P58(IPK) inhibition of endoplasmic reticulum stress in human retinal capillary endothelial cells in vitro.
OBJECTIVE
The goal of this research was to determine if P58(IPK), a member of the Hsp40 family that inhibits eukaryotic initiation factor 2alpha (eIF2alpha), inhibits endoplasmic reticulum (ER) stress and leads to downregulated expression of vascular endothelial growth factor (VEGF) and decreased apoptosis in human retinal capillary endothelial cells (HRCECs).


METHODS
Recombinant vectors were constructed using P58 in adeno-associated virus type 2 (rAAV2-P58 (IPK)) and P58 RNA in the plasmid pGIPZ (pGIPZ-P58(IPK)). The four experimental groups were: (1) non-transfected/non ER stressed control; (2) non-transfected/ER stressed; (3) rAAV2-P58(IPK)-transfected/ER stressed; and (4) pGIPZ- P58(IPK) RNAi transfected/ER stressed. ER stress was induced by treating cells with tunicamycin. Expression of P58(IPK) was determined in transfected cells. Expressions of the following factors were assessed: vascular endothelial growth factor (VEGF), C/EBP homologous protein (CHOP), activating transcription factor 4 (ATF4), and glucose-regulated protein 78 (GRP78). Apoptosis levels were also determined.


RESULTS
Significantly increased expression of P58(IPK) was detected in cells transfected with rAAV2-P58(IPK) (0.63+/-0.02) as compared to those transfected with pGIPZ-P58(IPK) RNAi (0.23+/-0.01). P58(IPK) expression was not different between the control transfected cells (rAAV2-GFP and pGIPZ-GFP). Following ER stress, expression levels of ATF-4, GRP78, CHOP, and VEGF in cells overexpressing P58(IPK) were not different from those in unstressed control cells. This inhibitory effect of P58(IPK) on the expression of ER stress-related factors was suppressed in cells transfected with pGIPZ-P58(IPK) RNAi. Apoptosis was significantly increased in cells transfected with pGIPZ-P58(IPK) RNAi but not with rAAV2-P58(IPK).


CONCLUSIONS
The study demonstrates that P58(IPK) inhibits ER stress and plays an important role in maintaining balance and stability of the ER in HRCECs.
Li
Bin
B
Department of Ophthalmology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, People's Republic of China. scotopsin@yahoo.com.cn
Li
Dong
D
Li
Gui-Gang
GG
Wang
Hao-Wen
HW
Yu
Ai-Xia
AX
D051701:Activating Transcription Factor 4 / Q000235:genetics / Q000378:metabolism; D000328:Adult; D017209:Apoptosis; D015153:Blotting, Western; D022762:CCAAT-Enhancer-Binding Proteins / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D004721:Endoplasmic Reticulum / Q000473:pathology*; D000091342:Endoplasmic Reticulum Chaperone BiP; D042783:Endothelial Cells / Q000473:pathology*; D005786:Gene Expression Regulation; D050956:HSP40 Heat-Shock Proteins / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism; D012160:Retina / Q000166:cytology*; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism
D022762:CCAAT-Enhancer-Binding Proteins; C494651:DNAJC3 protein, human; D000091342:Endoplasmic Reticulum Chaperone BiP; D050956:HSP40 Heat-Shock Proteins; C000717247:HSPA5 protein, human; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D051701:Activating Transcription Factor 4