Role of the unfolded protein response (UPR) in cataract formation
Introduction
Cataracts can be induced by a many different factors, such as senility, gender, physical and chemical stress conditions, diabetes, anorexia, uremia, nutritional deficiency, trauma, galactosemia, congenital defects, and exposure to radiation (Harding and Crabbe, 1984). The incidence of age-related cataracts increases with age and reaches 88% for people older than 75 years (Klein et al., 2002). One of the schemes proposed for the formation of cataracts in humans is the aggregation of crystallin protein into particles larger than 5 × 107 Da that occur in a highly oxidized environment, and these particles scatter light because of their high refractive index (Benedek, 1997).
The importance of the endoplasmic reticulum (ER) in the life of normal cells has been known for several decades, but the part it plays in the progression of human diseases has only recently been appreciated. Evidence has been accumulating that several common pathways are shared in the responses to multiple, seemingly divergent, stress conditions. A number of recent reports support the idea that one of the major stress-response pathways is the unfolded protein response (UPR; Kaufman, 1999, Kaufman, 2004, Szegezdi et al., 2003). The ER is a principal site for protein synthesis and is where the vast majority of secreted, glycosylated, modified lipid proteins are folded into their tertiary and quaternary structure. The concentration ratio of reduced glutathione (GSH):oxidized GSH (GSSG) is 3:1 in the lumen of the ER and 100:1 in the cytosol (Hwang et al., 1992). Thus, the ER is a highly oxidized compartment in all types of cells.
Only those proteins that are properly folded are formed as oligomers and transferred into the Golgi complex. Otherwise, Bip/GRP78, an ER protein, binds to improperly folded proteins, and the proteins are subsequently degraded by the ubiquitin-dependent pathway (Kaufman, 1999, Kaufman, 2004, Szegezdi et al., 2003).
In the early stage of the UPR, higher levels of reactive oxygen species (ROS), e.g., H2O2, O2·− and ·OH, are formed (Harding et al., 2003, Haynes et al., 2004, Tu and Weissman, 2004). In the UPR-regulated oxidative protein-folding machinery in the ER, two electrons are generated from disulfide bonds through the action of protein disulfide isomerase, and cellular flavin adenine dinucleotide (FAD) and oxygen molecules serve as terminal electron acceptors. These reactions can then lead to the production of ROS (Harding et al., 2003, Haynes et al., 2004, Tu and Weissman, 2004). A second source of ROS (O2·−) is the mitochondrial electron transport chain (Fribley et al., 2004). It is important to emphasize that depletion of GSH by the UPR induces mitochondrial dysfunction resulting in ROS accumulation (Fribley et al., 2004, Haynes et al., 2004).
Thus, the ER and mitochondria can contribute to the production of ROS through the UPR pathways. If this response is not capable of resolving the ER stress, i.e., removal of improperly folded proteins, an accumulation of unfolded proteins can activate cell death pathways. These pathways involve many different caspases that result ultimately in apoptosis.
As unfolded proteins accumulate in the ER, Bip/GRP78 is dissociated from the ER luminal surface and activates three ER bound proteins: (1) type-1 ER transmembrane protein kinase (IRE-1), (2) activating transcription factor 6 (ATF-6), and (3) PKR-like ER kinase (PERK) (Shi et al., 1998, Liu et al., 2000, Shen et al., 2002). Activated IRE-1 recruits Jun N-terminal inhibitory kinase (JIK) to activate apoptosis-signaling kinase 1, which in turn activates the c-jun N-terminal kinase (JNK) pathway. Then, JNK activates mitochondria/Apaf1-dependent caspases. Procaspase-12 is an ER-associated proximal effector of apoptosis. Activated caspase-12 activates caspase-9, which in turn activates caspase-3, leading to apoptosis (Xie et al., 2002, Mandic et al., 2003).
With ER stress, free ATF6 fragments migrate to the nucleus to activate transcription. In the nucleus, it interacts with the ER stress DNA element, thereby activating the transcription of many unfolded protein responsive genes, such as Bip/GRP78 and CHOP.
PERK is a kinase that phosphorylates a subunit of the translation initiation factor 2α (eIF2α) in response to ER stress. Phosphorylation of eIF2α activates a transcription factor, ATF4, and the up-regulation of ATF4 activates the transcription of Bip/GRP78 and CHOP, both proapoptotic factors. The elevation of CHOP results in a down-regulation of Bcl2, the activation of caspases, and cell death (McCullough et al., 2001).
Thus, Bip/GRP78, ATF4, CHOP, and caspase-12 are key enzymes that are activated by the UPR. The UPR is reversible and does not necessary lead to apoptosis under stress conditions; however, a protracted UPR induces apoptosis in most cell types (Harding et al., 2003, Haynes et al., 2004, Tu and Weissman, 2004).
Careful examination of many factors involved in cataractogenesis revealed that most were stressors that induce ER stress and activate the UPR in other cell types (Table 1; Shinohara et al., 2006). While there is much circumstantial evidence that the UPR can be generated in the crystalline lens, there is no evidence as to whether those stress conditions and agents induce the UPR, generate ROS production, decrease the levels of GSH, and cause apoptosis in LECs. Thus, the purpose of this study was to determine whether well-established ER stressors, e.g., homocysteine, tunicamycin, Ca2+ ionophore (A23187), and glucose deprivation, will induce ER stress and activate the UPR in cultured LECs.
Section snippets
LECs and lens culture
Transformed human LECs (from Dr. Venkat Reddy, University of Michigan) and primary mouse LECs were prepared as described (Singh et al., 2000). LECs (5 × 104 cells/ml in 6 cm or 10 cm dishes) were cultured in DMEM or glucose free DMEM with 10% FBS at 37 °C for the specified periods. Sprague–Dawley rats, 35–55 days old, were purchased from SASCO, Wilmington, MA. Animals were euthanized by CO2 and eyes were enucleated. All the animals were maintained and treated in accordance with the Association for
Higher doses of ER stressors inhibit cell growth and induce apoptosis in LECs
Four well-known ER stressors, homocysteine (Harris, 2002, Austin et al., 2004), tunicamycin (Kaufman, 1999), A23187 (Harding et al., 2003, Haynes et al., 2004, Tu and Weissman, 2004), and glucose deprivation (Doerrler and Lehrman, 1999; Gao et al., 2004), were selected for this study.
As a first step, we determined the optimal concentration and incubation period of each ER stressor to induce cell death. Human LECs were pre-cultured in DMEM with 10% FBS overnight. The cells attached to the Petri
Discussion
While there is considerable circumstantial evidence implicating the UPR in cataract formation (Table 1), our results provide critical evidence for the role of the UPR in inducing apoptosis of LECs. Treatment of LECs with a high dose of homocysteine, tunicamycin, A23187, or glucose deprivation blocked the cell cycle, and activated Bip/GRP78, ATF4, and caspase-12 and -3. Then, ROS were produced, levels of GSH were reduced, and apoptosis was induced. In contrast, cells treated with ER stressors
Acknowledgements
The authors are grateful to Dr. M.F. Lou for her important suggestion regarding the quantification of GSH and ROS, and Drs. J. Harding, J. Horwitz, and D. Hamasaki for critical reading of the manuscript and discussion prior to publication. This work was supported in part by the RPB and fund from the Department of Ophthalmology and Visual Sciences, UNMC.
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