Activation of the aryl hydrocarbon receptor sensitises human keratinocytes for CD95L- and TRAIL-induced apoptosis

Abstract

In this study, we have analysed the apoptotic effects of the ubiquitous environmental toxin benzo[a]pyrene (BP) in HaCaT cells and human keratinocytes. Although prolonged exposure to BP was not cytotoxic on its own, a strong enhancement of CD95 (Fas)-mediated apoptosis was observed with BP at concentrations activating the aryl hydrocarbon receptor (AhR). Importantly, the ultimately mutagenic BP-metabolite, that is, (+)-anti-BP-7,8-diol-9,10-epoxide (BPDE), failed to enhance CD95-mediated cell death, suggesting that the observed pro-apoptotic effect of BP is neither associated with DNA adducts nor DNA-damage related signalling. CD95-induced apoptosis was also enhanced by β-naphtoflavone, a well-known agonist of the AhR that does not induce DNA damage, thus suggesting a crucial role for AhR activation. Consistently, BP failed to sensitise for CD95L-induced apoptosis in AhR knockdown HaCaT cells. Furthermore, inhibition of CYP1A1 and/or 1B1 expression did not affect the pro-apoptotic crosstalk. Exposure to BP did not increase expression of CD95, but led to augmented activation of caspase-8. Enhancement of apoptosis was also observed with the TRAIL death receptors that activate caspase-8 and apoptosis by similar mechanisms as CD95. Together, these observations indicate an interference of AhR signalling with the activity of receptor-associated signalling intermediates that are shared by CD95 and TRAIL receptors. Our data thus suggest that AhR agonists can enhance cytokine-mediated adversity upon dermal exposure.

Figure 1

Induction of CYP1A1 by BP in NHEKs and HaCaT cells. (a) Transriptional induction of the CYP1A1 gene by BP: transcripts of CYP1A1 were measured by real-time PCR in normal (primary) human epidermal keratinocytes (NHEKs) and HepG2 cells after exposure to BP for 6 h. Control cells were treated with solvent only (0.1% DMSO). Total RNA was isolated and real-time PCR analysis was performed as described in Materials and Methods. Data are expressed as fold induction of transcripts. Values represent means±S.D. of three independent experiments. (b) Induction of CYP1A1 protein levels by BP: cells were treated with 3.5 μM BP as indicated and analysed by western blotting. Molecular size markers are shown on the left in kDa

Figure 2

BP does not induce apoptosis in HaCaT cells and NHEKs at exposure levels that trigger optimal induction of CYP1A1. (a) Cell viability and proliferation were determined by WST assays in the indicated cells after treatment with different concentrations of BP for 24 or 48 h. Values represent means±S.D. of three independent experiments. (b) Quantification of the apoptotic cell population by annexin V staining and flow cytometric analyses after BP treatment of cells for 24 or 48 h. Values represent means±S.D. of three independent experiments

Figure 3

BP sensitises HaCaT cells and NHEKs for CD95L-induced apoptosis. (a) HaCaT cells were pretreated with 3.5 μM BP (white bars) or 0.1% DMSO (black bars) for 48 h and subsequently exposed to different concentrations of CD95L for further 24 h with or without 20 μM pan-caspase inhibitor z-VAD (right). Cell viability was determined by crystal violet staining. Values represent means±S.D. of three independent experiments. (b and c) Cells were pretreated with 3.5 μM BP or solvent (0.1% DMSO) for 48 h and subsequently exposed to 2.5 ng/ml CD95L for 24 h. The populations of apoptotic cells were determined by annexin V staining and flow cytometric analyses. Data from three independent experiments are summarised in (b), and images obtained in a typical experiment with HaCaT cells are shown in (c). (d) Induction of cytochrome c release in CD95L/BP-treated cells was determined by western blotting as described in Materials and Methods. The increase of the cytosolic fraction was quantified by densiometry and average based on three independent experiments in (left panel). The columns shown in the right panel correspond to the bar diagram shown in the lower panel. Molecular size markers are shown on the left in kDa

Figure 4

Pro-apoptotic crosstalk between CD95L and BP is not triggered by BP metabolites. (a and b) Cells were pretreated with 3.5 μM BP, 2 μM 3-OH-BP or 2 μM BP-1.6-dione (a), 6 μM BPDE (b) or solvent (0.1% DMSO) for 48 h and subsequently exposed to 2.5 ng/ml CD95L for further 24 h. The populations of apoptotic cells were determined by annexin V staining and flow cytometric analyses. Values represent means±S.D. of three independent experiments

Figure 5

Sensitisation of HaCaT cells and NHEKs for CD95L-induced apoptosis by the non-genotoxic AhR agonist β-naphtoflavone (β-NF). (a) Cell viability in HaCaT cells was determined by crystal violet staining after treatment with β-NF for 24 and 48 h. Values represent means±S.D. of three independent experiments. (b) HaCaT cells were pretreated with 10 μM β-NF (white bars) or 0.1% DMSO (black bars) for 48 h and subsequently exposed to CD95L as indicated. Cell viability was then determined by crystal violet staining. Values represent means±S.D. of three independent experiments. (c) β-NF sensitises HaCaT cells and NHEKs for CD95L-induced apoptosis. Cells were pretreated with β-NF as indicated or with solvent (0.1% DMSO) for 48 h and subsequently exposed to 2.5 ng/ml CD95L for further 24 h. The populations of apoptotic cells were determined by annexin V staining and flow cytometric analyses. Values represent means±S.D. of three independent experiments

Figure 6

AhR agonists fail to promote apoptosis in AhR-deficient cells. (a) Expression of AhR in AhR-deficient (knockdown (k.d.)) HaCaT cells compared with wild-type (w.t.) and empty vector (e.v.) HaCaT cells, analysed by western blotting. Molecular size markers are shown on the left in kDa. (b) Sensitisation of HaCaT cells for CD95L-induced apoptosis depends on the presence of a functional AhR protein. AhR e.v., parental (empty) control vector (black bars); AhR k.d., shRNA AhR k.d. (white bars). Cells were pretreated with either 3.5 μM BP or 10 μM β-NF for 48 h and subsequently exposed to 2.5 ng/ml CD95L for further 24 h. The populations of apoptotic cells were determined by annexin V staining and flow cytometric analyses. Values represent means±S.D. of three independent experiments. (c) HaCaT cultures were transfected using HiPerfect (Qiagen, Hilden, Germany) and predesigned siRNAs against AhR (Applied Biosystems), CYP1A1 (Qiagen), CYP1B1 (Applied Biosystems) and a mixture of the CYP1A1 and CYP1B1 siRNAs. Cells were subsequently exposed to 3.5 mM BP for 48 h, followed by an exposure to 200 ng/ml TRAIL until significant cell death was observed (∼2–3 h). Cell viability was determined using crystal violet and compared with transfected cells not exposed to TRAIL. A control siRNA from Applied Biosystems was included as experimental control (right). Inhibition of EROD (CYP1A) activities after AhR, CYP1A1 and CYP1B1 k.d. was demonstrated for transfected HaCaT populations in parallel

Figure 7

AhR-dependent enhancement of death receptor-mediated apoptosis. (a) HaCaT cells were pretreated with 3.5 μM BP (white bars) or 0.1% DMSO (black bars) for 48 h and subsequently exposed to different concentrations of TRAIL for further 24 h with or without 20 μM pan-caspase inhibitor z-VAD (right). Cell viability was then determined by crystal violet staining. Values represent means±S.D. of three independent experiments. (b) HaCaT AhR parental (empty) control vector cells were pretreated with 3.5 μM BP (white bars) or 0.1% DMSO (black bars) for 48 h and subsequently exposed to different concentrations of CD95L or TRAIL for further 24 h with or without 20 μM pan-caspase inhibitor z-VAD (right). Cell viability was then determined by crystal violet staining. Values represent means±S.D. of three independent experiments. (c) HaCaT shRNA AhR k.d. cells were pretreated with 3.5 μM BP (white bars) or 0.1% DMSO (black bars) for 48 h and subsequently exposed to different concentrations of CD95L or TRAIL for further 24 h with or without 20 μM pan-caspase inhibitor z-VAD (right). Cell viability was then determined by crystal violet staining. Values represent means±S.D. of three independent experiments. (d) Sensitisation of undifferentiated (KBM) and differentiating (KBM−) NHEKs for TRAIL-induced apoptosis: to trigger differentiation, keratinocytes were switched to growth supplement deprived KBM− medium or maintained in KBM medium for 24 h. Expression of distinct differentiation markers was confirmed by real-time PCR (Supplementary Figure 5). Both populations were pretreated with 3.5 μM BP for 48 h and subsequently exposed to 250 ng/ml (left) or 100 ng/ml (right) TRAIL until signs of apoptosis were observed by light microscopy. Proportions of apoptotic cells were determined by annexin V staining and flow cytometric analyses. Values represent means±S.D. of three experiments

Figure 8

BP promotes activation of caspase-8 and caspase-3 in AhR-proficient but not in AhR-deficient cells. (a) HaCaT cells were pretreated with 3.5 μM BP or 0.1% DMSO for 48 h, subsequently exposed to indicated concentrations of CD95L (upper panel) or TRAIL (lower panel) and analysed by western blotting. Caspase cleavage products, FLIP long and actin are indicated. In addition, PARP cleavage was analysed in the TRAIL experiment. (b) HaCaT parental (empty) control vector cells (e.v.) and HaCaT shRNA AhR knockdown cells (k.d.) were pretreated with 3.5 μM BP or 0.1% DMSO for 48 h, subsequently exposed for different time intervals to 20 ng/ml of CD95L-Fc (upper panel) or 100 ng/ml TRAIL (lower panel) and analysed by western blotting. Caspase cleavage products, FLIP long and actin are indicated