11 Endoplasmic Reticulum Response to Oxidative Stress in RPE |
245 |
ER then elicits a series of cellular responses. Given the demonstrated ability of oxidative stress and redox changes to induce ER stress, this stress may mediate at least part of the pathology observed in AMD and other retinal diseases [21Ð23].
11.3ER Response to Oxidative Stress in RPE
Recent studies indicate that maintenance of ER homeostasis is closely linked to cellular redox status and to mitochondrial homeostasis [24]. While the mechanisms that link ER stress and oxidative stress are not yet well characterized, the ER response to oxidative stress is now being elucidated in RPE.
11.3.1 The Role of ROS and Redox Status in ER
During Oxidative Stress in RPE
Under aerobic conditions, ROS can be produced by exposure to toxic agents or can be a by-product of oxygen through enzymatic reactions, such as the mitochondrial respiratory chain [25]. Because oxidative protein folding occurs in the ER and perturbations in protein folding can cause deleterious consequences, accumulation of ROS or alteration of redox status could directly or indirectly affect ER homeostasis and protein folding [26]. Oxidative stress from aging, smoking, radiation, or other factors could enhance ROS levels in RPE cells [1, 27], and the generated ROS can accumulate in ER (Fig. 11.1). Lipofuscin, a product of phagocytosis of the rod outer segment disc membrane, is also a potential internal ER disruptor and an oxidative source that may result in degeneration of RPE and adjacent photoreceptors [28]. The autoßuorescent pigment N-retinylidene-N-retinylethanolamine (A2E), a primary component of RPE lipofuscin, might interact with cytosolic chaperone proteins to affect their proper protein folding [29]. More importantly, the mitochondrial burden caused by ROS as a consequence of A2E accumulation has been implicated in the disruption of ER homeostasis [30]. b-amyloid (Ab) peptide, a pathological marker shared by AlzheimerÕs disease and AMD, is found as a component of drusen in AMD patients. The cholesterol oxidation metabolite 27-hydroxycholesterol (27-OHC) is reported to cause Ab production, ER stress, and oxidative stress, which thereby promote RPE cell damage [31]. In addition, using human fetal RPE cells treated with the chemical oxidant tertbutylhydroperoxide (tBH), our laboratory showed that oxidative stress resulted in the accumulation of ROS in ER, suggesting its potent effect in ER homeostasis [6].
Excessive ROS is believed to cause Ca2+ inßux from ER into mitochondria, leading to more ROS production through stimulation of a sequence of events by mitochondrial Ca2+ loading [2, 32]. This ROS Ca2+ interactive loop between ER and mitochondria can cause mitochondrial DNA damage, loss of mitochondrial membrane permeability, and cytochrome c release, thereby exacerbating mitochondrial disruption and initiating cell death [33].
246 |
G. Dou et al. |
Fig. 11.1 Increased reactive oxygen species (ROS) production by oxidative stress in endoplasmic reticulum (ER). ROS was stained by carboxy-H2-DCFDA staining (green). ER was stained by ER tracker (red). (a) In control primary human fetal RPE cells, ROS production was negligible in ER. (b) In RPE cells treated with 40 mM tert-butylhydroperoxide for 24 h, ROS production in ER was prominently increased (yellow). Bar: 10 mm
Cells are equipped with a defense system to eliminate excessive ROS. Most ROS produced in normal physiological processes are eliminated immediately by endogenous antioxidant systems. Superoxide dismutase, glutathione peroxidase, catalase, and thioredoxin reductase are the main enzymatic antioxidant systems, while vitamins (e.g., ubiquinol and carotenoids) act as a nonenzymatic antioxidant defense by direct radical scavenging [4]. Notably, carotenoids such as lutein and zeaxanthin are abundant in RPE cells, especially in the macular region. They quench the reactive singlet oxygen and form an optical Þlter to shield the photoreceptors [34]. However, if ROS levels exceed antioxidant capacity, as a result of either excess ROS production or reduced antioxidant capability, oxidative damage may occur to both mitochondria and ER. Thus, the greater oxidizing environment of the ER may contribute to accumulation of unfolded proteins.
Oxidative stress could also be a crucial factor affecting heat shock proteins. Hydrogen peroxide (H2O2) is reported to block heat shock response and refolding activity, thereby leading to insufÞcient quality control and increasing ER stress. These uncontrolled stress responses may enhance oxidative stress-initiated signaling [35].
11.3.2Initiation of UPR to Alleviate ER Burden
As in all kinds of stress, the UPR is initially a cellular protective response to the aggregation of misfolded proteins in ER [36Ð38]. The UPR serves to limit the accumulation of unfolded proteins by reducing general protein synthesis and to
11 Endoplasmic Reticulum Response to Oxidative Stress in RPE |
247 |
selectively activate expression of speciÞc proteins that facilitate chaperone activities. The mechanisms mediating UPR are not yet clear; but in a well-stated theory, ER chaperone GRP78 is recognized to serve as a crucial regulator [15, 39]. Under normal conditions, GRP78 holds transmembrane ER proteins to keep them in an inactive state in the lumen of ER. When misfolded proteins accumulate, GRP78 translocates to bind to those misfolded proteins, thereby releasing these transmembrane proteins allowing oligomerization, and thus initiating the UPR. GRP78 may also promote cell survival by other signaling pathways. GRP78 can form a complex with caspase 12 and BH-only protein BIK that may contribute to the antiapoptotic activity [40]. Other protective proteins, such as sigma receptor 1, can bind to GRP78 under stress conditions to exert neuroprotection [41]. Several stimuli, including oxidative stress, chemical toxicity, and hypoxia, induce expression of GRP78. The increased expression of GRP78 that has been reported in many retinal diseases in animal models [42Ð45] could help to prevent retinal cell death, possibly by alleviating the misfolded protein burden [40, 46]. Similarly, we found that levels of GRP78 expression in RPE cells could be greatly increased by tBH-induced oxidative stress, suggesting that this ER chaperone may be induced to ease the ER burden caused by oxidative stress [6].
In the UPR process, three proximal stress sensors in signaling pathways have been identiÞed: activating transcription factor 6 (ATF6); type I transmembrane protein kinase and endoribonuclease (IRE1); and RNA-activated protein kinase-like ER kinase (PERK).
In the very early stages of UPR, an immediate transient attenuation of mRNA translation occurs, thereby preventing the continued inßux of newly synthesized polypeptides into the stressed ER lumen. This translational attenuation is conducted through PERK-mediated phosphorylation of the eukaryotic translation initiation factor 2 (eIF2), resulting in a global inhibition of protein synthesis and G1 arrest [47, 48]. PERK is an ER-associated transmembrane serine/threonine protein kinase. Once released from GRP78, PERK is dimerized and trans-autophosphorylated to activate eIF2 kinase function. PERK-mediated eIF2 then activates Activating transcription factor 4 (ATF4), which is responsible for genes encoding amino acid biosynthesis and transport functions, antioxidative stress response, and apoptosis. Notably, phosphorylation of eIF2 is closely associated with glutathione metabolism, which demonstrates how cells exploit multiple regulatory pathways.
IRE1 signaling is the Þrst component identiÞed in the UPR pathway [49]. Upon release, IRE1 undergoes oligomerization in ER membranes, resulting in production of X-box-binding protein 1(XBP-1), a transcriptional factor that induces expression of genes involved in restoring protein folding or degrading unfolded proteins [50]. Oligomerized IRE1 binds to TNF receptor-associated factor 2 (TRAF2), activating apoptosis signal-regulating kinase (ASK1) and downstream kinase that activates p38 MAPK (mitogen-activated protein kinase) and Jun N-terminal kinase (JNK) [51]. XBP1 also contributes to cellular resistance to oxidative stress. After exposure to H2O2 or the strong ROS inducer parthenolide, loss of mitochondrial permeability transition (MPT) and subsequent cell death occurred more extensively in XBP1-deÞcient cells. Overexpression of XBP1