High glucose activates PKC-ζ and NADPH oxidase through autocrine TGF-β1 signaling in mesangial cells

Abstract

Conversion of normally quiescent mesangial cells into extracellular matrix-overproducing myofibroblasts in response to high ambient glucose and transforming growth factor (TGF)-β1 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) -ζ activation. We investigated the role of TGF-β1 in this action of high glucose on primary rat mesangial cells within 1–48 h. Both high glucose and exogenous TGF-β1 stimulated PKC-ζ 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-β1 antibodies. Neutralizing TGF-β1 antibodies, or a TGF-β receptor kinase inhibitor (LY364947), or a phosphatidylinositol 3,4,5-trisphosphate (PI3) kinase inhibitor (wortmannin), prevented PKC-ζ activation by high glucose. TGF-β1 also stimulated cellular membrane translocation of PKC-α, -β1, -δ, and -ε, similar to high glucose. High glucose and TGF-β1 enhanced ROS generation by mesangial cell NADPH oxidase, as detected by 2,7-dichlorofluorescein immunofluorescence. This response was abrogated by neutralizing TGF-β1 antibodies, LY364947, or a specific PKC-ζ pseudosubstrate peptide inhibitor. Expression of constitutively active PKC-ζ in normal glucose caused upregulation of p22phox, a likely mechanism of NADPH oxidase activation. We conclude that very early responses of mesangial cells to high glucose include autocrine TGF-β1 stimulation of PKC isozymes including PI3 kinase activation of PKC-ζ and consequent generation of ROS by NADPH oxidase.