Pittsburgh Center for Pain Research, Department of Medicine, University of Pittsburgh, Pittsburgh, PA 15261, United States.
Neuroscience. 2013 Feb 12;231:28-37. doi: 10.1016/j.neuroscience.2012.11.034. Epub 2012 Nov 29.
Peripheral nerve injury evokes rapid and complex changes in gene transcription and cellular signaling pathways. Understanding how these changes are functionally related is essential for developing new approaches that accelerate and improve nerve regeneration. Toward this goal we found that nerve injury induces a rapid and significant up-regulation of the transcription factor Sox11 in dorsal root ganglia (DRG) neurons. Gain and loss of function studies have shown this increase is essential for normal axon regeneration. To determine how Sox11 impacts neuronal gene expression, DRG neurons were treated with Sox11 siRNA to identify potential transcriptional targets. One gene significantly reduced by Sox11 knockdown was TRAF (tumor necrosis factor (TNF) receptor-associated factor)-associated NF-κB activator (TANK). Here we show that TANK is expressed in DRG neurons, that TANK expression is increased in response to peripheral nerve injury and that Sox11 overexpression in vitro increases TANK expression. Injury and in vitro overexpression were also found to preferentially increase TANK transcript variant 3 and a larger TANK protein isoform. To determine if Sox11 regulates TANK transcription bioinformatic analysis was used to identify potential Sox-binding motifs within 5kbp of the TANK 5' untranslated region (UTR) across several mammalian genomes. Two sites in the mouse TANK gene were examined. Luciferase expression assays coupled with site-directed mutagenesis showed each site contributes to enhanced TANK promoter activity. In addition, chromatin immunoprecipitation assays showed direct Sox11 binding in regions containing the two identified Sox motifs in the mouse TANK 5'-UTR. These studies are the first to show that TANK is expressed in DRG neurons, that TANK is increased by peripheral nerve injury and that the regulation of TANK expression is, at least in part, controlled by the injury-associated transcription factor Sox11.
周围神经损伤会引起基因转录和细胞信号通路的快速而复杂的变化。了解这些变化如何在功能上相关对于开发新的方法来加速和改善神经再生至关重要。为此，我们发现神经损伤会迅速而显著地上调背根神经节（DRG）神经元中的转录因子 Sox11。增益和失能研究表明，这种增加对于正常轴突再生是必不可少的。为了确定 Sox11 如何影响神经元基因表达，我们用 Sox11 siRNA 处理 DRG 神经元，以鉴定潜在的转录靶标。一个基因的表达显著降低，该基因是 TRAF（肿瘤坏死因子（TNF）受体相关因子）相关 NF-κB 激活物（TANK）。在这里，我们显示 TANK 在 DRG 神经元中表达，外周神经损伤会导致 TANK 表达增加，体外 Sox11 过表达会增加 TANK 表达。损伤和体外过表达也被发现优先增加 TANK 转录变体 3 和更大的 TANK 蛋白同工型。为了确定 Sox11 是否调节 TANK 转录，我们进行了生物信息学分析，以鉴定跨几个哺乳动物基因组的 TANK 5'UTR 附近的潜在 Sox 结合基序。在小鼠 TANK 基因中检查了两个位点。荧光素酶表达分析与定点突变相结合表明，每个位点都有助于增强 TANK 启动子活性。此外，染色质免疫沉淀分析表明，直接 Sox11 结合在包含小鼠 TANK 5'-UTR 中两个鉴定的 Sox 基序的区域中。这些研究首次表明 TANK 在 DRG 神经元中表达，外周神经损伤会增加 TANK 的表达，并且 TANK 表达的调节至少部分受到损伤相关转录因子 Sox11 的控制。
