Response of VEGF expression to amino acid deprivation and inducers of endoplasmic reticulum stress.
OBJECTIVE
Vascular endothelial growth factor (VEGF) plays an important role in initiation of the angiogenesis that leads to proliferative retinopathy. Several environmental conditions and chemical agents that influence the expression of VEGF can also cause endoplasmic reticulum (ER) stress. The hypothesis for the current study was that expression of VEGF is responsive to conditions that cause ER stress, including amino acid deprivation.


METHODS
Confluent cultures of a human retinal pigmented epithelial cell line (ARPE-19) were deprived of amino acids or treated with chemical inducers of ER stress. Treatment with cobalt was used to mimic hypoxia-induced expression of VEGF. Northern blot analysis was used to measure intracellular VEGF mRNA, and ELISA was used to measure secreted VEGF protein. Glucose-regulated protein 78 (GRP78) mRNA levels were compared with those of VEGF. Glyceraldehyde-phosphate dehydrogenase (GAPDH) mRNA was used as a control.


RESULTS
Conditions and chemical agents known to activate ER stress response (ERSR) pathways also induced the expression of VEGF. Deprivation of amino acids in the culture medium increased VEGF mRNA expression by 1.3- to 6-fold. Glucose deprivation or treatment of ARPE-19 cells with tunicamycin, brefeldin A, the calcium ionophore A23187, or thapsigargin increased the expression of VEGF mRNA in these cells by 8- to 10-fold. Expression of GRP78 mRNA was well correlated with that of VEGF mRNA under all conditions. These treatments also increased the secretion of VEGF protein by up to twofold. The increase in VEGF mRNA level in response to glutamine deprivation was rapid (greater than 10-fold) and was observed in a physiologically relevant range of glutamine concentrations. The half-life of VEGF mRNA was increased 2.5-fold by glutamine starvation.


CONCLUSIONS
These results indicate that VEGF is an ER stress-responsive gene and suggest that cells can respond to nutrient deprivation by increasing VEGF expression through both transcriptional and posttranscriptional mechanisms.
Abcouwer
Steve F
SF
Department of Biochemistry and Molecular Biology, University of New Mexico, School of Medicine, Albuquerque, New Mexico 87131, USA. sabcouwer@salud.unm.edu
Marjon
Philip L
PL
Loper
Robyn K
RK
Vander Jagt
David L
DL
D000596:Amino Acids / Q000172:deficiency*; D015152:Blotting, Northern; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D015687:Cell Hypoxia / Q000502:physiology*; D002478:Cells, Cultured; D004721:Endoplasmic Reticulum / Q000502:physiology*; D000091342:Endoplasmic Reticulum Chaperone BiP; D016228:Endothelial Growth Factors / Q000235:genetics / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics / Q000378:metabolism; D006360:Heat-Shock Proteins; D006801:Humans; D008222:Lymphokines / Q000235:genetics / Q000378:metabolism*; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism; D010857:Pigment Epithelium of Eye / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D013312:Stress, Physiological / Q000378:metabolism*; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D000596:Amino Acids; D002352:Carrier Proteins; D000091342:Endoplasmic Reticulum Chaperone BiP; D016228:Endothelial Growth Factors; C000717247:HSPA5 protein, human; D006360:Heat-Shock Proteins; D008222:Lymphokines; D018832:Molecular Chaperones; D012333:RNA, Messenger; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases