Melatonin inhibits glucocorticoid receptor nuclear translocation in mouse thymocytes

PRESMAN, D.M.; HOIJMAN, E.; CEBALLOS, N.R.; GALIGNIANA, M.D.; PECCI, A

boston, USA

Congreso; Endo2006-Endocrine Society’s 88th Annual Meeting; 2006

Endocrine society

The antiapoptotic activity of melatonin (MEL) was previously described in several systems such as immune cells, cerebellar neurons and rat brain astrocytes. Our group has previously demonstrated that in the thymus MEL inhibits the release of cytochrome C from mitochondria and the dexamethasone (DEX)-dependent increase of bax mRNA levels. In this study we analyzed the ability of MEL to regulate the activation of the glucocorticoid receptor (GR) in mouse thymocytes. We found that even though MEL does not affect the binding of DEX to GR it impaired the DEX-dependent nuclear translocation of the GR when thymocytes were incubated in the presence of 10 nM DEX and 10 nM MEL during 30 minutes, as shown by immunofluorescence confocal microscopy (ICM) assays. MEL per se does not affect GR cytoplasm localization. Intriguingly, when MEL was co-incubated with DEX in cell types expressing a reporter gene codifying luciferase protein under control of Mouse Mammary Tumor Virus promoter no change in GR activity was observed. Results for L929 mouse fibroblast, mammary epithelial cells HC11 and GR expressing Cos-7 were as follow: Fold Induction vs. control: DEX= 4.8 ± 1.2, 4.4 ± 0.8 and 42.2 ± 14.8, respectively; MEL+DEX= 4.4 ± 1.0; 4.3 ± 0.5 and 31.9 ± 13.7, respectively. MEL alone had not significant effect on luciferase expression (0.8 ± 0.1; 1.1 ± 0.2 and 1.2 ± 0.1 fold induction over control, for L929, HC11 and Cos-7 cells, respectively). In those experiments, ICM studies confirmed the nuclear localization of activated GR. These results suggest that the inhibitory effect of MEL on GR is tissue-specific. Moreover, in thymocytes MEL also prevents GR transformation by impairing 90 kDa heat shock protein (hsp90) dissociation process as seen by co-inmunoprecipitation of GR followed by western blots of hsp90. In fact, treatment with DEX during 45 min shows that hsp90 is almost totally dissociated from GR-heterocomplex but, in the presence of MEL the heat shock protein is retained in the complex. These results suggest that MEL prevents GR transformation by blocking the dissociation of hsp90 protein. In summary, the present work shows for the first time the molecular mechanisms responsible for the antagonistic effect of MEL on the GR function, which involves an inhibitory effect of the methoxyindol on hsp90 dissociation and GR nuclear translocation. The apparent tissue specificity for this mechanism is the subject of our current studies.