PhD ThesisChloracne is a hyperkeratotic acneform skin disease caused by exposure to 2,3,7,8-tetrachlorodibenzo–p-dioxin (TCDD), a potent agonist of the aryl-hydrocarbon receptor (AhR). Despite the known role of TCDD exposure in the pathogenesis of chloracne, the molecular mechanisms mediating the disease remain poorly defined.
Using a previously optimised in vitro primary human keratinocyte epidermal equivalent model, we demonstrated the temporal regulation and development of a significantly reduced viable cell layer and compacted stratum corneum over 7 days with 10nM TCDD. These morphological changes were paralleled by cumulative AhR protein degradation and increased mRNA levels of CYP1A1. One of the key findings was a significant increase in the expression of the apoptotic marker, caspase-3 whilst there was no significant effect on Ki67 staining. Furthermore, TCDD treatment caused de-regulated epidermal differentiation as evidenced by increased mRNA expression but decreased protein expression of late differentiation markers. Treatment with TCDD also resulted in an induction of LC3 lipidation and endogenous LC3 protein expression as well as decreased P62 protein expression, well-established markers of autophagy induction. Interestingly, co-treatment of epidermal equivalents with TCDD and the lysosomal inhibitor, bafilomycin or cathepsin D inhibition (by pepstatin A or shRNA knockdown) resulted in restoration of the viable cell layer and reduction in TCDD-induced caspase-3 expression. Similar results were also seen after blockade of the autophagy pathway by ATG7 knockdown. Results also demonstrated lysosomal function and autophagy are required for TCDD-induced AhR degradation, indicating a potential role for chaperone-mediated autophagy. Collectively these data suggest exposure to TCDD results in deregulated epidermal differentiation, induction of autophagy, reduction of a viable cell layer and caspase-3 dependent cell death, likely mediated via lysosomal processing. Results provide an insight into the pathophysiology of chloracne, and demonstrate novel findings including TCDD-induced autophagy and potential role of lysosomal function in TCDD-induced death and AhR degradation.Biotechnology and Biological Sciences Research Council (BBSRC) and AstraZeneca
