Here although tibolone reduced the expression of inflammator
Here, although tibolone reduced the expression of inflammatory genes such as IL6, IL1β, TLR4 and MIR155-3p, in the presence of PA stimulation, it increased the expression of TNF gene. It should be noted that TNF has pleiotropic effects in the CNS, therefore its increased expression does not always involve negative effects (Probert, 2015). Inflammation is a process associated with several diseases, such as depression (Wohleb et al., 2016), Alzheimer and Parkinson's diseases, and stroke (Amor et al., 2014), whereby astrocytes play an important role on the induction of pro- and anti-inflammatory signals (Sofroniew, 2015). On the other hand, the Glutamate-ammonia ligase (GLUL) and APOE genes, that are involved in astrocyte functions, were affected by tibolone pretreatment. In here, differing from other studies demonstrating that ERs activation induced the expression of GLUL and APOE (Blutstein et al., 2006; Stone et al., 1997), we observed a significant reduction in the expression of these genes. These differences can be explained by a tibolone's inability to induce the transcriptional activity of ERs. Indeed, its effects can be explained by interaction with transcription factors that work as repressors or by the induction of kinase-activated transcription factors through activation of membrane receptors (ERα, ERβ and G protein-coupled ER (GPER) (Arevalo et al., 2015). Nevertheless, it should be noted that activation of ERα and ERβ induces a different effect on APOE expression (J. M. Wang, Irwin and Brinton, 2006). Telomeres are repetitive sequences in the end of GSK-3 Inhibitor IX synthesis and their main function is to preserve chromosome stability (Stewart et al., 2012). Telomere maintenance is performed by telomerase and other proteins conforming a shelterin complex, such as TERF1, TERF2 and TIN2 (Martinez and Blasco, 2011). Telomerase is encoded by TERT and TERC genes (Rebhan et al., 1997). As for TERT, several non-canonical functions have been identified, including reduction of oxidative damage and apoptosis in neurons (Gonzalez-Giraldo et al., 2016) and the regulation of gene expression (Martinez and Blasco, 2011), such as IL6 and TNF (Ghosh et al., 2012). In the current study, it was observed an increase of TERT and TERC expression induced by palmitic acid, which was attenuated by tibolone pretreatment (Fig. S5). The promoter region of TERT has binding sites for ERs, a mechanism by which ERα has been associated to increased expression of TERT (Cha et al., 2008). Against our initial hypothesis, we observed that stimulation of ERs with DPN, PPT and tibolone reduced TERT expression in cells treated with PA. This fact suggests that ER activation in our model might lead to induction of other signaling mechanisms in T98G cells, which do not involve a genomic action of ERs (Arevalo et al., 2015). It has been demonstrated that TERT is induced by inflammatory stimuli via activation of NF-kB (Gizard et al., 2011); therefore, NF-kB inactivation could lead to reduction of TERT expression (Zuo et al., 2011). Nonetheless, as this is the first study reporting a regulation of TERT by a fatty acid, further studies are necessary to address the role of telomerase under PA stimulation. We found that PA induced the overexpression of DNMT3B gene, which is responsible for the de novo methylation, and its expression was attenuated by treating cells with tibolone. Furthermore, we observed that tibolone reduced the expression of DNMT1 in comparison to PA only. DNA methylation is an important mechanism for the regulation of astrocytic functions and it has been associated with molecular mechanism in diseases. Increased DNA methylation of some genes is induced by stress and is reduced during astrocyte differentiation (Neal and Richardson, 2018). Changes in methylation status for genes related with glutamate ionotropic kainate receptors, actin-binding related proteins, adhesion molecules at junctions and signal transduction have been observed in astrocytic cells from patients with depression and suicide behavior (Nagy et al., 2015). Indeed, analysis of DNA methylation for pro-inflammatory cytokines has been performed in AD patients, whereby methylation status was inversely correlated to gene expression for IL6 and IL1β (Nicolia et al., 2017). In addition, it has been shown that PA induces changes on gene expression through DNA-related methylation mechanisms (Su et al., 2015), suggesting that DNA methylation may be involved in the detrimental effects of PA.