Angiotensin II-mediated expression of mitochondrial peroxiredoxin-3 in rat cardiac fibroblasts: Association with oxidative stress in myocardium
Yudi Purnomo, Yvette Piccart, Tamara Coenen, Jos Van Pelt, John S.Prihadi, Paul J.Lijnen
1Hypertension andCardiovascularRehabilitationUnit,Department ofCardiovascularResearch,CatholicUniversity of Leuven(KULeuven),Leuven,(BELGIUM)
2LiverFacility andLaboratoryHepatology,CatholicUniversity ofLeuven(KULeuven),Leuven, (BELGIUM)
The aim of this study was to determine whether angiotensin II (ANG II) affects the protein andmRNAexpression of themitochondrial antioxidant peroxiredoxin- 3 (Prx-3) in cardiac fibroblasts through inducing the phosphorylation of the proteins Akt and FOXO3a, thereby contributing to the oxidative stress in the myocardium.
Cardiac fibroblasts (passage 2) from normal male adult rats were cultured to confluency and incubated in serum-freeDulbecco’s modified Eagle’s medium for 24 h. The cells were then preincubated with (out) the tested inhibitors for 1 h and further incubated with (out) ANG II (1 μmol/l) for 24 h.
ANG II increased (p<0.001) the intracellular and mitochondrial reactive oxygen species (ROS) production in cardiac fibroblasts.ANG II also decreased (p<0.01) the mRNA and protein expression of Prx-3 by 36.9 ± 3.0% and 29.7 ± 2.7%, respectively. This ANG II-induced decrease in Prx-3 expression was blocked by the angiotensin II type 1 receptor blocker losartan and not by the angiotensin II type 2 blocker PD 123,319 The ANG II-released transforming growth factor-â1 did however not affect the ANG II-enhanced intracellular ROS generation. The mitochondrial complex II and IV activity were not affected by ANG II and the mitochondrial complex I and III activity were reduced (p<0.05) by ANG II.
The likelymechanism through whichANG II produces the effect of reducing Prx- 3 expression is by reducing the extent of binding of FOXO3a to the Prx-3 promoter. In control fibroblasts inhibition of FOXO3a transcription with small-interfering RNA (siRNA) led to a reduction in Prx-3 gene expression.
Our data also shown that when Akt is phosphorylated by ANG II, P-Akt is translocated from the cytoplasm to the nucleus, subsequently nuclear phosphorylation of FOXO3a by P-Akt leads to relocalization of FOXO3a from the nucleus to the cytosol, resulting in a decrease in its transcriptional activity, and consequently in Prx-3 expression. The likehood of such a mechanism is further strengthened by the fact that inhibition of phosphoinositide 3-kinase with wortmannin or LY 294002 was shown to lead to a decrease in P-Akt and to a consequent increase in Prx-3 expression.
Our data indicate that ANG II inactivates FOXO3a by activating Akt, leading to a reduction in the expression of the antioxidant Prx-3, and thereby potentially contributing to oxidative stress in the myocard.
Peroxiredoxin-3; Angiotensin II; Cardiac fibroblasts; Reactive oxygen species; Mitochondria; Complex I, II, III, IV activity.