Journal of Diabetes Research

Journal of Diabetes Research / 2012 / Article

Research Article | Open Access

Volume 2012 |Article ID 743780 | 11 pages |

Expression of Endoplasmic Reticulum Stress-Related Factors in the Retinas of Diabetic Rats

Academic Editor: Nils Welsh
Received18 Apr 2011
Revised18 Jun 2011
Accepted20 Jun 2011
Published28 Aug 2011


Recent reports show that ER stress plays an important role in diabetic retinopathy (DR), but ER stress is a complicated process involving a network of signaling pathways and hundreds of factors, What factors involved in DR are not yet understood. We selected 89 ER stress factors from more than 200, A rat diabetes model was established by intraperitoneal injection of streptozotocin (STZ). The expression of 89 ER stress-related factors was found in the retinas of diabetic rats, at both 1- and 3-months after development of diabetes, by quantitative real-time polymerase chain reaction arrays. There were significant changes in expression levels of 13 and 12 ER stress-related factors in the diabetic rat retinas in the first and third month after the development of diabetes, Based on the array results, homocysteine- inducible, endoplasmic reticulum stress-inducible, ubiquitin-like domain member 1(HERP), and synoviolin(HRD1) were studied further by immunofluorescence and Western blot. Immunofluorescence and Western blot analyses showed that the expression of HERP was reduced in the retinas of diabetic rats in first and third month. The expression of Hrd1 did not change significantly in the retinas of diabetic rats in the first month but was reduced in the third month.

1. Introduction

Diabetic retinopathy (DR) is one of the severe complications of diabetes leading to loss of vision. Although the pathogenic mechanism of DR has been investigated for many years and a number of theories have been proposed [1, 2], the mechanism of DR remains unknown and needs further exploration.

Some diabetic patients are susceptible to DR, while others are quite resistant or develop minimal pathological changes [3]. It may be supposed that such DR-resistant patients are protected genetically. The existence of a DR-resistant gene was proposed, and a comparative study was performed of the gene expression between susceptible and resistant DR patients [4]. It was found that many endoplasmic reticulum (ER) stress-related factors are highly expressed in non-DR diabetic patients.

In our earlier work, we found that , an ER stress-related factor, binds to the ER transmembrane protein PERK (protein kinase RNA-activated- (PKR-) like ER kinase), which is normally activated by the ER stress/unfolded protein response. By binding to PERK, thereby inhibits its phosphorylation of the -subunit of eukaryotic translation initiation factor 2 (eIF-2 ) and thus compromises eIF2/EIF2S3’s mediator role in the translation of mRNA [5]. In this way, inhibits ER stress in the endothelial cells of human retinal vessels. also downregulates the expression of vascular endothelial growth factor (VEGF), which is associated with regulation of the pathology of DR [6]. VEGF plays a key role in DR [7, 8] and is regulated at the transcriptional level by the unfolded protein response pathway [9]. Recent reports also show that ER stress plays an important role in DR [10, 11]. Li et al. [12] demonstrated that multiple ER stress markers, including 78 kDa glucose-regulated protein (GRP78), phosphoinositol-requiring transmembrane kinase (IRE)1 , and phosphor-eIF2 were significantly upregulated in the retinas of animal models of type 1 diabetes and oxygen-induced retinopathy. Our recent work suggests that early progression of DR may be mediated by ER stress, but probably does not involve changes in activating transcription factor (ATF)4 or GRP78 [13]. Together, these studies suggest that although ER stress is involved in the development of DR, its specific pathogenesis is not yet understood.

ER stress is a complicated process involving a network of signaling pathways and hundreds of factors that function by triggering the PERK, IRE1 and ATF6 signaling pathways [1416]. In order to delve into the effects of these ER stress-related factors on DR, we classified them into 11 categories according to function (Figure 1, Table 3), based on Jonikas et al. [17]. We selected 89 ER stress factors from more than 200, based on our work and that of others (Table 4) [13, 1721]. These factors contain the 11 categories of ER stress. Expression of these factors in the retinas of diabetic rats was determined by quantitative real-time PCR (Q-PCR) arrays to find the specific factors and the ER stress signaling pathways that may play a key role in the pathogenesis of DR.

2. Methods

2.1. Diabetic Rat Model

Two-month-old male Sprague Dawley rats weighing 150 to 200 g were obtained from the animal center of Huazhong University of Science and Technology. Care, use, and treatment of animals were approved by the laboratory animal center of Huazhong University of Science and Technology. Rats were randomly divided into diabetic and control groups ( per group). The diabetic model was created by intraperitoneal injection of a single dose of streptozotocin (STZ; 65 mg/kg in 0.01 M citrate buffer, pH 4.5) [22]. Nondiabetic rats (the control group) were injected with citrate buffer only. Fasting plasma glucose was examined 3 d after STZ injection, and diabetes was confirmed by a value ≥16.7 mmol/L using Touch Glucometer (Boehringer Mannheim Diagnostics, Indianapolis, IN). Our previous work [13] and that of others [23] have established that in the STZ-induced diabetes model, diabetic retinopathy develops within one month of the development of diabetes. Accordingly, one and three months after the STZ injection, the retinas were separated from the eyes of both the diabetic and control groups. RNA was extracted and assessed using Q-PCR arrays, with 9 rats in each group.

2.2. Quantitative Real-Time RNA Polymerase Chain Reaction (Q-PCR) Arrays

The mRNA levels of 96 factors (89 ER stress-related factors and 7 quality control factors) were measured using Q-PCR arrays. Total RNA was extracted from rat retinal tissue using Trizol reagent (Invitrogen, Carlsbad, USA) according to the manufacturer’s instructions. RNA was treated with DNAase (Invitrogen, Carlsbad, USA) and purified using Rneasy MinElute Clean-up Kit (Quiagen, Hilden, Germany). The cDNA was then synthesized using a SuperScript III kit (Invitrogen, Carlsbad, USA). Removing the plate seal from the PCR Array (SABioscience, Frederick, USA) and adding the cocktails to the PCR Array, Q-PCR was performed by using the Hot Star polymerase kit (Qiagen, Venlo, The Netherlands) with SYBR Green technology (ABI, Tampa, FL). PCR reaction buffer was added to a 384-well PCR array plate which was then tightly sealed with an optical adhesive cover. The thermocycling program consisted of 95°C for 10 min, then 40 cycles at 95°C for 15 s, and 60°C for one minute, then compared the differential expression of gene between the two groups.

2.3. Immunofluorescence

Immunofluorescence was performed on 5 μm frozen sections. Briefly, retinal sections were incubated with a rabbit anti-HERP (Santa Cruz Biotechnology, Santa Cruz, Calif) or anti-Hrd1 (Biosynthesis Biotechnology, Beijing, China) antibody (1 : 200) at 4°C overnight. This was followed by the secondary antibody, fluorescein-conjugated goat antirabbit IgG (Antigene, Wu Han, China), for one hour. The slides were visualized and photographed under a fluorescence microscope (Olympus, Hamburg, Germany).

2.4. Western Blot

Total protein was extracted from rat retinal tissue in 300  L lysis buffer (50 mM Tris pH 7.5, 0.5 M NaCl, 1% NP-40, 1% sodium deoxycholate monohydrate, 2 mM EDTA, and 0.1% SDS). After centrifugation at 1000 ×g for 3 min, protein extracts were diluted with sample buffer (126 mM Tris HCl pH 6.8, containing 20% glycerol, 4% SDS, 0.005% bromophenol blue, and 5% 2-mercaptoethanol) at a 1 : 1 ratio and boiled for 3 minutes. The samples were fractionated according to size on a 12.5% SDS-polyacrylamide gel, transferred to a nitrocellulose membrane (Millipore, Billerica, Mass), and probed with polyclonal anti-HERP (Santa Cruz Biotechnology, Santa Cruz, Calif) or polyclonal anti-Hrd1 (Biosynthesis Biotechnology, Beijing, China) antibodies. A secondary antibody, goat antirabbit IgG (Biosynthesis Biotechnology, Beijing, China) diluted 1 : 1000, was applied, and the chemiluminescent signal was detected. The same membrane was reused to detect -actin (the internal control) by incubating it with mouse antihuman -actin antibody (Gene, Hong Kong, China). Bands observed on the photography films were analyzed by automatic image analysis. The integrated optical density of each protein band was normalized to that of the corresponding -actin band from the same sample.

2.5. Rat Retinal Capillary Endothelial Cell (RRCEC) Culture

RRCECs cultured in vitro were prepared as previously described [24]. Two-month-old male Sprague Dawley rats weighing 150–200 g ( ) were obtained from the animal center of Huazhong University of Science and Technology. After anesthesia, the eyes were removed, and the retinas harvested and homogenized by two gentle up-and-down strokes in a 15 mL homogenizer (Dounce; Bellco Glass, Vineland, NJ). The homogenate was filtered through an 88 μm sieve. The retentate was digested in 0.066% collagenase for 45 min at 37°C. The homogenate was centrifuged (1000 ×g for 10 min), and the pellet was resuspended in endothelial basal growth medium (Invitrogen-Gibco, Grand Island, NY), supplemented with 20% fetal bovine serum, 50 U/mL endothelial cell growth factor (Sigma-Aldrich, St. Louis, Mo), and 1% insulin-transferrin-selenium. RRCECs were cultured in fibronectin-coated dishes and incubated at 37°C in a humidified atmosphere containing 5% CO2.

Cultured endothelial cells were characterized by evaluating expression of factor VIII antigen (von Willebrand factor) and determining unchanged morphology under culture conditions by light microscopy. The expression of acetyl-LDL (Ac-LDL) receptors in endothelial cells was measured by adding fluorescence-labeled AC-LDL (Biomedical Technologies, Palatine, Il). Only cells from passages 3 to 7 were used in the experiments.

2.6. Cell Immunofluorescence

The RRCECs were grown in 24-well plates in human endothelial serum-free material basal growth medium containing 8.3 mM glucose. Upon attaining 80%, confluency cells were treated with medium containing 25 mM glucose for 2 d. Cells were then fixed with 4% formaldehyde for 15 min and permeabilized in 0.1% Triton X-100 for 10 min. Cells were incubated with primary antibody at 4°C overnight followed by secondary antibody for one hour. The slides were visualized and photographed under a fluorescence microscope (Olympus, Hamburg, Germany).

2.7. Statistical Analysis

Normally distributed data were compared using Student’s independent samples t-test or one-way ANOVA where appropriate. When a significant difference was detected between groups, multiple comparisons of means were performed using the Bonferroni procedure, with type-I error rate at a maximum of 0.017 (0.05/3) adjustment. Statistical analyses were performed using Statistical Package for the Social Sciences (SPSS) 15.0 software (SPSS, Chicago, IL). Data were presented as the mean ± standard deviation (SD). A probability (P) value was considered statistically significant.

3. Results

3.1. Q-PCR Arrays

We detected 89 ER stress-related genes and found that the mRNA levels of 13 genes in the diabetic rats changed significantly during the first month (Table 1). We found that in the third month the levels of expression of 12 genes were changed significantly in these diabetic rats (Table 2). The changes in the expression levels of genes corresponded to 8 and 10 categories of signal pathways in the first and third months, respectively (Figure 1 and Table 3). The mRNA expressions of Erdj4 and HERP were lower both in the first and third months.

SymbolGene nameThe average ratio of gene expressiont-test
DRcontrolP value

CCT4Cctd 0.0134
DNAJB9Erdj4 0.0125
DNAJC3P58IPK 0.0173
Casp12Casp12 0.002
ERP44Pdia10 0.0337
GANABGluII 0.045
HERPUD1Herp 0.0006
HSPA1LHsp70-3 0.0183
HSPA2Hspt70 0.0183
MAPK8JNK 0.0391
NUCB1NUC 0.0289
OS9OS-9 0.0272
SELSAD-015 0.0486

SymbolGene nameThe average ratio of gene expression -test
DRcontrolP value

ATF4CREB-2 0.0178
DNAJB9Erdj4 0.0106
ERO1LEro1 0.0492
TRB3Trib3 0.0024
HERPUD1Herp 0.0008
HTRA2PARK13 0.0064
SYVN1/Hrd1HRD1 0.0067
UFD1LUFD1 0.0463
UGCGL1HUGT1 0.0833
USP14TGT 0.0405

Signaling pathwayFirst monthThird month

Unfolded protein bindingCctd, ERdj4, Hspt70-3ERdj4, OMI/PARK13, CYPA, HUGT1
ER protein folding quality controlGluII, PDIA10HUGT1
Regulation of cholesterol MetabolismSREBP1
Regulation of translation
ERADHerp, NUC, Os9, ADO15Herp, OMI/PARK13, Hrd1
UbiquitinationHerpHerp, UfD1, TGT
Transcription factorsATF4, SREBP1
Protein foldingCctd, ERdj4,  APG-1, PDIA10 ERdj4, Ero1l, CYPA
Protein disulfide isomerizationPDIA10SREBP1
Heat shock proteinsERdj4, P58IPK, Hspt70-3ERdj4
ApoptosisJNK/JNK1, Casp12OMI/PARK13, NIPK/Trib3

A01Rn.107561XM_341644AMFRAutocrine motility factor receptorAMFR
A02Rn.161941NM_001108183ARMETArginine rich, mutated in early-stage tumorsARMET
A03Rn.2423NM_024403ATF4Activating transcription factor 4 (tax-responsive enhancer element B67)CREB-2/CREB2
A04Rn.222130NM_001107196ATF6Activating transcription factor 6ATF6A
A05Rn.18179NM_001002809ATF6BActivating transcription factor 6 betaCREB-RP/CREBL1
A06Rn.42932NM_021702ATXN3Ataxin 3AT3/ATX3
A07Rn.10668NM_017059BAXBCL2-associated X proteinBCL2L4
A10Rn.97889NM_182814.2CCT4Chaperonin containing TCP1, subunit 4 (delta)CCT-DELTA/Cctd
A11Rn.62267NM_001106603.1CCT7Chaperonin containing TCP1, subunit 7 (eta)CCT-ETA/Ccth
A12Rn.6479NM_024125.4CEBPBCCAAT/enhancer binding protein (C/EBP), betaC/EBP-beta
B01Rn.104043NM_001013092.1CREB3CAMP responsive element binding protein 3LUMAN/LZIP
B02Rn.20059NM_001012115.1CREB3L3CAMP responsive element binding protein 3-like 3CREB-H/CREBH
B03Rn.11183NM_001109986DDIT3DNA-damage-inducible transcript 3CEBPZ/CHOP
B04Rn.110990NM_001014202.1DERL1Der1-like domain family, member 1DER-1/DER1
B05Rn.11209NM_031627CHOPRattus norvegicus nuclear receptor subfamily 1, group H, member 3LXRalpha/Nr1h3
B06Rn.40780NM_001109541DNAJB2DnaJ (Hsp40) homolog, subfamily B, member 2HSJ1/HSPF3
B07Rn.29778NM_012699DNAJB9DnaJ (Hsp40) homolog, subfamily B, member 9DKFZp564F1862/ERdj4
B08Rn.8642NM_001106486DNAJC10DnaJ (Hsp40) homolog, subfamily C, member 10DKFZp434J1813/ERdj5
B09Rn.162234NM_022232DNAJC3DnaJ (Hsp40) homolog, subfamily C, member 3HP58/P58
B10Rn.91398NM_001013196DNAJC4DnaJ (Hsp40) homolog, subfamily C, member 4DANJC4/HSPF2
B11Rn.107459NM_001033909Elf2E74-like factor 2Elf2
B12Rn.81078NM_130422Casp12Caspase 12Casp12
C01Rn.198593NM_001109339eIF2AEukaryotic translation initiation factor 2A, 65 kDaCDA02/EIF-2A
C02Rn.24897NM_031599EIF2AK3Eukaryotic translation initiation factor 2-alpha kinase 3DKFZp781H1925/HRI
C03Rn.19198NM_001037208CRELD2cysteine-rich with EGF-like domains 2Creld2
C04Rn.218563XM_344959.3ERN2Endoplasmic reticulum to nucleus signaling 2Ern2
C05Rn.64648NM_138528ERO1LERO1-like (S. cerevisiae)Ero1l
C06Rn.22325NM_144755TRB3Tribbles homolog 3NIPK/Trib3
C07Rn.2459NM_001008317ERP44Thioredoxin domain containing 4 (endoplasmic reticulum)PDIA10/TXNDC4
C08Rn.57325NM_138917FBXO6F-box protein 6FBG2/FBS2
C09Rn.99241NM_001106334GANABGlucosidase, alpha; neutral ABG2AN/GluII
C10Rn.23744NM_001145840GANCGlucosidase, alpha; neutral CMGC138256
C11Rn.4028NM_053523HERPUD1Homocysteine-inducible, endoplasmic reticulum stress-inducible, ubiquitin-like domain member 1Sup
C12Rn.1950NM_212504HSPA1BHeat shock 70 kDa protein 1BHSP70-1B/HSP70-2/Hsp72
D01Rn.187184NM_212546HSPA1L heat shock protein 1-likeHsp70-3/MGC112562/MGC114222
D02Rn.211303NM_021863HSPA2Heat shock protein 2Hspt70/Hst70/MGC93458
D03Rn.163092NM_153629HSPA4Heat shock protein 4Hsp110/ Hsp70/irp94
D04Rn.144829NM_001106428HSPA4LHeat shock protein 4-likeAPG-1; MGC187594; OSP94
D05Rn.11088NM_013083HSPA5Heat shock 70 kDa protein 5 (glucose-regulated protein, 78 kDa)BIP/GRP78
D06Rn.37805NM_001011901HSPH1Heat shock 105 kDa/110 kDa protein 1DKFZp686M05240/HSP105
D07Rn.107325NM_001106599HTRA2HtrA serine peptidase 2OMI/PARK13
D08Rn.163330NM_001107321HTRA4HtrA serine peptidase 4FLJ90724
D09Rn.772NM_022392INSIG1Insulin-induced gene 1CL-6
D10Rn.16736NM_178091INSIG2Insulin-induced gene 2MGC26273
D11Rn.9911NM_012806MAPK10Mitogen-activated protein kinase 10JNK3/JNK3A
D12Rn.4090XM_001056513MAPK8Mitogen-activated protein kinase 8JNK/JNK1
E01Rn.9910NM_017322MAPK9Mitogen-activated protein kinase 9JNK-55/JNK2
E02Rn.2362NM_053569MBTPS1Membrane-bound transcription factor peptidase, site 1PCSK8/S1P
E03Rn.212224NM_001035007MBTPS2Membrane-bound transcription factor peptidase, site 2S2P
E04Rn.144645NM_080577NPLOC4Nuclear protein localization 4 homolog (S. cerevisiae)NPL4
E05Rn.1492NM_053463NUCB1Nucleobindin 1DKFZp686A15286/NUC
E06Rn.1579NM_001007265OS9Osteosarcoma amplified 9, endoplasmic reticulum associated proteinOS-9
E07Rn.11527NM_017319PDIA3Protein disulfide isomerase family A, member 3ER60/ERp57
E08Rn.7627NM_001109476PFDN2Prefoldin subunit 2PFD2
E09Rn.3401NM_001106794PFDN5Prefoldin subunit 5MM-1/MM1
E10Rn.1463NM_017101PPIAPeptidylprolyl isomerase A (cyclophilin A)CYPA/CYPH
E11Rn.2232NM_133546PPP1R15AProtein phosphatase 1, regulatory (inhibitor) subunit 15AGADD34
E12Rn.104417NM_001106806PRKCSHProtein kinase C substrate 80K-HAGE-R2/G19P1
F01Rn.209127NM_001127545RNF139Ring finger protein 139HRCA1/RCA1
F02Rn.209127NM_006913RNF5Ring finger protein 5RING5/RMA1
F03Rn.4224NM_013067RPN1Ribophorin IDKFZp686B16177/OST1
F04Rn.99548NM_001100966SCAPSREBF chaperoneKIAA0199
F05Rn.98327NM_001034129SEC62SEC62 homolog (S. cerevisiae)Dtrp1/HTP1
F06Rn.24233NM_001107637SEC63SEC63 homolog (S. cerevisiae)ERdj2/PRO2507
F07Rn.20802NM_177933SEL1LSel-1 suppressor of lin-12-like (C. elegans)IBD2/PRO1063
F08Rn.4197NM_173120SELSSelenoprotein SAD-015/ADO15
F09Rn.2119NM_030835SERP1Stress-associated endoplasmic reticulum protein 1RAMP4
F10Rn.103851NM_199376SIL1SIL1 homolog, endoplasmic reticulum chaperone (S. cerevisiae)BAP/MSS
F11Rn.221929XM_001075680SREBF1Sterol regulatory element binding transcription factor 1SREBP-1c/SREBP1
F12Rn.41063NM_001033694SREBF2Sterol regulatory element binding transcription factor 2SREBP2/bHLHd2
G01Rn.162486NM_001100739SYVN1Synovial apoptosis inhibitor 1, synoviolinHRD1
G02Rn.7102NM_012670TCP1T-complex 1CCT-alpha/CCT1
G03Rn.20041NM_153303TOR1ATorsin family 1, member A (torsin A)DQ2/DYT1
G04Rn.139603NM_001106380UBE2G2Ubiquitin-conjugating enzyme E2G 2 (UBC7 homolog, yeast)UBC7
G05Rn.106299NM_001007655UBE2J2Ubiquitin-conjugating enzyme E2, J2 (UBC6 homolog, yeast)NCUBE2/PRO2121
G06Rn.2022NM_001012025UBXN4UBX domain protein 4UBXD2/UBXDC1
G07Rn.11946NM_053418UFD1LUbiquitin fusion degradation 1-like (yeast)UFD1
G08Rn.162227NM_133596UGCGL1UDP-glucose ceramide glucosyltransferase-like 1HUGT1
G09Rn.107678NM_019381BI-1Transmembrane BAX inhibitor motif containing 6Tmbim6
G10Rn.11790NM_001008301USP14Ubiquitin-specific peptidase 14 (tRNA-guanine transglycosylase)TGT
G11Rn.98891NM_053864VCPValosin-containing proteinIBMPFD/TERA
G12Rn.101044NM_001004210XBP1X-box binding protein 1TREB5/XBP2
H01Rn.973NM_001007604Rplp1Ribosomal protein, large, P1MGC72935
H02Rn.47NM_012583HprtHypoxanthine guanine phosphoribosyl transferaseHgprtase/Hprt1
H03Rn.92211NM_173340Rpl13aRibosomal protein L13ARpl13a
H04Rn.107896NM_017025LdhaLactate dehydrogenase ALdh1
H05Rn.94978NM_031144ActbActin, betaActx
H06N/AU26919RGDCRat genomic DNA contaminationRGDC
H07N/ASA_00104RTCReverse Transcription ControlRTC
H08N/ASA_00104RTCReverse transcription controlRTC
H09N/ASA_00104RTCReverse transcription controlRTC
H10N/ASA_00103PPCPositive PCR controlPPC
H11N/ASA_00103PPCPositive PCR controlPPC
H12N/ASA_00103PPCPositive PCR controlPPC

3.2. Expression of HERP and HRD1 in the Retinas of Diabetic Rats

We detected HERP and Hrd1 protein expression levels in the retinas of diabetic rats by Western blot and immunofluorescence in the first and the third months of diabetes development. The Western blot suggested that the HERP expression decreased significantly in the first month ( ) and third month ( ) compared with the nondiabetic control group. No significant change in the expression level of Hrd1 was observed in the first month ( ), while it decreased significantly in the third month compared with the control group ( ; Figures 2 and 3).

The results of immunofluorescence were consistent with the Western blot. The protein level of HERP decreased significantly at both the first and third months ( and 0.007, resp.; Figures 2 and 3). There was no significant change in the expression of retinal HRD1 in the first month, while it decreased significantly in the third month ( and 0.003, resp. Figures 2 and 3).

3.3. Expression of HERP and HRD1 in RRCECs in the Presence of High-Glucose Concentration

The expression levels of HERP and HRD1 in RRCECs in vitro in the presence of high glucose concentration were decreased significantly compared to the control group ( and 0.024, resp.; Figure 4).

4. Discussion

The STZ-induced rat diabetes model is an established animal model for studying DR. Although we did not verify the development of DR in this study, our previous studies and the publication from another group have demonstrated that DR develops within one month of STZ-induced diabetes [13, 22, 23]. Our results indicate that of 89 ER stress genes, the expression of 12 genes in the retinas of diabetic rats was downregulated by the third month of diabetes development, and the expression of CCT4 increased within the first month. We did not observe any change in the expression of AFT4 or GFP78 at either time point in our study, which is consistent with our earlier results [13].

The expression of genes belonging to 8 different categories of ER stress factors was altered in the first month, while those of 10 categories were changed by the third month, suggesting that with increasing time more categories of ER stress factors were involved in the pathogenic process of DR. The expression of a number of related factors of the ERAD signaling pathways was downregulated, indicating that the ERAD signaling pathway may play an important role in DR. The ERAD system is an important pathway of protein degradation in the ER [25, 26] and plays important physiological roles. The ER is the location of protein synthesis, and secretion [27, 28] and has strict quality control mechanisms which allow secretion of correctly folded protein into the cytoplasm. The wrongly folded protein will be degraded through ERAD. ERAD therefore is a quality control system of the ER.

Recent studies found that HRD1 plays a central role in the ERAD-luminal pathway [29] and that HERP coordinates and regulates HRD1-mediated ubiquitylation [28], so we selected HRD1 and HERP from the ERAD pathway for further study. HERP expression was downregulated significantly in the retinas of diabetic rats in the first and third months. HERP is a membrane-bound, ubiquitin-like protein that is located in the ER. It forms a complex with ubiquitinated proteins and with the 26S proteasome [3033]. HERP functions to degrade wrongly folded nonglycosylated proteins by forming a protein-enzyme complex with Derlin-1, HRD1, and p97 [34]. In our study, HRD1 expression in the retinas of diabetic rats remained unchanged in the first month, while it decreased in the third month. HRD1 is an E3 ubiquitin ligase and a key factor of ERAD [3537]. ERAD has three pathways in yeast [38]: ERAD-L, ERAD-M, and ERAD-C. Both ERAD-L and ERAD-M are the key enzymes of HRD1. In the mammalian ERAD, HRD1 plays a very broad role in the ubiquitination process of abnormal proteins in the ER. The ubiquitin ligase HRD1 is mainly involved in the degradation of glycosylation proteins [3941].

The decreased expression of HERP and HRD1 at both the mRNA and protein levels could lead to a decrease in function of ERAD’s ability to remove wrongly folded proteins in the cell. Misfolded protein accumulation in the ER induces ER stress and activates signaling pathways, including PERK, ATF6, and IRE1 [15]. Persistent ER stress leads to cell death and induction of inflammation [4245]. An inflammatory milieu is instrumental in breaking down the blood-retinal barrier in DR [46, 47].

In conclusion, we have shown by in vivo and in vitro experiments that an elevated concentration of glucose leads to downregulation of the ERAD signaling pathway. Such downregulation may result in local inflammation and DR.


This research was supported by the National Nature Science Foundation (Grant no. 30872823), China. S. Yan, C. Zheng, and Z.-q. Chen contributed equally to this paper.


  1. R. N. Frank, “Diabetic retinopathy,” The New England Journal of Medicine, vol. 350, no. 1, pp. 48–58, 2004. View at: Publisher Site | Google Scholar
  2. N. Cheung and T. Y. Wong, “Diabetic retinopathy and systemic vascular complications,” Progress in Retinal and Eye Research, vol. 27, no. 2, pp. 161–176, 2008. View at: Publisher Site | Google Scholar
  3. P. Cejkova, P. Novota, M. Cerna et al., “HLA DRB1, DQB1 and insulin promoter VNTR polymorphisms: interactions and the association with adult-onset diabetes mellitus in Czech patients,” International Journal of Immunogenetics, vol. 35, no. 2, pp. 133–140, 2008. View at: Publisher Site | Google Scholar
  4. B. Li, H. Q. Zhang, Y. Shi et al., “Overexpression of nuclear transport factor 2 may protect against diabetic retinopathy,” Molecular Vision, vol. 15, pp. 861–869, 2009. View at: Google Scholar
  5. W. Yan, C. L. Frank, M. J. Korth et al., “Control of PERK eIF2alpha kinase activity by the endoplasmic reticulum stress-induced molecular chaperone P58IPK,” Proceedings of the National Academy of Sciences of the United States of America, vol. 99, no. 25, pp. 15920–15925, 2002. View at: Publisher Site | Google Scholar
  6. B. Li, D. Li, G. G. Li, H. W. Wang, and A. X. Yu, “P58(IPK) inhibition of endoplasmic reticulum stress in human retinal capillary endothelial cells in vitro,” Molecular Vision, vol. 14, pp. 1122–1128, 2008. View at: Google Scholar
  7. T. Hattori, H. Shimada, H. Nakashizuka, Y. Mizutani, R. Mori, and M. Yuzawa, “Dose of intravitreal bevacizumab (avastin) used as preoperative adjunct therapy for proliferative diabetic retinopathy,” Retina, vol. 30, no. 5, pp. 761–764, 2010. View at: Publisher Site | Google Scholar
  8. S. Kant, G. Seth, and K. Anthony, “Vascular endothelial growth factor-A (VEGF-A) in vitreous fluid of patients with proliferative diabetic retinopathy,” Annals of Ophthalmology, vol. 41, no. 3-4, pp. 170–173, 2009. View at: Google Scholar
  9. R. Ghosh, K. L. Lipson, K. E. Sargent et al., “Transcriptional regulation of VEGF-A by the unfolded protein response pathway,” PLoS One, vol. 5, no. 3, Article ID e9575, 2010. View at: Publisher Site | Google Scholar
  10. T. Oshitari, N. Hata, and S. Yamamoto, “Endoplasmic reticulum stress and diabetic retinopathy,” Vascular Health and Risk Management, vol. 4, no. 1, pp. 115–122, 2008. View at: Publisher Site | Google Scholar
  11. C. S. McAlpine, A. J. Bowes, and G. H. Werstuck, “Diabetes, hyperglycemia and accelerated atherosclerosis: evidence supporting a role for endoplasmic reticulum (ER) stress signaling,” Cardiovascular and Hematological Disorders—Drug Targets, vol. 10, no. 2, pp. 151–157, 2010. View at: Google Scholar
  12. J. Li, J. J. Wang, Q. Yu, M. Wang, and S. X. Zhang, “Endoplasmic reticulum stress is implicated in retinal inflammation and diabetic retinopathy,” FEBS Letters, vol. 583, no. 9, pp. 1521–1527, 2009. View at: Publisher Site | Google Scholar
  13. B. Li, H. S. Wang, G. G. Li, M. J. Zhao, and M. H. Zhao, “The role of endoplasmic reticulum stress in the early stage of diabetic retinopathy,” Acta Diabetologica, vol. 48, pp. 103–111, 2011. View at: Google Scholar
  14. Y. Adachi, K. Yamamoto, T. Okada, H. Yoshida, A. Harada, and K. Mori, “ATF6 is a transcription factor specializing in the regulation of quality control proteins in the endoplasmic reticulum,” Cell Structure and Function, vol. 33, no. 1, pp. 75–89, 2008. View at: Publisher Site | Google Scholar
  15. J. H. Lin, H. Li, D. Yasumura et al., “IRE1 signaling affects cell fate during the unfolded protein response,” Science, vol. 318, no. 5852, pp. 944–949, 2007. View at: Publisher Site | Google Scholar
  16. H. Hirasawa, C. Jiang, P. Zhang, F. C. Yang, and H. Yokota, “Mechanical stimulation suppresses phosphorylation of eIF2alpha and PERK-mediated responses to stress to the endoplasmic reticulum,” FEBS Letters, vol. 584, no. 4, pp. 745–752, 2010. View at: Publisher Site | Google Scholar
  17. M. C. Jonikas, S. R. Collins, V. Denic et al., “Comprehensive characterization of genes required for protein folding in the endoplasmic reticulum,” Science, vol. 323, no. 5922, pp. 1693–1697, 2009. View at: Publisher Site | Google Scholar
  18. K. Kohno, “Stress-sensing mechanisms in the unfolded protein response: similarities and differences between yeast and mammals,” Journal of Biochemistry, vol. 147, no. 1, pp. 27–33, 2010. View at: Publisher Site | Google Scholar
  19. D. T. Rutkowski and R. S. Hegde, “Regulation of basal cellular physiology by the homeostatic unfolded protein response,” Journal of Cell Biology, vol. 189, no. 5, pp. 783–794, 2010. View at: Publisher Site | Google Scholar
  20. A. Kapoor and A. J. Sanyal, “Endoplasmic reticulum stress and the unfolded protein response,” Clinics in Liver Disease, vol. 13, no. 4, pp. 581–590, 2009. View at: Publisher Site | Google Scholar
  21. T. Hosoi and K. Ozawa, “Endoplasmic reticulum stress in disease: mechanisms and therapeutic opportunities,” Clinical Science, vol. 118, no. 1, pp. 19–29, 2010. View at: Publisher Site | Google Scholar
  22. R. M. Brucklacher, K. M. Patel, H. D. VanGuilder et al., “Whole genome assessment of the retinal response to diabetes reveals a progressive neurovascular inflammatory response,” BMC Medical Genomics, vol. 13, pp. 1–26, 2008. View at: Google Scholar
  23. H. Yang, R. Liu, Z. Cui et al., “Functional characterization of 58-kilodalton inhibitor of protein kinase in protecting against diabetic retinopathy via the endoplasmic reticulum stress pathway,” Molecular Vision, vol. 17, pp. 78–84, 2011. View at: Google Scholar
  24. B. Li, W. Yin, X. Hong et al., “Remodeling retinal neovascularization by ALK1 gene transfection in vitro,” Investigative Ophthalmology and Visual Science, vol. 49, no. 10, pp. 4553–4560, 2008. View at: Publisher Site | Google Scholar
  25. J. Hoseki, R. Ushioda, and K. Nagata, “Mechanism and components of endoplasmic reticulum-associated degradation,” Journal of Biochemistry, vol. 147, no. 1, pp. 19–25, 2010. View at: Publisher Site | Google Scholar
  26. H. Ando, M. Ichihashi, and V. J. Hearing, “Role of the ubiquitin proteasome system in regulating skin pigmentation,” International Journal of Molecular Sciences, vol. 10, no. 10, pp. 4428–4434, 2009. View at: Publisher Site | Google Scholar
  27. R. Sharma, M. Tsuchiya, and J. D. Bartlett, “Flouride induces endoplasmic reticulum stress and inhibits protein synthesis and secretion,” Environmental Health Perspectives, vol. 116, no. 9, pp. 1142–1146, 2008. View at: Publisher Site | Google Scholar
  28. H. Coe and M. Michalak, “Calcium binding chaperones of the endoplasmic reticulum,” General Physiology and Biophysics, vol. 28, pp. F96–F103, 2008. View at: Google Scholar
  29. P. Carvalho, A. M. Stanley, and T. A. Rapoport, “Retrotranslocation of a misfolded luminal ER protein by the ubiquitin-ligase Hrd1p,” Cell, vol. 143, no. 4, pp. 579–591, 2010. View at: Publisher Site | Google Scholar
  30. M. Kny, S. Standera, R. Hartmann-Petersen, P. M. Kloetzel, and M. Seeger, “Herp regulates Hrd1-mediated ubiquitylation in a ubiquitin-like domain-dependent manner,” Journal of Biological Chemistry, vol. 286, no. 7, pp. 5151–5156, 2011. View at: Publisher Site | Google Scholar
  31. K. Kokame, K. L. Agarwal, H. Kato, and T. Miyata, “Herp, a new ubiquitin-like membrane protein induced by endoplasmic reticulum stress,” Journal of Biological Chemistry, vol. 275, no. 42, pp. 32846–32853, 2000. View at: Google Scholar
  32. Y. Okuda-Shimizu and L. M. Hendershot, “Characterization of an ERAD pathway for nonglycosylated BiP substrates, which require Herp,” Molecular Cell, vol. 28, no. 4, pp. 544–554, 2007. View at: Publisher Site | Google Scholar
  33. S. Chigurupati, Z. Wei, C. Belal et al., “The homocysteine-inducible endoplasmic reticulum stress protein counteracts calcium store depletion and induction of CCAAT enhancer-binding protein homologous protein in a neurotoxin model of Parkinson disease,” Journal of Biological Chemistry, vol. 284, no. 27, pp. 18323–18333, 2009. View at: Publisher Site | Google Scholar
  34. A. Schulze, S. Standera, E. Buerger et al., “The ubiquitin-domain protein HERP forms a complex with components of the endoplasmic reticulum associated degradation pathway,” Journal of Molecular Biology, vol. 354, no. 5, pp. 1021–1027, 2005. View at: Publisher Site | Google Scholar
  35. K. Kanehara, W. Xie, and D. T. Ng, “Modularity of the Hrd1 ERAD complex underlies its diverse client range,” Journal of Cell Biology, vol. 188, no. 5, pp. 707–716, 2010. View at: Publisher Site | Google Scholar
  36. R. Bernasconi, C. Galli, V. Calanca, T. Nakajima, and M. Molinari, “Stringent requirement for HRD1, SEL1L, and OS-9/XTP3-B for disposal of ERAD-LS substrates,” Journal of Cell Biology, vol. 188, no. 2, pp. 223–235, 2010. View at: Publisher Site | Google Scholar
  37. A. Shmueli, Y. C. Tsai, M. Yang, M. A. Braun, and A. M. Weissman, “Targeting of gp78 for ubiquitin-mediated proteasomal degradation by Hrd1: cross-talk between E3s in the endoplasmic reticulum,” Biochemical and Biophysical Research Communications, vol. 390, no. 3, pp. 758–762, 2009. View at: Publisher Site | Google Scholar
  38. P. Carvalho, V. Goder, and T. A. Rapoport, “Distinct ubiquitin-ligase complexes define convergent pathways for the degradation of ER proteins,” Cell, vol. 126, no. 2, pp. 361–373, 2006. View at: Publisher Site | Google Scholar
  39. S. M. Carroll and R. Y. Hampton, “Usa1p is required for optimal function and regulation of the Hrd1p endoplasmic reticulum-associated degradation ubiquitin ligase,” Journal of Biological Chemistry, vol. 285, no. 8, pp. 5146–5156, 2010. View at: Publisher Site | Google Scholar
  40. B. K. Sato, D. Schulz, P. H. Do, and R. Y. Hampton, “Misfolded membrane proteins are specifically recognized by the transmembrane domain of the Hrd1p ubiquitin ligase,” Molecular Cell, vol. 34, no. 2, pp. 212–222, 2009. View at: Publisher Site | Google Scholar
  41. C. Hirsch, R. Gauss, S. C. Horn, O. Neuber, and T. Sommer, “The ubiquitylation machinery of the endoplasmic reticulum,” Nature, vol. 458, no. 7237, pp. 453–460, 2009. View at: Publisher Site | Google Scholar
  42. G. S. Hotamisligil, “Endoplasmic reticulum stress and the inflammatory basis of metabolic disease,” Cell, vol. 140, no. 6, pp. 900–917, 2010. View at: Publisher Site | Google Scholar
  43. H. Yoshida, “ER stress and diseases,” FEBS Journal, vol. 274, no. 3, pp. 630–658, 2007. View at: Publisher Site | Google Scholar
  44. K. Zhang and R. J. Kaufman, “From endoplasmic-reticulum stress to the inflammatory response,” Nature, vol. 454, no. 7203, pp. 455–462, 2008. View at: Publisher Site | Google Scholar
  45. T. Verfaillie, A. D. Garg, and P. Agostinis, “Targeting ER stress induced apoptosis and inflammation in cancer,” Cancer Letters. In press. View at: Publisher Site | Google Scholar
  46. T. S. Kern, “Contributions of inflammatory processes to the development of the early stages of diabetic retinopathy,” Experimental Diabetes Research, vol. 2007, Article ID 95103, 14 pages, 2007. View at: Publisher Site | Google Scholar
  47. M. Myśliwiec, A. Balcerska, K. Zorena, J. Myśliwska, P. Lipowski, and K. Raczyńska, “The role of vascular endothelial growth factor, tumor necrosis factor alpha and interleukin-6 in pathogenesis of diabetic retinopathy,” Diabetes Research and Clinical Practice, vol. 79, no. 1, pp. 141–146, 2008. View at: Publisher Site | Google Scholar

Copyright © 2012 Shu Yan et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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