Functional characterization of 58-kilodalton inhibitor of protein kinase in protecting against diabetic retinopathy via the endoplasmic reticulum stress pathway.
OBJECTIVE
58-kilodalton inhibitor of protein kinase (P58(IPK)) plays an important role in preventing endoplasmic reticulum (ER) stress. It is an interferon-induced kinase that targets the eukaryotic translation initiation factor eukaryotic initiation factor 2 alpha. The aim of this study was to determine the roles of P58(IPK) in protecting against diabetic retinopathy (DR) by inhibiting ER stress-signaling mediators.


METHODS
A rat diabetic model was established by intraperitoneal injection of streptozotocin. Overexpression of P58(IPK) was achieved by intravitreal injection of purified recombinant adeno-associated virus vector (rAAV2)-P58(IPK) or transfection into rat retinal capillary endothelial cells. Retinal vascular permeability was determined by assessing the Evans Blue retinal leakage. To downregulate the P58(IPK) level in cultured rat retinal capillary endothelial cells, pGIPZ-P58(IPK) RNA interference (P58(IPK)RNAi) was introduced in these cells. Real time reverse transcription (RT)-PCR and western blot analyses were performed to evaluate the mRNA and protein levels of Core/emopamil binding protein (C/EBP) homologous protein (CHOP), vascular endothelial growth factor (VEGF), and tumor necrosis factor-α (TNF-α).


RESULTS
Retinal blood vessel leakage was significantly decreased in rAAV2-P58(IPK)-transfected diabetic rats compared with the control diabetic rats. Both mRNA and protein levels of CHOP, TNF-α, and VEGF in the retina of diabetic rats were remarkably reduced in P58(IPK)-transfected rats. In vitro study further demonstrated that overexpression of P58(IPK) downregulated the expression of CHOP, TNF-α, and VEGF under high glucose conditions, whereas introduction of P58(IPK)RNAi enhanced the expression of CHOP, TNF-α, and VEGF.


CONCLUSIONS
These results revealed the protecting role of P58(IPK) against ER stress-mediated DR in diabetic rats, suggesting that P58(IPK) may act as a DR-resistant gene during diabetes.
Department of Ophthalmology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, People's Republic of China.
Hong
H
Yang
Rong
R
Liu
Zheng
Z
Cui
Zhi-Qi
ZQ
Chen
Shu
S
Yan
Han
H
Pei
Bin
B
Li
D000818:Animals; D003921:Diabetes Mellitus, Experimental / Q000378:metabolism; D003930:Diabetic Retinopathy / Q000201:enzymology* / Q000473:pathology; D004195:Disease Models, Animal; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D050956:HSP40 Heat-Shock Proteins / Q000378:metabolism*; D008297:Male; D018384:Oxidative Stress; D011494:Protein Kinases / Q000235:genetics*; D034622:RNA Interference; D051381:Rats; D017207:Rats, Sprague-Dawley; D012171:Retinal Vessels / Q000473:pathology; D015398:Signal Transduction; D051742:Transcription Factor CHOP / Q000096:biosynthesis; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis
C497249:DDIT3 protein, human; D015852:Eukaryotic Initiation Factor-2; D050956:HSP40 Heat-Shock Proteins; D014409:Tumor Necrosis Factor-alpha; D042461:Vascular Endothelial Growth Factor A; D051742:Transcription Factor CHOP; D011494:Protein Kinases; D005947:Glucose
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