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Alteration of signal transduction--associated gene expression in rat cardiac fibroblasts induced by blocking angiotensin Ⅱ receptors

Jiang Xiaoying, Gao Guangdao, Wang Xinfeng, Lin Yuanxi, Wang Yawen, Yang Yubai

Department of Genetics and Molecular Biology,Medical College of Xi'an Jiaotong University.Xi'an 710061,Shaanxi；China

Abstract

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 SpragneDawley 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 immunohistocbemical 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 Uniscandl000 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 upregulated. The maximum change was not beyond 20 folds. The following 9 pathways were activated： cAMP/PKA, Ca~(2+), PKC, PLC, MAPK, PI-3 kinase, NO-cGMP, Rho, NF-#kappa#B 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： Cypl9al （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-#kappa#B 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-1#beta# 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.

Key words: cardiac myoblasts;receptor, angiotensin, type 2;cDNA microarray

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Citing This Article：

Jiang Xiaoying, Gao Guangdao, Wang Xinfeng, Lin Yuanxi, Wang Yawen, Yang Yubai. Alteration of signal transduction--associated gene expression in rat cardiac fibroblasts induced by blocking angiotensin Ⅱ receptors. Acta Physiol Sin 2006; 58 (6): (in Chinese with English abstract).