Given the important role of Cx in

Given the important role of Cx43 in maintaining the function of gap junctions, to investigate the effects of stress on Cx43 may be an important step towards understanding the mechanism underlying CUS-induced dysfunction of gap junctions. Corticosterone (CORT), as an important stress hormone, has been used to induce depression model (Dwivedi et al., 2015), thus, CORT was used to simulate aspects of the stress response in the present study. First, we examined the effects of CORT on GJIC. Then, we detected the total level of Cx43 protein, the synthesis of Cx43, the degradation of Cx43, the stability of Cx43, the distribution of Cx43, and the phosphorylation of Cx43 in prefrontal cortical and hippocampal astrocytes.
Materials and methods
Results
Discussion Although gap junctions and Cx43 have been implicated in the progression of MDD, past studies only focused on the expression of Cx43 (Bernard et al., 2011, Ernst et al., 2011, Miguel-Hidalgo et al., 2014, Nagy et al., 2017, Sun et al., 2012). The present study clarified the mechanisms underlying gap junctions dysfunction induced by stress. We used CORT to stimulate aspects of stress conditions, observed the alternations of gap junctions and Cx43 life GSK-2118436 in prefrontal cortical and hippocampal astrocytes. As shown in Fig. 9, our data showed that CORT inhibited GJIC by decreasing the distribution of Cx43 in astrocyte membranes while simultaneously increasing the ratio of p-Cx43/Cx43. Further, CORT inhibited the synthesis of Cx43 while increasing the degradation of Cx43. Increased expression of N-Cadherin, and up-regulated association between Cx43 and ZO-1/drebrin in prefrontal cortical astrocytes may result in the formation of annular gap junctions; however, the expression of N-Cadherin and the association of Cx43 and ZO-1 in hippocampal astrocytes were downregulated. Given these results, we can postulate that both autophagy and the proteasome system participated in the degradation of Cx43 in prefrontal cortical astrocytes, but only the proteasome system was induced in hippocampal astrocytes. Moreover, CORT also decreased the concentration of Cx30. Though Cx30 was low abundant proteins in our cultured astrocytes, whether Cx43 and Cx30 perform the same role in CORT-induced gap junction dysfunction need further study. Previous study proved that the function of gap junctions is deteriorated by CUS, indicated by the decreased diffusion of LY in the rat prefrontal cortex (Sun et al., 2012). In accordance with this finding, CORT inhibited LY dye transfer, indicating the reduction of GJIC between astrocytes in the present study. Moreover, our results showed that CORT reduced expression of Cx43 protein, which has also been found in patients with depression and in rat with depressive behavior (Ernst et al., 2011, Miguel-Hidalgo et al., 2014, Sun et al., 2012). All these changes may be regulated by glucocorticoid receptor (GR), for CORT is an important steroid hormone and regulates downstream pathways by GR (Santos et al., 2014, Wang et al., 2015). Glucocorticoids (GC) suppressed Cx43 expression in osteoblasts by reducing phosphorylation of Akt/mTOR via GR (Shen et al., 2016). The inhibition of PI3K/Akt signaling reduced the stability of Cx43 mRNA in MC3T3E1 cells (Bhattacharjee et al., 2009). In addition, GC-induced autophagy in osteocytes was dependent on the inhibition of Akt-mTORC1 signaling, resulting in the degradation of Cx43 (Gao et al., 2016). In neural progenitor cell, membrane GR-mediated phosphorylation of ERK-1/2 contributed to the phosphorylation of Cx43, and reduction of GJIC (Samarasinghe et al., 2011). Membrane GR also activated protein kinase C (PKC) signaling cascade (Jozic et al., 2017), then PKC phosphorylates Cx43 at S368 (Lampe et al., 2000), inducing internalization and degradation of gap junction (Cone et al., 2014), resulting in the decrease of GJIC. Cx43 undergoes rapid dynamic changes, maintaining the balance between its synthesis and degradation (Beyer and Berthoud, 2002). In order to identify how CORT mediated the downregulation of Cx43 protein expression, we next detected the synthesis progress. CORT decreased the biosynthesis of Cx43 in both prefrontal cortical and hippocampal astrocytes. Similarly, a previous study found that DEX suppressed Cx43 mRNA in osteoblastic cells via the glucocorticoid receptor (Shen et al., 2016). Previous studies have also revealed that Cx43 mRNA was decreased in patients with depression (Bernard et al., 2011, Ernst et al., 2011, Nagy et al., 2017). Thus, according to our results, elevated CORT concentration may contribute to the downregulation of Cx43 mRNA.