Jiang Xiao-Ying, Gao Guang-Dao, Wang Xin-Feng, Lin Yuan-Xi, Wang Ya-Wen, Yang Yu-Bai
Department of Genetics and Molecular Biology, Medical College of Xi'an Jiaotong University, Xi'an 710061, China.
Sheng Li Xue Bao. 2006 Dec 25;58(6):556-66.
To investigate the molecular mechanism of angiotensin II (Ang II) receptor activation in adult rat cardiac fibroblasts, the expressions of cell signal transduction-associated genes were studied by using cDNA microarray. Cardiac fibroblasts of adult Sprague-Dawley rats (230~250 g) were isolated and cultured. The cells were divided into 4 groups: Ang II, Ang II + losartan, Ang II + PD123319, Ang II + losartan + PD123319. The expressions of Ang II receptors were studied by immunohistochemical staining. Total RNA was extracted and purified. After cDNA synthesis and biotin-16-dUTP labeling, the probes were denatured and hybridized with GEArray Q Series mouse G Protein-coupled Receptors Signaling Pathway Finder Gene Array (MM-025) containing 96 genes associated with 11 pathways. The arrays were scanned with a Uniscand1000 scanner and further analyzed with GEArray Analyzer software. RT-PCR was used to further confirm the results of gene microarray. The results of immunohistochemical staining showed that the expression of Ang II type 2 (AT2) receptor was evidently induced by Ang II stimulation when Ang II type 1 (AT1) receptor was blocked. The results of gene array indicated that blocking AT1 receptor changed 34 genes (more than 2 folds), 30 were down-regulated and 4 were up-regulated. The maximum change was not beyond 20 folds. The following 9 pathways were activated: cAMP/PKA, Ca2+, PKC, PLC, MAPK, PI-3 kinase, NO-cGMP, Rho, NF-kappaB pathways. Blockade of AT2 receptor caused 64 genes changing more than 2 folds (48 were down-regulated and 16 were up-regulated). Eleven pathways were basically activated. The change of the following 7 genes was over 30 folds: Cyp19a1 (37 folds), Il1r2 (42 folds), Cflar (53 folds), Bcl21 (31 folds), Pik3cg (278 folds), Cdkn1a (90 folds), Agt (162 folds). According to the activated extent, the signal transduction pathways in turn were PI-3 kinase, NF-kappaB and JAK-STAT pathways. Blocking both AT1 and AT2 receptors changed 46 genes more than 2 folds (36 were down-regulated and 10 were up-regulated). Eleven pathways were basically activated. The results of RT-PCR of IL-1beta and TNF-alpha confirmed the observations in gene microarray. Our results show that Ang II can induce a high expression of AT2 receptor in adult rat cardiac fibroblasts when AT1 receptor is blocked, and the signal mechanism of AT2 receptor is clearly different from that of AT1 receptor.
为研究血管紧张素II（Ang II）受体在成年大鼠心脏成纤维细胞中激活的分子机制，采用cDNA微阵列技术研究细胞信号转导相关基因的表达。分离并培养成年Sprague-Dawley大鼠（230~250 g）的心脏成纤维细胞。将细胞分为4组：Ang II组、Ang II + 氯沙坦组、Ang II + PD123319组、Ang II + 氯沙坦 + PD123319组。通过免疫组织化学染色研究Ang II受体的表达。提取并纯化总RNA。经cDNA合成及生物素-16-dUTP标记后，将探针变性并与包含与11条信号通路相关的96个基因的GEArray Q系列小鼠G蛋白偶联受体信号通路检测基因芯片（MM-025）杂交。用Uniscand1000扫描仪扫描芯片，并用GEArray Analyzer软件进一步分析。采用RT-PCR进一步验证基因芯片结果。免疫组织化学染色结果显示，当1型Ang II受体（AT1）被阻断时，Ang II刺激明显诱导2型Ang II受体（AT2）的表达。基因芯片结果表明，阻断AT1受体使34个基因发生变化（变化超过2倍），其中30个基因下调，4个基因上调。最大变化不超过20倍。激活了以下9条信号通路：cAMP/PKA、Ca2+、PKC、PLC、MAPK、PI-3激酶、NO-cGMP、Rho、NF-κB信号通路。阻断AT2受体导致64个基因变化超过2倍（48个基因下调，16个基因上调）。基本激活了11条信号通路。以下7个基因的变化超过30倍：Cyp19a1（37倍）、Il1r2（42倍）、Cflar（53倍）、Bcl21（31倍）、Pik3cg（278倍）、Cdkn1a（90倍）、Agt（162倍）。根据激活程度，信号转导通路依次为PI-3激酶、NF-κB和JAK-STAT信号通路。同时阻断AT1和AT2受体使46个基因变化超过2倍（36个基因下调，10个基因上调）。基本激活了11条信号通路。IL-1β和TNF-α的RT-PCR结果证实了基因芯片的观察结果。我们的结果表明，当AT1受体被阻断时，Ang II可诱导成年大鼠心脏成纤维细胞中AT2受体的高表达，且AT2受体的信号机制与AT1受体明显不同。
