Dozmorov Igor, Saban Marcia R, Gerard Norma P, Lu Bao, Nguyen Ngoc-Bich, Centola Michael, Saban Ricardo
Oklahoma Medical Research Foundation, Microarray Research Facility, Oklahoma City, USA.
Physiol Genomics. 2003 Feb 6;12(3):239-50. doi: 10.1152/physiolgenomics.00141.2002.
Neurokinin 1 (NK(1)) receptors play a fundamental role in neurogenic inflammation. We sought to determine the mechanisms downstream from NK(1) receptor (NK(1)R) activation using cDNA arrays and a novel statistical method to analyze gene expression. We used female NK(1)R(-/-) and wild-type (WT) mice that were sensitized actively by intraperitoneal injections of dinitrophenol 4 (DNP(4))-human serum albumin. Cystitis was induced by intravesical instillation of antigen of DNP(4)-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-(32)P]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, NK(1)R(-/-) 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 NK(1)R 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 NK(1)R and neprilysin in inflammation.
神经激肽1（NK(1)）受体在神经源性炎症中起重要作用。我们试图利用cDNA阵列和一种新颖的统计方法来分析基因表达，以确定NK(1)受体（NK(1)R）激活后的下游机制。我们使用了雌性NK(1)R基因敲除（-/-）小鼠和野生型（WT）小鼠，通过腹腔注射二硝基苯酚4（DNP(4)）-人血清白蛋白进行主动致敏。通过膀胱内灌注DNP(4)-卵清蛋白抗原诱导膀胱炎，对照组小鼠用生理盐水进行刺激。在灌注后1、4和24小时，取出膀胱用于：1）RNA提取（n = 3），2）RNA提取重复（n = 3），以及3）形态学分析（n = 6）。对于cDNA阵列实验，将每组的三个膀胱匀浆，获得总RNA。经脱氧核糖核酸酶处理的RNA逆转录为cDNA，用[α-(32)P]dATP标记并与Atlas Mouse 1.2阵列（Clontech公司）杂交。在计算背景点的平均值和标准差后，使用公式S' = (S - Av)/SD为每个实验值赋予一个标准化分数S，其中S'是原始像素值，Av和SD分别是背景点的平均值和标准差。仅使用表达值比背景高3个标准差的基因。通过聚类分析对高变基因进行分类。计算相关系数矩阵并以连通性镶嵌图表示。结果，我们发现在野生型小鼠中，最突出的基因簇以中性内肽酶处于中心位置，并且与一组激活蛋白-1（AP-1）反应性基因呈正相关，包括层粘连蛋白-α3、组织纤溶酶原激活物11、Fos-B和肿瘤坏死因子-β。在野生型小鼠中，抗原诱导的膀胱炎症导致中性内肽酶表达下调。相反，NK(1)R基因敲除小鼠未能引发炎症反应，且中性内肽酶与野生型小鼠中所述的相同基因呈负相关。总之，这项工作表明NK(1)R和中性内肽酶在膀胱炎症中起主要作用，为AP-1转录因子的参与提供了一个工作模型，并引发了关于NK(1)R和中性内肽酶在炎症中作用的可检验假设。
