In high glucose protein kinase C-zeta activation is required for mesangial cell generation of reactive oxygen species.
BACKGROUND
We postulated that in mesangial cells exposed to high glucose, protein kinase C-zeta (PKC-zeta) is necessary for the generation of reactive oxygen species (ROS) by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and that the requirement of PKC-zeta for filamentous (F)-actin disassembly may involve ROS. To identify signaling mechanisms relevant to PKC-zeta activation and ROS generation, including phosphoinositide 3 kinase (PI3 kinase), we examined mesangial cell stimulation with platelet-derived growth factor (PDGF).


METHODS
In primary rat mesangial cells cultured in 5.6 mmol/L or 30 mmol/L d-glucose, PKC-zeta expression was identified with immunoblotting and activity was analyzed in cell membrane immunoprecipitates and by confocal immunofluorescence imaging. ROS generation was measured by dichlorofluorescein fluorescence using confocal microscopy and was inhibited by transfection of antisense against NADPH subunits p22(phox) or p47(phox) or with Tempol. F-actin disassembly was observed by dual-channel confocal fluorescence imaging. PI3 kinase activity was detected by immunoblotting of phosphorylated Akt.


RESULTS
In high glucose, generation of NADPH oxidase-dependent ROS was dependent on PKC-zeta. Conversely, sustained PKC-zeta activity was dependent on ROS generation, suggesting a positive feedback. PKC-zeta-dependent F-actin disassembly in high glucose required ROS generation. PDGF stimulated NADPH oxidase generation of ROS through a PKC-zeta mechanism that was independent of Akt phosphorylation and remained unchanged in high glucose.


CONCLUSIONS
In high glucose, mesangial cell PKC-zeta is required for ROS generation from NADPH oxidase similar to PDGF stimulation of PKC-zeta-dependent ROS generation through a pathway independent of PI3 kinase. F-actin disassembly in high glucose also requires ROS. A positive feedback loop occurs between ROS and the activation of PKC-zeta in high glucose.
Kwan
Janice
J
Banting and Best Diabetes Centre, University of Toronto, Toronto, Canada.
Wang
Hong
H
Munk
Snezana
S
Xia
Ling
L
Goldberg
Howard J
HJ
Whiteside
Catharine I
CI
D000199:Actins / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D005947:Glucose / Q000494:pharmacology*; D008297:Male; D026901:Membrane Transport Proteins / Q000235:genetics; D050527:Mesangial Cells / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism; D016376:Oligonucleotides, Antisense; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D000099221:Protein Kinase C zeta
D000199:Actins; D005947:Glucose; D026901:Membrane Transport Proteins; D019255:NADPH Oxidases; D016376:Oligonucleotides, Antisense; D019869:Phosphatidylinositol 3-Kinases; D010750:Phosphoproteins; D010982:Platelet-Derived Growth Factor; D011493:Protein Kinase C; D017382:Reactive Oxygen Species; D000099221:Protein Kinase C zeta; C086986:CYBA protein, human; C060505:neutrophil cytosolic factor 1