F Neuroinflammation (2016) 13:Page 9 ofFig. 4 Role of Oxidative-ER stress in cocaine-mediated up-regulation of TLR2 and microglial activation in BV2 cells. a, b Effect of pharmacological CI-1011 cancer inhibitor of ER-stress 4-PBA on cocaine-mediated up-regulation of ATF4 and TLR2 PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/27486068 proteins in BV2 cells. c Effect of 4-PBA on cocaine-mediated induction of TLR2, TNF, IL-6, and MCP-1 mRNAs. Relative levels of TLR2, TNF, IL-6, and MCP-1 mRNAs were analyzed by qPCR using 18S rRNA as an internal control. d Effects of 4-PBA on cocaine-mediated induction of TNF and MCP-1 proteins assessed by ELISA. e 4-PBA did not ameliorate cocaine-mediated induction of ROS generation. f, g Effects of APO (NADPH oxidase inhibitor) and PBN (ROS scavenger) on cocaine-mediated up-regulation of ATF4 protein in BV2 cells. All data are presented as mean ?SD of three individual experiments. *p < 0.05, **p < 0.01, ***p < 0.001 vs control group. #p < 0.05, ##p < 0.01 vs cocaine group (Student's t test)sought to examine the molecular link between cocaine exposure and ER stress activation. Generation of ROS has been implied in the activation of PERKdependent ER stress signaling [40, 41]. Herein, we examined whether ROS was also the precedent signal for cocaine-mediated ER pathway activation. BV2 cells were pretreated with 4-PBA for 1 h followed by exposure of cells to cocaine and subsequent assessment of ROS production by DCFH-DA assay. As shown in Fig. 4e, pretreatment with 4-PBA had no effect on cocaine-mediated ROS production implying thereby that ROS production was upstream of ER-stress activation. Furthermore, both the ROS inhibitors (PBN and APO) blocked cocaine-mediated activation of ERstress as evidenced by the down-regulation of ATF4 levels (Fig. 4f, g). Taken together, our findings suggest that ROS lies upstream of ER signaling and participates in cocaine-mediated microglial activation.Role of ATF4 in cocaine-mediated up-regulation of TLR2 expression and microglial activationHaving established that blocking ROS production and ER-stress abolished cocaine-mediated up-regulation of ATF4, TLR2 protein and microglial activation, we next rationalized that ATF4 was critical for cocaine-mediated up-regulation of TLR2 as well as for microglial activation. To elucidate the role of ATF4, BV2 cells were transfected with either ATF4 siRNA or si-Con followed by exposure of cells to cocaine for 12 h and assessed subsequently for TLR2 levels. As shown in Fig. 5a, knocking down ATF4 significantly blocked cocainemediated up-regulation of TLR2 protein. Reciprocally, Fig. 5b showed that overexpression of ATF4 resulted in significant (1.9-fold, p = 0.00042) up-regulation of cocaine-mediated induction of TLR2. Similarly, the role of ATF4 was also examined in cocaine-mediated activation of microglia. As shown in Fig. 5c, cocaine-mediatedLiao et al. Journal of Neuroinflammation (2016) 13:Page 10 ofFig. 5 Role of ATF4 in cocaine-mediated up-regulation of TLR2 expression and microglial activation. a siRNA transfection was used to knock down ATF4. ATF4 siRNA but not si-Con inhibited cocaine-mediated induction of TLR2 protein. b Overexpression of ATF4 up-regulated TLR2 protein levels. c Role of ATF4 in cocaine-mediated induction of TNF and MCP-1 mRNAs. d, e Cocaine-mediated nuclear translocation of ATF4 in BV2 cells. f Schematic illustration of ATF4 binding sequence on the promoter region of TLR2. ChIP assay demonstrating cocaine-mediated binding of ATF4 to the TLR2 promoter. All data are presented as me.
