A role for PYK2 in regulation of ERK1/2 MAP kinases and PI 3-kinase by ANG II in vascular smooth muscle

Abnormal vascular smooth muscle cell (VSMC) growth plays a key role in the pathogenesis of hypertension and atherosclerosis. Angiotensin II (ANG II) elicits a hypertrophic growth response characterized by an increase in protein synthesis without cell proliferation. The present study investigated the role of the nonreceptor tyrosine kinase PYK2 in the regulation of ANG II-induced signaling pathways that mediate VSMC growth. Using coimmunoprecipitation analysis, the role of PYK2 as an upstream regulator of both extracellular signal-related kinase (ERK) 1/2 mitogen-activated protein kinase and phosphatidylinositol 3-kinase (PI 3-kinase) pathways was examined in cultured rat aortic VSMC. ANG II (100 nM) promoted the formation of a complex between PYK2 and the ERK1/2 regulators Shc and Grb2. ANG II caused a rapid and Ca(2+)-dependent tyrosine phosphorylation of the adapter molecule p130Cas, which coimmunoprecipitated both PYK2 and PI 3-kinase in ANG II-treated VSMC. Complex formation between PI 3-kinase and p130Cas and PYK2 was associated with a rapid phosphorylation of the ribosomal p70(S6) kinase in a Ca(2+)- and tyrosine kinase-dependent manner. These data suggest that PYK2 is an important regulator of multiple signaling pathways involved in ANG II-induced VSMC growth.     

Reactive oxygen species sensitivity of angiotensin II-dependent translation initiation in vascular smooth muscle cells

Translation initiation, the rate-limiting step in protein synthesis, is a key event in vascular smooth muscle cell growth, a major component of vascular disease. Translation initiation is regulated by interaction between PHAS-I and the eukaryotic initiation factor 4E (eIF4E). Although angiotensin II (Ang II)-induced vascular smooth muscle cell hypertrophy requires the generation of reactive oxygen species (ROS), the ROS sensitivity of these events and their upstream activators remain unclear. Here, we investigated the role of ROS in the regulation of PHAS-I phosphorylation on Thr-70 and Ser-65, an event required for the release of eIF4E from PHAS-I. Ang II-induced Ser-65 phosphorylation was ROS-dependent as assessed by pretreatment with ebselen (3.6 +/- 0.2 versus 1.1 +/- 0.2), diphenylene iodonium (3.6 +/- 0.2 versus 1.0 +/- 0.1), and N-acetyl cysteine (3.6 +/- 0.2 versus 1.2 +/- 0.1), but Ang II-stimulated phosphorylation of Thr-70 was ROS-insensitive. Although phosphatidylinositol 3-kinase pathway inhibition by LY294004 blocked both Ser-65 and Thr-70 phosphorylation (3.8 +/- 0.1 versus 0.8 +/- 0.1 and 3.2 +/- 0.2 versus 1.0 +/- 0.01, respectively), protein phosphatase 2A inhibition by okadaic acid selectively increased (3.3 +/- 0.1 versus 5.2 +/- 0.1) and p38 mitogen-activated protein kinase inhibition by SB203580 selectively decreased (3.8 +/- 0.1 versus 1.4 +/- 0.3) Ser-65 phosphorylation. Dominant negative Akt adenovirus also inhibited only Ser-65 phosphorylation (3.7 +/- 0.1 versus 1.0 +/- 0.03). These results demonstrate a unique differential ROS sensitivity of two separate residues on PHAS-I, which seems to be explained by the selective involvement of distinct signaling pathways in the regulation of these phosphorylation events.     

A role for PYK2 in ANG II-dependent regulation of the PHAS-1-eIF4E complex by multiple signaling cascades in vascular smooth muscle

Regulation of the PHAS-1-eukaryotic initiation factor-4E (eIF4E) complex is the rate-limiting step in the initiation of protein synthesis. This study characterized the upstream signaling pathways that mediate ANG II-dependent phosphorylation of PHAS-1 and eIF4E in vascular smooth muscle. ANG II-dependent PHAS-1 phosphorylation was maximal at 10 min (2.47 ± 0.3 fold vs. control). This effect was completely blocked by the specific inhibitors of phosphatidylinositol 3-kinase (PI3-kinase, LY-294002), mammalian target of rapamycin, and extracellular signal-regulated kinase 1/2 (ERK1/2, U-0126) or by a recombinant adenovirus encoding dominant-negative Akt. PHAS-1 phosphorylation was followed by dissociation of eIF4E. Increased ANG II-induced eIF4E phosphorylation was observed at 45 min (2.63 ± 0.5 fold vs. control), was maximal at 90 min (3.38 ± 0.3 fold vs. control), and was sustained at 2 h. This effect was blocked by inhibitors of the ERK1/2 and p38 mitogen-activated protein (MAP) kinase pathways, but not by PI3-kinase inhibition, and was dependent on PKC, intracellular Ca²⁺, and tyrosine kinases. Downregulation of proline-rich tyrosine kinase 2 (PYK2) by antisense oligonucleotides led to a near-complete inhibition of PHAS-1 and eIF4E phosphorylation in response to ANG II. Therefore, PYK2 represents a proximal signaling intermediate that regulates ANG II-induced vascular smooth muscle cell protein synthesis via regulation of the PHAS-1-eIF4E complex. tyrosine kinase; antisense oligonucleotides; protein synthesis     

Regulation of Akt by arachidonic acid and phosphoinositide 3-kinase in angiotensin II-stimulated vascular smooth muscle cells

Angiotensin (Ang) II stimulates cytosolic phospholipase A2(cPLA(2))-dependent release of arachidonic acid (ArAc) in vascular smooth muscle cells (VSMC). ArAc release and production of reactive oxygen species (ROS) lead to the activation of downstream kinases resulting in VSMC growth. To determine the role of Akt in this pathway, we used VSMC to link Ang II-induced ArAc release and ROS production to the activation of Akt and VSMC growth. We observed that Ang II, ArAc, or H(2)O(2) increased Akt activation. The Akt inhibitor SH6 blocked Ang II-, ArAc-, or H(2)O(2)-induced Akt activation, as did inhibition of phosphoinositide 3-kinase (PI(3)K). Inhibition of cPLA(2) blocked Ang II effects, while the ROS scavenger NaC decreased Ang II- and ArAc-induced Akt activation. Inhibition of Akt blocked the (3)H-thymidine incorporation induced by all three agonists. Thus, Ang II-induced ArAc release and ROS production leads to the PI(3)K-dependant activation of Akt and VSMC growth.     

The effects of thiazolidinediones on vascular smooth muscle cell activation by angiotensin II

Angiotensin II (Ang II) stimulates the activation of extracellular signal-regulated kinase (ERK), a subgroup of the mitogen-activated protein kinase (MAPK) family, in cultured vascular smooth muscle cells (VSMC). This ERK activation was recently shown to be a critical regulatory factor for Ang II-mediated migration and growth. It has been demonstrated that the thiazolidinedione troglitazone (TRO) blocked Ang II-induced DNA synthesis and migration in VSMC. Here we provide evidence for TRO to inhibit Ang II-induced ERK activation which was suggested to constitute the mechanism by which this agent blocks Ang II-induced VSMC growth and migration. We have found that pretreatment with PD98059, which selectively blocks the activity of ERK pathway at the level of MAPK kinase, decreased Ang II-induced AP-1 activation and that TRO is capable of inhibiting Ang II-induced AP-1 activation. On the other hand, the other thiazolidinediones pioglitazone (PIO) and rosiglitazone (ROSI) had little effect on Ang II-induced activation of ERK or AP-1, suggesting the inhibitory effects of TRO on VSMC activation by Ang II be independent of the peroxisome proliferator-activated receptor-gamma (PPARgamma) for which thiazolidinediones are ligands. Ang II-induced ERK activation was inhibited by protein kinase C (PKC)-specific inhibitor GF109203X, while TRO was also able to block PKC activator phorbol 12 myristate 13-acetate (PMA)-induced ERK activation. Accordingly, TRO may inhibit Ang II-induced MAPK activation at least partly by an inhibition of PKC. These results support the assumption that by targeting MAPK activation, TRO may inhibits the critical signaling steps leading to restenosis and atherosclerosis that may result in part from dysregulated VSMC growth and migration induced by Ang II.     

Anti-proliferative effect of rosiglitazone on angiotensin II-induced vascular smooth muscle cell proliferation is mediated by the mTOR pathway

VSMC (vascular smooth muscle cell) proliferation contributes significantly to intimal thickening in atherosclerosis, restenosis and venous bypass graft diseases. Ang II (angiotensin II) has been implicated in VSMC proliferation though the activation of multiple growth-promoting signals. Although TZDs (thiazolidinediones) can inhibit VSMC proliferation and reduce Ang II-induced fibrosis, the mechanism underlying the inhibition of VSMC proliferation and fibrosis needs elucidation. We have used primary cultured rat aortic VSMCs and specific antibodies to investigate the inhibitory mechanism of rosiglitazone on Ang II-induced VSMC proliferation. Rosiglitazone treatment significantly inhibited Ang II-induced rat aortic VSMC proliferation in a dose-dependent manner. Western blot analysis showed that rosiglitazone significantly lowered phosphorylated ERK1/2 (extracellular-signal-regulated kinase 1/2), Akt (also known as protein kinase B), mTOR (mammalian target of rapamycin), p70S6K (70 kDa S6 kinase) and 4EBP1 (eukaryotic initiation factor 4E-binding protein) levels in Ang II-treated VSMCs. In addition, PPAR-γ (peroxisome-proliferator-activated receptor γ) mRNA increased significantly and CTGF (connective tissue growth factor), Fn (fibronectin) and Col III (collagen III) levels decreased significantly. The results demonstrate that the rosiglitazone directly inhibits the pro-atherosclerotic effect of Ang II on rat aortic VSMCs. It also attenuates Ang II-induced ECM (extracellular matrix) molecules and CTGF production in rat aortic VSMCs, reducing fibrosis. Importantly, PPAR-γ activation mediates these effects, in part, through the mTOR-p70S6K and -4EBP1 system.     

Angiotensin II activates Akt/protein kinase B by an arachidonic acid/redox-dependent pathway and independent of phosphoinositide 3-kinase.

Angiotensin II (Ang II) exerts contractile and trophic effects in glomerular mesangial cells (MCs). One potential downstream target of Ang II is the protein kinase Akt/protein kinase B (PKB). We investigated the effect of Ang II on Akt/PKB activity in MCs. Ang II causes rapid activation of Akt/PKB (5-10 min) but delayed activation of phosphoinositide 3-kinase (PI3-K) (30 min). Activation of Akt/PKB by Ang II was not abrogated by the PI3-K inhibitors or by the introduction of a dominant negative PI3-K, indicating that in MCs, PI3-K is not an upstream mediator of Akt/PKB activation by Ang II. Incubation of MCs with phospholipase A2 inhibitors also blocked Akt/PKB activation by Ang II. AA mimicked the effect of Ang II. Inhibitors of cyclooxygenase-, lipoxyogenase-, and cytochrome P450-dependent metabolism did not influence AA-induced Akt/PKB activation. However, the antioxidants N-acetylcysteine and diphenylene iodonium inhibited both AA- and Ang II-induced Akt/PKB activation. Dominant negative mutant of Akt/PKB or antioxidants, but not the dominant negative form of PI3-K, inhibited Ang II-induced protein synthesis and cell hypertrophy. These data provide the first evidence that Ang II induces protein synthesis and hypertrophy in MCs through AA/redox-dependent pathway and Akt/PKB activation independent of PI3-K.     

Reactive oxygen species mediate the activation of Akt/protein kinase B by angiotensin II in vascular smooth muscle cells.

Angiotensin II, a hypertrophic/anti-apoptotic hormone, utilizes reactive oxygen species (ROS) as growth-related signaling molecules in vascular smooth muscle cells (VSMCs). Recently, the cell survival protein kinase Akt/protein kinase B (PKB) was proposed to be involved in protein synthesis. Here we show that angiotensin II causes rapid phosphorylation of Akt/PKB (6- +/- 0.4-fold increase). Exogenous H(2)O(2) (50-200 microM) also stimulates Akt/PKB phosphorylation (maximal 8- +/- 0.2-fold increase), suggesting that Akt/PKB activation is redox-sensitive. Both angiotensin II and H(2)O(2) stimulation of Akt/PKB are abrogated by the phosphatidylinositol 3-kinase (PI3-K) inhibitors wortmannin and LY294002 (2(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one), suggesting that PI3-K is an upstream mediator of Akt/PKB activation in VSMCs. Furthermore, diphenylene iodonium, an inhibitor of flavin-containing oxidases, or overexpression of catalase to block angiotensin II-induced intracellular H(2)O(2) production significantly inhibits angiotensin II-induced Akt/PKB phosphorylation, indicating a role for ROS in agonist-induced Akt/PKB activation. In VSMCs infected with dominant-negative Akt/PKB, angiotensin II-stimulated [(3)H]leucine incorporation is attenuated. Thus, our studies indicate that Akt/PKB is part of the remarkable spectrum of angiotensin II signaling pathways and provide insight into the highly organized signaling mechanisms coordinated by ROS, which mediate the hypertrophic response to angiotensin II in VSMCs.     

PYK2 signaling is required for PDGF-dependent vascular smooth muscle cell proliferation

Aberrant vascular smooth muscle cell (VSMC) growth is associated with many vascular diseases including atherosclerosis, hypertension, and restenosis. Platelet-derived growth factor-BB (PDGF) induces VSMC proliferation through control of cell cycle progression and protein and DNA synthesis. Multiple signaling cascades control VSMC growth, including members of the mitogen-activated protein kinase (MAPK) family as well as phosphatidylinositol 3-kinase (PI3K) and its downstream effector AKT/protein kinase B (PKB). Little is known about how these signals are integrated by mitogens and whether there are common receptor-proximal signaling control points that synchronize the execution of physiological growth functions. The nonreceptor proline-rich tyrosine kinase 2 (PYK2) is activated by a variety of growth factors and G protein receptor agonists in VSMC and lies upstream of both PI3K and MAPK cascades. The present study investigated the role of PYK2 in PDGF signaling in cultured rat aortic VSMC. PYK2 downregulation attenuated PDGF-dependent protein and DNA synthesis, which correlated with inhibition of AKT and extracellular signal-regulated kinases 1 and 2 (ERK1/2) but not p38 MAPK activation. Inhibition of PDGF-dependent protein kinase B (AKT) and ERK1/2 signaling by inhibitors of upstream kinases PI3K and MEK, respectively, as well as downregulation of PYK2 resulted in modulation of the G(1)/S phase of the cell cycle through inhibition of retinoblastoma protein (Rb) phosphorylation and cyclin D(1) expression, as well as p27(Kip) upregulation. Cell division kinase 2 (cdc2) phosphorylation at G(2)/M was also contingent on PDGF-dependent PI3K-AKT and ERK1/2 signaling. These data suggest that PYK2 is an important upstream mediator in PDGF-dependent signaling cascades that regulate VSMC proliferation.     

PPAR-gamma inhibits ANG II-induced cell growth via SHIP2 and 4E-BP1

The present study evaluated the effects of peroxisome proliferator-activated receptor (PPAR)-gamma activators on ANG II-induced signaling pathways and cell growth. Vascular smooth muscle cells (VSMC) derived from rat mesenteric arteries were treated with ANG II, with/without the AT1 receptor blocker valsartan or the AT2 receptor blocker PD-123319, after pretreatment for 24 h with the PPAR-gamma activators 15-deoxy-delta(12,14)-prostaglandin J2 (15d-PGJ2) or rosiglitazone. Both 15d-PGJ2 and rosiglitazone decreased ANG II-induced DNA synthesis. Rosiglitazone treatment increased nuclear PPAR-gamma expression and activity in VSMC. However, rosiglitazone did not alter expression of PPAR-alpha/beta, ERK 1/2, Akt, or ANG II receptors. 15d-PGJ2 and rosiglitazone decreased ERK 1/2 and Akt peak activity, both of which were induced by ANG II via the AT1 receptor. Rosiglitazone inhibited ANG II-enhanced phosphorylation of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), as well as Src homology (SH) 2-containing inositol phosphatase 2 (SHIP2). PPAR-gamma activation reduced ANG II-induced growth associated with inhibition of ERK 1/2, Akt, 4E-BP1, and SHIP2. Modulation of these pathways by PPAR-gamma activators may contribute to regression of vascular remodeling in hypertension.     

ERK1/2 activation by angiotensin II inhibits insulin-induced glucose uptake in vascular smooth muscle cells

Clinical evidence suggests a relationship between hypertension and insulin resistance, and cross-talk between angiotensin II (Ang II) and insulin signaling pathways may take place. We now report the effect of Ang II on insulin-induced glucose uptake and its intracellular mechanisms in vascular smooth muscle cells (VSMC). We examined the translocation of glucose transporter-4 (GLUT-4) and glucose uptake in rat aortic smooth muscle cells (RASMC). Mitogen-activated protein (MAP) kinases and Akt activities, and phosphorylation of insulin receptor substrate-1 (IRS-1) at the serine and tyrosine residues were measured by immunoprecipitation and immunoblotting. As a result, Ang II inhibited insulin-induced GLUT-4 translocation from cytoplasm to the plasma membrane in RASMC. Ang II induced extracellular signal-regulated kinase (ERK) 1/2 and c-Jun N-terminal kinase (JNK) activation and IRS-1 phosphorylation at Ser³⁰⁷ and Ser⁶¹⁶. Ang II-induced Ser³⁰⁷ and Ser⁶¹⁶ phophorylation of IRS-1 was inhibited by a MEK inhibitor, PD98059, and a JNK inhibitor, SP600125. Ang II inhibition of insulin-stimulated IRS-1 tyrosyl phophorylation and Akt activation were reversed by PD98059 but not by SP600125. Ang II inhibited insulin-induced glucose uptake, which was also reversed by PD98059 but not by SP600125. It is shown that Ang II-induced ERK1/2 activation inhibits insulin-dependent glucose uptake through serine phophorylation of IRS-1 in RASMC.     

Angiotensin II stimulates rat astrocyte mitogen-activated protein kinase activity and growth through EGF and PDGF receptor transactivation

We showed that the intracellular tyrosine kinases src and pyk2 mediate angiotensin II (Ang II) stimulation of growth and ERK1/2 mitogen-activated protein (MAP) kinase phosphorylation in astrocytes. In this study, we investigated whether the membrane-bound receptor tyrosine kinases platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) receptors mediate Ang II stimulation of ERK1/2 and astrocyte growth. Ang II significantly stimulated PDGF and EGF receptors in a dose- and time-dependent manner. The PDGF receptor and the EGF receptor were maximally stimulated with 100 nM Ang II (0.98+/-0.18- and 4.4+/-1.4-fold above basal, respectively). This stimulation occurred as early as 5 min, and was sustained for at least 15 min for both receptor tyrosine kinases. Moreover, 1 microM AG1478 and 0.25 microM PDGFRInhib attenuated Ang II stimulation of the EGF and PDGF receptors, respectively. Ang II-induced phosphorylation of ERK1/2 and astrocyte growth was mediated by both PDGF and EGF receptors. This report also provides novel findings that co-inhibiting EGF and PDGF receptors had a greater effect to decrease Ang II-induced ERK1/2 (90% versus 49% and 71% with PDGF receptor and EGF receptor inhibition, respectively), and astrocyte growth (60% versus 10% and 32% with PDGF receptor and EGF receptor inhibition, respectively). In conclusion we showed in astrocytes that the PDGF and the EGF receptors mediate Ang II-induced ERK1/2 phosphorylation and astrocyte growth and that these two receptors may exhibit synergism to regulate effects of the peptide in these cells.     

Expression and mechanism of spleen tyrosine kinase activation by angiotensin II and its implication in protein synthesis in rat vascular smooth muscle cells

Syk, a 72-kDa tyrosine kinase, is involved in development, differentiation, and signal transduction of hematopoietic and some non-hematopoietic cells. This study determined if Syk is expressed in vascular smooth muscle cells (VSMC) and contributes to angiotensin II (Ang II) signaling and protein synthesis. Syk was found in VSMC and was phosphorylated by Ang II through AT1 receptor. Ang II-induced Syk phosphorylation was inhibited by piceatannol and dominant negative but not wild type Syk mutant. Syk phosphorylation by Ang II was attenuated by cytosolic phospholipase A(2) (cPLA(2)) inhibitor pyrrolidine-1 and retrovirus carrying small interfering RNAs (shRNAs) of this enzyme. Arachidonic acid (AA) increased Syk phosphorylation, and AA- and Ang II-induced phosphorylation was diminished by inhibitors of AA metabolism (5,8,11,14-eicosatetraynoic acid) and lipoxygenase (LO; baicalein) but not cyclooxygenase (indomethacin). AA metabolites formed via LO, 5(S)-, 12(S)-, and 15(S)-hydroxyeicosatetraenoic acids, which activate p38 MAPK, increased Syk phosphorylation. p38 MAPK inhibitor SB202190, and dominant negative p38 MAPK mutant attenuated Ang II- and AA-induced Syk phosphorylation. Adenovirus dominant negative c-Src mutant abolished Ang II - and AA-induced Syk phosphorylation and SB202190, and dominant negative p38 MAPK mutant inhibited Ang II-induced c-Src phosphorylation. Syk dominant negative mutant but not epidermal growth factor receptor blocker AG1478 also inhibited Ang II-induced VSMC protein synthesis. These data suggest that Syk expressed in VSMC is activated by Ang II through p38 MAPK-activated c-Src subsequent to cytosolic phospholipase A(2) and generation of AA metabolites via LO, and it mediates Ang II-induced protein synthesis independent of epidermal growth factor receptor transactivation (Ang II --> cPLA(2) --> AA metabolites of LO --> p38 MAPK --> c-Src --> Syk --> protein synthesis).     

Early endosomal antigen 1 (EEA1) is an obligate scaffold for angiotensin II-induced, PKC-alpha-dependent Akt activation in endosomes

Akt/protein kinase B (PKB) activation/phosphorylation by angiotensin II (Ang II) is a critical signaling event in hypertrophy of vascular smooth muscle cells (VSMCs). Conventional wisdom asserts that Akt activation occurs mainly in plasma membrane domains. Recent evidence that Akt activation may take place within intracellular compartments challenges this dogma. The spatial identity and mechanistic features of these putative signaling domains have not been defined. Using cell fractionation and fluorescence methods, we demonstrate that the early endosomal antigen-1 (EEA1)-positive endosomes are a major site of Ang II-induced Akt activation. Akt moves to and is activated in EEA1 endosomes. The expression of EEA1 is required for phosphorylation of Akt at both Thr-308 and Ser-473 as well as for phosphorylation of its downstream targets mTOR and S6 kinase, but not for Erk1/2 activation. Both Akt and phosphorylated Akt (p-Akt) interact with EEA1. We also found that PKC-α is required for organizing Ang II-induced, EEA1-dependent Akt phosphorylation in VSMC early endosomes. EEA1 expression enables PKC-α phosphorylation, which in turn regulates Akt upstream signaling kinases, PDK1 and p38 MAPK. Our results indicate that PKC-α is a necessary regulator of EEA1-dependent Akt signaling in early endosomes. Finally, EEA1 down-regulation or expression of a dominant negative mutant of PKC-α blunts Ang II-induced leucine incorporation in VSMCs. Thus, EEA1 serves a novel function as an obligate scaffold for Ang II-induced Akt activation in early endosomes.     

Inhibition of AT1 receptor internalization by concanavalin A blocks angiotensin II-induced ERK activation in vascular smooth muscle cells. Involvement of epidermal growth factor receptor proteolysis but not AT1 receptor internalization

Recent studies of beta(2)-adrenergic receptor suggest that agonist-promoted receptor internalization may play an important role in extracellular signal-regulated kinase (ERK) activation by G protein-coupled receptors. In the present study, we explored the effects of angiotensin II (Ang II) type-1 receptor (AT(1)) internalization on Ang II-induced activation of ERK using the receptor internalization blocker concanavalin A (ConA) and the carboxyl terminus-truncated receptor mutants with impaired internalization. ConA inhibited AT(1) receptor internalization without affecting ligand binding to the receptor, Ang II-induced generation of second messengers, and activation of tyrosine kinases Src and Pyk2 in vascular smooth muscle cells (VSMC). ConA blocked ERK activation evoked by Ang II and the calcium ionophore A23187. Impairment of AT(1) receptor internalization by truncating the receptor carboxyl terminus did not affect Ang II-induced ERK activation. ConA induced proteolytic cleavage of the epidermal growth factor (EGF) receptor at carboxyl terminus and abolished Ang II-induced transactivation of the EGF receptor, which is critical for ERK activation by Ang II in VSMC. ConA also induced proteolysis of erbB-2 but not platelet-derived growth factor receptor. Thus, ConA blocks Ang II-induced ERK activation in VSMC through a distinct mechanism, the ConA-mediated proteolysis of the EGF receptor.     

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.     

Dual regulation of MMP-2 expression by the type 1 insulin-like growth factor receptor: the phosphatidylinositol 3-kinase/Akt and Raf/ERK pathways transmit opposing signals

The matrix metalloproteinase (MMP)-2 has been recognized as a major mediator of basement membrane degradation, angiogenesis, tumor invasion, and metastasis. The factors that regulate its expression have not, however, been fully elucidated. We previously identified the type I insulin-like growth factor (IGF-I) receptor as a regulator of MMP-2 synthesis. The objective of the present study was to investigate the signal transduction pathway(s) mediating this regulation. We show here that in Lewis lung carcinoma subline H-59 cells treated with IGF-I (10 ng/ml), the PI 3-kinase (phosphatidylinositol 3'-kinase) /protein kinase B (Akt) and C-Raf/ERK pathways were activated, and MMP-2 promoter activity, mRNA, and protein synthesis were induced. MMP-2 induction was blocked by the PI 3-kinase inhibitors LY294002 and wortmannin, by overexpression of a dominant-negative Akt or wild-type PTEN (phosphatase and tensin homologue deleted on chromosome 10), and by rapamycin. In contrast, a MEK inhibitor PD98059 failed to reduce MMP-2 promoter activation and actually increased MMP-2 mRNA and protein synthesis by up to 30%. Interestingly, suppression of PI 3-kinase signaling by a dominant-negative Akt enhanced ERK activity in cells stimulated with 10 ng/ml but not with 100 ng/ml IGF-I. Furthermore, at the higher (100 ng/ml) IGF-I concentration, C-Raf and ERK, but not PI 3-kinase activation, was enhanced, and this resulted in down-regulation of MMP-2 synthesis. This effect was reversed in cells expressing a dominant-negative ERK mutant. The results suggest that IGF-I can up-regulate MMP-2 synthesis via PI 3-kinase/Akt/mTOR (the mammalian target of rapamycin) signaling while concomitantly transmitting a negative regulatory signal via the Raf/ERK pathway. The outcome of IGF-IR (the receptor for IGF-I) activation may ultimately depend on factors, such as ligand bioavailability, that can shift the balance preferentially toward one pathway or the other.     

Activation of PPARδ counteracts angiotensin II-induced ROS generation by inhibiting rac1 translocation in vascular smooth muscle cells

Angiotensin II (Ang II)-mediated modification of the redox milieu of vascular smooth muscle cells (VSMCs) has been implicated in several pathophysiological processes, including cell proliferation, migration and differentiation. In this study, we demonstrate that the peroxisome proliferator-activated receptor (PPAR) δ counteracts Ang II-induced production of reactive oxygen species (ROS) in VSMCs. Activation of PPARδ by GW501516, a specific ligand for PPARδ, significantly reduced Ang II-induced ROS generation in VSMCs. This effect was, however, reversed in the presence of small interfering (si)RNA against PPARδ. The marked increase in ROS levels induced by Ang II was also eliminated by the inhibition of phosphatidylinositol 3-kinase (PI3K) but not of protein kinase C, suggesting the involvement of the PI3K/Akt signalling pathway in this process. Accordingly, ablation of Akt with siRNA further enhanced the inhibitory effects of GW501516 in Ang II-induced superoxide production. Ligand-activated PPARδ also blocked Ang II-induced translocation of Rac1 to the cell membrane, inhibiting the activation of NADPH oxidases and consequently ROS generation. These results indicate that ligand-activated PPARδ plays an important role in the cellular response to oxidative stress by decreasing ROS generated by Ang II in vascular cells.