Biological Characteristics of Lens Epithelial Cells from Grx1 and Grx2 Double Knockout Mice

Purpose: Glutaredoxins are glutathione (GSH) dependent enzymes that play an important role in repairing oxidized proteins, preventing subsequent protein misfolding and disrupting protein aggregation. The Grx system has two major isozymes: glutaredoxin 1 (Grx1) and the recently discovered glutaredoxin 2 (Grx2). To achieve a comprehensive understanding of the Grx system in the lens, our lab recently created a Grx1 and Grx2 double knockout (DKO) mouse model to observe how the double deletion of the enzymes may affect the lens epithelial cell (LEC) survival and lens transparency. Methods: Primary LECs were cultured from wild-type (WT) and DKO mice. Cell proliferation was tested via various assay kits, and cell cycle distribution was evaluated using flow cytometry analysis. Cell apoptotic markers including Bcl-2, Bax, and caspase 3 were detected using Western Blot. The mitochondrial function was evaluated via ATP concentration. Cytoskeletal arrangement and its intercellular connection were also examined by using fluorescent microscopy. Results: Compared to WT cells, DKO cells displayed a much slower growth. The number of DKO cells arrested in the M phase was twofold higher than that of WT cells. The population of DKO cells arrested in the S phase was 50% less than that of WT cells. For the apoptotic pathway, we found DKO cells have higher levels of Bax and cytochrome c with lower ATP production. Furthermore, we also found that DKO cells had higher levels of vimentin expression, which may lead to cytoskeleton reorganization and polarity. Conclusions: In conclusion, our data suggest that Grx function loss may inhibit cell proliferation, disrupt the normal cell cycle, trigger apoptosis pathway, and damage mitochondrial functions.