Cortisol synthesis by primary human keratinocytes

Abstract

Cortisol analogues have been used to treat skin disorders, such as psoriasis and atopic dermatitis, for over 50 years but the ability of normal human keratinocytes to synthesise cortisol has not been reported. Keratinocytes are capable of de novo cholesterol synthesis, they express P450 enzymes that are required for steroidogenesis and can metabolise androgens and estrogens. In addition, steroidogenic acute regulatory protein (StAR) that controls the rate determining step of acute steroidogenesis has been identified in the epidermis. The aim of this thesis was to identify de novo cortisol steroidogenesis by keratinocytes and investigate the function of cortisol in keratinocytes in vitro. Normal epidermis was shown to express three cholesterol transporters that are associated with promoting steroid synthesis; StAR was identified in the basal layer, metastatic lymph node 64 (MLN64) in the suprabasal layers and translocator protein (TSPO) was detected throughout the epidermal layers. In addition, the nuclear receptor DAX1, a negative regulator of StAR, was identified in the cytoplasm of cells that form normal epidermis. Comparatively, the expression of these proteins was altered in psoriasis and atopic dermatitis, where DAX1 was localised to the nucleus of most diseased tissue and StAR was not detected. This suggests that acute steroid synthesis is ablated in these hyperproliferative skin conditions. The ability of normal primary human keratinocytes to synthesise cortisol was investigated. Radioimmunoassay demonstrated keratinocytes were capable of de novo pregnenolone synthesis, which was promoted with the cortisol analogue dexamethasone (dex). Interestingly, 25-hydroxycholesterol, which bypasses StAR, did not further enhance steroid synthesis. This suggests that there is an alternative rate determining step of steroid synthesis in cultured primary keratinocytes. Thin layer chromatography demonstrated keratinocytes could metabolise pregnenolone to progesterone and progesterone to cortisol. Progesterone metabolism to cortisol was also confirmed with liquid chromatography/mass spectroscopy. 3 Dex was shown to maintain keratinocyte viability and was implicated in promoting cellular redox potential. Since redox potential is a critical regulator of steroidogenesis, this observation could provide a mechanism for dex-induced pregnenolone synthesis in cultured keratinocytes. These observations led to a hypothesis that local cortisol synthesis functions to regulate cellular redox potential to prevent cell death as part of a positive feedback system. Therefore, this thesis has identified the cortisol steroidogenic pathway in primary human keratinocytes and a potential functional mechanism for the pathway.