Satoru Eguchi

Calcium-dependent Epidermal Growth Factor Receptor Transactivation Mediates the Angiotensin II-induced Mitogen-activated Protein Kinase Activation in Vascular Smooth Muscle Cells

We have recently reported that angiotensin II (Ang II)-induced mitogen-activated protein kinase (MAPK) activation is mainly mediated by Ca2+-dependent activation of a protein tyrosine kinase through Gq-coupled Ang II type 1 receptor in cultured rat vascular smooth muscle cells (VSMC). In the present study, we found Ang II rapidly induced the tyrosine phosphorylation of the epidermal growth factor (EGF) receptor and its association with Shc and Grb2. These reactions were inhibited by the EGF receptor kinase inhibitor, AG1478. The Ang II-induced phosphorylation of the EGF receptor was mimicked by a Ca2+ionophore and completely inhibited by an intracellular Ca2+chelator. Thus, AG1478 abolished the MAPK activation induced by Ang II, a Ca2+ ionophore as well as EGF but not by a phorbol ester or platelet-derived growth factor-BB in the VSMC. Moreover, Ang II induced association of EGF receptor with catalytically active c-Src. This reaction was not affected by AG1478. These data indicate that Ang II induces Ca2+-dependent transactivation of the EGF receptor which serves as a scaffold for pre-activated c-Src and for downstream adaptors, leading to MAPK activation in VSMC.

Activation of MAPKs by Angiotensin II in Vascular Smooth Muscle Cells METALLOPROTEASE-DEPENDENT EGF RECEPTOR ACTIVATION IS REQUIRED FOR ACTIVATION OF ERK AND p38 MAPK BUT NOT FOR JNK

In cultured vascular smooth muscle cells (VSMC), the vasculotrophic factor, angiotensin II (AngII) activates three major MAPKs via the Gq-coupled AT1 receptor. Extracellular signal-regulated kinase (ERK) activation by AngII requires Ca2+-dependent “transactivation” of the EGF receptor that may involve a metalloprotease to stimulate processing of an EGF receptor ligand from its precursor. Whether EGF receptor transactivation also contributes to activation of other members of MAPKs such as p38MAPK and c-Jun N-terminal kinase (JNK) by AngII remains unclear. In the present study, we have examined the effects of a synthetic metalloprotease inhibitor BB2116, and the EGF receptor kinase inhibitor AG1478 on AngII-induced activation of MAPKs in cultured VSMC. BB2116 markedly inhibited ERK activation induced by AngII or the Ca2+ ionophore A23187 without affecting the activation by EGF or PDGF. BB2116 as well as HB-EGF neutralizing antibody inhibited the EGF receptor transactivation by AngII, suggesting a critical role of HB-EGF in the metalloprotease-dependent EGF receptor transactivation. In addition to the ERK activation, activation of p38MAPK and JNK by AngII was inhibited by an AT1 receptor antagonist, RNH6270.A23187 and EGF markedly activate p38MAPK, whereas A23187 but not EGF markedly activates JNK, indicating the possible contribution of the EGF receptor transactivation to the p38MAPK activation. The findings that both BB2116 and AG1478 specifically inhibited activation of p38MAPK but not JNK by AngII support this hypothesis. From these data, we conclude that ERK and p38MAPK activation by AngII requires the metalloprotease-dependent EGF receptor transactivation, whereas the JNK activation is regulated without involvement of EGF receptor transactivation.

Mechanotransduction of Rat Aortic Vascular Smooth Muscle Cells Requires RhoA and Intact Actin Filaments

The growth-promoting effect of mechanical stress on vascular smooth muscle cells (VSMCs) has been implicated in the progress of vascular disease in hypertension. Extracellular signal-regulated kinases (ERKs) have been implicated in cellular responses, such as vascular remodeling, induced by mechanical stretch. However, it remains to be determined how mechanical stretch activates ERKs. The cytoskeleton seems the most likely candidate for force transmission into the interior of the cell. Therefore, we examined (1) whether the cytoskeleton involves mechanical stretch-induced signaling, (2) whether Rho is activated by stretch, and (3) whether Rho mediates the stretch-induced signaling in rat cultured VSMCs. Mechanical stretch activated ERKs, with a peak response observed at 20 minutes, followed by a significant increase in DNA synthesis. Treatment with the ERK kinase-1 inhibitor, PD98059, inhibited the stretch-induced increase in DNA synthesis. Cytochalasin D, which selectively disrupts the network of actin filaments, markedly inhibited stretch-induced ERK activation. In the control state, RhoA was observed predominantly in the cytosolic fraction, but it was translocated in part to the particulate fraction in response to mechanical stretch. Botulinum C3 exoenzyme, which inactivates Rho p21 (known to participate in the reorganization of the actin cytoskeleton), attenuated stretch-induced ERK activation. Inhibition of Rho kinase (p160ROCK) also suppressed stretch-induced ERK activation dose dependently. Our results suggest that mechanotransduction in VSMCs is dependent on intact actin filaments, that Rho is activated by stretch, and that Rho/p160ROCK mediates stretch-induced ERK activation and vascular hyperplasia.

Intracellular Signaling of Angiotensin II-induced p70 S6 Kinase Phosphorylation at Ser411 in Vascular Smooth Muscle Cells POSSIBLE REQUIREMENT OF EPIDERMAL GROWTH FACTOR RECEPTOR, RAS, EXTRACELLULAR SIGNAL-REGULATED KINASE, AND AKT

Activation of p70 S6 kinase (p70S6K) by growth factors requires multiple signal inputs involving phosphoinositide 3-kinase (PI3K), its effector Akt, and an unidentified kinase that phosphorylates Ser/Thr residues (Ser411, Ser418, Ser424, and Thr421) clustered at its autoinhibitory domain. However, the mechanism by which G protein-coupled receptors activate p70S6K remains largely uncertain. By using vascular smooth muscle cells in which we have demonstrated Ras/extracellular signal-regulated kinase (ERK) activation through Ca2+-dependent, epidermal growth factor (EGF) receptor transactivation by Gq-coupled angiotensin II (Ang II) receptor, we present a unique cross-talk required for Ser411 phosphorylation of p70S6K by Ang II. Both p70S6K Ser411 and Akt Ser473phosphorylation by Ang II appear to involve EGF receptor transactivation and were inhibited by dominant-negative Ras, whereas the phosphorylation of p70S6K and ERK but not Akt was sensitive to the MEK inhibitor. By contrast, the phosphorylation of p70S6K and Akt but not ERK was sensitive to PI3K inhibitors. Similar inhibitory pattern on these phosphorylation sites by EGF but not insulin was observed. Taken together with the inhibition of Ang II-induced p70S6K activation by dominant-negative Ras and the MEK inhibitor, we conclude that Ang II-initiated activation of p70S6K requires both ERK cascade and PI3K/Akt cascade that bifurcate at the point of EGF receptor-dependent Ras activation.

Angiotensin II–Induced Cardiac Hypertrophy and Hypertension Are Attenuated by Epidermal Growth Factor Receptor Antisense

Background—Angiotensin II (Ang II) is a vasoconstrictor but also a growth factor. However, the Ang II type 1 receptor does not have a tyrosine kinase domain that mediates the cellular signals for mitosis. We have shown that Ang II acts via “trans”-activation of the epidermal growth factor receptor (EGFR) to induce activation of tyrosine kinase and mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) in vascular smooth muscle cells (VSMCs). To examine whether EGFR is involved in the development of left ventricular hypertrophy (LVH), we inhibited EGFR with a specific antisense oligodeoxynucleotide to attenuate the Ang II–induced cardiovascular hypertrophic effects. Methods and Results—The antisense oligodeoxynucleotide to EGFR (EGFR-AS) was designed and tested on Ang II–induced ERK activation in cultured VSMCs. We also investigated the effects of EGFR-AS on LVH and blood pressure (BP) in Ang II–infused hypertensive rats. In VSMCs, EGFR-AS (2.5 &mgr;mol/L) reduced EGFR expression and inhibited the Ang II–induced phosphorylation of ERK. In rats, Ang II (150 ng/h for 14 days) increased BP compared with controls (184±6 mm Hg versus 122±3 mm Hg; n=7;P <0.01). Continuous intravenous infusion of EGFR-AS (2 mg/kg) decreased BP (169±8 mm Hg; n=8;P <0.05). Ang II infusion increased the left ventricular/body weight (LV/BW) ratio compared with control rats (2.75±0.08 versus 2.33±0.07;P <0.01). EGFR-AS, but not EGFR-sense, normalized the LV/BW in Ang II–infused rats (2.32±0.06;P <0.01) and attenuated Ang II–enhanced EGFR expression and ERK phosphorylation. Conclusion—Ang II requires EGFR to mediate ERK activation in VSMCs and the heart. EGFR plays a critical role in the LVH induced by Ang II.

β-Adrenergic receptor-mediated DNA synthesis in cardiac fibroblasts is dependent on transactivation of the epidermal growth factor receptor and subsequent activation of extracellular signal-regulated kinases.

Cardiac hypertrophy often leads to heart failure and is associated with abnormal myocardial adrenergic signaling. This enlargement of myocardial mass can involve not only an increase in cardiomyocyte size, but increased proliferation of cardiac fibroblasts. A potential key player in the cardiac hypertrophic response is the ERK family of MAPKs. To gain mechanistic insight into adrenergic regulation of myocardial mitogenic signaling, we examined β-adrenergic receptor (β-AR) stimulation of ERK activation and DNA synthesis in cultured adult rat cardiac fibroblasts, including the involvement of tyrosine kinases in this signaling pathway. Addition of the β-AR agonist isoproterenol (ISO) to serum-starved cells induced DNA synthesis in a dose-dependent manner, and this was inhibited by selective inhibitors of the epidermal growth factor receptor (EGFR). Importantly and in agreement with the involvement of MAPKs and the EGFR in this response in cardiac fibroblasts, the EGFR inhibitor AG1478 attenuated ISO-induced ERK phosphorylation. Moreover, pretreatment with PP2, a selective inhibitor of the Src tyrosine kinase, attenuated both ISO-mediated EGFR phosphorylation and ERK activation. Furthermore, studies in these cardiac fibroblasts showed that phosphatidylinositol 3-kinase contributed to β-AR-mediated ERK activation, but not to EGFR activation. Finally, studies using selective inhibitors of matrix metalloproteases indicated that they and heparin-bound EGF shedding were involved in β-AR-induced ERK activation and subsequent DNA synthesis in cardiac fibroblasts. Because these cells primarily express the β2-AR subtype, our findings indicate that β2-AR-mediated EGFR transactivation of intracellular tyrosine kinase signaling pathways is the major signaling pathway responsible for the adrenergic stimulation of mitogenesis of cardiac fibroblasts.

Involvement of Reactive Oxygen Species in the Activation of Tyrosine Kinase and Extracellular Signal-Regulated Kinase by Angiotensin II.

Reactive oxygen species (ROS) have been proposed to mediate vascular hypertrophy induced by angiotensin II (Ang II). Recently, we and others have shown that growth-promoting signals by Ang II involve protein tyrosine kinase (PTK) and extracellular signal-regulated kinase (ERK). However, whether ROS contribute to the Ang II-induced PTK and/or ERK activation in vascular smooth muscle cells (VSMCs) remains largely unclear. Here, we have investigated the possible involvement of ROS in Ang II-induced PTK and ERK activation. In the presence of a NADH/NADPH oxidase inhibitor, diphenyleneiodonium (DPI) or an antioxidant,α -tocopherol, Ang II-induced protein tyrosine phosphorylation of two major proteins (p120, p70) and ERK activation were markedly reduced, whereas ERK activation by epidermal growth factor was unaffected. DPI also inhibited Ang II-induced H2O2 production and PTK activation. In this regard, H2O2 and a membrane permeable thiol-oxidizing agent, diamide, stimulated protein tyrosine phosphorylation of ...

Angiotensin II Type 2 Receptor Inhibits Cell Proliferation and Activates Tyrosine Phosphatase

The angiotensin II type 2 (AT2) receptor inhibits basic fibroblast growth factor-induced proliferation of R3T3 fibroblast cells and transiently stimulates a vanadate-sensitive phosphotyrosine phosphatase, strongly suggesting that AT2 is a mitogen inhibitor. We generated AT2 gene-null mice that showed increased blood pressure, indicating the hypotensive action of AT2. However, inhibition of renomedullary AT2 by selective antagonists, as reported by Sassard and associates, show that AT2 suppresses pressure natriuresis. Thus, both AT1 and AT2 work in the direction of sodium retention, suggesting a unique role for angiotensin II in the kidney in terms of blood pressure regulation and sodium metabolism.

ANGIOTENSIN RECEPTORS: MOLECULAR BIOLOGY AND SIGNALLING

1. The active peptide hormone angiotensin II (AngII) is formed from its prohormone angiotensinogen by way of inactive angiotensin I. The highly specific protease, renin, responsible for the initiation of this system was elusive and considered unstable. We isolated it in a pure and stable form from the kidney of the pig, human, rat, and land submandibular glands of the mouse. It was shown that there is only one type of renin with highly stringent substrate specificity, except certain strains of the mouse which have two gene products.

Metalloprotease inhibitor blocks angiotensin II-induced migration through inhibition of epidermal growth factor receptor transactivation.

In vascular smooth muscle cells (VSMCs), angiotensin II (AngII) induces transactivation of the EGF receptor (EGFR) which involves a metalloprotease that stimulates processing of heparin-binding EGF from its precursor. However, the identity and pharmacological sensitivity of the metalloprotease remain unclear. Here, we screened the effects of several metalloprotease inhibitors on AngII-induced EGFR transactivation in VSMCs. We found that an N-phenylsulfonyl-hydroxamic acid derivative [2R-[(4-biphenylsulfonyl)amino]-N-hydroxy-3-phenylpropinamide] (BiPS), previously known as matrix metalloprotease (MMP)-2/9 inhibitor, markedly inhibited AngII-induced EGFR transactivation, whereas the MMP-2 or -9 inhibition by other MMP inhibitors failed to block the transactivation. BiPS markedly inhibited AngII-induced ERK activation and protein synthesis without affecting AngII-induced intracellular Ca2+ elevation. VSMC migration induced by AngII was also inhibited not only by an EGFR inhibitor but also by BiPS. Thus, BiPS is a specific candidate to block AngII-induced EGFR transactivation and subsequent growth and migration of VSMCs, suggesting its potency to prevent vascular remodeling.