Article

The Unfolded Protein Response in Lens Epithelial Cells from Galactosemic Rat Lenses

Department of Ophthalmology and Visual Sciences, University of Nebraska, Omaha, NE 68198-5840, USA.
Investigative Ophthalmology & Visual Science (Impact Factor: 3.4). 10/2006; 47(9):3951-9. DOI: 10.1167/iovs.06-0193
Source: PubMed

ABSTRACT

Diabetic complications are associated with hypoglycemia and hyperglycemia. The purpose of this study was to investigate the effect of both glucose deprivation and hyperglycemia on the induction of endoplasmic reticulum (ER) stress and the subsequent activation of the unfolded protein response (UPR) that results in apoptosis in in vitro cultured lens epithelial cells (LECs) and in vivo cataract formation in galactose-fed rats.
Lenses from rats fed a standard diet containing 50% galactose with or without an aldose reductase inhibitor (ARI) were investigated. Transformed human LECs were cultured in standard 10% FCS-DMEM containing various concentrations of sugar. UPR-specific proteins from both the rat lenses and lens cultures were quantified by protein blot analysis. Cell death was evaluated with TUNEL staining and ethidium homodimer-1 (EthD) dyes. Reactive oxygen species (ROS) were quantified with H2-DCF, and free glutathione (GSH) levels were measured with a commercial GSH quantification kit.
Increased apoptosis of the LECs was observed in the lenses of rats fed the galactose diet for 5 to 9 days, and nuclear cataracts subsequently developed in these lenses after 13 to 15 days. Protein blot analysis of the LECs from these galactose-fed rats showed higher levels of the UPR-specific proteins Bip/GRP78, ATF4, and CHOP. These LECs also demonstrated activation of the UPR-specific procaspase-12 and the increased presence of ROS, whereas GSH was reduced. Because these results indicate that the UPR is activated in LECs along with the production of ROS and apoptosis during cataract formation in the galactose-fed rats, subsequent studies were conducted to determine the role of nonenzymatic glycation, osmotic stress, and oxidative stress on these biochemical processes. In vitro cultures of human LECs showed that the UPR was induced by osmotic and oxidative stress, but not by glycation. In addition, the UPR and apoptosis in LECs was induced by glucose deprivation. The ARI blocked the induction of the UPR, cell death, and cataract formation.
The UPR that is induced by abnormally high or low concentrations of sugar is linked to the production of ROS, increased apoptosis in LECs, and cataract formation. The inhibition of the UPR induction by ARI suggests that osmotic stress may be the primary inducer of the UPR. Modulation of the UPR pathways may offer novel methods for the development of therapeutic tools to delay cataracts.

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    • "Trudeau and colleagues have hypothesized that high glucose (HG) triggers mitochondrial morphological change and cytochrome c release in retinal endothelial cells (RECs) as well as retinal pericytes, further potentiating cell apoptosis [10] [11] [17], even leading to DR [18]. Extensive investigation on the role of ER stress in hyperglycemia has been shown that high glucose is linked to the unfolded protein response (UPR), production of ROS, and increased apoptosis [14]. Aldose reductase (AR), a member of aldo–keto reductase super family, catalyzes the conversion of glucose to sorbitol as the first step in the polyol pathway [19]. "
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    Full-text · Dataset · Jan 2015
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    • "Trudeau and colleagues have hypothesized that high glucose (HG) triggers mitochondrial morphological change and cytochrome c release in retinal endothelial cells (RECs) as well as retinal pericytes, further potentiating cell apoptosis [10] [11] [17], even leading to DR [18]. Extensive investigation on the role of ER stress in hyperglycemia has been shown that high glucose is linked to the unfolded protein response (UPR), production of ROS, and increased apoptosis [14]. Aldose reductase (AR), a member of aldo–keto reductase super family, catalyzes the conversion of glucose to sorbitol as the first step in the polyol pathway [19]. "
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    ABSTRACT: Chronic hyperglycemia is an important risk factor involved in the onset and progression of diabetic retinopathy (DR). Among other effectors, aldose reductase (AR) has been linked to the pathogenesis of this degenerative disease. The purpose of this study was to investigate whether the novel AR inhibitor, beta-glucogallin (BGG), can offer protection against various hyperglycemia-induced abnormalities in human adult retinal pigment epithelia (ARPE-19) cells. AR is an enzyme that contributes to cellular stress by production of reactive oxygen species (ROS) under high glucose conditions. A marked decrease in cell viability (from 100% to 78%) following long-term exposure (4 days) of RPE cells to high glucose (HG) was largely prevented by siRNA-mediated knockdown of AR gene expression (from 79% to 97%) or inhibition using sorbinil (from 66% to 86%). In HG, BGG decreased sorbitol accumulation (44%), ROS production (27%) as well as ER stress (22%). Additionally, we demonstrated that BGG prevented loss of mitochondrial membrane potential (MMP) under HG exposure. We also showed that AR inhibitor pretreatment reduced retinal microglia-induced apoptosis in APRE-19 cells. These results suggest that BGG may be useful as a therapeutic agent against retinal degeneration in the diabetic eye by preventing RPE cell death.
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    • "Furthermore, the same group showed that in cultured lens epithelial cell lines, the UPR can be induced by treatment with high concentrations (100-125 mM) of galactose, glucose, mannose, mannitol or NaCl. These results suggest that, in this model, the UPR is likely activated by osmotic stress and not by the accumulation of any specific metabolite such as galactose-1-phosphate (Mulhern et al., 2006). Recently, others have also proposed the use of chemical chaperones as drug candidates for classic galactosemia based on the protein structural instability caused by disease-associated mutations on the human GALT gene (McCorvie et al., 2013). "
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    ABSTRACT: Classic galactosemia is a human autosomal recessive disorder caused by mutations in the GALT gene - GAL7 in yeast - which encodes the enzyme galactose-1-phosphate uridyltransferase. Here we show that the unfolded protein response pathway is triggered by galactose in two yeast models of galactosemia: lithium-treated cells and the gal7Δ mutant. The synthesis of galactose-1-phosphate is essential to trigger the unfolded protein response under these conditions because the deletion of the galactokinase-encoding gene GAL1 completely abolishes unfolded protein response activation and galactose toxicity. Impairment of the unfolded protein response in both yeast models makes cells even more sensitive to galactose, unmasking its cytotoxic effect. These results indicate that endoplasmic reticulum stress is induced under galactosemic conditions and underscores the importance of the unfolded protein response pathway to the cellular adaptation in these models of classic galactosemia.
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