The mTOR kinase inhibitor rapamycin decreases iNOS mRNA stability in astrocytes

Lucia Lisi,Douglas L Feinstein,Cinzia Dello Russo,Pierluigi Navarra

doi:10.1186/1742-2094-8-1

Lucia Lisi, Douglas L Feinstein + Show 2 more

Open Access

https://doi.org/10.1186/1742-2094-8-1

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Abstract

BackgroundReactive astrocytes are capable of producing a variety of pro-inflammatory mediators and potentially neurotoxic compounds, including nitric oxide (NO). High amounts of NO are synthesized following up-regulation of inducible NO synthase (iNOS). The expression of iNOS is tightly regulated by complex molecular mechanisms, involving both transcriptional and post-transcriptional processes. The mammalian target of rapamycin (mTOR) kinase modulates the activity of some proteins directly involved in post-transcriptional processes of mRNA degradation. mTOR is a serine-threonine kinase that plays an evolutionarily conserved role in the regulation of cell growth, proliferation, survival, and metabolism. It is also a key regulator of intracellular processes in glial cells. However, with respect to iNOS expression, both stimulatory and inhibitory actions involving the mTOR pathway have been described. In this study the effects of mTOR inhibition on iNOS regulation were evaluated in astrocytes.MethodsPrimary cultures of rat cortical astrocytes were activated with different proinflammatory stimuli, namely a mixture of cytokines (TNFα, IFNγ, and IL-1β) or by LPS plus IFNγ. Rapamycin was used at nM concentrations to block mTOR activity and under these conditions we measured its effects on the iNOS promoter, mRNA and protein levels. Functional experiments to evaluate iNOS activity were also included.ResultsIn this experimental paradigm mTOR activation did not significantly affect astrocyte iNOS activity, but mTOR pathway was involved in the regulation of iNOS expression. Rapamycin did not display any significant effects under basal conditions, on either iNOS activity or its expression. However, the drug significantly increased iNOS mRNA levels after 4 h incubation in presence of pro-inflammatory stimuli. This stimulatory effect was transient, since no differences in either iNOS mRNA or protein levels were detected after 24 h. Interestingly, reduced levels of iNOS mRNA were detected after 48 hours, suggesting that rapamycin can modify iNOS mRNA stability. In this regard, we found that rapamycin significantly reduced the half-life of iNOS mRNA, from 4 h to 50 min when cells were co-incubated with cytokine mixture and 10 nM rapamycin. Similarly, rapamycin induced a significant up-regulation of tristetraprolin (TTP), a protein involved in the regulation of iNOS mRNA stability.ConclusionThe present findings show that mTOR controls the rate of iNOS mRNA degradation in astrocytes. Together with the marked anti-inflammatory effects that we previously observed in microglial cells, these data suggest possible beneficial effects of mTOR inhibitors in the treatment of inflammatory-based CNS pathologies.

Highlights

Reactive astrocytes are capable of producing a variety of pro-inflammatory mediators and potentially neurotoxic compounds, including nitric oxide (NO)
We recently showed that rapamycin and its analog, RAD001, reduce inducible NO synthase (iNOS) expression and activity in microglial cultures activated by proinflammatory cytokines while displaying minor effects on astrocyte iNOS [21], suggesting that mammalian target of rapamycin (mTOR) regulates glial inflammatory activation but with selective effects
Cytokine dependent mTOR activation does not significantly affect astrocyte iNOS activity Astrocytes were stimulated using a mixture of proinflammatory cytokines (TII), i.e. 10 ng/ml IL1b, 10 ng/ml tumor necrosis factor a (TNFa), 5 ng/ml interferon g (IFNg), and the activation of the mTOR pathway was examined by measurement of the phosphorylation level of mTOR at Ser2448 [21]

Summary

Introduction

Reactive astrocytes are capable of producing a variety of pro-inflammatory mediators and potentially neurotoxic compounds, including nitric oxide (NO). MTOR is a serine-threonine kinase that plays an evolutionarily conserved role in the regulation of cell growth, proliferation, survival, and metabolism. The mammalian target of rapamycin (mTOR) kinase modulates the activity of some proteins directly involved in post-transcriptional processes of mRNA degradation. The mammalian target of rapamycin (mTOR) kinase modulates the activity of some of the above mentioned RNA-BPs [12,13] mTOR is a serine-threonine kinase that plays an evolutionary conserved role in the regulation of cell growth, proliferation, survival, and metabolism, as well as of other physiological processes such as transcription, mRNA turnover and protein translation [14]. Rapamycin is a second generation immunosuppressant drug that blocks T-cell proliferation by inhibition of mTOR activity, and it is normally used to prevent transplant rejection in association with the older calcineurin inhibitors [17] mTORC1 activity is inhibited by rapamycin and its analogs, while mTORC2 is insensitive to the rapamycin inhibitory actions at least at immunosuppressive concentrations [18]

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