Inhibition of glucocorticoid-induced alteration of vimentin by a glucocorticoid receptor antagonist RU486 in the organ-cultured rat lens.

PURPOSE: Long-term application of glucocorticoids as a treatment for conditions such as allergy, autoimmune diseases, and transplantation presents a high risk of development of steroid-induced cataract. The presence of a functional glucocorticoid receptor (GR) in human and rat lens epithelial cells suggests a direct and specific targeting of these lens cells by glucocorticoids. One important cytoskeletal protein in lens epithelial cells is vimentin, which plays an important role in maintaining the normal lens morphology and function. Previous studies have shown that vimentin is involved in signal transduction, changes in cell structure and differentiation, and apoptosis. Based on a model of steroid-induced cataract from our previous study, the present study focuses on whether changes in vimentin can be induced in vitro through specific GR activation in glucocorticoid-induced cataracts of the rat lens. METHODS: Clear rat lenses, cultured in vitro, were treated with or without dexamethasone (Dex) or RU486 (a glucocorticoid receptor antagonist). Lenses were cultured for 7 days at 37 °C under 5% CO2, and were observed daily with an inverted microscope. Changes in morphology were followed by Hematoxylin-eosin (HE) staining, transmission electron microscopy, and immunohistochemistry. The expression of vimentin mRNA and protein was examined by reverse transcription polymerase chain reaction (RT-PCR) and western blot analysis, respectively, in the capsule-epithelium and fiber tissue of the lenses. RESULTS: Opacity was obviously present at day 7 in the Dex group. The lenses of the untreated group and the RU486+Dex group remained transparent throughout the incubation. Electron microscopy showed an orderly arrangement of fiber cells and normal cell junctions in the control group and the RU486+Dex group. However, in the Dex group, fiber cells were disarranged and the cell-cell junctions exhibited lacunae. The expression of vimentin protein in the lens capsule-epithelium and fiber tissue decreased in the Dex-treated group, but normal expression of vimentin mRNA was maintained. CONCLUSIONS: These results suggest that the GR-mediated reduction in vimentin may be involved in the formation of steroid-induced cataract.

Inhibition of glucocorticoid-induced changes of Na(+), K(+)-ATPase in rat lens by a glucocorticoid receptor antagonist RU486.

Cataract formation can be induced by prolonged use of glucocorticoids. The underlying mechanism is not fully understood yet. The presence of the functional glucocorticoid receptor (GR) in human and rat lens epithelial cells suggests that glucocorticoids target lens epithelial cells directly and specifically. Na(+), K(+)-ATPase has long been recognized for its role in regulating electrolyte concentration in the lens, contributing to lens transparency. We previously reported that the inactivation of Na(+), K(+)-ATPase induced by a glucocorticoid in rat lens. Therefore, the question is whether the changes of Na(+), K(+)-ATPase can be induced through the specific GR activation in glucocorticoid-induced cataract formation. Clear rat lenses were cultured in vitro and were treated with or without dexamethasone (Dex) or RU486 (a GR antagonist). The lenses were cultured for 7 days and photographed daily to record the development of opacity. The activity of Na(+), K(+)-ATPase was determined by using spectrophotometric analysis. The mRNA and protein level expressions of Na(+), K(+)-ATPase α1 were examined by reverse transcription polymerase chain reaction (RT-PCR), Western blot and immunohistochemistry analysis, respectively. Our findings are presented in this study and show that mist-like opacity of the lens was observed as early as 5 days after incubation with dexamethasone. The opacity was more obvious at day 7 in the Dex group. The lenses of the untreated group and the RU486+Dex group remained transparent throughout the incubation. The activity of Na(+), K(+)-ATPase in the Dex-treated group decreased in a time-dependent manner. There was no significant loss of enzyme activity in either the control or the RU486+Dex group throughout the incubation period. Both the protein and mRNA expression levels of Na(+), K(+)-ATPase α1 in the capsule-epithelium of lenses decreased in the Dex-treated group. The GR antagonist RU486 inhibited the decrease of the expression of Na(+), K(+)-ATPase α1 induced by Dex. All of the above results suggested that the GR-mediated reduction of Na(+), K(+)-ATPase may contribute to the formation of steroid-induced cataract. Intervention in this pathway maybe helpful to avoid glucocorticoids-cataract formation.

Collapsin Response Mediator Protein-2-induced Retinal Ischemic Injury in a Novel Mice Model of Ocular Ischemia Syndrome.

BACKGROUND: Collapsin response mediator protein-2 (CRMP2) has been shown to be involved in ischemia/hypoxia (IH) injury. We determined whether CRMP2 modulates ischemic injury in the retinal of Ocular ischemic syndrome (OIS). This study was to explore the molecular mechanisms underlying OIS in a novel mice model. METHODS: Experiments were performed on adult male C57/BL6 mice that received bilateral internal carotid arteries ligation for 1, 2, or 4 weeks. The mice received injection of calpeptin group before occlusion for 4 weeks or not. The expression of CRMP2 in the retinal was examined by western blotting (WB) analysis and immunohistochemical analysis (IHC). The effects of ischemic injury on retinal were evaluated by fundus examination, fundus fluorescein angiography, electroretinogram, cell counting of retinal ganglion cell (RGC), and measurement of the thickness of the retina. RESULTS: The veins dilated after chronic ischemia. In the electroretinography, the amplitudes of a- and b-waves kept diminishing in an ischemia time-dependent manner. Moreover, the tail vein-retinal circulation time prolonged in the 1- and 2-week group. In comparison, thickness of the retina decreased gradually with the ischemia time elapsed. WB analysis showed the CRMP2 and p-CRMP2 levels decreased in the 2- and 4-week groups. The results of IHC analysis were compatible with our results of WB. The loss of RGCs, decrease of the total reaction time and reduction of CRMP2 was alleviated by intravitreal injection of calpeptin. CONCLUSIONS: These results revealed that bilateral ligation of the internal carotid artery causes retinal ischemia in mice. Moreover, CRMP2 might play a pivotal role during the ischemic injury in the retina and inhibit the cleavage of CRMP2 can ameliorate the IH injury.