Oxidized phospholipids regulate expression of ATF4 and VEGF in endothelial cells via NRF2-dependent mechanism: novel point of convergence between electrophilic and unfolded protein stress pathways.
OBJECTIVE
The ATF4 arm of the unfolded protein response is increasingly recognized for its relevance to pathology, and in particular to angiogenic reactions. Oxidized phospholipids (OxPLs), known to accumulate in atherosclerotic vessels, were shown to upregulate vascular endothelial growth factor (VEGF) and induce angiogenesis via an ATF4-dependent mechanism. In this study, we analyzed the mechanism of ATF4 upregulation by OxPLs and more specifically the involvement of NRF2, the major transcriptional mediator of electrophilic stress response.


RESULTS
Using reverse transcription/real-time polymerase chain reaction and Western blotting, we found that OxPLs induced upregulation of ATF4 mRNA and protein in several types of endothelial cells and that these effects were suppressed by short interfering RNA (siRNA) against NRF2. Electrophilic (iso)prostaglandins and oxidized low-density lipoprotein, similarly to OxPLs, elevated ATF4 mRNA levels in an NRF2-dependent mode. Chromatin immunoprecipitation revealed OxPL-dependent binding of NRF2 to a putative antioxidant response element site in the ATF4 gene promoter. Knockdown of NRF2 inhibited OxPL-induced elevation of VEGF mRNA and endothelial cell sprout formation.


CONCLUSIONS
Our data characterize NRF2 as a positive regulator of ATF4 and identify a novel cross-talk between electrophilic and unfolded protein responses, which may play a role in stress-induced angiogenesis.
Afonyushkin
Taras
T
Department of Vascular Biology and Thrombosis Research, Center for Biomolecular Medicine and Pharmacology, Medical University of Vienna, Schwarzspanierstrasse 17, 1090 Vienna, Austria.
Oskolkova
Olga V
OV
Philippova
Maria
M
Resink
Therese J
TJ
Erne
Paul
P
Binder
Bernd R
BR
Bochkov
Valery N
VN
D051701:Activating Transcription Factor 4 / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D015153:Blotting, Western; D002478:Cells, Cultured; D047369:Chromatin Immunoprecipitation; D042783:Endothelial Cells / Q000378:metabolism*; D006801:Humans; D008077:Lipoproteins, LDL / Q000378:metabolism; D051267:NF-E2-Related Factor 2 / Q000235:genetics / Q000378:metabolism*; D018919:Neovascularization, Physiologic; D010084:Oxidation-Reduction; D010743:Phospholipids / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011453:Prostaglandins / Q000378:metabolism; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013312:Stress, Physiological; D013997:Time Factors; D015533:Transcriptional Activation; D056811:Unfolded Protein Response; D015854:Up-Regulation; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*
C497109:ATF4 protein, human; D008077:Lipoproteins, LDL; D051267:NF-E2-Related Factor 2; C495635:NFE2L2 protein, human; D010743:Phospholipids; D011453:Prostaglandins; D012333:RNA, Messenger; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; C099252:oxidized low density lipoprotein; D051701:Activating Transcription Factor 4