Differential effects of LY294002 and wortmannin on inducible nitric oxide synthase expression in glomerular mesangial cells

Nitric oxide (NO) that is produced by inducible nitric oxide synthase (iNOS) is associated with the pathophysiology of glomerulonephritis. Numerous studies have focused on the regulation of NO production by iNOS to reduce NO-mediated cytotoxicity. In the present study, we demonstrated the differential effects of two phosphatidylinositol 3-kinase (PI3K) inhibitors, LY294002 and wortmannin, on lipopolysaccharide- (LPS) and interferon (IFN)-γ-induced NO production in a glomerular mesangial cell line, MES-13 cells. At dosages without affecting cell viability of MES-13 cells, 5 μM LY294002 showed a more-significant inhibitory effect on LPS/IFN-γ-induced NO production, and iNOS protein and gene expressions than did 1 μM wortmannin. Akt phosphorylation in MES-13 cells declined upon the addition of wortmannin, but not upon treatment with LY294002. Suppression of PI3K expression by small interfering (si)RNA exhibited no effect on LPS/IFN-γ-stimulated NO production or iNOS protein expression in MES-13 cells. Neither LY294002 nor wortmannin reduced IFN-γ-induced STAT-1α phosphorylation. LY294002 exhibited a more-significant inhibitory effect on NF-κB luciferase activities than wortmannin in LPS/IFN-γ-stimulated MES-13 cells. Moreover, LY294002, but not wortmannin, accelerated iNOS protein degradation and reduced the iNOS dimer/monomer ratio in MES-13 cells. Although both LY294002 and wortmannin are known as PI3K inhibitors, their differential effects on iNOS expression in MES-13 cells indicate that the effects of LY294002 on inhibiting NF-κB activation and accelerating iNOS protein degradation are through a mechanism independent of PI3K.

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► LY294002 inhibited NO production and iNOS expression in MES-13 cells.
► PI3K siRNA showed no effect on NO production and iNOS expression in MES-13 cells.
► The effect of LY294002 on NO production in MES-13 cells is independent of PI3K.
► LY294002, but not wortmannin accelerated iNOS protein degradation in MES-13 cells.
► LY294002, but not wortmannin reduced iNOS dimer/monomer ratio in MES-13 cells.