retinal epithelial ( rpe ) cells perform multiple functions to maintain retinal homeostasis , including preserving the blood - retinal barrier , nourishing retinal cells by secreting growth factors [ 1 , 2 ] , phagocytosis of shed photoreceptor outer segments [ 3 , 4 ] , and maintenance of the visual cycle by resynthesizing 11-cis retinal . rpe cells located anterior to photoreceptors endure significant oxidative stress because they consume high levels of oxygen and polyunsaturated lipids and are subjected to long - term exposure to light . to protect against oxidative stress , rpe cells employ antioxidant systems , involving glutathione ( gsh ) s - transferases ( gst ) , heme oxygenase-1 ( ho-1 ) , superoxide dismutase ( sod ) , peroxiredoxin ( prdx1 ) , and thioredoxin ( trx ) . under physiological conditions , however , exposure to high levels of reactive oxygen species ( ros ) or free - radical - generating molecules can alter the redox balance . sulfhydryl groups are critical for the response to oxidative stress , and thioredoxin maintains cellular redox potential [ 3 , 9 , 10 ] . trx , originally identified in escherichia coli as a hydrogen donor for ribonucleotide reductase , contains two conserved cysteine residues within its active site [ 12 , 13 ] and scavenges intracellular ros . oxidized trx is reduced by trx reductase in the presence of nadph . thus , trx maintains the function of metabolic enzymes whose catalytic activity depends on the presence of disulfide bonds . these enzymes repair proteins damaged by oxidation , reduce dehydroascorbate , metabolize sulfur , ensure proper polypeptide chain folding , and regulate protein function . two classical trx isoforms , including trx1 in the cytosol / nucleus and trx2 in mitochondria , are both essential , and inactivating mutations in trx genes are embryonically lethal . we demonstrated the protective effect of trx2 against oxidative stress caused by 4-hydroxy nonenal ( 4-hne ) that involves upregulation of ib activity through its phosphorylation and translocation of nf-b to the nucleus . these findings led us to conclude that trx2 is localized to mitochondria , which are highly vulnerable to oxidative damage . however , trx2 had no effect on cell death caused by high concentrations of 4-hne . specifically , 50  m 4-hne induced 78% cell death and 41% at 25  m , and trx2 only protected against relatively low concentrations ( 25  m ) of 4-hne . therefore , there may be a fundamental difference in how oxidative stress affects cells in the presence of 50  m or 25  m 4-hne . in the present study , we investigated whether trx2 protects against oxidative stress caused by 4-hne or c2-ceramide in the human retinal pigmented epithelial cell line ( arpe-19 ) . we also studied whether exponential cell death caused by 4-hne as a function of its concentration is common in oxidative stress and how trx2 affects this process . the human arpe-19 ( arpe ) cell line was provided by l. hjelmeland ( department of ophthalmology , section of molecular ) and was maintained in dulbecco 's modified eagle 's medium ( dmem ) supplemented with 10% fetal bovine serum ( fbs ) and antibiotics , all from life technologies ( tokyo , japan ) as described previously [ 3 , 15 ] . the medium was changed every three days , and cultured cells were passaged after treatment with a solution containing 0.125% trypsin and 0.01% edta . the aav-293 cell line was obtained from agilent technologies ( tokyo , japan ) , and ht1080 cells were supplied by the cell resource center of tohoku university ( sendai , japan ) and were maintained in dmem supplemented with 10% fbs and passaged with 0.05% trypsin-0.53  mm edta or 0.125% trypsin-0.01% edta , respectively . an adeno - associated virus ( aav ) vector carrying trx1 or trx2 was constructed as previously described . the paav - rc and phelper plasmids were obtained from the aav helper - free system ( agilent technologies , tokyo , japan ) . the aav vectors were produced according to a published method . to determine the virus titer , the level of aav2-specific capsid proteins was measured using an enzyme - linked immunosorbent assay ( progen biotechnik , heidelberg , germany ) , and virus titer was defined as number of capsids per ml . successful transgenic expression of trx1 or trx2 was confirmed by assessing the expression of pmcherry fluorescence by microscope ( axiovert 40 ; carl zeiss , oberkochen , germany ) and confocal microscope ( lsm700 ; carl zeiss ) . the localization of the trx2 in the cells was also further investigated using mitotracker dye ( life technologies ) as a mitochondrial marker . one day after plating , the medium was exchanged for a medium containing various concentrations of 4-hne ( emd millipore , billerica , ma , usa ) or c2-ceramide ( enzo life sciences , tokyo , japan ) to induce oxidative stress in the absence of fbs . after incubating the cells for 3 , 6 , and 24 hours , their viability was assessed by measuring the reduction of 3-(4,5-dimethlthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2h - tetrazolium , inner salt ( mts ) to an insoluble formazan ( mts assay , celltiter 96 aqueous non - radioactive cell proliferation assay , promega , madison , wi , usa ) . to study the effect of trx1 or trx2 on stress , arpe cells were infected with aav - trx1 , aav - trx2 , or aav - pmcherry . after three days , the expression of pmcherry fluorescence was confirmed using fluorescence microscope , and the cells were added to the wells of a 96-well plate . one day after plating , oxidative reagents were added , and their effects on cell viability were determined using the mts assay described above . uninfected arpe cells and cells infected with aav - trx1 or -trx2 were plated on a 24-well culture plate at 1  10 cells / well , and the number of cells was counted at 6 , 24 , and 48 hours after seeding to the plate using a tc20 cell counter ( bio - rad , hercules , ca , usa ) . cells were exposed to 25  m of 4-hne or medium for 3 hours in the absence of fbs and were then lysed with ripa buffer ( thermo fisher scientific , suwanee , ga , usa ) and kept on ice for 5  min . the lysates were centrifuged , and the supernatant was used for western blot analysis according to the manufacturer 's protocol . twenty - five micrograms of the lysate was subjected to 415% sds - page ( mini - protean system , bio - rad ) , and the proteins were transferred to a polyvinylidene difluoride membrane ( bio - rad ) . the blots were probed with antibodies against hsp70 and hsp90 ( cell signaling technology , boston , ma , usa ) and -actin ( santa cruz biotechnology , inc . , santa cruz , ca , usa ) . for reblotting with primary antibodies , re - blot plus ( millipore , darmstadt , germany ) protein bands were detected using a flour - s max chemiluminescence imager ( bio - rad ) . densitometric analysis of these proteins was performed using quantity one software ( bio - rad ) . statistical analysis was performed using graphpad prism ( graphpad software , san diego , ca , usa ) . the unpaired t - test or bonferroni 's multiple comparison test after three , six , and 24 hours of exposure to 4-hne or c2-ceramide , using the mts assay , the survival of untreated cells was compared with that of treated cells . after six hours , there was a little protective effect against 4-hne- or c2-ceramide - induced stress . after 24 hours of exposure , both agents caused significant cell death in a dose - dependent manner ( figure 1 ) . expression of pmcherry and trx1-pmcherry was observed in the cytoplasm , while that of trx2-pmcherry mainly colocalized with a mitochondrial marker , mitotracker dye ( figures 2(a ) and 2(b ) ) , which is consistent with the localization of endogenous trx2 in arpe cells . the transgene reduced cell proliferation ; however , there was no significant difference between infected and uninfected cells ( figure 3 ) . we used 4-hne or c2-ceramide to induce oxidative stress in arpe cells expressing transgenic trx1 or trx2 . trx2 protected cells exposed to 12.5  m and 25  m of 4-hne as well as to 12.5  m of c2-ceramide but not those exposed to 25  m c2-ceramide ( figure 4 ) . hsp90 expression was increased in cells expressing trx2 three hours after exposure to 4-hne ( figure 5 ) . hsp70 expression increased three hours after exposure to 4-hne in all cells ; however , its expression in arpe - trx2 cells was lower compared with uninfected and arpe - pmcherry cells . we show here the relation between trx - mediated protection against oxidative stress and hsp expression . trx2 protected cells from oxidative stress caused by low but high concentrations of stressors , indicating that different pathways of cell death were involved . the oxidative stressor 4-hne is a diffusible aldehyde product of membrane - lipid peroxidation ( lpo ) , which produces relatively stable and toxic electrophiles . moreover , 4-hne may act as a key mediator of oxidative - stress - induced cell death . elevated levels of 4-hne were detected in patients with oxidative - stress - related degenerative diseases . in the retina , ros - induced lpo of polyunsaturated fatty acids in rpe cells produces higher amounts of lpo products , including 4-hne . reported that 4-hne is a major oxidant in the retina [ 20 , 21 ] . ceramide is an endogenous mediator of apoptosis , and when its intracellular concentration is increased under oxidative stress   cell proliferation is inhibited and cell death is induced . ceramide is converted by ceramidases to sphingosine , the levels of which increase in the early steps of apoptosis . in the retina , photoreceptors are susceptible to ceramide , and during the phagocytosis of photoreceptors ceramide attacks rpe cells . in the present study , we used n - acetylsphingosine ( c2-ceramide ) , a synthetic cell - permeable ceramide analog . here we show that oxidative stress induced by both stressors caused cell death in a concentration - dependent manner ( figure 1 ) . our previous study demonstrated that trx2 , a mitochondrial redox protein , rescued cells from 4-hne - induced oxidative stress and showed that it plays a key role in mitochondrial function . reported that c2-ceramide caused mitochondrial dysfunction through direct reduction of the mitochondrial respiratory chain complex iii . thus , we hypothesized that trx2 exerts a protective effect against c2-ceramide - induced oxidative stress . this hypothesis is supported by our present data showing that trx2 protected arpe cells from the effects of exposure to 12.5  m c2-ceramide , indicating that the trx2 protects mitochondrial function ( figure 4 ) . consistent with our data published previously , trx1 expressed in the cytoplasm was less protective against c2-ceramide - induced oxidative stress than trx2 . in contrast , trx2 did not protect cells from exposure to 25  m c2-ceramide . figure 1 shows that oxidative stress induced by a high concentration of c2-ceramide caused rapid cell death . the differences in the cellular response at the threshold concentrations may involve either apoptosis [ 26 , 27 ] or necrotic cell death . in the present study , we attempted to determine whether trx2 protected against oxidative stress induced by a low concentration of 4-hne . to study the effects of oxidative stress on arpe cells , we measured the activity of mitochondrial dehydrogenases using the mts assay . these results show that low concentrations of oxidative stressors upregulated mitochondrial function early after exposure ( three to six hours ) but not at 24 hours ( figure 1 ) . therefore , we assume that 3 hours of exposure caused protection and 24 hours of exposure led to excess stress . thus , if trx2 functionally affected cell survival only at a low concentration , 3 hours may be the key time point to rescue the cells . thus , we focused on endoplasmic reticulum ( er ) stress because of its function as a double - edged sword to combat environmental insults and intrinsic stress . suggested that er stress plays a potent role in age - related macular degeneration ( amd ) . in er stress , cells activate a self - protective system termed the unfolded protein response , which includes elevation of molecular chaperone expression and er - associated protein degradation . we hypothesized that er stress activates mitochondrial metabolic activity early after exposure to 4-hne , and prolonged exposure ( 24 hours ) causes er - mediated cell death . to study er stress induced by a low concentration of 4-hne ( 25  m ) , expression of atf4 and phosphorylated - jnk ( p - jnk ) was analyzed using western blotting . however , 4-hne did not induce atf4 expression and did not increase p - jnk expression ( data not shown ) . to study er stress caused by misfolding , we analyzed hsp expression . hsps prevent the accumulation of cytotoxic protein aggregates and assist in the refolding of misfolded proteins . hsp90 is one of the most abundant proteins in eukaryotic cells , and its expression can increase severalfold after stress [ 30 , 31 ] . both hsps regulate the activation of the hsf1 transcription factor , which is linked to the transcriptional stress response [ 33 , 34 ] . therefore , we determined whether these hsps are involved in the protective effects of trx2 and found that 4-hne increased hsp90 expression but inhibited the increase of hsp70 expression . reported that inhibition of hsp90 led to an increase of hsp70 expression and cell damage . thus , inhibition of hsp70 by trx2 might protect against oxidative stress , and its protective effect was limited to the stress caused by a low concentration of 4-hne . however , our experiments were performed using an overexpression model , and several nonspecific reactions might have occurred . further experiments will be required to reveal how endogenous trx2 functions to counteract oxidative stress . oxidative stress is a trigger for amd . in the present study , two inducers of oxidative stress , 4-hne and c2-ceramide , were added to arpe cells , and the protective effect of trx2 expression was investigated . both stressors caused cell death in a concentration - dependent manner and induced rapid cell death at a high concentration . these results suggest that there are concentration - dependent differences in the types of cell death . trx2 only protected arpe cells from low concentrations that upregulated mitochondrial activity early after exposure . trx2 protected cells by inhibiting the increase in hsp70 expression induced by oxidative stress .
the retina is constantly subjected to oxidative stress , which is countered by potent antioxidative systems present in retinal pigment epithelial ( rpe ) cells . 
 disruption of these systems leads to the development of age - related macular degeneration . 
 thioredoxin 2 ( trx2 ) is a potent antioxidant , which acts directly on mitochondria . in the present study , 
 oxidative stress was induced in the human rpe cell line ( arpe-19 ) using 4-hydroxynonenal ( 4-hne ) or c2-ceramide . 
 the protective effect of trx2 against oxidative stress was investigated by assessing cell viability , the kinetics of cell death , mitochondrial metabolic activity , and expression of heat shock proteins ( hsps ) in trx2-overexpressing cell lines generated by transfecting arpe cells with an adeno - associated virus vector encoding trx2 . 
 we show that overexpression of trx2 reduced cell death induced by both agents when they were present in low concentrations . moreover , early after the induction of oxidative stress trx2 played a key role in the maintenance of the cell viability through upregulation of mitochondrial metabolic activity and inhibition of hsp70 expression .