Transcription factors Sp1 and Sp4 regulate TRPV1 gene expression in rat sensory neurons.
Transcription factors Sp1 and Sp4 regulate TRPV1 gene expression in rat sensory neurons.
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DOI：
10.1186/1744-8069-7-44
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发表时间：
2011-06-06
期刊：
影响因子：
3.3
通讯作者：
Schumacher MA
中科院分区：
文献类型：
--
作者：
Chu C;Zavala K;Fahimi A;Lee J;Xue Q;Eilers H;Schumacher MA
辣椒素受体瞬时受体电位-1 （TRPV1）在信号转导中起着复杂的作用，包括检测由细胞损伤和炎症引起的有害刺激。在病理生理条件下，TRPV1 mRNA和受体蛋白在背根神经节（DRG）神经元中表达升高，持续数周至数月，并与痛觉过敏有关。在我们之前分离的大鼠TRPV1基因启动子系统的基础上，我们研究了TRPV1近端p2启动子，首先使用染色质免疫沉淀（ChIP）试验鉴定了体内结合在p2启动子上的候选sp1样转录因子。然后，我们进行了GC-box结合位点的缺失分析，并量化了Sp1 / Sp4过表达与抑制/敲低条件下启动子的活性。在培养的DRG神经元Sp1/Sp4过表达或siRNA介导敲低的情况下，采用qRT-PCR方法定量Sp1、Sp4和TRPV1的mRNA编码。通过对DRG组织的ChIP分析，我们发现Sp1和Sp4与内源性TRPV1 p2启动子内的候选GC-box位点区域结合。P2-启动子内GC-box“a”或“a + b”的缺失导致转录活性完全丧失，这表明GC-box“a”是启动子激活的关键位点。在培养的DRG神经元中，共转染Sp1增加了p2启动子活性，而米特霉素-a （Sp1样功能抑制剂）在PC12细胞中剂量依赖性地阻断了NGF和Sp1依赖性启动子活性。针对Sp1或Sp4的siRNA共转染降低了DRG神经元和NGF处理的PC12细胞的启动子活性。最后，将Sp1或Sp4 cDNA电穿孔到DRG神经元培养物中，导致Sp1/Sp4 mRNA的增加，重要的是TRPV1 mRNA的增加。相反，联合si-RNA定向敲除Sp1/Sp4导致TRPV1 mRNA减少。基于这些研究，我们现在提出了一个TRPV1表达依赖于sp1样转录因子的模型，其中Sp4在激活DRG神经元TRPV1 RNA转录中起主导作用。鉴于TRPV1表达的增加与包括持续性疼痛在内的多种病理生理状态有关，阻断sp1样转录因子代表了治疗策略的新方向。
The capsaicin receptor, transient receptor potential vanilloid type -1 (TRPV1) directs complex roles in signal transduction including the detection of noxious stimuli arising from cellular injury and inflammation. Under pathophysiologic conditions, TRPV1 mRNA and receptor protein expression are elevated in dorsal root ganglion (DRG) neurons for weeks to months and is associated with hyperalgesia. Building on our previous isolation of a promoter system for the rat TRPV1 gene, we investigated the proximal TRPV1 P2-promoter by first identifying candidate Sp1-like transcription factors bound in vivo to the P2-promoter using chromatin immunoprecipitation (ChIP) assay. We then performed deletion analysis of GC-box binding sites, and quantified promoter activity under conditions of Sp1 / Sp4 over-expression versus inhibition/knockdown. mRNA encoding Sp1, Sp4 and TRPV1 were quantified by qRT-PCR under conditions of Sp1/Sp4 over-expression or siRNA mediated knockdown in cultured DRG neurons. Using ChIP analysis of DRG tissue, we demonstrated that Sp1 and Sp4 are bound to the candidate GC-box site region within the endogenous TRPV1 P2-promoter. Deletion of GC-box "a" or "a + b" within the P2- promoter resulted in a complete loss of transcriptional activity indicating that GC-box "a" was the critical site for promoter activation. Co-transfection of Sp1 increased P2-promoter activity in cultured DRG neurons whereas mithramycin-a, an inhibitor of Sp1-like function, dose dependently blocked NGF and Sp1-dependent promoter activity in PC12 cells. Co-transfection of siRNA directed against Sp1 or Sp4 decreased promoter activity in DRG neurons and NGF treated PC12 cells. Finally, electroporation of Sp1 or Sp4 cDNA into cultures of DRG neurons directed an increase in Sp1/Sp4 mRNA and importantly an increase in TRPV1 mRNA. Conversely, combined si-RNA directed knockdown of Sp1/Sp4 resulted in a decrease in TRPV1 mRNA. Based on these studies, we now propose a model of TRPV1 expression that is dependent on Sp1-like transcription factors with Sp4 playing a predominant role in activating TRPV1 RNA transcription in DRG neurons. Given that increases of TRPV1 expression have been implicated in a wide range of pathophysiologic states including persistent painful conditions, blockade of Sp1-like transcription factors represents a novel direction in therapeutic strategies.
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影响因子：
4.8
作者：
Liu, A;Prenger, MS;Bai, G
通讯作者：
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11.4
作者：
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DOI：
10.1006/bbrc.1998.9964
发表时间：
1999-01-19
影响因子：
3.1
作者：
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Todisco, A