Vanita Gupta1, B J Wagner. 1. Department of Biochemistry & Molecular Biology, the University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey 07101-1709, USA.
Abstract
PURPOSE: Studies have questioned the reported presence of a classic glucocorticoid receptor (GR) in the mammalian lens. The purpose of this study is to determine whether the functional GR is expressed in human and mouse lens epithelial cells. METHODS: GR mRNA was determined by RT-PCR in freshly isolated human lens epithelia, mouse lens, immortalized human (HLE B-3) and mouse (alphaTN4) lens epithelial cells and in mouse lung, NIH-3T3 cells, and HeLa cells, which served as positive controls. Western blot analysis with the GR-specific antibody H-300 was performed on protein extracts from human lens epithelia, HLE B-3 cells, and alphaTN4 cells and from HeLa cells, NIH-3T3 cells, and partially purified GR, which served as positive controls. pGRE.Luc drives the expression of firefly luciferase. HLE B-3 and alphaTN4 cells were transfected with pGRE.Luc and cotreated with dexamethasone, with and without the competitive inhibitor RU-486. RESULTS: PCR products of the expected size were detected in all samples, sequenced in both directions, and found to have 97% to 100% homology with the GR. A band in the appropriate molecular weight range was identified by Western blot analysis in the lens extracts. Active GR binding to the GRE was demonstrated by an increase in firefly luciferase expression in transfected cells treated with dexamethasone. The dexamethasone-induced increase in luciferase activity was inhibited with the addition of RU-486. CONCLUSIONS: These results demonstrate expression of the functional glucocorticoid receptor in mouse and human lens epithelial cells. This finding suggests that glucocorticoids may act on the mouse and human lens directly during normal lens development and/or cataractogenesis.
PURPOSE: Studies have questioned the reported presence of a classic glucocorticoid receptor (GR) in the mammalian lens. The purpose of this study is to determine whether the functional GR is expressed in human and mouse lens epithelial cells. METHODS:GR mRNA was determined by RT-PCR in freshly isolated humanlens epithelia, mouse lens, immortalized human (HLE B-3) and mouse (alphaTN4) lens epithelial cells and in mouse lung, NIH-3T3 cells, and HeLa cells, which served as positive controls. Western blot analysis with the GR-specific antibody H-300 was performed on protein extracts from humanlens epithelia, HLE B-3 cells, and alphaTN4 cells and from HeLa cells, NIH-3T3 cells, and partially purified GR, which served as positive controls. pGRE.Luc drives the expression of firefly luciferase. HLE B-3 and alphaTN4 cells were transfected with pGRE.Luc and cotreated with dexamethasone, with and without the competitive inhibitor RU-486. RESULTS: PCR products of the expected size were detected in all samples, sequenced in both directions, and found to have 97% to 100% homology with the GR. A band in the appropriate molecular weight range was identified by Western blot analysis in the lens extracts. Active GR binding to the GRE was demonstrated by an increase in firefly luciferase expression in transfected cells treated with dexamethasone. The dexamethasone-induced increase in luciferase activity was inhibited with the addition of RU-486. CONCLUSIONS: These results demonstrate expression of the functional glucocorticoid receptor in mouse and human lens epithelial cells. This finding suggests that glucocorticoids may act on the mouse and human lens directly during normal lens development and/or cataractogenesis.
Authors: Viktória Szabó; Gábor Borgulya; Tamás Filkorn; Judit Majnik; Ilona Bányász; Zoltán Zsolt Nagy Journal: Mol Vis Date: 2007-04-27 Impact factor: 2.367