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 CEP-37440 web 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:http://www.ncbi.nlm.nih.gov/pubmed/27107493 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 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.
