Aryl hydrocarbon receptor activation in primary human keratinocytes and epidermal equivalents

Abstract

The Aryl hydrocarbon Receptor (AhR) mediates the toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) resulting in the human specific toxicity, chloracne. To test whether the chloracnegenic potential of AhR-agonists depends upon binding affinity for the AhR, residency and/or down-regulation of the AhR, we investigated the effects of different AhR agonists in primary human keratinocytes and epidermal equivalents. The AhR agonists used were high-affinity, high-residency and high-potency TCDD, and two agonists not known to induce chloracne; low-affinity, low-residency and low-potency β-naphthoflavone (β-NF) and the low-affinity, low-residency and high-potency physiological agonist 2-(1‟H-indole-3‟-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE). -NF, a partial agonist was used to test AhR dependency. The effects of these agonists on AhR activation, terminal differentiation, autophagy and expression of cathepsin D (CTSD) in primary human keratinocytes and epidermal equivalents were determined. All three agonists induced AhR activation by XRE-luciferase assay, which was inhibited by α-NF, demonstrating AhR dependence of the ligands. AhR degradation was induced by all ligands and CYP1A1 was induced strongly by TCDD but weakly by β-NF and ITE. CYP1A1 and XRE-luciferase induction correlated with ligand binding affinity; ranking levels of binding affinity as TCDD>β-NF>ITE. TCDD treatment induced a chloracne-like phenotype in epidermal equivalents, with a decrease in viable cell layer thickness and compacted stratum corneum. This was not induced by β-NF or ITE. To investigate the differential effects of AhR-ligands on epidermal equivalent phenotype, we studied differentiation markers filaggrin, involucrin and TGM-1. TGM-1 expression was induced specifically by TCDD while aberrant expression of involucrin and filaggrin were induced by TCDD, β-NF and ITE. AhR activation was not associated with increased apoptosis. Caspase-3 independent cell death has been implicated as a mechanism of decreased thickness of the viable cell layer, so we studied the effects of AhR-agonists on autophagy. Autophagy in keratinocytes and epidermal equivalents was characterised by induction of LC3 II, p62 degradation and transmission electron microscopy. TCDD robustly induced active autophagy, while ITE induced lower levels and β-NF blocked autophagy. TCDD- and ITE-induced autophagy in epidermal equivalents appeared to result in decreased numbers of lamellar bodies, which may account at least in part for the compacted stratum corneum phenotype shown by the TCDD-induced phenotype in epidermal equivalents and chloracne. As CTSD has been implicated in keratinocyte differentiation and an XRE domain has been identified upstream of CTSD, we studied the effects of ligand-dependent AhR activation on lysosomal aspartic protease CTSD expression. CTSD was increased by AhR activity in epidermal equivalents. Induction of CYP1A1 did not appear to be a specific biomarker of chloracnegenic potential of AhR agonists. The data presented have shown differential effects by TCDD, β-NF and ITE on autophagy that we hypothesise contributes to the chloracne phenotype. In this thesis, potential biomarkers specific to chloracne were identified in keratinocytes, TGM-1, CTSD, autophagy and decreased lamellar bodies, although further validation is required.