An ?SD of three individual experiments. *p < 0.05, **p < 0.01, ***p < 0.001 vs control
An ?SD of three individual experiments. *p < 0.05, **p < 0.01, ***p < 0.001 vs control group (Student's t test)induction of TNF and MCP-1 expression was attenuated in cells transfected with ATF4 siRNA but not in cells transfected with si-Con. These findings underscored the role of ATF4 in cocaine-mediated induction of microglial activation. We next sought to enquire whether cocaine could also mediate nuclear translocation of ATF4 resulting ultimately in increased transcription of TLR2. BV2 cells were exposed to cocaine for varying time points (0 to 6 h) followed by assessment of ATF4 translocation in the nuclear fractions. As shown in Fig. 5d, e, exposure of BV2 cells to cocaine resulted in a time-dependent increase in translocation of ATF4 into the nucleus with a maximal response at 1 h (2.3-fold, p = 0.0013) and a concomitant decrease in the cytoplasm. It is well-recognized that nuclear translocation of transcription factors is necessary for accessing and binding to the promoter region of a gene. Intriguingly, using the TFSEARCH software we found a predicted ATF4 binding site in the intron of the gene instead of its presence at a traditional promoter site upstream of the transcription start site. This led to a speculation that ATF4 could be binding to the intronic promoter of the TLR2 gene. Adding credence to this hypothesis are reports identifying an intronic promoter in the murine proteinase 3 gene [42] and another report that described the binding of ATF4 to an intronic promoter leading, in turn, to regulated expression of Siah2 mRNA in response to ERstress [43]. Interestingly, ATF4-binding site is also present in the first intron of the human VEGFA gene [44]. To assess whether cocaine mediated the binding of ATF4 to the intronic region of TLR2, we performed the ATF4 ChIP assay. BV2 cells were treated with cocaine for 1 h followed by RNA extraction and processed using a ChIP assay kit. As shown in Fig. 5f, exposure of BV2 cells to cocaine resulted in enhanced binding of ATF4 to the TLR2 intronic promoter.TLR2 phosphorylation, NF-B translocation, and MyD88 are involved in cocaine-mediated microglial activationHaving demonstrated that cocaine mediated up-regulation of TLR2 protein and microglial activation, we next sought to examine whether cocaine could also PubMed ID: activate the TLR2 signaling pathway(s) leading, in turn, to downstream activation of microglia. To explore the possibility that cocaine exposure could also increase phosphorylation of TLR2, lysates from cocaine-exposed BV2 cells were immunoprecipitated with TLR2 antibody and subsequently assessed for TLR2 tyrosine phosphorylation using the specific tyrosine antibody 4G10. As shown in Fig. 6a, cocaine-induced phosphorylation of TLR2 was Stattic custom synthesis observed as early as 15 min after cocaine exposure with a peak at 30 min. Next, we sought to investigate whether cocaine could also promote nuclear translocation of NF-B. For this, following exposure of BV2 cells to cocaine, cytosolic and nuclear protein extracts were monitored for levels of NF-B at theLiao et al. Journal of Neuroinflammation (2016) 13:Page 11 ofFig. 6 Cocaine-mediated induction of phosphorylation-dependent TLR2 signaling pathway and microglial activation. a Cocaine-mediated induction of phosphorylation of TLR2 in BV2 cells. b Cocaine-mediated induction of NF-B nuclear translocation in BV2 cells. c siRNA transfection was used to knock down Myd88. Myd88 siRNA but not si-Con inhibited cocaine-mediated induction of IL-6, MCP-1, and T.

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