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.     

Phosphorylation of p47phox sites by PKC alpha,beta II,delta, and zeta: effect on binding to p22phox and on NADPH oxidase activation

Production of superoxide anions by the multicomponent enzyme of human neutrophil NADPH oxidase is accompanied by extensive phosphorylation of p47(phox), one of its cytosolic components. p47(phox) is an excellent substrate for protein kinase C (PKC), but the respective contribution of each PKC isoform to this process is not clearly defined. In this study, we found that PKC isoforms known to be present in human neutrophils (PKC alpha, beta, delta, and zeta) phosphorylate p47(phox) in a time- and concentration-dependent manner, with apparent K(m) values of 10.33, 3.37, 2.37, and 2.13 microM for PKC alpha, beta II, delta, and zeta, respectively. Phosphopeptide mapping of p47(phox) showed that, as opposed to PKC zeta, PKC alpha, beta II, and delta are able to phosphorylate all the major PKC sites. The use of p47(phox) mutants identified serines 303, 304, 315, 320, 328, 359, 370, and 379 as targets of PKC alpha, beta II, and delta. Comparison of the intensity of phosphopeptides suggests that Ser 328 is the most phosphorylated serine. The ability of each PKC isoform to induce p47(phox) to associate with p22(phox) was tested by using an overlay technique; the results showed that all the PKC isoforms that were studied induce p47(phox) binding to the cytosolic fragment of p22(phox). In addition, PKC alpha, beta II, delta, and zeta were able to induce production of superoxide anions in a cell-free system using recombinant cytosolic proteins. Surprisingly, PKC zeta, which phosphorylates a subset of selective p47(phox) sites, induced stronger activation of the NADPH oxidase. Taken together, these results suggest that PKC alpha, beta II, delta, and zeta expressed in human neutrophils can individually phosphorylate p47(phox) and induce both its translocation and NADPH oxidase activation. In addition, phosphorylation of some serines could have an inhibitory effect on oxidase activation.     

Angiotensin II-induced NADPH oxidase activation impairs insulin signaling in skeletal muscle cells

The renin-angiotensin system (RAS) and reactive oxygen species (ROS) have been implicated in the development of insulin resistance and its related complications. There is also evidence that angiotensin II (Ang II)-induced generation of ROS contributes to the development of insulin resistance in skeletal muscle, although the precise mechanisms remain unknown. In the present study, we found that Ang II markedly enhanced NADPH oxidase activity and consequent ROS generation in L6 myotubes. These effects were blocked by the angiotensin II type 1 receptor blocker losartan, and by the NADPH oxidase inhibitor apocynin. Ang II also promoted the translocation of NADPH oxidase cytosolic subunits p47phox and p67phox to the plasma membrane within 15 min. Furthermore, Ang II abolished insulin-induced tyrosine phosphorylation of insulin receptor substrate 1 (IRS1), activation of protein kinase B (Akt), and glucose transporter-4 (GLUT4) translocation to the plasma membrane, which was reversed by pretreating myotubes with losartan or apocynin. Finally, small interfering RNA (siRNA)-specific gene silencing targeted specifically against p47phox (p47siRNA), in both L6 and primary myotubes, reduced the cognate protein expression, decreased NADPH oxidase activity, restored Ang II-impaired IRS1 and Akt activation as well as GLUT4 translocation by insulin. These results suggest a pivotal role for NADPH oxidase activation and ROS generation in Ang II-induced inhibition of insulin signaling in skeletal muscle cells.     

Phosphoinositide 3-kinase regulates the phosphorylation of NADPH oxidase component p47(phox) by controlling cPKC/PKCdelta but not Akt

Superoxide production by NADPH oxidase is essential for the bactericidal properties of phagocytes. Phosphorylation of p47(phox), one of the cytosolic components of NADPH oxidase, is a crucial step of the oxidase activation. Some evidences suggest that phosphoinositide 3-kinase (PI3K) is involved in p47(phox) phosphorylation, but it has not been fully understood how PI3K regulates it. The aim of this study was to examine the mechanism underlying the PI3K regulation of p47(phox) phosphorylation. Pharmacological inhibition of PI3K attenuated both fMLP-stimulated p47(phox) phosphorylation and NADPH oxidase activity in HL-60 cells differentiated to a neutrophil-like phenotype. Although fMLP elicited Akt activation in a PI3K-dependent manner, an Akt inhibitor had no effect on the oxidase activity triggered by fMLP. In vitro kinase assay revealed that Akt was unable to catalyze p47(phox) phosphorylation. Interestingly, the activation of cPKC and PKCdelta after fMLP stimulation was dependent on PI3K. Furthermore, PI3K inhibitors reduced the activation of phospholipase Cgamma2 without affecting tyrosine phosphorylation on it. These results suggest that PI3K regulates the phosphorylation of NADPH oxidase component p47(phox) by controlling diacylglycerol-dependent PKCs but not Akt.     

Protein kinase C delta is required for p47phox phosphorylation and translocation in activated human monocytes

Our laboratory is interested in understanding the regulation of NADPH oxidase activity in human monocyte/macrophages. Protein kinase C (PKC) is reported to be involved in regulating the phosphorylation of NADPH oxidase components in human neutrophils; however, the regulatory roles of specific isoforms of PKC in phosphorylating particular oxidase components have not been determined. In this study calphostin C, an inhibitor for both novel PKC (including PKCdelta, -epsilon, -theta;, and -eta) and conventional PKC (including PKCalpha and -beta), inhibited both phosphorylation and translocation of p47phox, an essential component of the monocyte NADPH oxidase. In contrast, GF109203X, a selective inhibitor of classical PKC and PKCepsilon, did not affect the phosphorylation or translocation of p47phox, suggesting that PKCdelta, -theta;, or -eta is required. Furthermore, rottlerin (at doses that inhibit PKCdelta activity) inhibited the phosphorylation and translocation of p47phox. Rottlerin also inhibited O2 production at similar doses. In addition to pharmacological inhibitors, PKCdelta-specific antisense oligodeoxyribonucleotides were used. PKCdelta antisense oligodeoxyribonucleotides inhibited the phosphorylation and translocation of p47phox in activated human monocytes. We also show, using the recombinant p47phox-GST fusion protein, that p47phox can serve as a substrate for PKCdelta in vitro. Furthermore, lysate-derived PKCdelta from activated monocytes phosphorylated p47phox in a rottlerin-sensitive manner. Together, these data suggest that PKCdelta plays a pivotal role in stimulating monocyte NADPH oxidase activity through its regulation of the phosphorylation and translocation of p47phox.     

Involvement of protein kinase Cdelta in the activation of NADPH oxidase and the phagocytosis of neutrophils

This experiment was performed to clarify the role of protein kinase C (PKC) delta in NADPH oxidase-dependent O(2-) production and actin polymerization followed by phagocytosis in neutrophils. Bovine neutrophils and human neutrophil-like differentiated HL-60 (dHL-60) cells were stimulated with serum-opsonized zymosan (OZ) and fMet-Leu-Phe (fMLP), respectively. Rottlerin, a specific inhibitor of PKCdelta, attenuated the production of O(2-) from NADPH oxidase in both neutrophils and dHL-60 cells. However, it did not inhibit the translocation of p47(phox) from the cytosol to the membrane in either type of cell or the phosphorylation of p47(phox) in dHL-60 cells. GF109203X (GFX), an inhibitor of cPKC, attenuated not only the production of O(2-) but also the translocation of p47(phox) in both cells. Furthermore, rottlerin significantly attenuated the ingestion of opsonized particles and the formation of F-actin in OZ-stimulated neutrophils, whereas, GFX did not affect those phagocytic processes. These results suggest that both PKCdelta and cPKC regulate NADPH oxidase through different pathways, but only PKCdelta regulates the phagocytic function in neutrophils.     

Protein kinase C zeta phosphorylates a subset of selective sites of the NADPH oxidase component p47phox and participates in formyl peptide-mediated neutrophil respiratory burst

Generation of superoxide anion by the multiprotein complex NADPH phagocyte oxidase is accompanied by extensive phosphorylation of its 47-kDa protein component, p47(phox), a major cytosolic component of this oxidase. Protein kinase C zeta (PKC zeta), an atypical PKC isoform expressed abundantly in human polymorphonuclear leukocytes (PMN), translocates to the PMN plasma membrane upon stimulation by the chemoattractant fMLP. We investigated the role of PKC zeta in p47(phox) phosphorylation and in superoxide anion production by human PMN. In vitro incubation of recombinant p47(phox) with recombinant PKC zeta induced a time- and concentration-dependent phosphorylation of p47(phox) with an apparent K(m) value of 2 microM. Phosphopeptide mapping analysis of p47(phox) showed that PKC zeta phosphorylated fewer selective sites in comparison to "conventional" PKCs. Serine 303/304 and serine 315 were identified as targets of PKC zeta by site-directed mutagenesis. Stimulation of PMN by fMLP induced a rapid and sustained plasma membrane translocation of PKC zeta that correlated to that of p47(phox). A cell-permeant-specific peptide antagonist of PKC zeta inhibited both fMLP-induced phosphorylation of p47(phox) and its membrane translocation. The antagonist also inhibited the fMLP-induced production of oxidant (IC(50) of 10 microM), but not that induced by PMA. The inhibition of PKC zeta expression in HL-60 neutrophil-like cells using antisense oligonucleotides (5 and 10 microM) inhibited fMLP-promoted oxidant production (27 and 50%, respectively), but not that induced by PMA. In conclusion, p47(phox) is a substrate for PKC zeta and participates in the signaling cascade between fMLP receptors and NADPH oxidase activation.     

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.     

Diosgenin inhibits superoxide generation in FMLP-activated mouse neutrophils via multiple pathways

Abstract

Diosgenin possesses anti-inflammatory and anticancer properties. Activated neutrophils produce high concentrations of the superoxide anion which is involved in the pathophysiology of inflammation-related diseases and cancer. In the present study, the inhibitory effect and possible mechanisms of diosgenin on superoxide generation were investigated in mouse bone marrow neutrophils. Diosgenin potently and concentration-dependently inhibited the extracellular and intracellular superoxide anion generation in Formyl-Met-Leu-Phe (FMLP)- activated neutrophils, with IC₅₀ values of 0.50 ± 0.08 μM and 0.66 ± 0.13 μM, respectively. Such inhibition was not mediated by scavenging the superoxide anion or by a cytotoxic effect. Diosgenin inhibited the phosphorylation of p47phox and membrane translocation of p47phox and p67phox, and thus blocking the assembly of nicotinamide adenine dinucleotide phosphate oxidase. Moreover, cellular cyclic adenosine monophosphate (cAMP) levels and protein kinase A (PKA) expression were also effectively increased by diosgenin. It attenuated FMLP-induced increase of phosphorylation of cytosolic phospholipase A (cPLA₂), p21-activated kinase (PAK), Akt, p38 mitogen-activated protein kinase (p38MAPK), extracellular signal-regulated kinase (ERK1/2), and c-Jun N-terminal kinase (JNK). Our data indicate that diosgenin exhibits inhibitory effects on superoxide anion production through the blockade of cAMP, PKA, cPLA₂, PAK, Akt and MAPKs signaling pathways. The results may explain the clinical implications of diosgenin in the treatment of inflammation-related disorders.     

Lipopolysaccharide-induced cytokine expression in alveolar epithelial cells: role of PKCζ-mediated p47phox phosphorylation

Chronic inflammation incited by bacteria in the saccular lung of premature infants contributes to the pathogenesis of bronchopulmonary dysplasia (BPD). LPS-mediated type II alveolar epithelial cell (AEC) injury induces the expression of pro-inflammatory cytokines that trigger pulmonary neutrophil influx, alveolar matrix degradation and lung remodeling. We hypothesized that NADPH oxidase (Nox)-dependent mechanisms mediate LPS-induced cytokine expression in AEC. We examined the role of p47phox in mediating LPS-dependent inflammatory cytokine expression in A549 cells (which exhibit phenotypic features characteristic of type II AEC) and elucidated the proximal signaling events by which Nox is activated by LPS. LPS-induced ICAM-1 and IL-8 expression was associated with increased superoxide formation in AEC. LPS-mediated oxidative stress and cytokine expression was inhibited by apocynin and augmented by PMA demonstrating that Nox-dependent redox signaling regulates LPS-dependent pro-inflammatory signaling in AEC. In LPS-treated cells, p47phox translocated from the cytoplasm to the perinuclear region and co-localized with gp91phox. LPS also induced a temporal increase in p47phox serine304 phosphorylation in AEC. While inhibition of classical PKC and novel PKC with calphostin and rottlerin did not inhibit ICAM-1 or IL-8 expression, the myristolyated PKCζ pseudosubstrate peptide (a specific inhibitor of PKCζ) inhibited LPS-induced cytokine expression in AEC. Inhibition of PKCζ also attenuated LPS-mediated p47phox phosphorylation and perinuclear translocation in AEC. Consistent with these data, LPS activated PKCζ in AEC as evidenced by increased threonine410 phophorylation. We conclude that PKCζ-mediated p47phox activation regulates LPS-dependent cytokine expression in AEC. Selective inhibition of PKCζ or p47phox might attenuate LPS-mediated inflammation and alveolar remodeling in BPD.     

Akt phosphorylates p47phox and mediates respiratory burst activity in human neutrophils

Respiratory burst activity and phosphorylation of an NADPH oxidase component, p47(phox), during neutrophil stimulation are mediated by phosphatidylinositol 3-kinase (PI-3K) activation. Products of PI-3K activate several kinases, including the serine/threonine kinase Akt. The present study examined the ability of Akt to regulate neutrophil respiratory burst activity and to interact with and phosphorylate p47(phox). Inhibition of Akt activity in human neutrophils by an inhibitory peptide significantly attenuated fMLP-stimulated, but not PMA-stimulated, superoxide release. Akt inhibitory peptide also inhibited hydrogen peroxide generation stimulated by bacterial phagocytosis. A direct interaction between p47(phox) and Akt was shown by the ability of GST-p47(phox) to precipitate recombinant Akt and to precipitate Akt from neutrophil lysates. Active recombinant Akt phosphorylated recombinant p47(phox) in vitro, as shown by (32)P incorporation, by a mobility shift change detected by two-dimensional gel electrophoresis, and by immunoblotting with phospho-Akt substrate Ab. Mutation analysis indicated that 2 aa residues, Ser(304) and Ser(328), were phosphorylated by Akt. Inhibition of Akt activity also inhibited fMLP-stimulated neutrophil chemotaxis. We propose that Akt mediates PI-3K-dependent p47(phox) phosphorylation, which contributes to respiratory burst activity in human neutrophils.     

NADPH oxidase activation and assembly during phagocytosis

Generation of superoxide (O2-) by the NADPH-dependent oxidase of polymorphonuclear leukocytes is an essential component of the innate immune response to invading microorganisms. To examine NADPH oxidase function during phagocytosis, we evaluated its activation and assembly following ingestion of serum-opsonized Neisseria meningitidis, serogroup B (NMB), and compared it with that elicited by serum-opsonized zymosan (OPZ). Opsonized N. meningitidis- and OPZ-dependent generation of reactive oxygen species by polymorphonuclear leukocytes peaked early and then terminated. Phosphorylation of p47phox coincided with peak generation of reactive oxygen species by either stimulus, consistent with a role for p47phox phosphorylation during NADPH oxidase activation, and correlated with phagosomal colocalization of flavocytochrome b558 (flavocytochrome b) and p47phox and p67phox (p47/67phox). Termination of respiratory burst activity did not reflect dephosphorylation of plasma membrane- and/or phagosome-associated p47phox; in contrast, the specific activity of phosphorylated p47phox at the phagosomal membrane increased. Most significantly, termination of oxidase activity paralleled the loss of p47/67phox from both NMB and OPZ phagosomes despite the continued presence of flavocytochrome b. These data suggest that 1) the onset of respiratory burst activity during phagocytosis is linked to the phosphorylation of p47phox and its translocation to the phagosome; and 2) termination of oxidase activity correlates with loss of p47/67phox from flavocytochrome b-enriched phagosomes and additional phosphorylation of membrane-associated p47phox.     

Modulation of microglial superoxide production by α-tocopherol in vitro: attenuation of p67phox translocation by a protein phosphatase-dependent pathway

As in other phagocytic cells, the NADPH-oxidase system in microglia is thought to be primarily responsible for the production of superoxide anion radicals (O2(-.), a potentially cytotoxic reactive oxygen species. The assembly of a functional NADPH-oxidase complex at the plasma membrane depends on the phosphorylation and subsequent translocation of several cytosolic subunits. Immunocytochemical and subcellular fractionation experiments performed during the present study revealed that the NADPH-oxidase subunit p67(phox) translocates from the cytosol to the plasma membrane upon stimulation. Pre-incubation of microglia in alpha-tocopherol (alphaTocH) containing medium decreased O2(-.) production in a time- and concentration-dependent manner, findings attributed to attenuated p67(phox) translocation to the plasma membrane. Moreover, alphaTocH-supplementation of the culture medium resulted in decreased microglial protein kinase C (PKC) activities, an effect that could be partially or completely reversed by the addition of protein phosphatase inhibitors (okadaic acid and calyculin A). The addition of the PKC-inhibitor staurosporine inhibited the microglial respiratory burst in a manner comparable to alphaTocH. The addition of okadaic acid or calyculin A completely restored O2(-.) production in alphaTocH-supplemented cells. The present findings suggest that alphaTocH inactivates PKC via a PP1 or PP2A-mediated pathway and, as a consequence, blocks the phosphorylation-dependent translocation of p67(phox) to the plasma membrane. As a result, O2(-.) production by the microglial NADPH-oxidase system is substantially inhibited.     

Src-mediated tyrosine phosphorylation of p47phox in hyperoxia-induced activation of NADPH oxidase and generation of reactive oxygen species in lung endothelial cells

Superoxide (O(2)(-)) production by nonphagocytes, similar to phagocytes, is by activation of the NADPH oxidase multicomponent system. Although activation of neutrophil NADPH oxidase involves extensive serine phosphorylation of p47(phox), the role of tyrosine phosphorylation of p47(phox) in NADPH oxidase-dependent O(2)(-) production is unclear. We have shown recently that hyperoxia-induced NADPH oxidase activation in human pulmonary artery endothelial cells (HPAECs) is regulated by mitogen-activated protein kinase signal transduction. Here we provided evidence on the role of nonreceptor tyrosine kinase, Src, in hyperoxia-induced tyrosine phosphorylation of p47(phox) and NADPH oxidase activation in HPAECs. Exposure of HPAECs to hyperoxia for 1 h resulted in increased O(2)(-) and reactive oxygen species (ROS) production and enhanced tyrosine phosphorylation of Src as determined by Western blotting with phospho-Src antibodies. Pretreatment of HPAECs with the Src kinase inhibitor PP2 (1 mum) or transient expression of a dominant-negative mutant of Src attenuated hyperoxia-induced tyrosine phosphorylation of Src and ROS production. Furthermore, exposure of cells to hyperoxia enhanced tyrosine phosphorylation of p47(phox) and its translocation to cell peripheries that were attenuated by PP2. In vitro, Src phosphorylated recombinant p47(phox) in a time-dependent manner. Src immunoprecipitates of cell lysates from control cells revealed the presence of immunodetectable p47(phox) and p67(phox), suggesting the association of oxidase components with Src under basal conditions. Moreover, exposure of HPAECs to hyperoxia for 1 h enhanced the association of p47(phox), but not p67(phox), with Src. These results indicated that Src-dependent tyrosine phosphorylation of p47(phox) regulates hyperoxia-induced NADPH oxidase activation and ROS production in HPAECs.     

The mitogen-activated protein kinase extracellular signal-regulated kinase 1/2 pathway is involved in formyl-methionyl-leucyl-phenylalanine-induced p47phox phosphorylation in human neutrophils

Phosphorylation of p47 phagocyte oxidase, (p47(phox)), one of the NADPH oxidase components, is essential for the activation of this enzyme and for superoxide production. p47(phox) is phosphorylated on multiple serine residues, but the kinases involved in this process in vivo remain to be characterized. We examined the role of extracellular signal-regulated kinase (ERK1/2) and p38 mitogen-activated protein kinase in p47(phox) phosphorylation. Inhibition of ERK1/2 activation by PD98059, a specific inhibitor of ERK kinase 1/2, inhibited the fMLP-induced phosphorylation of p47(phox). However, PD98059 weakly affected PMA-induced p47(phox) phosphorylation, even though ERK1/2 activation was abrogated. This effect was confirmed using U0126, a second ERK kinase inhibitor. Unlike PD98059 and U0126, the p38 mitogen-activated protein kinase inhibitor SB203580 did not inhibit the phosphorylation of p47(phox) induced either by fMLP or by PMA. Two-dimensional phosphopeptide mapping analysis showed that, in fMLP-induced p47(phox) phosphorylation, PD98059 affected the phosphorylation of all the major phosphopeptides, suggesting that ERK1/2 may regulate p47(phox) phosphorylation either directly or indirectly via other kinases. In PMA-induced p47(phox) phosphorylation, GF109203X, a protein kinase C inhibitor, strongly inhibits p47(phox) phosphorylation. However, in fMLP-induced p47(phox) phosphorylation, PD98059 and GF109203X partially inhibited the phosphorylation of p47(phox) when tested alone, and exerted additive inhibitory effects on p47(phox) phosphorylation when tested together. These results show for the first time that the ERK1/2 pathway participates in the phosphorylation of p47(phox). Furthermore, they strongly suggest that p47(phox) is targeted by several kinase cascades in intact neutrophils activated by fMLP and is therefore a converging point for ERK1/2 and protein kinase C.     

利多卡因对人离体中性白细胞超氧阴离子和p47^phox磷酸化的影响

目的用人离体中性白细胞研究利多卡因对刺激剂诱导超氧阴离子产生,蛋白质酪氨酸磷酸化和NADPH氧化酶组成因子p47^phox和p67^phox从细胞质向细胞膜移动的影响。方法用细胞色素C还原法测定不同浓度利多卡因对3种刺激剂介导的中性白细胞释放超氧阴离子量。用Western blot检测中性白细胞蛋白质的磷酸化及NADPH氧化酶细胞质因子p47^phox和p67^phox的磷酸化。结果利多卡因可呈浓度依赖性抑制f MLP（N-formyl-methionyl-leucyl-phenylalanine）介导的中性白细胞释放超氧阴离子,而对PMA（phorbol 12-myristate 13-acetate）或AA（arachidonic acid）介导的中性白细胞释放超氧阴离子并无影响。利多卡因呈浓度依赖性抑制f MLP介导的中性白细胞蛋白质（86.0,58.0,45.0 kDa）的磷酸化,与利多卡因对中性白细胞释放超氧阴离子的影响相一致,另外利多卡因还可抑制细胞质因子p47^phox和p67^phox的从细胞质向细胞膜的移动,从而抑制NADPH氧化酶释放超氧阴离子。结论利多卡因呈浓度依赖性抑制f MLP介导的中性白细胞产生超氧阴离子,这一作用与抑制细胞的一些蛋白质磷酸化及p47^phox和p67^phox从细胞质向细胞膜移动有关。     

Regulation of hyperoxia-induced NADPH oxidase activation in human lung endothelial cells by the actin cytoskeleton and cortactin

Although the actin cytoskeleton has been implicated in the control of NADPH oxidase in phagocytosis, very little is known about the cytoskeletal regulation of endothelial NADPH oxidase assembly and activation. Here, we report a role for cortactin and the tyrosine phosphorylation of cortactin in hyperoxia-induced NADPH oxidase activation and ROS production in human pulmonary artery ECs (HPAECs). Exposure of HPAECs to hyperoxia for 3 h induced NADPH oxidase activation, as demonstrated by enhanced superoxide production. Hyperoxia also caused a thickening of the subcortical dense peripheral F-actin band and increased the localization of cortactin in the cortical regions and lamellipodia at cell-cell borders that protruded under neighboring cells. Pretreatment of HPAECs with the actin-stabilizing agent phallacidin attenuated hyperoxia-induced cortical actin thickening and ROS production, whereas cytochalasin D and latrunculin A enhanced basal and hyperoxia-induced ROS formation. In HPAECs, a 3-h hyperoxic exposure enhanced the tyrosine phosphorylation of cortactin and interaction between cortactin and p47(phox), a subcomponent of the EC NADPH oxidase, when compared with normoxic cells. Furthermore, transfection of HPAECs with cortactin small interfering RNA or myristoylated cortactin Src homology domain 3 blocking peptide attenuated ROS production and the hyperoxia-induced translocation of p47(phox) to the cell periphery. Similarly, down-regulation of Src with Src small interfering RNA attenuated the hyperoxia-mediated phosphorylation of cortactin tyrosines and blocked the association of cortactin with actin and p47(phox). In addition, the hyperoxia-induced generation of ROS was significantly lower in ECs expressing a tyrosine-deficient mutant of cortactin than in vector control or wild-type cells. These data demonstrate a novel function for cortactin and actin in hyperoxia-induced activation of NADPH oxidase and ROS generation in human lung endothelial cells.     

A critical role of protein kinase C delta activation loop phosphorylation in formyl-methionyl-leucyl-phenylalanine-induced phosphorylation of p47(phox) and rapid activation of nicotinamide adenine dinucleotide phosphate oxidase

Generation of superoxide by professional phagocytes is an important mechanism of host defense against bacterial infection. Several protein kinase C (PKC) isoforms have been found to phosphorylate p47(phox), resulting in its membrane translocation and activation of the NADPH oxidase. However, the mechanism by which specific PKC isoforms regulate NADPH oxidase activation remains to be elucidated. In this study, we report that PKCdelta phosphorylation in its activation loop is rapidly induced by fMLF and is essential for its ability to catalyze p47(phox) phosphorylation. Using transfected COS-7 cells expressing gp91(phox), p22(phox), p67(phox), and p47(phox) (COS-phox cells), we found that a functionally active PKCdelta is required for p47(phox) phosphorylation and reconstitution of NADPH oxidase. PKCbetaII cannot replace PKCdelta for this function. Characterization of PKCdelta/PKCbetaII chimeras has led to the identification of the catalytic domain of PKCdelta as a target of regulation by fMLF, which induces a biphasic (30 and 180 s) phosphorylation of Thr(505) in the activation loop of mouse PKCdelta. Mutation of Thr(505) to alanine abolishes the ability of PKCdelta to catalyze p47(phox) phosphorylation in vitro and to reconstitute NADPH oxidase in the transfected COS-phox cells. A correlation between fMLF-induced activation loop phosphorylation and superoxide production is also established in the differentiated PLB-985 human myelomonoblastic cells. We conclude that agonist-induced PKCdelta phosphorylation is a novel mechanism for NADPH oxidase activation. The ability to induce PKCdelta phosphorylation may distinguish a full agonist from a partial agonist for superoxide production.     

Hyperoxia-induced NAD(P)H oxidase activation and regulation by MAP kinases in human lung endothelial cells

Hyperoxia increases reactive oxygen species (ROS) production in vascular endothelium; however, the mechanisms involved in ROS generation are not well characterized. We determined the role and regulation of NAD(P)H oxidase in hyperoxia-induced ROS formation in human pulmonary artery endothelial cells (HPAECs). Exposure of HPAECs to hyperoxia for 1, 3, and 12 h increased the generation of superoxide anion, which was blocked by diphenyleneiodonium but not by rotenone or oxypurinol. Furthermore, hyperoxia enhanced NADPH- and NADH-dependent and superoxide dismutase- or diphenyleneiodonium-inhibitable ROS production in HPAECs. Immunohistocytochemistry and Western blotting revealed the presence of gp91, p67 phox, p22 phox, and p47 phox subcomponents of NADPH oxidase in HPAECs. Transfection of HPAECs with p22 phox antisense plasmid inhibited hyperoxia-induced ROS production. Exposure of HPAECs to hyperoxia activated p38 MAPK and ERK, and inhibition of p38 MAPK and MEK1/2 attenuated the hyperoxia-induced ROS generation. These results suggest a role for MAPK in regulating hyperoxia-induced NAD(P)H oxidase activation in HPAECs.