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Published on: August 9, 2019
Differential effects of LY294002 and wortmannin on inducible nitric oxide synthase expression in glomerular mesangial
Kuen-Daw Tsai1, Wen-Wei Chang, Chia-Ching Lin
1Department of Internal Medicine, China Medical University Beigang Hospital, 123 Sinde Road, Beigang Township, Yunlin County 65152, Taiwan, ROC.
Abstract:
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, 5μM LY294002 showed a more-significant inhibitory effect on LPS/IFN-γ-induced NO production, and iNOS protein and gene expressions than did 1μM 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.
Insights
The study found LY294002 more effectively inhibits nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in kidney cells than wortmannin. LY294002
Area of Science:
- Nephrology
- Molecular Biology
- Biochemistry
Background:
- Nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) plays a role in glomerulonephritis pathophysiology.
- Targeting iNOS-mediated NO production is a strategy to mitigate NO-induced cytotoxicity.
Purpose of the Study:
- To investigate the differential effects of phosphatidylinositol 3-kinase (PI3K) inhibitors LY294002 and wortmannin on NO production in glomerular mesangial cells.
- To elucidate the mechanisms underlying the differential inhibition of iNOS expression by these PI3K inhibitors.
Main Methods:
- Utilized MES-13 glomerular mesangial cells stimulated with lipopolysaccharide (LPS) and interferon-gamma (IFN-γ).
- Assessed NO production, iNOS protein and gene expression, Akt and STAT-1α phosphorylation, NF-κB activity, and iNOS protein degradation.
- Employed PI3K inhibitors LY294002 and wortmannin, and small interfering RNA (siRNA) for PI3K suppression.
Main Results:
- 5μM LY294002 significantly inhibited LPS/IFN-γ-induced NO production, iNOS expression, and NF-κB activity more than 1μM wortmannin.
- Wortmannin reduced Akt phosphorylation, while LY294002 did not; neither affected IFN-γ-induced STAT-1α phosphorylation.
- LY294002 accelerated iNOS protein degradation and reduced the iNOS dimer/monomer ratio, an effect not observed with wortmannin.
- PI3K suppression via siRNA did not impact LPS/IFN-γ-stimulated NO production or iNOS expression.
Conclusions:
- LY294002 demonstrates superior inhibition of NO production and iNOS expression compared to wortmannin in MES-13 cells.
- LY294002's inhibitory effects on NF-κB activation and iNOS protein degradation appear to be independent of PI3K inhibition.
- These findings suggest distinct mechanisms of action for LY294002 and wortmannin in regulating iNOS in the context of glomerulonephritis.
