Inhibitory and Stimulatory Effects of Lactacystin on Expression of Nitric Oxide Synthase Type 2 in Brain Glial Cells: THE ROLE OF IκB-β

Mariusz Stasiolek,Vitaliy Gavrilyuk,Anthony Sharp,Peter Horvath,Kris Selmaj,Douglas L Feinstein

doi:10.1074/jbc.m910284199

Mariusz Stasiolek, Vitaliy Gavrilyuk + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m910284199

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Abstract

Expression of inflammatory nitric oxide synthase (NOS2) is mediated by transcription factor NFkappaB. By using the specific proteasome inhibitor lactacystin to examine IkappaB degradation, we observed a paradoxical increase in lipopolysaccharide- and cytokine-dependent NOS2 expression at low concentrations or when lactacystin was added subsequent to cytokines. Lactacystin reduced the initial accumulation of NOS2 mRNA but reduced its subsequent decrease. Lactacystin increased NOS2 promoter activation after 24 h, but not after 4 h, and similarly prevented initial NFkappaB activation and at later times caused NFkappaB reactivation. Lactacystin reduced initial degradation of IkappaB-alpha and IkappaB-beta, however, at later times selectively increased IkappaB-beta, which was predominantly non-phosphorylated. Expression of full-length rat IkappaB-beta, but not a carboxyl-terminal truncated form, inhibited NOS2 induction and potentiation by lactacystin. Lactacystin increased IkappaB-beta expression in the absence of NOS2 inducers, as well as expression of heat shock protein 70, and the heat shock response due to hyperthermia increased IkappaB-beta expression. These results suggest that IkappaB-beta contributes to persistent NFkappaB activation and NOS2 expression in glial cells, that IkappaB-beta is a stress protein inducible by hyperthermia or proteasome inhibitors, and that delayed addition of proteasome inhibitors can have stimulatory rather than inhibitory actions.

Highlights

Expression of inflammatory nitric oxide synthase (NOS2) is mediated by transcription factor NF␬B
These results confirm that NOS2-dependent nitrite production in astrocytes requires proteasome activation, as previously concluded using less specific proteasome inhibitors, and suggest that low doses of proteasome inhibitors potentiate, rather than inhibit, NOS2 expression
We have found that incubation with low doses of the highly specific proteasome inhibitor lactacystin led to a paradoxical increase, rather than decrease, in NOS2 expression

Summary

Introduction

Expression of inflammatory nitric oxide synthase (NOS2) is mediated by transcription factor NF␬B. Our results suggest that sustained NF␬B activation, mediated by increases in I␬B-␤ expression, is important for glial cell NOS2 expression, which may be potentiated by proteasome inhibitors due to induction of a HSR.

Results

Conclusion