assessed by immunostaining in enteric neuronal cells isolated from WT mice but not in neural crest cell-specific Ahr deletion mutant mice (Wnt1Cre^+/-/Ahr^b(fl/fl)), emphasized the pivotal role of AHR in this process. Importantly, the apoptosis in IM-FEN cells treated with TCDD was mediated through a ceramide-dependent pathway, independent of endoplasmic reticulum stress, as evidenced by increased ceramide synthesis and the reversal of cytotoxic effects with myriocin, a potent inhibitor of ceramide biosynthesis. We identified Sptlc2 and Smpd2 as potential gene targets of AHR in ceramide regulation by a chromatin immunoprecipitation (ChIP) assay in IM-FEN cells. Additionally, TCDD downregulated phosphorylated Akt and phosphorylated Ser9-GSK-3β levels, implicating the PI3 kinase/AKT pathway in TCDD-induced neurotoxicity. Overall, this study provides important insights into the mechanisms underlying TCDD-induced enteric neurotoxicity and identifies potential targets for the development of therapeutic interventions.

Keywords: AHR; ENS; TCDD; apoptosis; ceramides; cytotoxicity.

Figure 1

TCDD induces apoptosis in IM-FEN cells. Cells were treated with vehicle or various doses of TCDD (0.1, 1, and 10 nM) for 24 h. Representative images of cleaved caspase-3 and cleaved PARP Western blots are included (A,B). Results from the protein bands’ density normalized to GAPDH have been included in the correspondent graphs. (C) Representative images of neuronal marker TUJ1 (red), TUNEL (green), and DAPI (blue) immunostaining in IM-FEN cells treated with vehicle and 10 nM TCDD for 24 h. Arrows point to the apoptotic cells (yellow) with condensed nuclei. Magnified image of the neuron shows the DNA fragmentation seen during apoptosis. Scale bar, 90 μm. The data represent three independent experiments. Results are mean ± SEM, **** p < 0.0001.

Figure 1

TCDD induces apoptosis in IM-FEN cells. Cells were treated with vehicle or various doses of TCDD (0.1, 1, and 10 nM) for 24 h. Representative images of cleaved caspase-3 and cleaved PARP Western blots are included (A,B). Results from the protein bands’ density normalized to GAPDH have been included in the correspondent graphs. (C) Representative images of neuronal marker TUJ1 (red), TUNEL (green), and DAPI (blue) immunostaining in IM-FEN cells treated with vehicle and 10 nM TCDD for 24 h. Arrows point to the apoptotic cells (yellow) with condensed nuclei. Magnified image of the neuron shows the DNA fragmentation seen during apoptosis. Scale bar, 90 μm. The data represent three independent experiments. Results are mean ± SEM, **** p < 0.0001.

Figure 2

Cytotoxicity and apoptosis induced by TCDD are AHR-dependent. IM-FEN cells were pretreated with an AHR antagonist, CH-223191 (10 µM), for 1 h and then treated with 10 nM TCDD at different time points (3, 6, 12, and 24 h). (A) Cytotoxicity was assessed by an LDH release assay. The percentage of cytotoxicity was calculated relative to the maximum LDH release control (10% Triton^® X-100). (B) Cell death by apoptosis was assessed by measuring Caspase-3/7 activity 1 h after adding the Caspase−Glo-3/7 reagent. Statistical analysis of LDH cytotoxicity assay data shows significant differences between vehicle and 10 nM TCDD after 6, 12, and 24 h of treatment. Not significant (ns) differences were observed between the vehicle and 10 nM TCDD + CH-223191 experimental groups. Results from the Caspase 3/7 assay show significant differences between vehicle and 10 nM TCDD treatment at all timepoints studied, whereas n.s. differences were observed when comparing the vehicle and 10 nM TCDD + CH-223191 experimental groups. Apoptosis was assessed by TUJ1/cleaved caspase-3 immunostaining of myenteric neurons isolated from (C) Wnt1Cre^−/−/Ahr^b(fl/fl) (control) mice and (D) Wnt1Cre^+/−/Ahr^b(fl/fl) (neural crest-specific Ahr^−/−) mice treated with vehicle and 10 nM TCDD for 24 h. Representative images show TUJ1 (red) and cleaved caspase-3 (green). The white arrow points to Tuj1⁺ cell bodies, and the white arrowhead points to the axons. The yellow arrowhead points to cleaved caspase-3-positive neurons. Scale bar, 90 μm. Results are mean ± SEM; * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 2

Cytotoxicity and apoptosis induced by TCDD are AHR-dependent. IM-FEN cells were pretreated with an AHR antagonist, CH-223191 (10 µM), for 1 h and then treated with 10 nM TCDD at different time points (3, 6, 12, and 24 h). (A) Cytotoxicity was assessed by an LDH release assay. The percentage of cytotoxicity was calculated relative to the maximum LDH release control (10% Triton^® X-100). (B) Cell death by apoptosis was assessed by measuring Caspase-3/7 activity 1 h after adding the Caspase−Glo-3/7 reagent. Statistical analysis of LDH cytotoxicity assay data shows significant differences between vehicle and 10 nM TCDD after 6, 12, and 24 h of treatment. Not significant (ns) differences were observed between the vehicle and 10 nM TCDD + CH-223191 experimental groups. Results from the Caspase 3/7 assay show significant differences between vehicle and 10 nM TCDD treatment at all timepoints studied, whereas n.s. differences were observed when comparing the vehicle and 10 nM TCDD + CH-223191 experimental groups. Apoptosis was assessed by TUJ1/cleaved caspase-3 immunostaining of myenteric neurons isolated from (C) Wnt1Cre^−/−/Ahr^b(fl/fl) (control) mice and (D) Wnt1Cre^+/−/Ahr^b(fl/fl) (neural crest-specific Ahr^−/−) mice treated with vehicle and 10 nM TCDD for 24 h. Representative images show TUJ1 (red) and cleaved caspase-3 (green). The white arrow points to Tuj1⁺ cell bodies, and the white arrowhead points to the axons. The yellow arrowhead points to cleaved caspase-3-positive neurons. Scale bar, 90 μm. Results are mean ± SEM; * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 3

TCDD-induced apoptosis in IM-FEN cells is independent of ER stress. Cells were treated with Veh and 10 nM TCDD for 24 h. Some cells received pre-treatment with the AHR antagonist CH-223191 (10 µM) for one hour before a 24 h exposure to 10 nM TCDD. Thapsigargin (300 nM, 5 h treatment) was used as a positive control for ER stress. Representative images of GRP78, IRE1α, and CHOP Western blots are included. Results from the protein bands’ density normalized to GAPDH have been included in the correspondent graphs. Results are mean ± SEM. Not significant (ns) differences were observed between vehicle and 10 nM TCDD with or without CH-223191 experimental groups.

Figure 4

Regulation of ceramide biosynthesis and sphingolipid metabolism by TCDD in IM-FEN cells. Quantitative PCR (qPCR), neutral sphingomyelinase (N-SMase) activity assays, lipidomics, and chromatin immunoprecipitation (ChIP) assays to elucidate the effects of TCDD treatment on ceramide metabolism and gene regulation. (A) Heat map visualization shows the normalized expression levels of key genes involved in ceramide biosynthesis (Sptlc1, Sptlc2, Cers2, Cser5, Cers6, Degs1, Smpd1, Smpd2, Smpd3, and Smpd4) in IM-FEN cells treated with vehicle or 10 nM TCDD over various time points (30 min, 1, 3, 6, 12, and 24 h), with upregulated genes in red and downregulated genes in blue. Columns represent the timepoint, and rows represent individual genes. (B) N-SMase activity of vehicle and 10 nM TCDD-treated IM-FEN cells for 24 h. (C) Lipidomics analysis presented through a heatmap, illustrating the log2 mean-centered normalized data of sphingolipids in vehicle and 10 nM TCDD-treated IM-FEN cells, with tiles colored red for high abundance and blue for low abundance. (D) Statistically significant changes in sphingolipids, sorted into categories, are highlighted to show the alterations in ceramide, sphingomyelin, and hexosylceramide levels. log2 mean-centered data were imported into R, and the Complex Heatmap package was used to create the heatmap. (E,F) IM-FEN cells were treated with vehicle and 10 nM TCDD for 1 h. (E) ChIP-qPCR result for Cyp1a1 gene promoter. (F) ChIP-qPCR results for Sptlc2 and Smpd2 gene promoters that were quantified by normalization with the corresponding input signal. Results are mean ± SEM, * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 4

Regulation of ceramide biosynthesis and sphingolipid metabolism by TCDD in IM-FEN cells. Quantitative PCR (qPCR), neutral sphingomyelinase (N-SMase) activity assays, lipidomics, and chromatin immunoprecipitation (ChIP) assays to elucidate the effects of TCDD treatment on ceramide metabolism and gene regulation. (A) Heat map visualization shows the normalized expression levels of key genes involved in ceramide biosynthesis (Sptlc1, Sptlc2, Cers2, Cser5, Cers6, Degs1, Smpd1, Smpd2, Smpd3, and Smpd4) in IM-FEN cells treated with vehicle or 10 nM TCDD over various time points (30 min, 1, 3, 6, 12, and 24 h), with upregulated genes in red and downregulated genes in blue. Columns represent the timepoint, and rows represent individual genes. (B) N-SMase activity of vehicle and 10 nM TCDD-treated IM-FEN cells for 24 h. (C) Lipidomics analysis presented through a heatmap, illustrating the log2 mean-centered normalized data of sphingolipids in vehicle and 10 nM TCDD-treated IM-FEN cells, with tiles colored red for high abundance and blue for low abundance. (D) Statistically significant changes in sphingolipids, sorted into categories, are highlighted to show the alterations in ceramide, sphingomyelin, and hexosylceramide levels. log2 mean-centered data were imported into R, and the Complex Heatmap package was used to create the heatmap. (E,F) IM-FEN cells were treated with vehicle and 10 nM TCDD for 1 h. (E) ChIP-qPCR result for Cyp1a1 gene promoter. (F) ChIP-qPCR results for Sptlc2 and Smpd2 gene promoters that were quantified by normalization with the corresponding input signal. Results are mean ± SEM, * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 4

Regulation of ceramide biosynthesis and sphingolipid metabolism by TCDD in IM-FEN cells. Quantitative PCR (qPCR), neutral sphingomyelinase (N-SMase) activity assays, lipidomics, and chromatin immunoprecipitation (ChIP) assays to elucidate the effects of TCDD treatment on ceramide metabolism and gene regulation. (A) Heat map visualization shows the normalized expression levels of key genes involved in ceramide biosynthesis (Sptlc1, Sptlc2, Cers2, Cser5, Cers6, Degs1, Smpd1, Smpd2, Smpd3, and Smpd4) in IM-FEN cells treated with vehicle or 10 nM TCDD over various time points (30 min, 1, 3, 6, 12, and 24 h), with upregulated genes in red and downregulated genes in blue. Columns represent the timepoint, and rows represent individual genes. (B) N-SMase activity of vehicle and 10 nM TCDD-treated IM-FEN cells for 24 h. (C) Lipidomics analysis presented through a heatmap, illustrating the log2 mean-centered normalized data of sphingolipids in vehicle and 10 nM TCDD-treated IM-FEN cells, with tiles colored red for high abundance and blue for low abundance. (D) Statistically significant changes in sphingolipids, sorted into categories, are highlighted to show the alterations in ceramide, sphingomyelin, and hexosylceramide levels. log2 mean-centered data were imported into R, and the Complex Heatmap package was used to create the heatmap. (E,F) IM-FEN cells were treated with vehicle and 10 nM TCDD for 1 h. (E) ChIP-qPCR result for Cyp1a1 gene promoter. (F) ChIP-qPCR results for Sptlc2 and Smpd2 gene promoters that were quantified by normalization with the corresponding input signal. Results are mean ± SEM, * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 5

Short-Chain Ceramides induce cytotoxicity and apoptosis in IM-FEN cells. IM-FEN cells were treated with vehicle, 25 µM C₂-ceramide, C₆-ceramide, C₂-DHC, and C₆-DHC for 30 min, 1-, 3-, 6-, 12-, and 24-h. (A) Cytotoxicity was assessed by the LDH release assay. The percentage of cytotoxicity was calculated relative to the maximum LDH release control (10% Triton^® X-100). (B) Apoptosis was assessed by measuring Caspase 3/7 activity. The statistical significance of the C₂-ceramide and C₆-ceramide treatment groups is shown with respect to vehicle. Not significant (ns) differences were observed between the vehicle and C₂- and C₆-DHC experimental groups. Results are mean ± SEM, * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 5

Short-Chain Ceramides induce cytotoxicity and apoptosis in IM-FEN cells. IM-FEN cells were treated with vehicle, 25 µM C₂-ceramide, C₆-ceramide, C₂-DHC, and C₆-DHC for 30 min, 1-, 3-, 6-, 12-, and 24-h. (A) Cytotoxicity was assessed by the LDH release assay. The percentage of cytotoxicity was calculated relative to the maximum LDH release control (10% Triton^® X-100). (B) Apoptosis was assessed by measuring Caspase 3/7 activity. The statistical significance of the C₂-ceramide and C₆-ceramide treatment groups is shown with respect to vehicle. Not significant (ns) differences were observed between the vehicle and C₂- and C₆-DHC experimental groups. Results are mean ± SEM, * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 6

Ceramide involvement in TCDD-induced cytotoxicity in IM-FEN cells. Cells were pretreated with 10 µM myriocin, an inhibitor of ceramide synthesis, for an hour and then treated with 10 nM TCDD at different time points (3, 6, 12, and 24 h). Cytotoxicity was assessed by the LDH release assay. The percentage of cytotoxicity was calculated relative to the maximum LDH release control (10% Triton^® X-100). The statistical significance of 10 nM TCDD with or without myriocin is shown with respect to vehicle. Results are mean ± SEM, * p < 0.05; *** p < 0.001. ns: Not significant.

Figure 7

Impact of TCDD and ceramide on neuronal survival pathways via Akt and GSK-3β modulation. IM-FEN cells were treated with vehicle and various doses of TCDD (0.1, 1, and 10 nM) and C₂ ceramide (25 µM) for 24 h. Representative images of Phospho-Akt (A,C) and Phospho-GSK-3β (B,D) Western blots are included. Results from the protein bands’ density normalized to corresponding Akt and GSK-3β have been included in the correspondent graphs. Results are mean ± SEM, *** p < 0.001; **** p < 0.0001.

Figure 8

A proposed model showing enhanced ceramide synthesis leading to apoptosis in IM-FEN cells. Key components showing activators in blue arrows and inhibitory associations in red lines.