Galpha12/13-mediated production of reactive oxygen species is critical for angiotensin receptor-induced NFAT activation in cardiac fibroblasts.
Angiotensin II (Ang II) activates multiple signaling pathways leading to hyperplasia of cardiac fibroblasts. Reactive oxygen species (ROS) produced by Ang II stimulation are assumed to play pivotal roles in this process. Here, we show that ROS mediate Ang II-induced activation of nuclear factor of activated T cells (NFAT) in rat cardiac fibroblasts. Ang II-induced NFAT activation was suppressed by diphenyleneiodonium (an NADPH oxidase inhibitor), dominant negative (DN)-Rac, DN-p47(phox), and an inhibitor of Galpha(12/13) (Galpha(12/13)-specific regulator of G protein signaling domain of p115RhoGEF, p115-regulator of G protein signaling (RGS)). Stimulation of Ang II receptor increased the intracellular ROS level in a Rac- and p47(phox)-dependent manner. Because p115-RGS suppressed Ang II-induced Rac activation, Ang II receptor-coupled Galpha(12/13) mediated NFAT activation through ROS production by Rac activation. Ang II-induced nuclear translocation of the green fluorescent protein (GFP)-tagged amino-terminal region of NFAT4 (GFP-NFAT4) was suppressed by p115-RGS or BAPTA but not by diphenyleneiodonium. The expression of constitutively active (CA)-Galpha(12/13), CA-G translocation alpha(13), or CA-Rac increased the nuclear of GFP-NFAT4. These results suggest that NFAT activity is regulated by both Ca(2+)-dependent and ROS-dependent pathways. Furthermore, activation of c-Jun NH(2)-terminal kinase (JNK) induced by Ang II stimulation is required for NFAT activation because Ang II-induced NFAT activation was inhibited by SP600125, a selective JNK inhibitor. These results indicate that Ang II stimulates the nuclear translocation and activation of NFAT by integrated pathways including the activation of Galpha(12/13), Rac, NADPH oxidase, and JNK and that Galpha(12/13)-mediated ROS production is essential for NFAT transcriptional activation.
Fujii
Tomomi
T
Department of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan.
Onohara
Naoya
N
Maruyama
Yoshiko
Y
Tanabe
Shihori
S
Kobayashi
Hiroyuki
H
Fukutomi
Masashi
M
Nagamatsu
Yuichi
Y
Nishihara
Naoki
N
Inoue
Ryuji
R
Sumimoto
Hideki
H
Shibasaki
Futoshi
F
Nagao
Taku
T
Nishida
Motohiro
M
Kurose
Hitoshi
H
D021581:Active Transport, Cell Nucleus; D000256:Adenoviridae / Q000235:genetics; D000804:Angiotensin II / Q000737:chemistry; D000818:Animals; D000873:Anthracenes / Q000494:pharmacology; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000378:metabolism*; D044365:GTP-Binding Protein alpha Subunits, G12-G13 / Q000378:metabolism*; D005799:Genes, Dominant; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000378:metabolism; D006352:Heart Ventricles / Q000166:cytology; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D048670:MAP Kinase Kinase 4; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D008954:Models, Biological; D008956:Models, Chemical; D009206:Myocardium / Q000378:metabolism*; D019255:NADPH Oxidases / Q000378:metabolism; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins / Q000378:metabolism*; D009861:Onium Compounds / Q000494:pharmacology; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species; D011945:Receptors, Angiotensin / Q000378:metabolism*; D015398:Signal Transduction; D013997:Time Factors; D014157:Transcription Factors / Q000378:metabolism*; D015533:Transcriptional Activation; D020744:rac GTP-Binding Proteins / Q000235:genetics
D000873:Anthracenes; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D009861:Onium Compounds; D010750:Phosphoproteins; D017382:Reactive Oxygen Species; D011945:Receptors, Angiotensin; D014157:Transcription Factors; D000804:Angiotensin II; D049452:Green Fluorescent Proteins; C432165:pyrazolanthrone; C007517:diphenyleneiodonium; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D048031:JNK Mitogen-Activated Protein Kinases; D048670:MAP Kinase Kinase 4; D020929:Mitogen-Activated Protein Kinase Kinases; D044365:GTP-Binding Protein alpha Subunits, G12-G13; D020744:rac GTP-Binding Proteins; D002118:Calcium