In vitro effects of dexamethasone on human corneal keratocytes.

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Abstract

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