High glucose activates PKC-zeta and NADPH oxidase through autocrine TGF-beta1 signaling in mesangial cells.
Conversion of normally quiescent mesangial cells into extracellular matrix-overproducing myofibroblasts in response to high ambient glucose and transforming growth factor (TGF)-beta(1) is central to the pathogenesis of diabetic nephropathy. Previously, we reported that mesangial cells respond to high glucose by generating reactive oxygen species (ROS) from NADPH oxidase dependent on protein kinase C (PKC) -zeta activation. We investigated the role of TGF-beta(1) in this action of high glucose on primary rat mesangial cells within 1-48 h. Both high glucose and exogenous TGF-beta(1) stimulated PKC-zeta kinase activity, as measured by an immune complex kinase assay and immunofluorescence confocal cellular imaging. In high glucose, Akt Ser473 phosphorylation appeared within 1 h and Smad2/3 nuclear translocation was prevented with neutralizing TGF-beta(1) antibodies. Neutralizing TGF-beta(1) antibodies, or a TGF-beta receptor kinase inhibitor (LY364947), or a phosphatidylinositol 3,4,5-trisphosphate (PI3) kinase inhibitor (wortmannin), prevented PKC-zeta activation by high glucose. TGF-beta(1) also stimulated cellular membrane translocation of PKC-alpha, -beta(1), -delta, and -epsilon, similar to high glucose. High glucose and TGF-beta(1) enhanced ROS generation by mesangial cell NADPH oxidase, as detected by 2,7-dichlorofluorescein immunofluorescence. This response was abrogated by neutralizing TGF-beta(1) antibodies, LY364947, or a specific PKC-zeta pseudosubstrate peptide inhibitor. Expression of constitutively active PKC-zeta in normal glucose caused upregulation of p22(phox), a likely mechanism of NADPH oxidase activation. We conclude that very early responses of mesangial cells to high glucose include autocrine TGF-beta(1) stimulation of PKC isozymes including PI3 kinase activation of PKC-zeta and consequent generation of ROS by NADPH oxidase.
Xia
Ling
L
University Health Network, Univ. of Toronto, Toronto, ON, Canada M5S 1A8.
Wang
Hong
H
Munk
Snezana
S
Kwan
Janice
J
Goldberg
Howard J
HJ
Fantus
I George
IG
Whiteside
Catharine I
CI
D000818:Animals; D017931:DNA Primers; D004789:Enzyme Activation; D005920:Glomerular Mesangium / Q000502:physiology* / Q000503:physiopathology; D006943:Hyperglycemia / Q000503:physiopathology*; D007700:Kinetics; D019255:NADPH Oxidases / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism; D053773:Transforming Growth Factor beta1 / Q000502:physiology*; D000099221:Protein Kinase C zeta
D017931:DNA Primers; D019255:NADPH Oxidases; D011493:Protein Kinase C; D017382:Reactive Oxygen Species; D053773:Transforming Growth Factor beta1; D000099221:Protein Kinase C zeta