Human immunodeficiency virus type 1 Tat regulates endothelial cell actin cytoskeletal dynamics through PAK1 activation and oxidant production

Human immunodeficiency virus type 1 Tat exerts prominent angiogenic effects which may lead to a variety of vasculopathic conditions in AIDS patients. Because endothelial cells undergo prominent cytoskeletal rearrangement during angiogenesis, we investigated the specific effects of Tat on the endothelial cell actin cytoskeleton. Glutathione S-transferase (GST)-Tat, at a level of 200 ng/ml (equivalent to 52 ng of Tat/ml), caused stress fiber disassembly, peripheral retraction, and ruffle formation in human umbilical vein endothelial cells (HUVEC) and human lung microvascular endothelial cells. At 600 ng of GST-Tat/ml (157 ng of Tat/ml), actin structures were lost, and severe cytoskeletal collapse occurred. In contrast, GST-Tat harboring mutations within either the cysteine-rich or basic domains exerted minimal effects on the endothelial cytoskeleton. HUVEC expressing a DsRed-Tat fusion protein displayed similar actin rearrangements, followed by actin collapse, whereas neighboring nontransfected cells retained normal actin structures. Because active mutants of p21-activated kinase 1 (PAK1) induce identical changes in actin dynamics, we hypothesized that Tat exerts its cytoskeletal effects through PAK1. GST-Tat activated PAK1 within 5 min, and adenovirus delivery of a kinase-dead PAK1 [PAK1(K298A)] completely prevented cytoskeletal collapse induced by GST-Tat or DsRed-Tat and also blocked downstream activation of c-Jun N-terminal kinase. Further, GST-Tat increased phosphorylation of the NADPH oxidase subunit p47(phox) and caused its rapid redistribution to membrane ruffles. PAK1(K298A) blocked p47(phox) phosphorylation, and interference with NADPH oxidase function through superoxide scavenging or through expression of a transdominant inhibitor, p67(V204A), prevented GST-Tat-induced alterations in the actin cytoskeleton. We conclude that Tat induces actin cytoskeletal rearrangements through PAK1 and downstream activation of the endothelial NADPH oxidase.     

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.     

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.     

Rac activation induces NADPH oxidase activity in transgenic COSphox cells, and the level of superoxide production is exchange factor-dependent

Transient expression of constitutively active Rac1 derivatives, (G12V) or (Q61L), was sufficient to induce phagocyte NADPH oxidase activity in a COS-7 cell model in which human cDNAs for essential oxidase components, gp91(phox), p22(phox), p47(phox), and p67(phox), were expressed as stable transgenes. Expression of constitutively active Rac1 in "COS(phox)" cells induced translocation of p47(phox) and p67(phox) to the membrane. Furthermore, translocation of p47(phox) was induced in the absence of p67(phox) expression, even though Rac does not directly bind p47(phox). Rac effector domain point substitutions (A27K, G30S, D38A, Y40C), which can selectively eliminate interaction with different effector proteins, impaired Rac1V12-induced superoxide production. Activation of endogenous Rac1 by expression of constitutively active Rac-guanine nucleotide exchange factor (GEF) derivatives was sufficient to induce high level NADPH oxidase activity in COS(phox) cells. The constitutively active form of the hematopoietic-specific GEF, Vav1, was the most effective at activating superoxide production, despite detection of higher levels of Rac1-GTP upon expression of constitutively active Vav2 or Tiam1 derivatives. These data suggest that Rac can play a dual role in NADPH oxidase activation, both by directly participating in the oxidase complex and by activating signaling events leading to oxidase assembly, and that Vav1 may be the physiologically relevant GEF responsible for activating this Rac-regulated complex.     

Rac GTPase interacts with GAPs and target proteins through multiple effector sites.

Rac, a small GTPase in the ras superfamily, regulates at least two biological processes in animal cells: (i) the polymerization of actin and the assembly of integrin complexes to produce lamellipodia and ruffles; and (ii) the activity of an NADPH oxidase in phagocytic cells. NADPH oxidase activation is mediated through a rac effector protein, p67phox, and using chimeras made between rac and the closely related GTPase, rho, we have identified two distinct effector sites in rac, one N-terminal and one C-terminal, both of which are required for activation of p67phox. The same two effector sites are essential for rac-induced actin polymerization in fibroblasts. p65PAK, a ubiquitous serine/threonine kinase, interacts with rac at both the N- and C-terminal effector sites, but the GTPase-activating protein, bcr interacts with rac at a different region. This makes p65PAK, but not bcr, a candidate effector of rac-induced lamellipodium formation.     

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.     

The guanine nucleotide exchange factor trio activates the phagocyte NADPH oxidase in the absence of GDP to GTP exchange on Rac. "The emperor's nw clothes"

The superoxide-generating NADPH oxidase complex of phagocytes consists of a membrane-associated flavocytochrome b(559) and four cytosolic components as follows: p47(phox), p67(phox), p40(phox), and the small GTPase Rac (1 or 2). Activation of the oxidase is the result of assembly of the cytosolic components with cytochrome b(559) and can be mimicked in vitro by mixtures of membrane and cytosolic components exposed to an anionic amphiphile, serving as activator. We reported that prenylation of Rac1 endows it with the ability to support oxidase activation in conjunction with p67(phox) but in the absence of amphiphile and p47(phox). We now show the following 6 points. 1) The Rac guanine nucleotide exchange factor Trio markedly potentiates oxidase activation by prenylated Rac1-GDP. 2) This occurs in the absence of exogenous GTP or any other source of GTP generation, demonstrating that the effect of Trio does not involve GDP to GTP exchange on Rac1. 3) Trio does not potentiate oxidase activation by prenylated Rac1-GTP, by nonprenylated Rac1-GDP in the presence or absence of amphiphile, and by a prenylated [p67(phox)-Rac1] chimera in GDP-bound form. 4) Rac1 mutants defective in the ability to bind Trio or to respond to Trio by nucleotide exchange fail to respond to Trio by enhanced oxidase activation. 5) A Trio mutant with conserved Rac1-binding ability but lacking nucleotide exchange activity fails to enhance oxidase activation. 6) The effect of Trio is mimicked by displacement of Mg(2+) from Rac1-GDP. These results reveal the existence of a novel mechanism of Rac activation by a guanine nucleotide exchange factor and suggest that the induction by Trio of a conformational change in Rac1, in the absence of nucleotide exchange, is sufficient for enhancing its effector function.     

Angiotensin II-induced stimulation of p21-activated kinase and c-Jun NH2-terminal kinase is mediated by Rac1 and Nck

p21-activated kinase (PAK) has been shown to be an upstream mediator of JNK in angiotensin II (AngII) signaling. Little is known regarding other signaling molecules involved in activation of PAK and JNK by AngII. Rho family GTPases Rac and Cdc42 have been shown to enhance PAK activity by binding to p21-binding domain of PAK (PAK-PBD). In vascular smooth muscle cells (VSMC) AngII stimulated Rac1 binding to GST-PAK-PBD fusion protein. Pretreatment of VSMC by genistein inhibited AngII-induced Rac1 activation, whereas Src inhibitor PP1 had no effect. Inhibition of protein kinase C by phorbol 12,13-dibutyrate pretreatment also decreased AngII-mediated activation of Rac1. The adaptor molecule Nck has been shown previously to mediate PAK activation by facilitating translocation of PAK to the plasma membrane. In VSMC AngII stimulated translocation of Nck and PAK to the membrane fraction. Overexpression of dominant-negative Nck in Chinese hamster ovary (CHO) cells, stably expressing the AngII type I receptor (CHO-AT1), inhibited both PAK and JNK activation by AngII, whereas it did not affect ERK1/2. Finally, dominant-negative Nck inhibited AngII-induced DNA synthesis in CHO-AT1 cells. Our data provide evidence for Rac1 and Nck as upstream mediators of PAK and JNK in AngII signaling and implicate JNK in AngII-induced growth responses.     

Interleukin-8-induced priming of neutrophil oxidative burst requires sequential recruitment of NADPH oxidase components into lipid rafts

The superoxide-producing phagocyte NADPH oxidase consists of a membrane-bound flavocytochrome b(558), the cytosol factors p47(phox), p67(phox), p40(phox), and the small GTPase Rac2, which translocate to the membrane to assemble the active complex following neutrophil activation. Interleukin-8 (IL-8) does not activate NADPH oxidase, but potentiates the oxidative burst induced by stimuli such as formyl-methionyl-leucyl-phenylalanine (fMLP) via a priming mechanism. The effect of IL-8 on the components of NADPH oxidase during the priming process has never been investigated in human neutrophils. Here we showed that within 3 min, IL-8 treatment enhanced the Btk- and ERK1/2-dependent phosphorylation of p47(phox), as well as the recruitment of flavocytochrome b(558), p47(phox), and Rac2 into cholesterol-enriched detergent-resistant microdomains (or lipid rafts). Conversely, IL-8 treatment lasting 15 min failed to recruit flavocytochrome b(558), p47(phox), or Rac2, but did enhance the Btk- and p38 MAPK-dependent phosphorylation and the translocation of p67(phox) into detergent-resistant microdomains. Moreover, methyl-beta-cyclodextrin, which disrupts lipid rafts, inhibited IL-8-induced priming in response to fMLP. Our findings indicate that IL-8-induced priming of the oxidative burst in response to fMLP involves a sequential assembly of the NADPH oxidase components in the lipid rafts of neutrophils.     

NADPH oxidase activity selectively modulates vascular endothelial growth factor signaling pathways

Vascular endothelial growth factor (VEGF) and reactive oxygen species (ROS) play critical roles in vascular physiology and pathophysiology. We have demonstrated previously that NADPH oxidase-derived ROS are required for VEGF-mediated migration and proliferation of endothelial cells. The goal of this study was to determine the extent to which VEGF signaling is coupled to NADPH oxidase activity. Human umbilical vein endothelial cells and/or human coronary artery endothelial cells were transfected with short interfering RNA against the p47(phox) subunit of NADPH oxidase, treated in the absence or presence of VEGF, and assayed for signaling, gene expression, and function. We show that NADPH oxidase activity is required for VEGF activation of phosphoinositide 3-kinase-Akt-forkhead, and p38 MAPK, but not ERK1/2 or JNK. The permissive role of NADPH oxidase on phosphoinositide 3-kinase-Akt-forkhead signaling is mediated at post-VEGF receptor levels and involves the nonreceptor tyrosine kinase Src. DNA microarrays revealed the existence of two distinct classes of VEGF-responsive genes, one that is ROS-dependent and another that is independent of ROS levels. VEGF-induced, thrombomodulin-dependent activation of protein C was dependent on NADPH oxidase activity, whereas VEGF-induced decay-accelerating factor-mediated protection of endothelial cells against complement-mediated lysis was not. Taken together, these findings suggest that NADPH oxidase-derived ROS selectively modulate some but not all the effects of VEGF on endothelial cell phenotypes.     

Relationship between p38 mitogen-activated protein kinase and small GTPase Rac for the activation of NADPH oxidase in bovine neutrophils

Superoxide production by NADPH oxidase is essential for bactericidal properties of neutrophils. However, molecular mechanisms underlying the activation of this enzyme remain largely unknown. Here, using bovine neutrophils we examined the role of p38 mitogen-activated protein kinase (p38 MAPK) in the signaling pathways of the NADPH oxidase activation. Superoxide production was induced by stimulation with serum-opsonized zymosan (OZ) and attenuated by p38 MAPK inhibitor, SB203580. OZ stimulation induced the translocation of p47(phox) and Rac to the plasma membrane and SB203580 completely blocked the translocation of Rac, but only partially blocked that of p47(phox). Furthermore, SB203580 abolished the OZ-elicited activation of Rac, which was assessed by detecting the GTP-bound form of this protein. Phosphatidylinositol 3-kinase (PI3K) inhibitors, wortmannin and LY294002, blocked not only p38 MAPK activation but also Rac activation. However, SB203580 showed no effect on the PI3K activity. These results suggested that PI3K/p38 MAPK/Rac pathway was present in the activation of NADPH oxidase in bovine neutrophils.     

NADPH oxidase is required for NMDA receptor-dependent activation of ERK in hippocampal area CA1

Previous studies have shown that N-methyl-D-aspartate (NMDA) receptor activation results in production of reactive oxygen species (ROS) and activation of extracellular signal-regulated kinase (ERK) in hippocampal area CA1. In addition, application of ROS to hippocampal slices has been shown to result in activation of ERK in area CA1. To determine whether these events were linked causally, we investigated whether ROS are required for NMDA receptor-dependent activation of ERK. In agreement with previous studies, we found that treatment of hippocampal slices with NMDA resulted in activation of ERK in area CA1. The NMDA receptor-dependent activation of ERK was either blocked or attenuated by a number of antioxidants, including the general antioxidant N-acetyl-L-cysteine (L-NAC), the superoxide-scavenging enzyme superoxide dismutase (SOD), the membrane-permeable SOD mimetic Mn(III) tetrakis (4-benzoic acid) porphyrin (MnTBAP), the hydrogen peroxide-scavenging enzyme catalase, and the catalase mimetic ebselen. The NMDA receptor-dependent activation of ERK also was blocked by the NADPH oxidase inhibitor diphenylene iodonium (DPI) and was absent in mice that lacked p47(phox), one of the required protein components of NADPH oxidase. Taken together, our results suggest that ROS production, especially superoxide production via NADPH oxidase, is required for NMDA receptor-dependent activation of ERK in hippocampal area CA1.     

Human monocytes use Rac1, not Rac2, in the NADPH oxidase complex

Phagocyte NADPH oxidase is critical for defense against pathogens and contributes to inflammatory tissue injury. One component of the NADPH oxidase complex is the small GTP-binding protein Rac. There are two isoforms of Rac, and Rac2 is the predominant isoform in neutrophils and has been shown to be essential for NADPH oxidase activity. In primary human monocytes we report that in contrast to neutrophils, Rac1 is the predominantly expressed isoform. Upon monocyte activation by a variety of agents, we found that Rac1 dissociates from Rho GDP dissociation inhibitor (RhoGDI) and translocates to the membrane. We also found that Rac1 interacts with two other NADPH oxidase components, p67phox and p47phox, upon monocyte activation. These data indicate that Rac1, and not Rac2, is a component of the activated NADPH oxidase in monocytes. This finding suggests that it may be possible to selectively interfere with either monocyte or neutrophil NADPH oxidase activity, thereby selectively targeting chronic versus acute inflammatory processes.     

A prodigiosin analogue inactivates NADPH oxidase in macrophage cells by inhibiting assembly of p47phox and Rac

Prodigiosins are natural red pigments that have multi-biological activities. Recently, we discovered a marine bacterial strain, which produces a red pigment. Extensive two-dimensional nuclear magnetic resonance and mass spectrometry analysis showed that the pigment is a prodigiosin analogue (PG-L-1). Here, we investigated the effect of PG-L-1 on NADPH oxidase activity in macrophage cells. PG-L-1 significantly inhibited superoxide anion (O(2)(-)) production by phorbol 12-myristate 13-acetate (PMA)-stimulated RAW 264.7 cells, a mouse macrophage cell line. The ED(50) value was estimated to be approximately 0.3 microM. PG-L-1 had no direct scavenging effect on O(2)(-) generated by hypoxanthine/xanthine oxidase system in electron spin resonance-spin trapping determinations, suggesting that this compound directly acts on the O(2)(-) production system, NADPH oxidase, in macrophage cells. We further investigated the effect of PG-L-1 on the behaviour of the cytosolic components of the NADPH oxidase, p67(phox), p47(phox), p40(phox), Rac and protein kinase C (PKC), in PMA-stimulated RAW 264.7 cells. Although PG-L-1 showed no effect on the activation of PKC, the immunoblotting analysis using specific antibodies showed that PG-L-1 strongly inhibits the association of p47(phox) and Rac in the plasma membrane of PMA-stimulated RAW 264.7 cells. These results suggest that PG-L-1 inactivates NADPH oxidase through the inhibition of the binding of p47(phox) and Rac to the membrane components of NADPH oxidase.     

Activation of the superoxide-generating NADPH oxidase by chimeric proteins consisting of segments of the cytosolic component p67(phox) and the small GTPase Rac1.

Activation of the superoxide (O2(-))-generating NADPH oxidase of phagocytes is the consequence of the assembly of a membrane-associated flavocytochrome b(559) with the cytosolic proteins p47(phox) and p67(phox) and the small GTPase Rac (1 or 2). We proposed that Rac1 serves as a membrane-targeting molecule for p67(phox). This hypothesis was tested by constructing recombinant chimeric proteins, joining various functional domains of p67(phox) and Rac1, and expressing these in Escherichia coli. Chimeras were assayed for the ability to support O2(-) production by phagocyte membranes in an amphiphile-activated cell-free system in the presence or absence of p47(phox). A chimera consisting of p67(phox) truncated at residue 212 and fused to a full-length Rac1 [p67(phox)(1-212)-Rac1(1-192)] was a potent NADPH oxidase activator. A p67(phox)(1-212)-Rac1(178-192) chimera, to which Rac1 contributed only the C-terminal polybasic domain, was a weaker but consistent activator. Chimeras comprising the full length of Rac1 bound GTP/GDP, like bona fide GTPases. The activity of p67(phox)-Rac1 chimeras was dependent on the presence of the tetratricopeptide repeat and activation domains, in the p67(phox) segment, and on an intact polybasic region, at the C terminus of the Rac1 segment, but not on the insert region of Rac1. Partial activation by chimeras, in the GTP-bound form, was also possible in the absence of p47(phox). Evidence is offered in support of the proposal that the GTP- and GDP-bound forms of chimera p67(phox)(1-212)-Rac1(1-192) have distinct conformations, corresponding to the presence and absence of intrachimeric bonds, respectively.     

Dual role of Rac in the assembly of NADPH oxidase, tethering to the membrane and activation of p67phox: a study based on mutagenesis of p67phox-Rac1 chimeras

NADPH oxidase activation involves the assembly of membrane-localized cytochrome b559 with the cytosolic components p47phox, p67phox, and the small GTPase Rac. Assembly is mimicked by a cell-free system consisting of membranes and cytosolic components, activated by an anionic amphiphile. We reported that a chimeric construct, consisting of residues 1-212 of p67phox and full-length Rac1, activates the oxidase in vitro in an amphiphile-dependent manner, and when prenylated, in the absence of amphiphile and p47phox. We subjected chimera p67phox-(1-212)-Rac1 to mutational analysis and found that: 1) replacement of a single basic residue at the C terminus of the Rac1 moiety by glutamine is sufficient for loss of activity by the non-prenylated chimera; replacement of all six basic residues by glutamines is required for loss of activity by the prenylated chimera. 2) A V204A mutation in the activation domain of the p67phox moiety leads to a reduction in activity. 3) Mutating residues, known to participate in the interaction between free p67phox and Rac1, in the p67phox-(R102E) or Rac1 (A27K, G30S) moieties of the chimera, leads to a marked decrease in activity, indicating a requirement for intrachimeric bonds, in addition to the engineered fusion. 4) Chimeras, inactive because of mutations A27K or G30S in the Rac1 moiety, are reactivated by supplementation with exogenous Rac1-GTP but not with exogenous p67phox. This demonstrates that Rac has a dual role in the assembly of NADPH oxidase. One is to tether p67phox to the membrane; the other is to induce an "activating" conformational change in p67phox.     

Puerarin attenuates high-glucose-and diabetes-induced vascular smooth muscle cell proliferation by blocking PKCβ2/Rac1-dependent signaling

Oxidative stress has been implicated in several steps leading to the development of diabetic vascular complications. The purpose of this study was to determine the efficacy and the possible mechanism of puerarin on high-glucose (HG; 25 mM)-induced proliferation of cultured rat vascular smooth muscle cells (VSMCs) and neointimal formation in a carotid arterial balloon injury model of obese Zucker rats. Our data demonstrated that puerarin significantly inhibited rat VSMC proliferation as well as reactive oxygen species (ROS) generation and NADPH oxidase activity induced by HG treatment. Further studies revealed that HG treatment resulted in phosphorylation and membrane translocation of PKCβ2 as well as Rac1, p47phox, and p67phox subunits, leading to NADPH oxidase activation. Puerarin treatment remarkably disrupted the phosphorylation and membrane translocation of PKCβ2 as well as Rac1, p47phox, and p67phox subunits. Blocking PKCβ2 by infection with AdDNPKCβ2 also abolished HG-induced phosphorylation and membrane translocation of Rac1, p47phox, and p67phox subunits as well as ROS production and NADPH oxidase activation in VSMCs. In vivo neointimal formation of obese Zucker rats evoked by balloon injury was evidently attenuated by the administration of puerarin. These results demonstrate that puerarin may exert inhibitory effects on HG-induced VSMC proliferation via interfering with PKCβ2/Rac1-dependent ROS pathways, thus resulting in the attenuation of neointimal formation in the context of hyperglycemia in diabetes mellitus.     

Antagonistic cross-talk between Rac and Cdc42 GTPases regulates generation of reactive oxygen species

Cross-talk between Rho GTPase family members (Rho, Rac, and Cdc42) plays important roles in modulating and coordinating downstream cellular responses resulting from Rho GTPase signaling. The NADPH oxidase of phagocytes and nonphagocytic cells is a Rac GTPase-regulated system that generates reactive oxygen species (ROS) for the purposes of innate immunity and intracellular signaling. We recently demonstrated that NADPH oxidase activation involves sequential interactions between Rac and the flavocytochrome b(558) and p67(phox) oxidase components to regulate electron transfer from NADPH to molecular oxygen. Here we identify an antagonistic interaction between Rac and the closely related GTPase Cdc42 at the level of flavocytochrome b(558) that regulates the formation of ROS. Cdc42 is unable to stimulate ROS formation by NADPH oxidase, but Cdc42, like Rac1 and Rac2, was able to specifically bind to flavocytochrome b(558) in vitro. Cdc42 acted as a competitive inhibitor of Rac1- and Rac2-mediated ROS formation in a recombinant cell-free oxidase system. Inhibition was dependent on the Cdc42 insert domain but not the Switch I region. Transient expression of Cdc42Q61L inhibited ROS formation induced by constitutively active Rac1 in an NADPH oxidase-expressing Cos7 cell line. Inhibition of Cdc42 activity by transduction of the Cdc42-binding domain of Wiscott-Aldrich syndrome protein into human neutrophils resulted in an enhanced fMetLeuPhe-induced oxidative response, consistent with inhibitory cross-talk between Rac and Cdc42 in activated neutrophils. We propose here a novel antagonism between Rac and Cdc42 GTPases at the level of the Nox proteins that modulates the generation of ROS used for host defense, cell signaling, and transformation.