Stress Modifies the Expression of Glucocorticoid-Responsive Genes by Acting at Epigenetic Levels in the Rat Prefrontal Cortex: Modulatory Activity of Lurasidone

Epigenetics is one of the mechanisms by which environmental factors can alter brain function and may contribute to central nervous system disorders. Alterations of DNA methylation and miRNA expression can induce long-lasting changes in neurobiological processes. Hence, we investigated the effect of chronic stress, by employing the chronic mild stress (CMS) and the chronic restraint stress protocol, in adult male rats, on the glucocorticoid receptor (GR) function. We focused on DNA methylation specifically in the proximity of the glucocorticoid responsive element (GRE) of the GR responsive genes Gadd45β, Sgk1, and Gilz and on selected miRNA targeting these genes. Moreover, we assessed the role of the antipsychotic lurasidone in modulating these alterations. Chronic stress downregulated Gadd45β and Gilz gene expression and lurasidone normalized the Gadd45β modification. At the epigenetic level, CMS induced hypermethylation of the GRE of Gadd45β gene, an effect prevented by lurasidone treatment. These stress-induced alterations were still present even after a period of rest from stress, indicating the enduring nature of such changes. However, the contribution of miRNA to the alterations in gene expression was moderate in our experimental conditions. Our results demonstrated that chronic stress mainly affects Gadd45β expression and methylation, effects that are prolonged over time, suggesting that stress leads to changes in DNA methylation that last also after the cessation of stress procedure, and that lurasidone is a modifier of such mechanisms.

Glucocorticoid Receptor Regulates TNFSF11 Transcription by Binding to Glucocorticoid Responsive Element in TNFSF11 Proximal Promoter Region

Glucocorticoid osteoporosis is a serious side effect of long term glucocorticoid uptake and it is caused by osteoblast apoptosis and imbalance in the major bone remodeling pathway RANK/RANKL/OPG. The impact of glucocorticoid on the maintenance of RANK/RANKL/OPG is well explored; dexamethasone was shown to disturb the ratio between OPG and RANKL level by decreasing the expression level of OPG and increasing level of RANKL. Here, were aimed to decipher whether glucocorticoid receptor directly influences RANKL promoter activity and its transcriptional regulation. We demonstrate that overexpression of glucocorticoid receptor (GR) NR3C1 increased RANKL promoter activity in human osteosarcoma, cervical cancer (2-fold) and adenocarcinoma cells (4.5-fold). Mutational analysis revealed that +352 site in the RANKL promoter is functional glucocorticoid responsive element (GRE) since the effect of GR on RANKL promoter activity was diminished by mutation at this site. Overexpression of NR3C1 upregulated RANKL mRNA expression 1.5-fold in human A549 and HOS cells. On the other hand silencing of NR3C1 caused slight decrease in RANKL mRNA level, suggesting that NR3C1 directly accounts for RANKL transcriptional regulation. Using electrophoretic mobility shift assay we demonstrate that NR3C1 binds to the proximal RANKL promoter region. Our study provides evidences that NR3C1 directly upregulates RANKL transcription in human cell lines and connects the missing link in the mechanism of RANK/RANKL/OPG imbalance of glucocorticoid induced osteoporosis.

Impaired miR449a-induced downregulation of Crhr1 expression in low-birth-weight rats

Low birth weight (LBW) is related to increased incidence of common cardiovascular and metabolic disorders, and psychopathologies later in life. Recent studies have suggested that maternal malnutrition affects fetal hypothalamic–pituitary–adrenal (HPA) axis programing although the mechanism is unknown. We demonstrated that LBW offspring delivered from malnourished dams showed prolonged elevated plasma corticosterone concentrations when compared with those of normal-birth-weight (NBW) offspring and impaired downregulation of corticotropin-releasing factor receptor type 1 (CRF-R1, Crhr1) in the anterior pituitary in restraint. Restraint increased expression of miR449a, which we had previously demonstrated to be involved in Crhr1 downregulation, in the anterior pituitary and serum exosomal miR449a contents through glucocorticoids in NBW offspring, but not in LBW offspring. Although plasma corticosterone concentrations were higher at 2000 h than at 0800 h in both LBW and NBW offspring, they were significantly higher in LBW offspring than in NBW offspring at 2000 and 0200 h. There were no significant diurnal changes in miR449a expression levels in the anterior pituitary of either NBW or LBW offspring, but the expression was significantly lower in LBW offspring than in NBW offspring at 1400, 2000, and 0200 h. The expression levels of GAS5, which inhibits glucocorticoid receptor (GR) binding to glucocorticoid-responsive element, in the anterior pituitary of LBW offspring were elevated when compared with those of NBW offspring. The downregulation of GR found in NBW offspring did not occur in restrained LBW offspring. These results indicate that impaired miR449a expression, probably induced by increased GAS5 expression, causes dysregulation of Crhr1 expression in the anterior pituitary, resulting in prolonged HPA axis activation in restrained LBW offspring.