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Mahita Kadmiel, John Cidlowski, Molecular Endocrinology; Functional characterization of the glucocorticoid receptor in the cornea. Invest. Ophthalmol. Vis. Sci. 2013;54(15):974.
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Glucocorticoids have long been in use for the treatment of ophthalmic diseases for their anti-inflammatory and anti-angiogenic effects. However, chronic use of glucocorticoids can cause serious side effects. For example, glucocorticoids are associated with poor prognosis in wound healing. Nevertheless, the underlying mechanisms for this complication are poorly understood. The purpose of this investigation is to determine the precise function of glucocorticoid receptor (GR) signaling in corneal epithelial wound healing.
For nuclear trafficking experiments, Human corneal epithelial cells (HCE-T) cells were stimulated with 100nM dexamethasone (Dex; synthetic glucocorticoid) or vehicle (Veh) for 1 hour, fixed, incubated overnight with hGRα antibody and DAPI, and imaged. For gene expression profiling, HCE-T were treated with 100nM Dex or Veh for 6 hours. Total RNA was extracted, DNase-treated and subjected to whole-genome expression profiling using the Agilent 4x44 arrays. For in vitro wound healing assays, HCE-T cells were pre-treated with 100nM Dex or Veh for 24hours. A scratch-wound was made and imaged at time zero and after 24hours. Images were analyzed using Image J software.
Human corneal epithelial cells expressed the glucocorticoid receptor, which trafficked to the nucleus upon stimulation by Dex. Microarray analysis in HCE-T cells revealed that glucocorticoids significantly regulated 2905 probes. Ingenuity Pathway Analysis of the data identified cell development, movement, morphology and cell-to-cell signaling as the top significant glucocorticoid-regulated biological functions. Moreover, in vitro wound healing assays with HCE-T cells exhibited a remarkable delay in wound healing in cells treated with Dex. This effect of Dex was inhibited by the GR antagonist RU486, indicating that the Dex-induced delay in wound healing is mediated through GR.
Glucocorticoids significantly regulate gene expression in human corneal epithelial cells. One of the major functions of glucocorticoids in the corneal epithelium is to regulate wound healing. The goal of this project is to dissect the molecular mechanism involved in glucocorticoid/GR-mediated delay in wound healing. Furthermore, we are generating a mouse model with cornea-specific deletion of GR to elucidate the in vivo role of GR in the cornea during embryonic and postnatal development.
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