Reactive oxygen species amplify protein kinase C signaling in high glucose-induced fibronectin expression by human peritoneal mesothelial cells

Abstract

We previously demonstrated that high glucose up-regulates fibronectin mRNA and protein expression by human peritoneal mesothelial cells (HPMC) through activation of protein kinase C (PKC). PKC is known to induce cellular reactive oxygen species (ROS) and PKC-dependent activation of the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase has recently been shown to be responsible, in part, for increased oxidative stress in diabetes. On the other hand, high glucose-induced mitochondrial overproduction of superoxide anion was found to activate PKC. We, therefore, hypothesized that high glucose-induced activation of PKC in HPMC may increase cellular ROS and ROS, in turn, may activate PKC and thus provide signal amplification in high glucose-induced fibronectin up-regulation in HPMC. The role of ROS in high glucose- and PKC-induced fibronectin expression was examined by quantification of cellular ROS after stimulation with high glucose and phorbol 12-myristate 13-acetate (PMA), by the effect of hydrogen peroxide (H(2)O(2)) and PMA on fibronectin expression, and finally by inhibition of ROS and PKC. The source of cellular ROS was further examined by inhibition of NADPH oxidase and mitochondrial metabolism. D-glucose increased dichlorofluorescein (DCF)-sensitive cellular ROS in HPMC in a dose-dependent manner. l-glucose did not induce ROS generation and cytochalasin B completely blocked high glucose-induced ROS generation, suggesting that glucose uptake, but not media hyperosmolality, is required in ROS generation in HPMC. PMA increased cellular ROS and fibronectin secretion. A single dose of H(2)O(2) or H(2)O(2) continuously generated by glucose oxidase up-regulated fibronectin expression [corrected]. Antioxidants trolox and catalase inhibited high glucose- and PMA-induced fibronectin mRNA and protein expression. Inhibition of PKC inhibited high glucose-and H(2)O(2)-induced fibronectin secretion. NADPH oxidase inhibitors (diphenyleneiodinium and apocynin) and an inhibitor of mitochondrial electron transport chain subunit I (rotenone) all effectively inhibited high glucose-induced cellular ROS generation and fibronectin secretion. The present data demonstrate that high glucose increases cellular ROS in HPMC through activation of PKC, NADPH oxidase, and mitochondrial metabolism and that ROS, thus generated, up-regulate fibronectin expression by HPMC. ROS are not only downstream but also upstream signaling molecules to PKC and provide signal amplification in high glucose-induced fibronectin expression by HPMC. The present data imply that cellular ROS may be potential therapeutic targets in progressive accumulation of extracellular matrix in the peritoneal tissue of long-term peritoneal dialysis patients using high glucose-containing peritoneal dialysis solutions.