Role of neutrophil NADPH oxidase in the mechanism of tumor necrosis factor-alpha -induced NF-kappa B activation and intercellular adhesion molecule-1 expression in endothelial cells.

In this study, we explored a novel function of polymorphonuclear neutrophils (PMN) NAD(P)H oxidase in the mechanism of tumor necrosis factor-alpha (TNFalpha)-induced NF-kappaB activation and intercellular adhesion molecule-1 (ICAM-1) expression in endothelial cells. Studies were made in mice lacking the p47(phox) subunit of NAD(P)H oxidase as well as in cultured mouse lung vascular endothelial cells (MLVEC) from these mice. In response to TNFalpha challenge, NF-kappaB activation and ICAM-1 expression were significantly attenuated in lungs of p47(phox)(-/-) mice as compared with wild-type (WT) mice. The attenuated NF-kappaB activation in p47(phox)(-/-) mice was secondary to inhibition of NIK activity and subsequent IkappaBalpha degradation. Induction of neutropenia using anti-PMN serum prevented the initial TNFalpha-induced NF-kappaB activation and ICAM-1 expression in WT mice, indicating the involvement of PMN NAD(P)H oxidase in signaling these responses. Moreover, the responses were restored upon repletion with PMN obtained from WT mice but not with PMN from p47(phox)(-/-) mice. These findings were recapitulated in MLVEC co-cultured with PMN, suggesting that NF-kappaB activation and resultant ICAM-1 expression in endothelial cells occurred secondary to oxidants generated by the PMN NAD(P)H oxidase complex. The functional relevance of the PMN NAD(P)H oxidase in mediating TNFalpha-induced ICAM-1-dependent endothelial adhesivity was evident by markedly reduced adhesion of p47(phox)(-/-) PMN in co-culture experiments. Thus, oxidant signaling by the PMN NAD(P)H oxidase complex is an important determinant of TNFalpha-induced NF-kappaB activation and ICAM-1 expression in endothelial cells.     

TNFalpha activates c-Jun amino terminal kinase through p47(phox).

Reactive oxygen intermediates have been implicated in the transduction of TNFalpha signals, although the source of such oxidants has not been established. We found that activation of ECV-304 cells by TNFalpha was accompanied by a transient burst of oxidants and activation of JNK, both of which were suppressed by two distinct inhibitors of the phagocyte NADPH oxidase and the thiol antioxidant N-acetyl cysteine (NAC). We cloned partial and full-length cDNA sequences from ECV-304 cells and human umbilical vein endothelial cells (HUVEC), respectively, for p47(phox), demonstrating that these nonphagocytic cells express this adapter protein known to specifically initiate assembly of the NADPH oxidase in professional phagocytes. A mutant p47(phox), defective in the first Src homology 3 (SH3) domain (p47W(193)R), diminished JNK activation by TNFalpha. Surprisingly, p47(phox) resided entirely in the particulate, not cytosolic, fraction of cells. Immunostaining suggested partial colocalization with cytoskeletal elements, and cytoskeletal disrupters decreased both oxidant production and JNK activation by TNFalpha. A p47-GFP fusion protein localized to the cortical cytoskeleton in living cells; further, stimulation of cells with TNFalpha caused a marked concentration of p47-GFP in membrane ruffles, actin-rich structures associated with intense respiratory burst activity in stimulated neutrophils. We conclude that nonphagocytic cells express p47(phox), which appears to localize to the cytoskeleton and participate in TNFalpha signaling. We speculate that this physical targeting may prove important in conferring signal specificity and enhancing signaling efficiency of unstable oxidants.     

Acute tumor necrosis factor alpha signaling via NADPH oxidase in microvascular endothelial cells: role of p47phox phosphorylation and binding to TRAF4

Tumor necrosis factor alpha (TNF-alpha) receptor-associated factors (TRAFs) play important roles in TNF-alpha signaling by interacting with downstream signaling molecules, e.g., mitogen-activated protein kinases (MAPKs). However, TNF-alpha also signals through reactive oxygen species (ROS)-dependent pathways. The interrelationship between these pathways is unclear; however, a recent study suggested that TRAF4 could bind to the NADPH oxidase subunit p47phox. Here, we investigated the potential interaction between p47phox phosphorylation and TRAF4 binding and their relative roles in acute TNF-alpha signaling. Exposure of human microvascular endothelial cells (HMEC-1) to TNF-alpha (100 U/ml; 1 to 60 min) induced rapid (within 5 min) p47phox phosphorylation. This was paralleled by a 2.7- +/- 0.5-fold increase in p47phox-TRAF4 association, membrane translocation of p47phox-TRAF4, a 2.3- +/- 0.4-fold increase in p47phox-p22phox complex formation, and a 3.2- +/- 0.2-fold increase in NADPH-dependent O2- production (all P < 0.05). TRAF4-p47phox binding was accompanied by a progressive increase in extracellular signal-regulated kinases 1 and 2 (ERK1/2) and p38(MAPK) activation, which was inhibited by an O2- scavenger, tiron. TRAF4 predominantly bound the phosphorylated form of p47phox, in a protein kinase C-dependent process. Knockdown of TRAF4 expression using siRNA had no effect on p47phox phosphorylation or binding to p22phox but inhibited TNF-alpha-induced ERK1/2 activation. In coronary microvascular EC from p47phox-/- mice, TNF-alpha-induced NADPH oxidase activation, ERK1/2 activation, and cell surface intercellular adhesion molecule 1 (ICAM-1) expression were all inhibited. Thus, both p47phox phosphorylation and TRAF4 are required for acute TNF-alpha signaling. The increased binding between p47phox and TRAF4 that occurs after p47phox phosphorylation could serve to spatially confine ROS generation from NADPH oxidase and subsequent MAPK activation and cell surface ICAM-1 expression in EC.     

Vascular endothelial growth factor causes translocation of p47phox to membrane ruffles through WAVE1

Growth factors initiate cytoskeletal rearrangements tightly coordinated with nuclear signaling events. We hypothesized that the angiogenic growth factor, vascular endothelial growth factor (VEGF), may utilize oxidants that are site-directed to a complex critical to both cytoskeletal and mitogenic signaling. We identified the WASP-family verprolin homologous protein-1 (WAVE1) as a binding partner for the NADPH oxidase adapter p47phox within membrane ruffles of VEGF-stimulated cells. Within 15 min of VEGF stimulation, p47phox coprecipitated with WAVE1, with the ruffle and oxidase agonist Rac1, and with the Rac1 effector PAK1. VEGF also increased p47phox phosphorylation, oxidant production, and ruffle formation, all of which were dependent upon PAK1 kinase activity. The antioxidant Mn (III) tetrakis(4-benzoic acid) porphyrin and ectopic expression of either the p47-binding WAVE1 domain or the WAVE1-binding p47phox domain decreased VEGF-induced ruffling, whereas the active mutant p4-(S303D, S304D,S328D) stimulated oxidant production and formation of circular dorsal ruffles. Both kinase-dead PAK1-(K298A) and Mn (III) tetrakis(4-benzoic acid) porphyrin decreased c-Jun N-terminal kinase (JNK) activation by VEGF, whereas dominant-negative JNK did not block ruffle formation, suggesting a bifurcation of mitogenic and cytoskeletal signaling events at or distal to the oxidase but proximal to JNK. Thus, WAVE1 may act as a scaffold to recruit the NADPH oxidase to a complex involved with both cytoskeletal regulation and downstream JNK activation.     

Subcellular targeting of oxidants during endothelial cell migration

Endogenous oxidants participate in endothelial cell migration, suggesting that the enzymatic source of oxidants, like other proteins controlling cell migration, requires precise subcellular localization for spatial confinement of signaling effects. We found that the nicotinamide adenine dinucleotide phosphate reduced (NADPH) oxidase adaptor p47(phox) and its binding partner TRAF4 were sequestered within nascent, focal complexlike structures in the lamellae of motile endothelial cells. TRAF4 directly associated with the focal contact scaffold Hic-5, and the knockdown of either protein, disruption of the complex, or oxidant scavenging blocked cell migration. An active mutant of TRAF4 activated the NADPH oxidase downstream of the Rho GTPases and p21-activated kinase 1 (PAK1) and oxidatively modified the focal contact phosphatase PTP-PEST. The oxidase also functioned upstream of Rac1 activation, suggesting its participation in a positive feedback loop. Active TRAF4 initiated robust membrane ruffling through Rac1, PAK1, and the oxidase, whereas the knockdown of PTP-PEST increased ruffling independent of oxidase activation. Our data suggest that TRAF4 specifies a molecular address within focal complexes that is targeted for oxidative modification during cell migration.     

Phosphatidylinositol 3-kinase gamma signaling through protein kinase Czeta induces NADPH oxidase-mediated oxidant generation and NF-kappaB activation in endothelial cells

We addressed the role of class 1B phosphatidylinositol 3-kinase (PI3K) isoform PI3Kgamma in mediating NADPH oxidase activation and reactive oxidant species (ROS) generation in endothelial cells (ECs) and of PI3Kgamma-mediated oxidant signaling in the mechanism of NF-kappaB activation and intercellular adhesion molecule (ICAM)-1 expression. We used lung microvascular ECs isolated from mice with targeted deletion of the p110gamma catalytic subunit of PI3Kgamma. Tumor necrosis factor (TNF) alpha challenge of wild type ECs caused p110gamma translocation to the plasma membrane and phosphatidylinositol 1,4,5-trisphosphate production coupled to ROS production; however, this response was blocked in p110gamma-/- ECs. ROS production was the result of TNFalpha activation of Ser phosphorylation of NADPH oxidase subunit p47(phox) and its translocation to EC membranes. NADPH oxidase activation failed to occur in p110gamma-/- ECs. Additionally, the TNFalpha-activated NF-kappaB binding to the ICAM-1 promoter, ICAM-1 protein expression, and PMN adhesion to ECs required functional PI3Kgamma. TNFalpha challenge of p110gamma-/- ECs failed to induce phosphorylation of PDK1 and activation of the atypical PKC isoform, PKCzeta. Thus, PI3Kgamma lies upstream of PKCzeta in the endothelium, and its activation is crucial in signaling NADPH oxidase-dependent oxidant production and subsequent NF-kappaB activation and ICAM-1 expression.     

Treatment with 17-beta-estradiol reduces superoxide production in aorta of ovariectomized rats

OBJECTIVE: Oxidant stress contributes to vascular injury and atherosclerosis. We hypothesized that estrogen treatment of ovariectomized rats decreases O(2)(-) by decreasing the activity of NAD(P)H oxidase and this reduction in O(2)(-) could have a vasculoprotective effect. METHODS AND RESULTS: Ovariectomized rats were treated with 17-beta-estradiol E2 (0.25mg) or oil placebo for 21 days. Aorta were removed for contractility studies and O(2)(-) production was measured by lucigenin enhanced chemiluminescence (230 and 5microM). E2 treatment decreased basal O(2)(-) production but did not alter NADH or NADPH stimulated O(2)(-) production. Total p47phox and p47phox in membrane fractions of cardiac tissue were decreased, which suggests less activation of NAD(P)H oxidase in E2 treated rats. E2 did not change expression of other components of NAD(P)H oxidase in heart, lung, spleen and diaphragm. Expression of eNOS was also lower in E2 treated rats. E2 did not affect the contractile response to phenylepherine, dilation with acetylcholine, dilation with superoxide dismutase or constriction with l-NAME. This argues against changes in bioavailable NO. CONCLUSIONS: E2 decreases activation of p47phox and O(2)(-) production by NAD(P)H oxidase. This did not affect contractile properties of the vessel, but could still potentially alter cell signaling from oxidant increasing stresses.     

NAD(P)H oxidase mediates the endothelial barrier dysfunction induced by TNF-alpha

We tested the hypothesis that the NAD(P)H oxidase-dependent generation of superoxide anion (O2-*) mediates tumor necrosis factor-alpha (TNF)-induced alterations in the permeability of pulmonary microvessel endothelial monolayers (PMEM). The permeability of PMEM was assessed by the clearance rate of Evans blue-labeled albumin. The NAD(P)H oxidase subcomponents p47phox and p22phox were assessed by immunofluorescent microscopy and Western blot. The reactive oxygen species O2-* was measured by the fluorescence of 6-carboxy-2',7'-dichlorodihydrofluorescein diacetatedi(acetoxymethyl ester), 5 (and 6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate-acetyl ester, and dihydroethidium. TNF treatment (50 ng/ml for 4.0 h) induced 1) p47phox translocation, 2) an increase in p22phox protein, 3) increased localization of p47phox with p22phox, 4) O2-* generation, and 5) increased permeability to albumin. p22phox antisense oligonucleotide prevented the TNF-induced effect on p22phox, p47phox, O2-*, and permeability. The scrambled nonsense oligonucleotide had no effect. The TNF-induced increase in O2-* and permeability to albumin was also prevented by the O2-* scavenger Cu-Zn superoxide dismutase (100 U/ml). The results indicate that the activation of NAD(P)H oxidase, via the generation of O2-*, mediates TNF-induced barrier dysfunction in PMEM.     

Membrane localization of TRAF 3 enables JNK activation

Members of the tumor necrosis factor receptor family as well as other receptors achieve their diverse biological effects through the activation of intracellular signals including the c-Jun N-terminal kinase (JNK) pathway. Such signals are believed to be delivered through mediators known as TNF receptor-associated factors (TRAFs). Although the N-terminal zinc finger region of TRAFs has been shown to be essential for downstream signaling, there is no indication yet as to the nature of its role or of the factors that distinguish the N terminus of TRAF 3, which does not activate JNK in the systems examined thus far, from those of other TRAFs, which do activate this pathway. In the present study, it is shown that, among the known TRAFs, localization to the insoluble cell pellet fraction consistently correlates with JNK activation and that both characteristics map to the TRAF N terminus. Furthermore, it is demonstrated that forced localization of TRAF 3 to the cell membrane is sufficient to convert this molecule into an activator of JNK. This suggests that one of the roles of the TRAF N terminus may be to participate in interactions that promote the recruitment of TRAFs to the membrane and that this localization effect plays an important role in TRAF-mediated JNK activation.     

Reactive oxygen species as mediators of angiogenesis signaling. Role of NAD(P)H oxidase

Angiogenesis, a process of new blood vessel growth, contributes to various pathophysiologies such as cancer, diabetic retinopathy and atherosclerosis. Accumulating evidence suggests that cardiovascular diseases are associated with increased oxidative stress in blood vessels. Reactive oxygen species (ROS) such as superoxide and H2O2 cause blood vessels to thicken, produce inflammation in the vessel wall, and thus are regarded as "risk factors" for vascular disease, whereas ROS also act as signaling molecules in many aspects of growth factor-mediated physiological responses. Recent reports suggest that ROS play an important role in angiogenesis; however, its underlying molecular mechanisms remain unknown. Vascular endothelial growth factor (VEGF) induces angiogenesis by stimulating endothelial cell (EC) proliferation and migration primarily through the receptor tyrosine kinase VEGF receptor2 (Flk1/KDR). VEGF binding initiates tyrosine phosphorylation of KDR, which results in activation of downstream signaling enzymes including ERK1/2, Akt and eNOS, which contribute to angiogenic-related responses in EC. Importantly, the major source of ROS in EC is a NAD(P)H oxidase and EC express all the components of phagocytic NAD(P)H oxidase including gp91phox, p22phox, p47phox, p67phox and the small G protein Rac1. We have recently demonstrated that ROS derived from NAD(P)H oxidase are critically important for VEGF signaling in vitro and angiogenesis in vivo. Furthermore, a peptide hormone, angiotensin II, a major stimulus for vascular NAD(P)H oxidase, also plays an important role in angiogenesis. Because EC migration and proliferation are primary features of the process of myocardial angiogenesis, we would like to focus on the recent progress that has been made in the emerging area of NAD(P)H oxidase-derived ROS-dependent signaling in ECs, and discuss the possible roles in angiogenesis. Understanding these mechanisms may provide insight into the components of NAD(P)H oxidase as potential therapeutic targets for treatment of angiogenesis-dependent diseases such as cancer and atherosclerosis and for promoting myocardial angiogenesis in ischemic heart diseases.     

Inhibition of mixed lineage kinase 3 prevents HIV-1 Tat-mediated neurotoxicity and monocyte activation

The HIV-1 gene products Tat and gp120 are toxic to neurons and can activate cells of myeloid origin, properties that are thought to contribute to the clinical manifestations of HIV-1-associated dementia (HAD). To investigate the intracellular signaling mechanisms involved in these events, the effect of Tat and gp120 on mixed lineage kinase (MLK) 3 activation was examined. Tat and gp120 were shown to induce autophosphorylation of MLK3 in primary rat neurons; this was abolished by the addition of an inhibitor of MLK3 (CEP1347). CEP1347 also enhanced survival of both rat and human neurons and inhibited the activation of human monocytes after exposure to Tat and gp120. Furthermore, overexpression of wild-type MLK3 led to the induction of neuronal death, whereas expression of a dominant negative MLK3 mutant protected neurons from the toxic effects of Tat. MLK3-dependent downstream signaling events were implicated in the neuroprotective and monocyte-deactivating pathways triggered by CEP1347. Thus, the inhibition of p38 MAPK and JNK protected neurons from Tat-induced apoptosis, whereas the inhibition of p38 MAPK, but not of JNK, was sufficient to prevent Tat- and gp120-mediated activation of monocytes. These results suggest that the normal function of MLK3 is compromised by HIV-1 neurotoxins (Tat, gp120), resulting in the activation of downstream signaling events that result in neuronal death and monocyte activation (with release of inflammatory cytokines). In aggregate, our data define MLK3 as a promising therapeutic target for intervention in HAD.     

Hydrogen peroxide signaling through tumor necrosis factor receptor 1 leads to selective activation of c-Jun N-terminal kinase

Binding of tumor necrosis factor-alpha (TNFalpha) to its receptor, TNF-R1, results in the activation of inhibitor of kappaB kinase (IKK) and c-Jun N-terminal kinase (JNK) pathways that are coordinately regulated and important in survival and death. We demonstrated previously that in response to hydrogen peroxide (H2O2), the ability of TNFalpha to activate IKK in mouse lung epithelial cells (C10) was inhibited and that H2O2 alone was sufficient to activate JNK and induce cell death. In the current study, we investigated the involvement of TNF-R1 in H2O2-induced JNK activation. In lung fibroblasts from TNF-R1-deficient mice the ability of H2O2 to activate JNK was inhibited compared with fibroblasts from control mice. Additionally, in C10 cells expressing a mutant form of TNF-R1, H2O2-induced JNK activation was also inhibited. Immunoprecipitation of TNF-R1 revealed that in response to H2O2, the adapter proteins, TRADD and TRAF2, and JNK were recruited to the receptor. However, expression of the adaptor protein RIP, which is essential for IKK activation by TNFalpha, was decreased in cells exposed to H2O2, and its chaperone Hsp90 was cleaved. Furthermore, data demonstrating that expression of TRAF2 was not affected by H2O2 and that overexpression of TRAF2 was sufficient to activate JNK provide an explanation for the inability of H2O2 to activate IKK and for the selective activation of JNK by H2O2. Our data demonstrate that oxidative stress interferes with IKK activation while promoting JNK signaling, creating a signaling imbalance that may favor apoptosis.     

HIV-1 Tat protein-induced VCAM-1 expression in human pulmonary artery endothelial cells and its signaling

Expression of cell adhesion molecule in endothelial cells upon activation by human immunodeficiency virus (HIV) infection is associated with the development of atherosclerotic vasculopathy. We postulated that induction of vascular cell adhesion molecule-1 (VCAM-1) by HIV-1 Tat protein in endothelial cells might represent an early event that could culminate in inflammatory cell recruitment and vascular injury. We determined the role of HIV-1 Tat protein in VCAM-1 expression in human pulmonary artery endothelial cells (HPAEC). HIV-1 Tat protein treatment significantly increased cell-surface expression of VCAM-1 in HPAEC. Consistently, mRNA expression of VCAM-1 was also increased by HIV-1 Tat protein as measured by RT-PCR. HIV-1 Tat protein-induced VCAM-1 expression was abolished by the NF-kappaB inhibitor pyrrolidine dithiocarbamate (PDTC) and the p38 MAPK inhibitor SB-203580. Furthermore, HIV-1 Tat protein enhanced DNA binding activity of NF-kappaB, facilitated nuclear translocation of NF-kappaB subunit p65, and increased production of reactive oxygen species (ROS). Similarly to VCAM-1 expression, HIV-1 Tat protein-induced NF-kappaB activation and ROS generation were abrogated by PDTC and SB-203580. These data indicate that HIV-1 Tat protein is able to induce VCAM-1 expression in HPAEC, which may represent a pivotal early molecular event in HIV-induced vascular/pulmonary injury. These data also suggest that the molecular mechanism underlying the HIV-1 Tat protein-induced VCAM-1 expression may involve ROS generation, p38 MAPK activation, and NF-kappaB translocation, which are the characteristics of pulmonary endothelial cell activation.     

p47phox participates in activation of RelA in endothelial cells

Activation of endothelial cell NF-kappaB by interleukin (IL)-1 constitutes an event critical to the progression of the innate immune response. In this context, oxidants have been associated with NF-kappaB activation, although the molecular source and mechanism of targeting have remained obscure. We found that RelA, essential for NF-kappaB activation by IL-1, was associated with the NADPH oxidase adapter protein p47(phox) in yeast two-hybrid, coprecipitation, and in vitro binding studies. RelA and p47-GFP also colocalized in endothelial cells in focal submembranous dorsoventral protrusions. Overexpression of p47(phox) synergized with IL-1beta in the activation of an artificial kappaB-luciferase reporter and specifically augmented IL-1beta-induced RelA transactivation activity. p47(phox) overexpression also greatly increased IL-1beta-stimulated RelA phosphorylation, whereas it had no effect on I-kappaB degradation or on RelA nuclear translocation or kappaB binding. The tandem SH3 domains of p47(phox) were found to associate with a proline-rich mid-region of RelA (RelA-PR) located between the Rel homology and transactivation domains. The RelA-PR peptide blocked interaction of p47(phox) and RelA, and ectopic expression of RelA-PR abrogated IL-1beta-induced transactivation of the NF-kappaB-dependent E-selectin promoter. Further, suppression of NADPH oxidase function through the inhibitor diphenylene iodonium, the superoxide dismutase mimetic Mn(III) tetrakis(4-benzoic acid)porphyrin (MnTBAP), or expression of a dominant interfering mutant of a separate NADPH oxidase subunit (p67(V204A)) decreased IL-1beta-induced E-selectin promoter activation, suggesting that p47(phox) facilitates NF-kappaB activation through linkage with the NADPH oxidase. IL-1beta rapidly increased tyrosine phosphorylation of IL-1 type I receptor-associated proteins, suggesting that oxidants may operate through inactivation of local protein-tyrosine phosphatases in the proximal IL-1beta signaling pathway leading to RelA activation.     

NADPH oxidase-derived superoxide anion-induced apoptosis is mediated via the JNK-dependent activation of NF-κB in cardiomyocytes exposed to high glucose

Hyperglycemia-induced generation of reactive oxygen species (ROS) can lead to cardiomyocyte apoptosis and cardiac dysfunction. However, the mechanism by which high glucose causes cardiomyocyte apoptosis is not clear. In this study, we investigated the signaling pathways involved in NADPH oxidase-derived ROS-induced apoptosis in cardiomyocytes under hyperglycemic conditions. H9c2 cells were treated with 5.5 or 33 mM glucose for 36 h. We found that 33 mM glucose resulted in a time-dependent increase in ROS generation as well as a time-dependent increase in protein expression of p22(phox), p47(phox), gp91(phox), phosphorylated IκB, c-Jun N-terminal kinase (JNK) and p38, as well as the nuclear translocation of NF-kB. Treatment with apocynin or diphenylene iodonium (DPI), NADPH oxidase inhibitors, resulted in reduced expression of p22(phox), p47(phox), gp91(phox), phosphorylated IκB, c-Jun N-terminal kinase (JNK) and p38. In addition, treatment with JNK and NF-kB siRNAs blocked the activity of caspase-3. Furthermore, treatment with JNK, but not p38, siRNA inhibited the glucose-induced activation of NF-κB. Similar results were obtained in neonatal cardiomyocytes exposed to high glucose concentrations. Therefore, we propose that NADPH oxidase-derived ROS-induced apoptosis is mediated via the JNK-dependent activation of NF-κB in cardiomyocytes exposed to high glucose.     

Role of the actin cytoskeleton in angiotensin II signaling in human vascular smooth muscle cells

Angiotensin II (Ang II) regulates vascular smooth muscle cell (VSMC) function by activating signaling cascades that promote vasoconstriction, growth, and inflammation. Subcellular mechanisms coordinating these processes are unclear. In the present study, we questioned the role of the actin cytoskeleton in Ang II mediated signaling through mitogen-activated protein (MAP) kinases and reactive oxygen species (ROS) in VSMCs. Human VSMCs were studied. Cells were exposed to Ang II (10-7 mol/L) in the absence and presence of cytochalasin B (10-6 mol/L, 60 min), which disrupts the actin cytoskeleton. Phosphorylation of p38MAP kinase, JNK, and ERK1/2 was assessed by immuno blotting. ROS generation was measured using the fluoroprobe chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (4 micromol/L). Interaction between the cytoskeleton and NADPH oxidase was determined by evaluating the presence of p47phox in the Triton X-100 insoluble membrane fraction. Ang II significantly increased phosphorylation of p38MAP kinase, JNK, and ERK1/2 (two- to threefold above control, p < 0.05). Cytochalasin B pretreatment attenuated p38MAP kinase and JNK effects (p < 0.05) without altering ERK1/2 phosphorylation. ROS formation, which was increased in Ang II stimulated cells, was significantly reduced by cytochalasin B (p < 0.01). p47phox, critically involved in NADPH oxidase activation, colocalized with the actin cytoskeleton in Ang II stimulated cells. Our data demonstrate that Ang II mediated ROS formation and activation of p38MAP kinase and JNK, but not ERK1/2, involves the actin cytoskeleton in VSMCs. In addition, Ang II promotes interaction between actin and p47phox. These data indicate that the cytoskeleton is involved in differential MAP kinase signaling and ROS generation by Ang II in VSMCs. Together, these studies suggest that the cytoskeleton may be a central point of crosstalk in growth- and redox-signaling pathways by Ang II, which may be important in the regulation of VSMC function.