Inhibitors of Protein Phosphatase 1 and 2A Differentially Regulate the Expression of Inducible Nitric-oxide Synthase in Rat Astrocytes and Macrophages

Kalipada Pahan,Faruk G Sheikh,Aryan M.S Namboodiri,Inderjit Singh

doi:10.1074/jbc.273.20.12219

Kalipada Pahan, Faruk G Sheikh + Show 2 more

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https://doi.org/10.1074/jbc.273.20.12219

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Abstract

Nitric oxide produced by inducible nitric-oxide synthase (iNOS) in different cells including brain cells in response to proinflammatory cytokines plays an important role in the pathophysiology of stroke and other neurodegenerative diseases. The present study underlines the importance of protein phosphatase (PP) 1 and 2A in the regulation of the differential expression of iNOS in rat primary astrocytes and macrophages. Compounds (calyculin A, microcystin, okadaic acid, and cantharidin) that inhibit PP 1 and 2A were found to stimulate the lipopolysaccharide (LPS)- and cytokine-mediated expression of iNOS and production of NO in rat primary astrocytes and C6 glial cells. However, these inhibitors inhibited the LPS- and cytokine-mediated expression of iNOS and production of NO in rat resident macrophages and RAW 264.7 cells. Similarly, okadaic acid, an inhibitor of PP 1/2A, stimulated the iNOS promoter-derived chloramphenicol acetyltransferase activity in astrocytes and inhibited the iNOS promoter-derived chloramphenicol acetyltransferase activity in macrophages, indicating that okadaic acid also differentially regulates the transcription of the iNOS gene in astrocytes and macrophages. The observed stimulation of the expression of iNOS in astrocytes and the inhibition of the expression of iNOS in macrophages with the inhibition of PP 1/2A activity clearly delineate a novel role of PP 1/2A in the differential regulation of iNOS in rat astrocytes and macrophages. Because the activation of NF-kappaB is necessary for the induction of iNOS and the expression of tumor necrosis factor (TNF)-alpha also depends on the activation of NF-kappaB, we examined the effect of okadaic acid on the LPS-mediated activation of NF-kappaB and production of TNF-alpha in rat primary astrocytes and macrophages. Interestingly, in both cell types, okadaic acid stimulated the LPS-mediated DNA binding as well as transcriptional activity of NF-kappaB and production of TNF-alpha. This study suggests that the stimulation of iNOS expression in astrocytes by inhibitors of PP 1/2A is possibly due to the stimulation of NF-kappaB activation; however, activation of NF-kappaB is not sufficient for the induction of iNOS in macrophages and that apart from NF-kappaB some other signaling pathway(s) sensitive to PP 1 and/or PP 2A is/are possibly involved in the regulation of iNOS in macrophages. This differential induction of iNOS as compared with similar activation of NF-kappaB by inhibitors of PP 1/2A indicates the involvement of different intracellular signaling events for the induction of iNOS in two cell types of the same animal species.

Highlights

Inhibitors of PP 1/2A indicates the involvement of different intracellular signaling events for the induction of inducible nitric-oxide synthase (iNOS) in two cell types of the same animal species
To understand the mechanism of stimulatory effect of inhibitors of PP 1/2A on the LPS-mediated nitrite production in astrocytes, we examined the effect of these inhibitors on the protein and mRNA level of iNOS
Consistent with the production of nitrite (Fig. 1A), Western blot analysis with antibodies against murine macrophage iNOS and Northern blot analysis for iNOS mRNA of LPS-stimulated astrocytes clearly show that inhibitors of PP 1/2A enhanced the LPS-mediated induction of iNOS protein (Fig. 1B) and mRNA (Fig. 1C)

Summary

Introduction

Inhibitors of PP 1/2A indicates the involvement of different intracellular signaling events for the induction of iNOS in two cell types of the same animal species. MEK is a dual specificity kinase whose activation requires Ser/Thr phosphorylation (19 – 21) These observations suggest that cellular regulation of this signaling pathway may utilize Ser/Thr phosphatases to modulate the phosphorylation state of critical phosphoproteins associated with the activation of NF-␬B and the induction of iNOS. Our results clearly demonstrate that calyculin A, microcystin, cantharidin, and okadaic acid, inhibitors of PP 1 and PP 2A, stimulate the LPS- and cytokine-mediated expression of iNOS and production of NO in astrocytes and C6 glial cells, whereas the same inhibitors inhibit the LPS- and cytokine-mediated expression of iNOS and production of NO in macrophages and RAW 264.7 cells This differential regulation of the induction of iNOS in astrocytes and macrophages by inhibitors of PP 1/2A suggests that different intracellular signaling events may be involved for the induction of iNOS in astrocytes and macrophages. Despite this differential regulation of the induction of iNOS in astrocytes and macrophages, inhibitors of PP 1/2A stimulate the activation of NF-␬B and the production of TNF-␣ in both astrocytes and macrophages

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