Reactive oxygen species, PKC-beta1, and PKC-zeta mediate high-glucose-induced vascular endothelial growth factor expression in mesangial cells.
Vascular endothelial growth factor (VEGF) is implicated in the development of proteinuria in diabetic nephropathy. High ambient glucose present in diabetes stimulates VEGF expression in several cell types, but the molecular mechanisms are incompletely understood. Here primary cultured rat mesangial cells served as a model to investigate the signal transduction pathways involved in high-glucose-induced VEGF expression. Exposure to high glucose (25 mM) significantly increased VEGF mRNA evaluated by real-time PCR by 3 h, VEGF cellular protein content assessed by immunoblotting or immunofluorescence within 24 h, and VEGF secretion by 24 h. High-glucose-induced VEGF expression was blocked by an antioxidant, Tempol, and antisense oligonucleotides directed against p22(phox), a NADPH oxidase subunit. Inhibition of protein kinase C (PKC)-beta(1) with the specific pharmacological inhibitor LY-333531 or inhibition of PKC-zeta with a cell permeable specific pseudosubstrate peptide also prevented enhanced VEGF expression in high glucose. Enhanced VEGF secretion in high glucose was prevented by Tempol, PKC-beta(1), or PKC-zeta inhibition. In normal glucose (5.6 mM), overexpression of p22(phox) or constitutively active PKC-zeta enhanced VEGF expression. Hypoxia inducible factor-1alpha protein was significantly increased in high glucose only by 24 h, suggesting a possible contribution to high-glucose-stimulated VEGF expression at later time points. Thus reactive oxygen species generated by NADPH oxidase, and both PKC-beta(1) and -zeta, play important roles in high-glucose-stimulated VEGF expression and secretion by mesangial cells.
Xia
Ling
L
University Health Network, University of Toronto, Toronto, ON, Canada.
Wang
Hong
H
Munk
Snezana
S
Frecker
Helena
H
Goldberg
Howard J
HJ
Fantus
I George
IG
Whiteside
Catharine I
CI
D000818:Animals; D003497:Cyclic N-Oxides / Q000494:pharmacology; D003928:Diabetic Nephropathies / Q000201:enzymology / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D005947:Glucose / Q000008:administration & dosage* / Q000378:metabolism; D007211:Indoles / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D050527:Mesangial Cells / Q000378:metabolism*; D018832:Molecular Chaperones / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology; D020319:Oligodeoxyribonucleotides, Antisense / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D064546:Protein Kinase C beta; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D013113:Spin Labels; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis* / Q000235:genetics; D000099221:Protein Kinase C zeta
D003497:Cyclic N-Oxides; D005947:Glucose; D007211:Indoles; D008301:Maleimides; D018832:Molecular Chaperones; D019255:NADPH Oxidases; D020319:Oligodeoxyribonucleotides, Antisense; D011493:Protein Kinase C; D064546:Protein Kinase C beta; D047428:Protein Kinase Inhibitors; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D013113:Spin Labels; D042461:Vascular Endothelial Growth Factor A; C490392:Prkcz protein, rat; D000099221:Protein Kinase C zeta; C467498:vascular endothelial growth factor A, rat; C099154:ruboxistaurin; C515768:Cyba protein, rat; C001803:tempol