PT  - JOURNAL ARTICLE
AU  - Glenn Dorsam
AU  - Mohinddin M. Taher
AU  - Kristoffer C. Valerie
AU  - Nancy B. Kuemmerle
AU  - James C. Chan
AU  - Richard C. Franson
TI  - Diphenyleneiodium Chloride Blocks Inflammatory Cytokine-Induced Up-Regulation of Group IIA Phospholipase A<sub>2</sub> in Rat Mesangial Cells
DP  - 2000 Jan 01
TA  - Journal of Pharmacology and Experimental Therapeutics
PG  - 271--279
VI  - 292
IP  - 1
4099  - http://jpet.aspetjournals.org/content/292/1/271.short
4100  - http://jpet.aspetjournals.org/content/292/1/271.full
SO  - J Pharmacol Exp Ther2000 Jan 01; 292
AB  - Inflammatory cytokines, interleukin 1β and tumor necrosis factor-α, potently stimulate rat mesangial cells to express and secrete group IIA phospholipase A2 (PLA2). Cytokine-induced up-regulation of PLA2 has been blocked by inhibitors (antioxidants) of the transcription factor, nuclear factor-κB (NF-κB), suggesting a role for NF-κB in the regulation of group IIA PLA2 expression. Reactive oxygen species such as H2O2, which are elevated in mesangial cells after cytokine activation, can mimic cytokine-induced NF-κB activation. However, the source of reactive oxygen species generation in mesangial cells, produced by cytokine stimulation, has yet to be clarified. Recently, tumor necrosis factor-α has been demonstrated to increase superoxide radical generation in mesangial cells. Therefore, we hypothesized that a selective NADPH oxidase inhibitor, diphenyleneiodium chloride (DPI), could block cytokine-induced group IIA PLA2 up-regulation by attenuating NF-κB binding. To test this hypothesis, we isolated rat mesangial cells and characterized them by ultrastructural and immunochemical methods. This homogeneous mesangial cell population was responsive to cytokine as evidenced by an increase in steady-state levels of group IIA PLA2 mRNA and extracellular enzymatic activity over time. DPI (0.02–20 μM), added 90 min before cytokine activation, inhibited both group IIA PLA2 mRNA and enzymatic activity in a concentration-dependent manner. By electrophoretic mobility shift analysis, cytokine activation also increased specific NF-κB binding to one of two NF-κB consensus elements in the rat group IIA PLA2 promoter and also was suppressed by DPI pretreatment. Antibodies to NF-κB p65 (Rel A) and p50 (but not normal rabbit IgG) supershifted this retardation signal and verified the type of NF-κB species as the classical p50/p65 heterodimer. The American Society for Pharmacology and Experimental Therapeutics