In vitro effects of dexamethasone on human corneal keratocytes

To investigate whether cultured human keratocytes express the glucocorticoid receptor (GR) and to assess the influence of dexamethasone (DEX) on these cells.

Human keratocytes were cultured in medium supplemented with various concentrations of DEX (ranging from 10(-10) to 10(-4) M). Cell proliferation was analyzed by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-s ulfophenyl)-2H-tetrazolium inner salt (MTS) assay at 2, 4, and 6 days of culture. Some experiments were performed in the presence of mifepristone (RU38486), an antiglucocorticoid molecule. The early phase of apoptosis was studied by means of keratocyte staining with a fluorescein conjugate of annexin V and propidium iodide, and cells were analyzed by flow cytometry. Glucocorticoid receptor mRNA was detected in keratocytes by means of reverse transcription-polymerase chain reaction (RT-PCR). Immunocytochemical staining of the cells was performed with a monoclonal anti-human GR.

RT-PCR and immunocytochemistry showed the expression of GR (mRNA and protein) in cultured keratocytes. Dexamethasone significantly increased keratocyte proliferation with concentrations ranging from 10(-9) to 10(-5) M, with a maximum effect at 10(-7) M (P < 0.005). Dexamethasone's proproliferative effect was inhibited by RU38486. However, DEX also induced apoptosis of cultured keratocytes at any concentration used.

These results indicate that cultured human keratocytes express the GR and proliferate in response to DEX stimulation (10(-9)-10(-5) M), which also induces keratocyte apoptosis.