Mechanism of RPE Cell Death in α-Crystallin Deficient Mice: A Novel and Critical Role for MRP1-Mediated GSH Efflux

Abstract

Absence of α-crystallins (αA and αB) in retinal pigment epithelial (RPE) cells renders them susceptible to oxidant-induced cell death. We tested the hypothesis that the protective effect of α-crystallin is mediated by changes in cellular glutathione (GSH) and elucidated the mechanism of GSH efflux. In α-crystallin overexpressing cells resistant to cell death, cellular GSH was >2 fold higher than vector control cells and this increase was seen particularly in mitochondria. The high GSH levels associated with α-crystallin overexpression were due to increased GSH biosynthesis. On the other hand, cellular GSH was decreased by 50% in murine retina lacking αA or αB crystallin. Multiple multidrug resistance protein (MRP) family isoforms were expressed in RPE, among which MRP1 was the most abundant. MRP1 was localized to the plasma membrane and inhibition of MRP1 markedly decreased GSH efflux. MRP1-suppressed cells were resistant to cell death and contained elevated intracellular GSH and GSSG. Increased GSH in MRP1-supressed cells resulted from a higher conversion of GSSG to GSH by glutathione reductase. In contrast, GSH efflux was significantly higher in MRP1 overexpressing RPE cells which also contained lower levels of cellular GSH and GSSG. Oxidative stress further increased GSH efflux with a decrease in cellular GSH and rendered cells apoptosis-prone. In conclusion, our data reveal for the first time that 1) MRP1 mediates GSH and GSSG efflux in RPE cells; 2) MRP1 inhibition renders RPE cells resistant to oxidative stress-induced cell death while MRP1 overexpression makes them susceptible and 3) the antiapoptotic function of α-crystallin in oxidatively stressed cells is mediated in part by GSH and MRP1. Our findings suggest that MRP1 and α crystallin are potential therapeutic targets in pathological retinal degenerative disorders linked to oxidative stress.

αB-Crystallin Regulates Subretinal Fibrosis by Modulation of Epithelial-Mesenchymal Transition

“…8,53,54 Therefore, the novel property of aB-crystallin as an EMT inducer of RPE might account for the known functional roles of aB-crystallin in AMD, such as cytoprotection and VEGF production in response to stress stimuli. 21,22 The physiological RPE monolayer with cell-cell contact showed no difference in the expression of EMT markers between WT and aB-crystallin knockout mice. RPE cells with cell-cell contact are known to preferentially express E-cadherin.…”

“…17e20 Furthermore, RPE-overexpressing aB-crystallin shows resistance to apoptosis. 21 These findings suggest that aB-crystallin plays a cytoprotective role against multiple stress stimuli that can cause RPE cell death, resulting in drusen formation and geographic atrophy. In addition, we previously demonstrated that aB-crystallin plays a regulatory role by functioning as a chaperone for VEGF in ocular angiogenesis and may play a part in CNV formation in nAMD.…”

Antiapoptotic Properties of α-Crystallin–Derived Peptide Chaperones and Characterization of Their Uptake Transporters in Human RPE Cells

“…In this context, it is of interest that our previous work showed that aB-crystallin modulated glutathione (GSH) levels in mitochondria and mediated transport of GSH via MRP1, the recently identified transporter of GSH in RPE cells. 19 Thus, whether a-crystallin mini-chaperones participate in the regulation of redox proteins could be worthy of investigation.…”

“…Primary cultures of hfRPE cells were established as described previously and used at passages 3 to 4. 19 …”

Nucleotides in the Eye: Focus on Functional Aspects and Therapeutic Perspectives

“…Oxidative stress is also an important contributing factor to RPE apoptosis during the development of retinal diseases such as age-related macular degeneration or diabetic retinopathy (Cai et al, 2000;Hao et al, 2012;Sreekumar et al, 2012). Interestingly, overproduction of reactive oxygen species (ROS) and increased P2X7-mediated permeabilization of YO-PRO-1 were reported in cultured human RPE cells treated with tert-butyl hydroperoxide (Dutot et al, 2008), a chemical oxidant.…”