Experimental Diabetes ResearchVolume 2012 (2012), Article ID 743780, 11 pagesdoi:10.1155/2012/743780

Research Article

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

Shu Yan, Cui Zheng, Zhi-qi Chen, Rong Liu, Gui-gang Li, Wei-kun Hu, Han Pei, and Bin Li

Department of Ophthalmology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1095 Jie-fang Road, Wuhan, Hubei Province 430030, China

Received 18 April 2011; Revised 18 June 2011; Accepted 20 June 2011

Academic Editor: Nils Welsh

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.

Abstract.

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 [14–16]. 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, 17–21]. 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.

(a)

(b)

Figure 1: Assessment of the expression of ER stress-related factors in diabetic retinas in the first and third months after the development of diabetes by Q-PCR arrays. (a) the histogram of the expression of different genes in 11 signaling pathways related to ER stress after the first month; (b) the histogram of the expression of different genes in 11 signaling pathways related to ER stress after the third month. Unfolded Protein Binding: UPB, ER Protein Folding Quality Control: ERPFQC, Regulation of Cholesterol Metabolism: RCM, ER-associated degradation: ERAD, Ubiquitination: Ub, Transcription Factors: TF, Protein Folding: PF, Protein Disulfide Isomerization: PDI, Heat Shock Proteins: HSP, Apoptosis: Ap°.

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.

Table 1: Q-PCR arrays showed that the expression of the ER stress factor had significant differences in the first and the third month in diabetic rat retina: The ER stress factor of differential gene expression in the first month.

Symbol
Gene name
The average ratio of gene expression
t-test

DR
control
P value

CCT4
Cctd

0.0134

DNAJB9
Erdj4

0.0125

DNAJC3
P58IPK

0.0173

Casp12
Casp12

0.002

ERP44
Pdia10

0.0337

GANAB
GluII

0.045

HERPUD1
Herp

0.0006

HSPA1L
Hsp70-3

0.0183

HSPA2
Hspt70

0.0183

MAPK8
JNK

0.0391

NUCB1
NUC

0.0289

OS9
OS-9

0.0272

SELS
AD-015

0.0486

Table 2: Q-PCR arrays showed that the expression of the ER stress factor had significant differences in the first and the third month in diabetic rat retina: The ER stress factor of differential gene expression in the third month.

Symbol
Gene name
The average ratio of gene expression
-test

DR
control
P value

ATF4
CREB-2

0.0178

DNAJB9
Erdj4

0.0106

ERO1L
Ero1

0.0492

TRB3
Trib3

0.0024

HERPUD1
Herp

0.0008

HTRA2
PARK13

0.0064

PPIA
CYPA

0.0238

SREBF1
SREBP1

0.0187

SYVN1/Hrd1
HRD1

0.0067

UFD1L
UFD1

0.0463

UGCGL1
HUGT1

0.0833

USP14
TGT

0.0405

Table 3: Q-PCR arrays showed that the expression of the ER stress factor had significant differences in the first and the third month in diabetic rat retina: the ER stress factor of significant differences belongs to different ER stress signaling pathways.

Signaling pathway
First month
Third month

Unfolded protein binding
Cctd, ERdj4, Hspt70-3
ERdj4, OMI/PARK13, CYPA, HUGT1

ER protein folding quality control
GluII, PDIA10
HUGT1

Regulation of cholesterol Metabolism
—
SREBP1

Regulation of translation
—
—

ERAD
Herp, NUC, Os9, ADO15
Herp, OMI/PARK13, Hrd1

Ubiquitination
Herp
Herp, UfD1, TGT

Transcription factors
—
ATF4, SREBP1

Protein folding
Cctd, ERdj4,  APG-1, PDIA10
ERdj4, Ero1l, CYPA

Protein disulfide isomerization
PDIA10
SREBP1

Heat shock proteins
ERdj4, P58IPK, Hspt70-3
ERdj4

Apoptosis
JNK/JNK1, Casp12
OMI/PARK13, NIPK/Trib3

Table 4: Q-PCR array gene table. We selected 89 ER stress-related factors, and other 6 genes as a quality control a total; of 96 genes were detected in Q-PCR arrays.

A01
Rn.107561
XM_341644
AMFR
Autocrine motility factor receptor
AMFR

A02
Rn.161941
NM_001108183
ARMET
Arginine rich, mutated in early-stage tumors
ARMET

A03
Rn.2423
NM_024403
ATF4
Activating transcription factor 4 (tax-responsive enhancer element B67)
CREB-2/CREB2

A04
Rn.222130
NM_001107196
ATF6
Activating transcription factor 6
ATF6A

A05
Rn.18179
NM_001002809
ATF6B
Activating transcription factor 6 beta
CREB-RP/CREBL1

A06
Rn.42932
NM_021702
ATXN3
Ataxin 3
AT3/ATX3

A07
Rn.10668
NM_017059
BAX
BCL2-associated X protein
BCL2L4

A08
Rn.974
NM_022399
CALR
Calreticulin
CRT/RO

A09
Rn.1762
NM_172008.2
CANX
Calnexin
CNX/IP90

A10
Rn.97889
NM_182814.2
CCT4
Chaperonin containing TCP1, subunit 4 (delta)
CCT-DELTA/Cctd

A11
Rn.62267
NM_001106603.1
CCT7
Chaperonin containing TCP1, subunit 7 (eta)
CCT-ETA/Ccth

A12
Rn.6479
NM_024125.4
CEBPB
CCAAT/enhancer binding protein (C/EBP), beta
C/EBP-beta

B01
Rn.104043
NM_001013092.1
CREB3
CAMP responsive element binding protein 3
LUMAN/LZIP

B02
Rn.20059
NM_001012115.1
CREB3L3
CAMP responsive element binding protein 3-like 3
CREB-H/CREBH

B03
Rn.11183
NM_001109986
DDIT3
DNA-damage-inducible transcript 3
CEBPZ/CHOP

B04
Rn.110990
NM_001014202.1
DERL1
Der1-like domain family, member 1
DER-1/DER1

B05
Rn.11209
NM_031627
CHOP
Rattus norvegicus nuclear receptor subfamily 1, group H, member 3
LXRalpha/Nr1h3

B06
Rn.40780
NM_001109541
DNAJB2
DnaJ (Hsp40) homolog, subfamily B, member 2
HSJ1/HSPF3

B07
Rn.29778
NM_012699
DNAJB9
DnaJ (Hsp40) homolog, subfamily B, member 9
DKFZp564F1862/ERdj4

B08
Rn.8642
NM_001106486
DNAJC10
DnaJ (Hsp40) homolog, subfamily C, member 10
DKFZp434J1813/ERdj5

B09
Rn.162234
NM_022232
DNAJC3
DnaJ (Hsp40) homolog, subfamily C, member 3
HP58/P58

B10
Rn.91398
NM_001013196
DNAJC4
DnaJ (Hsp40) homolog, subfamily C, member 4
DANJC4/HSPF2

B11
Rn.107459
NM_001033909
Elf2
E74-like factor 2
Elf2

B12
Rn.81078
NM_130422
Casp12
Caspase 12
Casp12

C01
Rn.198593
NM_001109339
eIF2A
Eukaryotic translation initiation factor 2A, 65 kDa
CDA02/EIF-2A

C02
Rn.24897
NM_031599
EIF2AK3
Eukaryotic translation initiation factor 2-alpha kinase 3
DKFZp781H1925/HRI

C03
Rn.19198
NM_001037208
CRELD2
cysteine-rich with EGF-like domains 2
Creld2

C04
Rn.218563
XM_344959.3
ERN2
Endoplasmic reticulum to nucleus signaling 2
Ern2

C05
Rn.64648
NM_138528
ERO1L
ERO1-like (S. cerevisiae)
Ero1l

C06
Rn.22325
NM_144755
TRB3
Tribbles homolog 3
NIPK/Trib3

C07
Rn.2459
NM_001008317
ERP44
Thioredoxin domain containing 4 (endoplasmic reticulum)
PDIA10/TXNDC4

C08
Rn.57325
NM_138917
FBXO6
F-box protein 6
FBG2/FBS2

C09
Rn.99241
NM_001106334
GANAB
Glucosidase, alpha; neutral AB
G2AN/GluII

C10
Rn.23744
NM_001145840
GANC
Glucosidase, alpha; neutral C
MGC138256

C11
Rn.4028
NM_053523
HERPUD1
Homocysteine-inducible, endoplasmic reticulum stress-inducible, ubiquitin-like domain member 1
Sup

C12
Rn.1950
NM_212504
HSPA1B
Heat shock 70 kDa protein 1B
HSP70-1B/HSP70-2/Hsp72

D01
Rn.187184
NM_212546
HSPA1L
heat shock protein 1-like
Hsp70-3/MGC112562/MGC114222

D02
Rn.211303
NM_021863
HSPA2
Heat shock protein 2
Hspt70/Hst70/MGC93458

D03
Rn.163092
NM_153629
HSPA4
Heat shock protein 4
Hsp110/ Hsp70/irp94

D04
Rn.144829
NM_001106428
HSPA4L
Heat shock protein 4-like
APG-1; MGC187594; OSP94

D05
Rn.11088
NM_013083
HSPA5
Heat shock 70 kDa protein 5 (glucose-regulated protein, 78 kDa)
BIP/GRP78

D06
Rn.37805
NM_001011901
HSPH1
Heat shock 105 kDa/110 kDa protein 1
DKFZp686M05240/HSP105

D07
Rn.107325
NM_001106599
HTRA2
HtrA serine peptidase 2
OMI/PARK13

D08
Rn.163330
NM_001107321
HTRA4
HtrA serine peptidase 4
FLJ90724

D09
Rn.772
NM_022392
INSIG1
Insulin-induced gene 1
CL-6

D10
Rn.16736
NM_178091
INSIG2
Insulin-induced gene 2
MGC26273

D11
Rn.9911
NM_012806
MAPK10
Mitogen-activated protein kinase 10
JNK3/JNK3A

D12
Rn.4090
XM_001056513
MAPK8
Mitogen-activated protein kinase 8
JNK/JNK1

E01
Rn.9910
NM_017322
MAPK9
Mitogen-activated protein kinase 9
JNK-55/JNK2

E02
Rn.2362
NM_053569
MBTPS1
Membrane-bound transcription factor peptidase, site 1
PCSK8/S1P

E03
Rn.212224
NM_001035007
MBTPS2
Membrane-bound transcription factor peptidase, site 2
S2P

E04
Rn.144645
NM_080577
NPLOC4
Nuclear protein localization 4 homolog (S. cerevisiae)
NPL4

E05
Rn.1492
NM_053463
NUCB1
Nucleobindin 1
DKFZp686A15286/NUC

E06
Rn.1579
NM_001007265
OS9
Osteosarcoma amplified 9, endoplasmic reticulum associated protein
OS-9

E07
Rn.11527
NM_017319
PDIA3
Protein disulfide isomerase family A, member 3
ER60/ERp57

E08
Rn.7627
NM_001109476
PFDN2
Prefoldin subunit 2
PFD2

E09
Rn.3401
NM_001106794
PFDN5
Prefoldin subunit 5
MM-1/MM1

E10
Rn.1463
NM_017101
PPIA
Peptidylprolyl isomerase A (cyclophilin A)
CYPA/CYPH

E11
Rn.2232
NM_133546
PPP1R15A
Protein phosphatase 1, regulatory (inhibitor) subunit 15A
GADD34

E12
Rn.104417
NM_001106806
PRKCSH
Protein kinase C substrate 80K-H
AGE-R2/G19P1

F01
Rn.209127
NM_001127545
RNF139
Ring finger protein 139
HRCA1/RCA1

F02
Rn.209127
NM_006913
RNF5
Ring finger protein 5
RING5/RMA1

F03
Rn.4224
NM_013067
RPN1
Ribophorin I
DKFZp686B16177/OST1

F04
Rn.99548
NM_001100966
SCAP
SREBF chaperone
KIAA0199

F05
Rn.98327
NM_001034129
SEC62
SEC62 homolog (S. cerevisiae)
Dtrp1/HTP1

F06
Rn.24233
NM_001107637
SEC63
SEC63 homolog (S. cerevisiae)
ERdj2/PRO2507

F07
Rn.20802
NM_177933
SEL1L
Sel-1 suppressor of lin-12-like (C. elegans)
IBD2/PRO1063

F08
Rn.4197
NM_173120
SELS
Selenoprotein S
AD-015/ADO15

F09
Rn.2119
NM_030835
SERP1
Stress-associated endoplasmic reticulum protein 1
RAMP4

F10
Rn.103851
NM_199376
SIL1
SIL1 homolog, endoplasmic reticulum chaperone (S. cerevisiae)
BAP/MSS

F11
Rn.221929
XM_001075680
SREBF1
Sterol regulatory element binding transcription factor 1
SREBP-1c/SREBP1

F12
Rn.41063
NM_001033694
SREBF2
Sterol regulatory element binding transcription factor 2
SREBP2/bHLHd2

G01
Rn.162486
NM_001100739
SYVN1
Synovial apoptosis inhibitor 1, synoviolin
HRD1

G02
Rn.7102
NM_012670
TCP1
T-complex 1
CCT-alpha/CCT1

G03
Rn.20041
NM_153303
TOR1A
Torsin family 1, member A (torsin A)
DQ2/DYT1

G04
Rn.139603
NM_001106380
UBE2G2
Ubiquitin-conjugating enzyme E2G 2 (UBC7 homolog, yeast)
UBC7

G05
Rn.106299
NM_001007655
UBE2J2
Ubiquitin-conjugating enzyme E2, J2 (UBC6 homolog, yeast)
NCUBE2/PRO2121

G06
Rn.2022
NM_001012025
UBXN4
UBX domain protein 4
UBXD2/UBXDC1

G07
Rn.11946
NM_053418
UFD1L
Ubiquitin fusion degradation 1-like (yeast)
UFD1

G08
Rn.162227
NM_133596
UGCGL1
UDP-glucose ceramide glucosyltransferase-like 1
HUGT1

G09
Rn.107678
NM_019381
BI-1
Transmembrane BAX inhibitor motif containing 6
Tmbim6

G10
Rn.11790
NM_001008301
USP14
Ubiquitin-specific peptidase 14 (tRNA-guanine transglycosylase)
TGT

G11
Rn.98891
NM_053864
VCP
Valosin-containing protein
IBMPFD/TERA

G12
Rn.101044
NM_001004210
XBP1
X-box binding protein 1
TREB5/XBP2

H01
Rn.973
NM_001007604
Rplp1
Ribosomal protein, large, P1
MGC72935

H02
Rn.47
NM_012583
Hprt
Hypoxanthine guanine phosphoribosyl transferase
Hgprtase/Hprt1

H03
Rn.92211
NM_173340
Rpl13a
Ribosomal protein L13A
Rpl13a

H04
Rn.107896
NM_017025
Ldha
Lactate dehydrogenase A
Ldh1

H05
Rn.94978
NM_031144
Actb
Actin, beta
Actx

H06
N/A
U26919
RGDC
Rat genomic DNA contamination
RGDC

H07
N/A
SA_00104
RTC
Reverse Transcription Control
RTC

H08
N/A
SA_00104
RTC
Reverse transcription control
RTC

H09
N/A
SA_00104
RTC
Reverse transcription control
RTC

H10
N/A
SA_00103
PPC
Positive PCR control
PPC

H11
N/A
SA_00103
PPC
Positive PCR control
PPC

H12
N/A
SA_00103
PPC
Positive PCR control
PPC

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).

(a)

(b)

Figure 2: Western blot and immunofluorescence detected the expression of HERP and HRD1 in the first month after the development of diabetes: (a) Western blot detection of the expression of HERP and HRD1 in the first month. The expression of HERP in the diabetic group was less than that of the control group (); Hrd1 expression was similar in both groups (). (b) Immunofluorescence detection of the expression of HERP and HRD1 in the first month. The expression of HERP in the diabetic group was less than that of the control group () Hrd1 expression was similar in both groups ().

(a)

(b)

Figure 3: Western blot and immunofluorescence detected the expression of HERP and Hrd1 in the third month after the development of diabetes: (a) Western blot detection of the expression of HERP and Hrd1 in the third month. The expression of HERP and Hrd1in the diabetic group was less than that of the control group ( and , resp.). (b) immunofluorescence detection of the expression of HERP and HRD1 in the third month. The expression of HERP and Hrd1 in the diabetic group was less than that of the control group ( and , resp.).

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).

Figure 4: Immunofluorescence detection of the expression of HERP and HRD1 in RRCECs. The expression of HERP in the high glucose group was decreased compared to the control group, . The expression of HRD1 in the high glucose group was also decreased compared to the control group, .

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 [30–33]. 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 [35–37]. 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 [39–41].

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 [42–45]. 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.

Acknowledgments

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.

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