Vascular endothelial growth factor expression and secretion by retinal pigment epithelial cells in high glucose and hypoxia is protein kinase C-dependent.
Retinal pigment epithelial (RPE) cells express vascular endothelial growth factor (VEGF) in response to high glucose or hypoxia. We hypothesised that VEGF expression and secretion by RPE cells in high glucose and hypoxia are regulated by protein kinase C (PKC). Primary cultured RPE cells from Sprague-Dawley rats were growth-arrested for 48 hr in 0.5% FBS in 5.6 or 30 mm D-glucose. Cells were exposed to hypoxic conditions (<1% O(2), 5% CO(2)) for the last 15-18 hr of growth-arrest. PKC -alpha, -beta(1), -delta, -epsilon, and -zeta were expressed by RPE cells and exposure to high glucose for 48 hr had no effect on expression as demonstrated by Western immunoblotting. High glucose, hypoxia or VEGF stimulated translocation of a number of the PKC isozymes to the membrane or particulate fractions implying activation. In response to high glucose or acute phorbol myristate acetate (PMA) stimulation, VEGF mRNA analysed by RT-PCR was increased. Intracellular VEGF protein identified by immunoblotting and confocal immunofluorescence imaging was significantly increased by high glucose, hypoxia or acute PMA stimulation. Calphostin C or a specific inhibitor of PKC-zeta prevented high glucose-stimulated VEGF expression in high glucose. VEGF secretion, as measured by ELISA in the culture medium, was enhanced in hypoxia but not in high glucose. Following exposure of RPE cells to PMA for 24 hr, PKC-delta was significantly down regulated, whereas PKC-alpha, -beta, -epsilon and -zeta remained unchanged. Secretion of VEGF in normal or high glucose, or hypoxia was significantly reduced following treatment with PMA for 24 hr but not with the PKC-zeta inhibitor. We conclude that in high glucose and hypoxia PKC isozymes are activated and are necessary for VEGF expression. Secretion of VEGF is enhanced in hypoxia and appears to be regulated by PKC-delta. RPE cells may contribute to the pathogenesis of retinopathy caused by high glucose and hypoxia through the expression and secretion of VEGF that are regulated by PKC isozymes.
Young
Tara A
TA
Institute of Medical Science, University of Toronto, 1 King's College Circle, Medical Sciences Bldg, Room 2113, Toronto, ON, Canada M5S 1A8.
Wang
Hong
H
Munk
Snezana
S
Hammoudi
Dena S
DS
Young
Diana S
DS
Mandelcorn
Mark S
MS
Whiteside
Catharine I
CI
D000818:Animals; D015153:Blotting, Western / Q000379:methods; D015687:Cell Hypoxia; D002478:Cells, Cultured; D004789:Enzyme Activation; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005947:Glucose / Q000494:pharmacology*; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008297:Male; D018613:Microscopy, Confocal; D009281:Naphthalenes / Q000494:pharmacology; D010857:Pigment Epithelium of Eye / Q000737:chemistry* / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051745:Protein Kinase C-delta; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis* / Q000378:metabolism
D007527:Isoenzymes; D009281:Naphthalenes; D012333:RNA, Messenger; D042461:Vascular Endothelial Growth Factor A; C497260:Prkcd protein, rat; C087857:protein kinase C gamma; D011493:Protein Kinase C; D051745:Protein Kinase C-delta; C058819:calphostin C; D005947:Glucose; D013755:Tetradecanoylphorbol Acetate