The effects of testosterone and lipopolysaccharide on gene expression in immortalized ocular surface and adnexal cells

Immortalized human corneal, conjunctival, and meibomian gland epithelial cells were pre-treated with dihydrotesteosterone (DHT) or vehicle, then exposed to lipopolysaccharide (LPS) and ligand binding protein (LBP) or vehicle, lysed, and subjected to microarray analysis of RNA expression. Cells were cultured in keratinocyte serum-free medium (KSFM) supplemented with bovine pituitary extract (BPE; 25 ug/mL HCEC and HCjE, 50 ug/mL HMGEC), 5 ng/mL epidermal growth factor (EGF), penicillin and streptomycin. On reaching confluence, cells were rinsed twice with a phosphate-buffered saline solution and exposed to a stratification medium consisting of DMEM and F12 (Mediatech, Inc) with 10% fetal bovine serum, epidermal growth factor (10 ng/mL), penicillin, and streptomycin for 2 days. After this time period, cells were incubated in serum-free DMEM/F12 and exposed to vehicle (1% bovine serum albumin [BSA]), or LPS (15 μg/ml) and LBP (150 ng/ml), for six hours. The LPS and LBP were dissolved in DMEM and the BSA in PBS. For androgen-related studies, the stratification medium contained 10% charcoal and dextran-treated fetal bovine serum with ethanol or DHT (10nM); the poststratification medium also contained ethanol or DHT.