Vol. 292, Issue 1, 271-279, January 2000

Diphenyleneiodium Chloride Blocks Inflammatory Cytokine-Induced Up-Regulation of Group IIA Phospholipase A₂ in Rat Mesangial Cells¹

Glenn Dorsam, Mohinddin M. Taher, Kristoffer C. Valerie, Nancy B. Kuemmerle, James C. Chan and Richard C. Franson

Departments of Biochemistry and Molecular Biophysics (G.D., R.C.F.), Radiation Oncology (M.M.T., K.C.V.), and Pediatric Nephrology (N.B.K., J.C.C.), School of Medicine, Virginia Commonwealth University, Richmond, Virginia

Inflammatory cytokines, interleukin 1 and tumor necrosis factor-, 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-B (NF-B), suggesting a role for NF-B 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-B 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- 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-B 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-B binding to one of two NF-B consensus elements in the rat group IIA PLA₂ promoter and also was suppressed by DPI pretreatment. Antibodies to NF-B p65 (Rel A) and p50 (but not normal rabbit IgG) supershifted this retardation signal and verified the type of NF-B species as the classical p50/p65 heterodimer.