Lens Epithelial Cells Contain a Transcriptionally Active Alpha Glucocorticoid Receptor

Abstract

Abstract: : Purpose: Reports concerning the existence of a glucocorticoid receptor (GR) in lens epithelial cells (LEC) are contradictory. Studies aimed at identifying a possible mechanism for the induction of steroid-mediated cataract would benefit from resolution of this controversy. The aim of this study was to determine whether LEC contain a GR and whether this GR is transcriptionally active. Methods: Protein and mRNA were obtained from human, rabbit and bovine lens epithelia and from cultured human lens epithelial cells (B3 – hLEC) and rabbit lens epithelial cells (N/N1003A – rLEC). Paraffin sections were prepared from human lenses for immunohistochemical localization of GR. RT-PCR was performed to amplify portions of GR, and the products were sequenced. Protein samples were analyzed by Western blot. hLEC and rLEC were transfected with pTAT3-luc, and assayed for luciferase activity following treatment with dexamethasone (Dex) and/or RU486. Dex-treated hLEC were also analyzed by Western blot for expression of alpha-B-crystallin, cIAP2 and Hsp70. Results: By PCR and sequencing, products consistent with GR sequences were obtained from human, rabbit and bovine lenses and from hLEC and rLEC. The complete GR-alpha sequence was obtained from rLEC. By Western blot, bands of approximately 94 kDa, the expected size of GR, were identified from human, rabbit and bovine lens samples and from hLEC and rLEC using four anti-GR antibodies. Anti-GR antisera localized GR to both the cytosol of anterior and bow region lens epithelial cells and to the nuclei of early differentiating lens fiber cells. Luciferase expression was induced in pTAT3-luc transfected rLEC and hLEC by Dex treatment and this expression was partially (hLEC) or completely (rLEC) blocked by pre-treatment with RU486. alpha-B-crystallin and c-IAP2 mRNA and protein levels were upregulated following Dex exposure while Hsp70 levels were downregulated. Conclusions: Our data indicate that a transcriptionally active GR-alpha is present in lens epithelial cells and that the expression levels of proteins known to be regulated by glucocorticoids could be modified in these cells by glucocorticoid treatment.

Keywords: receptors: pharmacology/physiology • apoptosis/cell death • cataract