In vitro effects of dexamethasone on human corneal keratocytes

PURPOSE. To investigate whether cultured human keratocytes express the gluc ocorticoid receptor (GR) and to assess the influence of dexamethasone (DEX) on these cells. METHODS. Human keratocytes were cultured in medium supplemented with variou s concentrations of DEX (ranging from 10(-10) to 10(-4) M). Cell proliferat ion was analyzed by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl )-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) assay at 2, 4, and 6 da ys of culture. Some experiments were performed in the presence of mifeprist one (RU38486), an antiglucocorticoid molecule. The early phase of apoptosis was studied by means of keratocyte staining with a fluorescein conjugate o f annexin V and propidium iodide, and cells were analyzed by flow cytometry . Glucocorticoid receptor mRNA was detected in keratocytes by means of reve rse transcription-polymerase chain reaction (RT-PCR). Immunocytochemical st aining of the cells was performed with a monoclonal anti-human GR. RESULTS. RT-PCR and immunocytochemistry showed the expression of GR (mRNA a nd 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 proprolif erative effect was inhibited by RU38486. However, DEX also induced apoptosi s of cultured keratocytes at any concentration used. CONCLUSIONS. These results indicate that cultured human keratocytes express the GR and proliferate in response to DEX stimulation (10(-9)-10(-5) M), w hich also induces keratocyte apoptosis.