Neurokinin 1 receptors and neprilysin modulation of mouse bladder gene regulation
Neurokinin 1 receptors and neprilysin modulation of mouse bladder gene regulation
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DOI：
10.1152/physiolgenomics.00141.2002
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发表时间：
2003-02-06
影响因子：
4.6
通讯作者：
Saban, R
中科院分区：
文献类型：
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作者：
Dozmorov, I;Saban, MR;Saban, R
神经激肽1（NK1）受体在神经源性炎症中起着重要作用。我们试图确定从NK 1受体（NK 1R）激活下游的机制，使用cDNA阵列和一种新的统计方法来分析基因表达。我们使用雌性NK1R-/-和野生型（WT）小鼠，通过腹腔注射二硝基苯酚4（DNP4）-人血清白蛋白主动致敏。膀胱内滴注DNP4-卵清蛋白抗原诱导膀胱炎，对照组用生理盐水攻击。在滴注后1、4和24小时，取出膀胱用于1）RNA提取（n = 3），2）重复RNA提取（n = 3）和3）形态学分析（n = 6）。对于cDNA阵列实验，将来自每组的三个膀胱均质化，并获得总RNA。将DNA酶处理的RNA逆转录为cDNA，用[α-P-32] dATP标记并与Atlas小鼠1.2阵列（Clontech）杂交。在计算背景斑点的平均值和SD之后，使用公式S '=（S-Av）/SD为每个实验值指定归一化得分S，其中S'是原始像素值，并且Av和SD分别是背景斑点的平均值和标准偏差。仅使用表达高于背景3个SD值的基因。通过聚类分析对高变基因进行排序。计算相关系数矩阵，并以连接性镶嵌图表示。作为结果，我们发现在WT小鼠中，最突出的基因簇在中心位置具有脑啡肽酶，并且与一组激活蛋白-1（AP-1）应答基因正相关，包括层粘连蛋白-α 3、组织纤溶酶原激活物11、fos-B和TNF-β。在WT小鼠中，抗原诱导的膀胱炎症导致脑啡肽酶表达下调。相比之下，NK 1 R-/-小鼠未能产生炎症反应，并且脑啡肽与WT中描述的相同基因呈负相关。总之，这项工作表明了压倒一切的参与NK1R和neprilysin在膀胱炎症，AP-1转录因子的参与提供了一个工作模型，并唤起可检验的假设NK1R和neprilysin在炎症中的作用。
Neurokinin 1 (NK1) receptors play a fundamental role in neurogenic inflammation. We sought to determine the mechanisms downstream from NK1 receptor (NK1R) activation using cDNA arrays and a novel statistical method to analyze gene expression. We used female NK1R-/- and wild-type (WT) mice that were sensitized actively by intraperitoneal injections of dinitrophenol 4 (DNP4)-human serum albumin. Cystitis was induced by intravesical instillation of antigen of DNP4-ovalbumin, and control mice were challenged with saline. At 1, 4, and 24 h after instillation, bladders were removed for 1) RNA extraction (n = 3), 2) replicate of RNA extraction (n = 3), and 3) morphological analysis (n = 6). For cDNA array experiments, three bladders from each group were homogenized, and total RNA was obtained. DNase-treated RNA was reverse-transcribed to cDNA, labeled with [alpha-P-32] dATP and hybridized to Atlas Mouse 1.2 Arrays (Clontech). After calculating the mean and SD for background spots, each experimental value was assigned a normalized score S using the formula S' = ( S - Av)/SD, where S' is the original pixel value, and Av and SD are the mean and standard deviation of background spots, respectively. Only genes that expressed 3 SD values above background were used. Hypervariable genes were sorted by cluster analysis. Matrices of correlation coefficients were calculated and represented in a connectivity mosaic. As results, we found that in WT mice the most prominent gene cluster had neprilysin in a central position and positively correlated to a group of activator protein-1 (AP-1)-responsive genes, including laminin-alpha3, tissue plasminogen activator 11, fos-B, and TNF-beta. In WT mice, antigen-induced bladder inflammation led to a downregulation in neprilysin expression. In contrast, NK1R-/- mice failed to mount an inflammatory reaction and presented neprilysin negatively correlated with the same genes described in WT. In conclusion, this work indicates an overriding participation of NK1R and neprilysin in bladder inflammation, provides a working model for the involvement of AP-1 transcription factor, and evokes testable hypotheses regarding the role of NK1R and neprilysin in inflammation.