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Molecular mechanisms underlying dexamethasone inhibition of iNOS expression and activity in C6 glioma cells
Jun Shinoda1, Katherine E McLaughlin, Helen S Bell
1Department of Clinical Neurosciences, Western General Hospital, University of Edinburgh, Scotland, United Kingdom. junshino@cc.gifu-u.ac.jp
Abstract:
The synthetic glucocorticoid dexamethasone is routinely used to stabilize patients with malignant gliomas. One putative target for glucocorticoid action is inducible nitric oxide synthase (iNOS), which is produced by the tumor cells as well as the host immune cells. In this study, we characterize the stimulatory effects of lipopolysaccharide (LPS) and the cytokine, tumor necrosis factor-alpha (TNFalpha), as well as the inhibitory effect of glucocorticoids, on iNOS gene expression and activity in C6 glioma cells cultured in vitro. LPS significantly increased iNOS mRNA expression, peaking at 6 h, while nitrite formation increased with time up to 72 h. Although TNFalpha alone induced neither iNOS mRNA expression nor nitrite formation, it significantly potentiated the effect of LPS on both. iNOS activity induced by LPS with or without TNFalpha was dose-dependently inhibited by dexamethasone, reaching a maximum of approximately 83% inhibition. This was completely reversed by the addition of RU38486, an antagonist of glucocorticoid receptors (GR). Dexamethasone inhibited iNOS mRNA expression; however, the maximum inhibition obtained was only 10%. These results suggest that as for induction of iNOS activity in C6 cells in vitro, the stimulatory effect of LPS is mainly due to an action at the transcriptional level. TNFalpha does not have intrinsic inducing activity, but has potentiating effects at the transcriptional and possibly at the posttranscriptional levels in the presence of LPS. The inhibitory effect of dexamethasone is GR-mediated and is mainly due to action at the posttranscriptional level.
Insights
Dexamethasone inhibits inducible nitric oxide synthase (iNOS) activity in glioma cells, primarily at the post-transcriptional level. This action, mediated by glucocorticoid receptors (GR), is reversed by antagonists, suggesting therapeutic potential for malignant gliomas.
Area of Science:
- Neuroscience
- Molecular Biology
- Pharmacology
Background:
- Malignant gliomas are often treated with dexamethasone, a synthetic glucocorticoid.
- Inducible nitric oxide synthase (iNOS) is implicated in glioma pathogenesis and is a potential target for glucocorticoids.
- Understanding glucocorticoid effects on iNOS in glioma cells is crucial for optimizing treatment.
Purpose of the Study:
- To investigate the effects of lipopolysaccharide (LPS), tumor necrosis factor-alpha (TNFalpha), and dexamethasone on iNOS gene expression and activity in C6 glioma cells.
- To elucidate the mechanisms underlying these modulations, including transcriptional and post-transcriptional regulation.
- To determine the role of glucocorticoid receptors (GR) in dexamethasone's inhibitory action.
Main Methods:
- C6 glioma cells were cultured in vitro and treated with LPS, TNFalpha, and dexamethasone.
- iNOS mRNA expression was measured using quantitative methods.
- Nitrite formation (an indicator of iNOS activity) was assessed over time.
- The effect of RU38486, a GR antagonist, was evaluated.
Main Results:
- LPS significantly increased iNOS mRNA and nitrite formation, with TNFalpha potentiating LPS effects.
- Dexamethasone dose-dependently inhibited iNOS activity by up to 83%, an effect reversed by RU38486.
- Dexamethasone showed only minor inhibition (10%) of iNOS mRNA expression, indicating post-transcriptional regulation.
Conclusions:
- LPS primarily stimulates iNOS activity at the transcriptional level in C6 glioma cells.
- TNFalpha enhances LPS-induced iNOS expression at transcriptional and possibly post-transcriptional levels.
- Dexamethasone's inhibition of iNOS activity is GR-mediated and occurs mainly at the post-transcriptional level.