Characterisation of Pre–Teceptor Glucocorticoid Regulation in the Rabbit Eye

Abstract

Purpose: : Glucocorticoids (GCs) activate receptors that translocate from the cytoplasm to the nuclear chromatin thereby modulating transcription of specific GC responsive genes. The preceptor regulation of GCs has emerged as a novel determinant of GC function in systemic tissues such as adipose and bone, but is less well described in the eye. Regulation is dependent on the expression of a bidirectional isozyme, 11ß–hydroxysteroid dehydrogenase (11ß–HSD1) that interconverts active cortisol and inactive cortisone. Recent descriptive studies have localised this isozyme to the human corneal epithelium and non–pigmented epithelium of the ciliary body. In this study we characterise preceptor regulation of GCs in an animal model – the New Zealand White Albino rabbit (NZWAR).

Methods: : Using in–house generated primary antibody to human 11ß–HSD1, immunohistochemical analyses were performed on paraffin embedded sections of whole NZWAR eyes. 11ß–HSD1 enzyme assays for oxo–reductase (cortisone to cortisol) and dehydrogenase (cortisol to cortisone) activity were conducted on NZWAR primary cultures (corneal stromal keratocytes (CSK), corneal epithelium (CEC), conjunctival fibroblasts (CF)) and dissected ciliary body tissue (CBT). Further examination of 11ß–HSD1 expression was undertaken using immunocytochemistry of CEC and Western Blot analyses of CBT.

Results: : Immunohistochemical studies showed that 11ß–HSD1 localised to the corneal epithelium and NPE of the ciliary body. Specific activity assays indicated predominant 11ß–HSD1 oxo–reductase activity from both the CBT (n=12, median, 44.4% conversion; range, 26.9–61.7%; p=0.003) and CEC (median, 3.0±1.9 pmol/mg/ml, p=0.006) compared to dehydrogenase activity (median, 18.6% conversion; range, 10.9–44.9% and 0.5±0.8 pmol/mg/ml respectively). These findings were supported by expression of the 11ß–HSD1 protein as visualised by Western Blotting of CBT and immunocytochemistry of CEC. 11ß–HSD1 activity was not demonstrated in basal CF or CSK.

Conclusions: : Our results indicate that pre–receptor GC regulation by 11ß–HSD1 is a component of GC function in rabbit corneal and uveal tissues. 11ß–HSD1 manipulation may represent a novel therapeutic mechanism for GC responsive eye disease.

Keywords: corticosteroids • cornea: epithelium • enzymes/enzyme inhibitors