Angiotensin II upregulates Kv1.5 expression through ROS-dependent transforming growth factor-beta1 and extracellular signal-regulated kinase 1/2 signalings in neonatal rat atrial myocytes

Kv1.5 potassium channel represents a promising target for atrial fibrillation (AF) therapy. During AF, the renin–angiotensin system is markedly activated. Recent evidence indicates that angiotensin II (Ang II) can upregulate Kv1.5 channel, but the mechanism remains unknown. In this study, we report that Ang II-mediated transforming growth factor-beta1 (TGF-β₁)/Smad2/3 and extracellular signal-regulated kinase (ERK) 1/2 signalings are involved in atrial Kv1.5 expression. In neonatal rat atrial myocytes, quantitative PCR and Western blotting revealed that Ang II upregulated TGF-β₁, synapse-associated protein 97 (SAP97) and Kv1.5 expression in a time- and concentration-dependent manner. The Ang II-induced upregulation of Kv1.5, SAP97 and phosphorylated Smad2/3 (P-Smad2/3) were reversed by the Ang II type 1 (AT₁) receptor antagonist losartan, an anti-TGF-β₁ antibody and the ERK 1/2 inhibitor PD98059 but not by the AT₂ receptor antagonist PD123319. mRNA knockdown of either Smad2 or Smad3 blocked Ang II-induced expression of Kv1.5 and SAP97. These data suggest that AT₁ receptor/TGF-β₁/P-Smad2/3 and ERK 1/2 signalings are involved in Ang II-induced Kv1.5 and SAP97 expression. Flow cytometry and Western blotting revealed that losartan and the anti-TGF-β₁ antibody diminished Ang II-induced reactive oxygen species (ROS) generation and that the antioxidants diphenyleneiodonium and N-acetyl cysteine inhibited Ang II-induced expression of P-Smad2/3, phosphorylated ERK (P-ERK) 1/2, Kv1.5, SAP97, suggesting that ROS participate in Kv1.5 and SAP97 regulation by modulating Ang II-induced P-Smad2/3 and P-ERK 1/2 expression. In conclusion, we demonstrate that ROS-dependent Ang II/AT₁ receptor/TGF-β₁/P-Smad2/3 and Ang II/ERK 1/2 signalings are involved in atrial Kv1.5 and SAP97 expression. Antioxidants would be beneficial for AF treatment through inhibiting atrial Kv1.5 expression.     

Angiotensin II downregulates catalase expression and activity in vascular adventitial fibroblasts through an AT1R/ERK1/2-dependent pathway

Angiotensin II (Ang II) plays a profound regulatory effect on NADPH oxidase and the functional features of vascular adventitial fibroblasts, but its role in antioxidant enzyme defense remains unclear. This study investigated the effect of Ang II on expressions and activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) in adventitial fibroblasts and the possible mechanism involved. Ang II decreased the expression and activity of CAT in a dose- and time-dependent manner, but not that of SOD and GPx. The effects were abolished by the angiotensin II type 1 receptor (AT1R) blocker losartan and AT1R small-interfering RNA (siRNA). Incubation with polyethylene glycol-CAT prevented the Ang II-induced effects on reactive oxygen species (ROS) generation and myofibroblast differentiation. Moreover, Ang II rapidly induced phosphorylation of ERK1/2, which was reversed by losartan and AT1R siRNA. Pharmacological blockade of ERK1/2 improved Ang II-induced decrease in CAT protein expression. These in vitro results indicate that Ang II induces ERK1/2 activation, contributing to the downregulation of CAT as well as promoting oxidative stress and adventitial fibroblast phenotypic differentiation in an AT1R-mediated manner.     

Roscovitine inhibits ERK1/2 activation induced by angiotensin II in vascular smooth muscle cells

Roscovitine is a potent CDK inhibitor often used as a biological tool in cell-cycle studies, but its working mechanism and real targets in vascular smooth muscle cells (VSMCs) remain unclear. In this study, we observed that ERK1/2 phosphorylation induced by Ang II was abrogated by pretreating VSMCs with roscovitine for 15h. Pretreating VSMCs with roscovitine also inhibited Ang II-induced c-Jun expression and phosphorylation. We further demonstrated that roscovitine could suppress the DNA binding activity of c-Jun and activation of angiotensinogen promoter by Ang II. These results suggest that roscovitine represses Ang II-induced angiotensinogen expression by inhibiting activation of ERK1/2 and c-Jun.     

Redox-dependent MAP kinase signaling by Ang II in vascular smooth muscle cells: role of receptor tyrosine kinase transactivation

We investigated the role of receptor tyrosine kinases in Ang II-stimulated generation of reactive oxygen species (ROS) and assessed whether MAP kinase signaling by Ang II is mediated via redox-sensitive pathways. Production of ROS and activation of NADPH oxidase were determined by DCFDA (dichlorodihydrofluorescein diacetate; 2 micromol/L) fluorescence and lucigenin (5 micromol/L) chemiluminescence, respectively, in rat vascular smooth muscle cells (VSMC). Phosphorylation of ERK1/2, p38MAP kinase and ERK5 was determined by immunoblotting. The role of insulin-like growth factor-1 receptor (IGF-1R) and epidermal growth factor receptor (EGFR) was assessed with the antagonists AG1024 and AG1478, respectively. ROS bioavailability was manipulated with Tiron (10(-5) mol/L), an intracellular scavenger, and diphenylene iodinium (DPI; 10(-6) mol/L), an NADPH oxidase inhibitor. Ang II stimulated NADPH oxidase activity and dose-dependently increased ROS production (p < 0.05). These actions were reduced by AG1024 and AG1478. Ang II-induced ERK1/2 phosphorylation (276% of control) was decreased by AG1478 and AG1024. Neither DPI nor tiron influenced Ang II-stimulated ERK1/2 activity. Ang II increased phosphorylation of p38 MAP kinase (204% of control) and ERK5 (278% of control). These effects were reduced by AG1024 and AG1478 and almost abolished by DPI and tiron. Thus Ang II stimulates production of NADPH-inducible ROS partially through transactivation of IGF-1R and EGFR. Inhibition of receptor tyrosine kinases and reduced ROS bioavaliability attenuated Ang II-induced phosphorylation of p38 MAP kinase and ERK5, but not of ERK1/2. These findings suggest that Ang II activates p38MAP kinase and ERK5 via redox-dependent cascades that are regulated by IGF-1R and EGFR transactivation. ERK1/2 regulation by Ang II is via redox-insensitive pathways.     

Rho plays an important role in angiotensin II-induced hypertrophic responses in cardiac myocytes

Angiotensin II (Ang II) evokes a variety of hypertrophic responses such as activation of protein kinases, reprogramming of gene expressions and an increase in protein synthesis in cardiac myocytes. In this study, we examined the role of Rho family small GTP binding proteins (G proteins) in Ang II-induced cardiac hypertrophy. Ang II strongly activated extracellular signal-regulated protein kinases (ERKs) in cardiac myocytes of neonatal rats. Although Ang II-induced activation of ERKs was completely suppressed by an Ang II type 1 receptor antagonist, CV-11974, this activation was not inhibited by the pretreatment with C3 exoenzyme, which abrogates Rho functions. Overexpression of Rho GDP dissociation inhibitor (Rho-GDI), dominant negative mutants of Rac1 (D.N.Rac1), or D.N.Cdc42 had no effects on Ang II-induced activation of transfected ERK2. The promoter activity of skeletal a-actin and c-fos genes was increased by Ang II, and the increase was partly inhibited by overexpression of Rho-GDI and the pretreatment with C3 exoenzyme. Ang II increased phenylalanine incorporation into cardiac myocytes by approximately 1.4 fold as compared with control, and this increase was also significantly suppressed by the pretreatment with C3 exoenzyme. These results suggest that the Rho family small G proteins play important roles in Ang II-induced hypertrophic responses in cardiac myocytes.     

Angiotensin II induces the production of MMP-3 and MMP-13 through the MAPK signaling pathways via the AT1 receptor in osteoblasts

Angiotensin II (Ang II) plays an important role in the maintenance of bone mass and integrity by activation of the mitogen-activated protein kinases (MAPKs) and by modulation of balance between resorption by osteoclasts and formation by osteoblasts. However, the role of Ang II in the turnover of extracellular matrix (ECM) in osteoid by osteoblasts remains unclear. Therefore, we examined the effect of Ang II on the expression of matrix metalloproteinases (MMPs), plasminogen activators (PAs), and their inhibitors [i.e., tissue inhibitors of metalloproteinases (TIMPs) and PA inhibitor-1 (PAI-1)] using osteoblastic ROS17/2.8 cells. Treatment with Ang II strikingly increased the expressions of MMP-3 and -13 and promoted cell proliferation associated with reduced alkaline phosphatase activity as well as enhanced phosphorylated expression of extracellular signal-regulated kinase (ERK)1/2, p38 MAPK, and stress-activated protein kinases/c-jun N-terminal kinases (SAPK/JNK) in ROS17/2.8 cells. However, Ang II had no effect on the expression of MMP-2, -9, -14, urokinase-type PA, tissue-type PA, TIMP-1, -2, -3, and PAI-1 in cells. Losartan (AT₁ receptor blocker) blocked Ang II-induced expression of MMP-3 and -13, whereas PD123319 (AT₂ receptor blocker) did not completely block these responses. Losartan also blocked the Ang II-induced phosphorylation of ERK1/2, p38 MAPK, and SAPK/JNK. MAPK kinase 1/2 inhibitor PD98059 and JNK inhibitor SP600125 suppressed Ang II-induced expression of MMP-3 and -13. These results suggested that Ang II stimulated the degradation process that occurs during ECM turnover in osteoid by increasing the production of MMP-3 and -13 through MAPK signaling pathways via the AT₁ receptor in osteoblasts. Furthermore, our findings suggest that Ang II does not influence the plasminogen/plasmin pathway in osteoblasts.     

Independence of angiotensin II-induced MAP kinase activation from angiotensin type 1 receptor internalization in clone 9 hepatocytes

The agonist-induced internalization of several G protein-coupled receptors is an obligatory requirement for their activation of MAPKs. Studies on the relationship between endocytosis of the angiotensin II (Ang II) type 1 receptor (AT1-R) and Ang II-induced ERK1/2 activation were performed in clone 9 (C9) rat hepatic cells treated with inhibitors of endocytosis [sucrose, phenylarsine oxide (PAO), and concanavalin A]. Although Ang II-induced endocytosis of the AT1-R was prevented by sucrose and PAO, and was partially inhibited by concanavalin A, there was no impairment of Ang II-induced ERK activation. However, the specific epidermal growth factor receptor (EGF-R) kinase inhibitor, AG1478, abolished Ang II-induced activation of ERK1/2. Sucrose and PAO also inhibited EGFinduced internalization of the EGF-R in C9 cells, and the inability of these agents to impair EGF-induced ERK activation suggested that the latter is also independent of receptor endocytosis. In COS-7 cells transiently expressing the rat AT1A-R, Ang II also caused ERK activation through EGF-R transactivation. Furthermore, a mutant AT1A-R with truncated carboxyl terminus and impaired internalization retained full ability to activate ERK1/2 in response to Ang II stimulation. These findings demonstrate that Ang II-induced ERK1/2 activation in C9 hepatocytes is independent of both AT1-R and EGF-R endocytosis and is mediated by transactivation of the EGF-R.     

Candesartan attenuates Angiotensin II-induced mesangial cell apoptosis via TLR4/MyD88 pathway

Angiotensin II (Ang II) can stimulate Toll-like receptor 4 (TLR4) expression in mesangial cells (MCs), but the role of TLR4 in the Ang II-induced apoptosis and the effect of candesartan on TLR4 expression remain unclear. Here, we report that Ang II-induced MC apoptosis in a time-dependent manner and up-regulated TLR4/MyD88 expression, and that the intracellular ROS was subsequently increased. We also show that candesartan attenuated the Ang II-induced MC apoptosis, and that this protective effect was dependent on decreased TLR4/MyD88 expression as well as reduced intracellular ROS formation. Furthermore, Ang II increased the apoptosis inducing factor protein level, while candesartan markedly reduced this increase. These results demonstrate that TLR4/MyD88 pathway was involved in the Ang II promoted MC apoptosis, which was related to TLR4/MyD88 mediated oxidative stress. These data also suggest that candesartan exerted anti-apoptotic effect as an antioxidant by modulating this pathway.     

Nox4 NAD(P)H oxidase mediates Src-dependent tyrosine phosphorylation of PDK-1 in response to angiotensin II: role in mesangial cell hypertrophy and fibronectin expression

Activation of glomerular mesangial cells (MCs) by angiotensin II (Ang II) leads to hypertrophy and extracellular matrix accumulation. Here, we demonstrate that, in MCs, Ang II induces an increase in PDK-1 (3-phosphoinositide-dependent protein kinase-1) kinase activity that required its phosphorylation on tyrosine 9 and 373/376. Introduction into the cells of PDK-1, mutated on these tyrosine residues or kinase-inactive, attenuates Ang II-induced hypertrophy and fibronectin accumulation. Ang II-mediated PDK-1 activation and tyrosine phosphorylation (total and on residues 9 and 373/376) are inhibited in cells transfected with small interfering RNA for Src, indicating that Src is upstream of PDK-1. In cells expressing oxidation-resistant Src mutant C487A, Ang II-induced hypertrophy and fibronectin expression are prevented, suggesting that the pathway is redox-sensitive. Ang II also up-regulates Nox4 protein, and siNox4 abrogates the Ang II-induced increase in intracellular reactive oxygen species (ROS) generation. Small interfering RNA for Nox4 also inhibits Ang II-induced activation of Src and PDK-1 tyrosine phosphorylation (total and on residues 9 and 373/376), demonstrating that Nox4 functions upstream of Src and PDK-1. Importantly, inhibition of Nox4, Src, or PDK-1 prevents the stimulatory effect of Ang II on fibronectin accumulation and cell hypertrophy. This work provides the first evidence that Nox4-derived ROS are responsible for Ang II-induced PDK-1 tyrosine phosphorylation and activation through stimulation of Src. Importantly, this pathway contributes to Ang II-induced MC hypertrophy and fibronectin accumulation. These data shed light on molecular processes underlying the oxidative signaling cascade engaged by Ang II and identify potential targets for intervention to prevent renal hypertrophy and fibrosis.     

LPS regulate ERK1/2-dependent signaling in cardiac fibroblasts via PKC-mediated MKP-1 induction

Activation of MAPK pathways by angiotensin II (Ang II) is important for cardiac fibroblast (CFB) proliferation and migration. Activity of MAP-kinases is closely controlled by a group of dual-specific MAP kinase phosphatases (MKPs). Lipopolysaccharides (LPS) and cytokines are elevated in patients with heart failure and may contribute to disease progression. In this study, we investigate the effect of LPS on Ang II-induced CFB function. Pretreatment of CFBs with LPS (1 microg/mL; 30 min) almost completely inhibited Ang II-induced DNA-synthesis and inhibited Ang II directed chemotaxis by more than 80%. Compared to controls, LPS pretreatment significantly reduced phosphorylation levels of ERK1/2- and p38 MAPK and induced MKP-1 levels. Silencing MKP-1 with antisense oligodesoxynucleotides reversed the antimitogenic effect of LPS on Ang II-induced CFB DNA-synthesis and migration. Induction of MKP-1 by LPS was inhibited by the protein kinase C (PKC)-inhibitor calphostin C, but not by the ERK1/2-pathway inhibitor PD98059, suggesting that PKC but not ERK1/2 is required for LPS-mediated MKP-1 induction in CFBs. Our data demonstrate that LPS have direct cellular effects in CFBs through an inhibition of Ang II-induced MAPK activity via PKC-mediated induction of MKP-1. This might be relevant with regard to the decreased MAPK activity and increased levels in MKPs reported during chronic heart failure in humans.     

Angiotensin II stimulates ERK via two pathways in epithelial cells: protein kinase C suppresses a G-protein coupled receptor-EGF receptor transactivation pathway.

In GN4 rat liver epithelial cells, angiotensin II (Ang II) produces intracellular calcium and protein kinase C (PKC) signals and stimulates ERK and JNK activity. JNK activation appears to be mediated by a calcium-dependent tyrosine kinase (CADTK). To define the ERK pathway, we established GN4 cells expressing an inhibitory Ras(N17). Induction of Ras(N17) blocked EGF- but not Ang II- or phorbol ester (TPA)-dependent ERK activation. In control cells, Ang II and TPA produced minimal increases in Ras-GTP level and Raf kinase activity. PKC depletion by chronic TPA exposure abolished TPA-dependent ERK activation but failed to diminish the effect of Ang II. In PKC-depleted cells, Ang II increased Ras-GTP level and activated Raf and ERK in a Ras-dependent manner. In PKC depleted cells, Ang II stimulated Shc and Cbl tyrosine phosphorylation, suggesting that without PKC, Ang II activates another tyrosine kinase. PKC-depletion did not alter Ang II-dependent tyrosine phosphorylation or activity of p125(FAK), CADTK, Fyn or Src, but PKC depletion or incubation with GF109203X resulted in Ang II-dependent EGF receptor tyrosine phosphorylation. In PKC-depleted cells, EGF receptor-specific tyrosine kinase inhibitors blocked Ang II-dependent EGF receptor and Cbl tyrosine phosphorylation, and ERK activation. In summary, Ang II can activate ERK via two pathways; the latent EGF receptor, Ras-dependent pathway is equipotent to the Ras-independent pathway, but is masked by PKC action. The prominence of this G-protein coupled receptor to EGF receptor pathway may vary between cell types depending upon modifiers such as PKC.     

Propofol depresses angiotensin II-induced cardiomyocyte hypertrophy in vitro

Cardiomyocyte hypertrophy is formed in response to pressure or volume overload, injury, or neurohormonal activation. The most important vascular hormone that contributes to the development of hypertrophy is angiotensin II (Ang II). Accumulating studies have suggested that reactive oxygen species (ROS) may play an important role in cardiac hypertrophy. Propofol is a general anesthetic that possesses antioxidant action. We therefore examined whether propofol inhibited Ang II-induced cardiomyocyte hypertrophy. Our results showed that both ROS formation and hypertrophic responses induced by Ang II in cardiomyocytes were partially blocked by propofol. Further studies showed that propofol inhibited the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and mitogen-activated protein kinase/ERK kinase 1/2 (MEK1/2) induced by Ang II via a decrease in ROS production. In addition, propofol also markedly attenuated Ang II-stimulated nuclear factor-kappaB (NF-kappaB) activation via a decrease in ROS production. In conclusion, propofol prevents cardiomyocyte hypertrophy by interfering with the generation of ROS and involves the inhibition of the MEK/ERK signaling transduction pathway and NF-kappaB activation.     

YS 49, 1-(alpha-naphtylmethyl)-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline, regulates angiotensin II-stimulated ROS production, JNK phosphorylation and vascular smooth muscle cell proliferation via the induction of heme oxygenase-1

Overexpression of the gene for heme oxygenase (HO)-1 leads to a reduction in pressor responsiveness to angiotensin II (Ang II) in experimental animals. Using rat vascular smooth muscle cells (VSMCs), we tested whether YS 49 [1-(alpha-naphtylmethyl)-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline] inhibits Ang II-stimulated proliferation of VSMCs via induction of HO-1. YS 49 induced HO-1 protein production in a dose-and time-dependent manner in VSMCs. Treatment with YS 49 significantly and dose-dependently inhibited Ang II-induced VSMC proliferation, ROS production, and phosphorylation of JNK, but not P38 MAP kinase or ERK1/2. The antiproliferation effect of YS 49 was reversed by pretreatment with the HO-1 inhibitor zinc protoporphyrin IX (ZnPPIX), or with hemoglobin, a carbon monoxide (CO) scavenger. Similarly, VSMC proliferation, ROS production and phosphorylation of JNK by Ang II were significantly inhibited in VSMCs transfected with the HO-1 gene. Thus, HO-1 and the HO-1 product CO play, at least in part, a crucial role in Ang II-stimulated VSMC proliferation through the regulation of ROS production and JNK phosphorylation. Therefore, YS 49 has potential as a therapeutic strategy for the pathogenesis of Ang II-related vascular diseases such as hypertension and atherosclerosis, via the induction of HO-1 gene activity.     

ERK1/2 and JNKs, but not p38 kinase, are involved in reactive oxygen species-mediated induction of osteopontin gene expression by angiotensin II and interleukin-1beta in adult rat cardiac fibroblasts

Osteopontin (OPN), also called cytokine Eta-1, expressed in the myocardium co-incident with heart failure plays an important role in post myocardial infarction (MI) remodeling by promoting collagen synthesis and accumulation. Angiotensin II (Ang II) and inflammatory cytokines are increased in the heart following MI. We studied the involvement of mitogen-activated protein kinases (ERK1/2, JNKs, p38 kinase) and reactive oxygen species (ROS) in Ang II- and cytokine-induced OPN gene expression in adult rat cardiac fibroblasts. Ang II alone increased OPN mRNA (3.3 +/- 0.3-folds; P < 0.05; n = 7), while interleukin-1beta (IL-1beta), tumor necrosis factor (TNF-alpha), and interferon-gamma (IFN-gamma) had no effect. A combination of Ang II with IL-1beta or TNF-alpha, not IFN-gamma, increased OPN mRNA more than Ang II alone. Nitric oxide donor, S-nitrosoacetylpenicillamine (SNAP), alone or in combination with Ang II had no effect. Diphenylene iodonium (DPI), inhibitor of NAD(P)H oxidase, and tiron, superoxide scavenger, inhibited Ang II- and Ang II+ IL-1beta-stimulated increases in OPN mRNA. Ang II activated ERK1/2 within 5 min of treatment, not JNKs. IL-1beta activated ERK1/2 and JNKs within 15 min of treatment. A combination of Ang II and IL-1beta activated ERK1/2 within 5 min of treatment. None of these stimuli activated p38 kinase. DPI almost completely inhibited Ang II + IL-1beta-stimulated activation of ERK1/2, while partially inhibiting JNKs. PD98059, ERK1/2 pathway inhibitor, and SP600125, JNKs inhibitor, partially inhibited Ang II + IL-1beta-stimulated increases in OPN mRNA. A combination of PD98059 and SP600125 almost completely inhibited Ang II + IL-1beta-stimulated increases in OPN mRNA. Thus, Ang II alone increases OPN expression, while IL-1beta and TNF-alpha act synergistically with Ang II to increase OPN mRNA possibly via NO independent mechanisms. The synergistic increase in OPN mRNA involves ROS-mediated activation of ERK1/2 and JNKs, not P38 kinase, pathways in cardiac fibroblasts.     

Activation of ERK5 in angiotensin II-induced hypertrophy of human aortic smooth muscle cells

Extracellular signal-regulated kinase 5 (ERK5), a recently discovered mitogen-activated protein kinase (MAPK), plays a key role in the development and pathogenesis of cardiovascular disease. In order to clarify the pathophysiological significance of ERK5 in vascular remodeling, we investigated ERK5 phosphorylation in hypertrophy of human aortic smooth muscle cells (HASMCs) induced by angiotensin II (Ang II). The AT1 receptor was involved in Ang II-induced ERK5 activity. Hypertrophy was detected by the measurement of protein synthesis with [³H]-Leu incorporation in cultured HASMCs. Ang II rapidly induced phosphorylation of ERK5 at Thr218/Tyr220 residues in a time- and dose-dependent manner. Activation of myocyte enhancer factor-2C (MEF2C) by ERK5 was inhibited by PD98059. Transfecting HASMCs with small interfering RNA (siRNA) to silence ERK5 inhibited Ang II-induced cell hypertrophy. Thus, ERK5 phosphorylation contributes to MEF2C activation and subsequent HASMC hypertrophy induced by Ang II, for a novel molecular mechanism in cardiovascular diseases induced by Ang II.