Fujii T, Onohara N, Maruyama Y, Tanabe S, Kobayashi H, Fukutomi M, Nagamatsu Y, Nishihara N, Inoue R, Sumimoto H, Shibasaki F, Nagao T, Nishida M, Kurose H.
Galpha12/13-mediated production of reactive oxygen species is critical for angiotensin receptor-induced NFAT activation in cardiac fibroblasts.
J. Biol. Chem.
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.
Active Transport, Cell Nucleus
Adenoviridae / genetics
Angiotensin II / chemistry
Anthracenes / pharmacology
Calcium / metabolism
Cell Nucleus / metabolism
DNA-Binding Proteins / metabolism*
Enzyme Inhibitors / pharmacology
Fibroblasts / metabolism*
GTP-Binding Protein alpha Subunits, G12-G13 / metabolism*
Green Fluorescent Proteins / chemistry
Green Fluorescent Proteins / metabolism
Heart Ventricles / cytology
JNK Mitogen-Activated Protein Kinases / metabolism
MAP Kinase Kinase 4
Mitogen-Activated Protein Kinase Kinases / metabolism
Myocardium / metabolism*
NADPH Oxidases / metabolism
NFATC Transcription Factors
Nuclear Proteins / metabolism*
Onium Compounds / pharmacology
Phosphoproteins / metabolism
Plasmids / metabolism
Protein Structure, Tertiary
Reactive Oxygen Species*
Receptors, Angiotensin / metabolism*
Transcription Factors / metabolism*
rac GTP-Binding Proteins / genetics
BIOCHEMISTRY & MOLECULAR BIOLOGY