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.     

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.     

Role of protein kinase C in NADPH oxidase derived O2-mediated regulation of KV-LVOCC axis under U46619 induced increase in [Ca]i in pulmonary smooth muscle cells

Treatment of bovine pulmonary smooth muscle cells with the TxA₂ mimetic, U46619 stimulated [Ca²⁺]_i, which was inhibited upon pretreatment with apocynin (NADPH oxidase inhibitor). Pretreatment with cromakalim (K_V channel opener) or nifedepine (L-VOCC inhibitor) inhibited U46619 induced increase in [Ca²⁺]_i, indicating a role of K_V-LVOCC axis in this scenario. Neither cromakalim nor nifedepine inhibited U46619 induced increase in NADPH oxidase activity, suggesting that the NADPH oxidase activation is proximal to the K_V-LVOCC axis in the cells. Pretreatment with calphostin C (PKC inhibitor) markedly reduced U46619 induced increase in NADPH oxidase activity and [Ca²⁺]_i in the cells. Calphostin C pretreatment also markedly reduced p^47phox phosphorylation and translocation to the membrane and association with p^22phox, a component of Cyt.b₅₅₈ of NADPH oxidase in the membrane. Overall, PKC plays an important role in NADPH oxidase derived O₂⁻-mediated regulation of K_V-LVOCC axis leading to an increase in [Ca²⁺]_i by U46619 in the cells.     

Mechanism of endothelial cell NADPH oxidase activation by angiotensin II. Role of the p47phox subunit

Endothelial cells express a constitutively active phagocyte-type NADPH oxidase whose activity is augmented by agonists such as angiotensin II. We recently reported (Li, J.-M., and Shah, A. M. (2002) J. Biol. Chem. 277, 19952-19960) that in contrast to neutrophils a substantial proportion of the NADPH oxidase in unstimulated endothelial cells exists as preassembled intracellular complexes. Here, we investigate the mechanism of angiotensin II-induced endothelial NADPH oxidase activation. Angiotensin II (100 nmol/liter)-induced reactive oxygen species production (as measured by dichlorohydrofluorescein fluorescence or lucigenin chemiluminescence) was completely absent in coronary microvascular endothelial cells isolated from p47(phox) knockout mice. Transfection of p47(phox) cDNA into p47(phox-/-) cells restored the angiotensin II response, whereas transfection of antisense p47(phox) cDNA into wild-type cells depleted p47(phox) and inhibited the angiotensin II response. In unstimulated human microvascular endothelial cells, there was significant p47(phox)-p22(phox) complex formation but minimal detectable p47(phox) phosphorylation. Angiotensin II induced rapid serine phosphorylation of p47(phox) (within 1 min, peaking at approximately 15 min), a 1.9 +/- 0.1-fold increase in p47(phox)-p22(phox) complex formation and a 1.6 +/- 0.2-fold increase in NADPH-dependent O(2)-* production (p < 0.05). p47(phox) was redistributed to "nuclear" and membrane-enriched cell fractions. These data indicate that angiotensin II-stimulated endothelial NADPH oxidase activity is regulated through serine phosphorylation of p47(phox) and its enhanced binding to p22(phox).     

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.     

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.     

Glycated albumin activates NADPH oxidase in rat mesangial cells through up-regulation of p47phox

Glycated albumin, an early-glycation Amadori-modified protein, stimulates transforming growth factor-β (TGF-β) expression and increases the production of the extracellular matrix proteins in mesangial cells, contributing to the pathogenesis of diabetic nephropathy. Glycated albumin has been shown to increase NADPH oxidase-dependent superoxide formation in mesangial cells. However, the mechanisms are not well understood. Therefore, in the present studies, we determined the mechanisms by which glycated albumin activates NADPH oxidase in primary rat mesangial cells and its contribution to glycated albumin-induced TGF-β expression and extracellular matrix protein production. Our data showed that glycated albumin treatment stimulated NADPH oxidase activity and increased the formation of superoxide formation in rat mesangial cells. Moreover, glycated albumin treatment stimulated the expression and phosphorylation of p47phox, one of the cytosolic regulatory subunits of the NADPH oxidase. However, the levels of other NADPH oxidase subunits including Nox1, Nox2, Nox4, p22phox, and p67phox were not altered by glycated albumin. Moreover, siRNA-mediated knockdown of p47phox inhibited glycated albumin-induced NADPH oxidase activity and superoxide formation. Glycated albumin-induced TGF-β expression and extracellular matrix production (fibronectin) was also inhibited by p47phox knock down. Taken together, these data suggest that up-regulation of p47phox is involved in glycated albumin-mediated activation of NADPH oxidase, leading to glycated albumin-induced expression of TGF-β and extracellular matrix proteins in mesangial cells and contributing to the development of diabetic nephropathy.     

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.     

NADPH oxidase and apoptosis in cerulein-stimulated pancreatic acinar AR42J cells

Apoptosis linked to oxidative stress has been implicated in pancreatitis. We investigated whether NADPH oxidase mediates apoptosis in cerulein-stimulated pancreatic acinar AR42J cells. We report here that cerulein treatment resulted in the activation of NADPH oxidase, as determined by ROS production, translocation of cytosolic subunits p 47(phox) and p 67(phox) to the membrane, and interaction between NADPH oxidase subunits. Cerulein induced Ca(2+) oscillation, the expression of apoptotic genes p53 and bax, and apoptotic indices (DNA fragmentation, TUNEL staining, caspase 3 activity, decrease in cell viability) in AR42J cells. Treatment with a Ca(2+) chelator, BAPTA-AM, or transfection with antisense oligonucleotides for NADPH oxidase subunits p22(phox) and p 47(phox) inhibited cerulein-induced ROS production, translocation of NADPH oxidase cytosolic subunits p 47(phox) and p 67(phox) to the membrane, and the expression of apoptotic genes and apoptotic indices, as compared to the cells without treatment and those transfected with the corresponding sense oligonucleotides. These results indicate that NADPH oxidase may mediate ROS-induced apoptosis in pancreatic acinar cells in a Ca(2+)-dependent manner.     

Homocysteine stimulates phosphorylation of NADPH oxidase p47phox and p67phox subunits in monocytes via protein kinase Cbeta activation

Hyperhomocysteinaemia is an independent risk factor for cardiovascular diseases due to atherosclerosis. The development of atherosclerosis involves reactive oxygen species-induced oxidative stress in vascular cells. Our previous study [Wang and O (2001) Biochem. J. 357, 233-240] demonstrated that Hcy (homocysteine) treatment caused a significant elevation of intracellular superoxide anion, leading to increased expression of chemokine receptor in monocytes. NADPH oxidase is primarily responsible for superoxide anion production in monocytes. In the present study, we investigated the molecular mechanism of Hcy-induced superoxide anion production in monocytes. Hcy treatment (20-100 microM) caused an activation of NADPH oxidase and an increase in the superoxide anion level in monocytes (THP-1, a human monocytic cell line). Transfection of cells with p47phox siRNA (small interfering RNA) abolished Hcy-induced superoxide anion production, indicating the involvement of NADPH oxidase. Hcy treatment resulted in phosphorylation and subsequently membrane translocation of p47phox and p67phox subunits leading to NADPH oxidase activation. Pretreatment of cells with PKC (protein kinase C) inhibitors Ro-32-0432 (bisindolylmaleimide XI hydrochloride) (selective for PKCalpha, PKCbeta and PKCgamma) abolished Hcy-induced phosphorylation of p47phox and p67phox subunits in monocytes. Transfection of cells with antisense PKCbeta oligonucleotide, but not antisense PKCalpha oligonucleotide, completely blocked Hcy-induced phosphorylation of p47phox and p67phox subunits as well as superoxide anion production. Pretreatment of cells with LY333531, a PKCbeta inhibitor, abolished Hcy-induced superoxide anion production. Taken together, these results indicate that Hcy-stimulated superoxide anion production in monocytes is regulated through PKC-dependent phosphorylation of p47phox and p67phox subunits of NADPH oxidase. Increased superoxide anion production via NADPH oxidase may play an important role in Hcy-induced inflammatory response during atherogenesis.     

Ceramide-induced activation of NADPH oxidase and endothelial dysfunction in small coronary arteries

We tested the hypothesis that ceramide induces endothelial dysfunction in small coronary arteries via NADPH oxidase-mediated superoxide and resulting peroxynitrite formation. With the use of dihydroethidium as a superoxide indicator, C(2)-ceramide was found to increase superoxide production in the endothelial cells of small coronary arteries, which was inhibited by the NADPH oxidase inhibitors N-vanillylnonanamide, apocynin, and diphenylene iodonium. NADPH oxidase expression was confirmed in endothelial cells, as indicated by the immunoblotting of its subunits gp91(phox) and p47(phox). C(2)-ceramide increased NADPH oxidase activity by 52%, which was blocked by NADPH oxidase inhibitors but not by inhibitors of NO synthase, xanthine oxidase, and mitochondrial electron transport chain enzymes. By Western blot analysis, ceramide-induced NADPH oxidase activation was found to be associated with the translocation of p47(phox) to the membrane. In isolated and pressurized small coronary arteries, N-vanillylnonanamide, apocynin, or uric acid, a peroxynitrite scavenger, largely restored the inhibitory effects of ceramide on bradykinin- and A-23187-induced vasorelaxation. With the use of nitrotyrosine as a marker, C(2)-ceramide was found to increase peroxynitrite in small coronary arteries, which could be blocked by uric acid. We conclude that NADPH oxidase-mediated superoxide production and subsequent peroxynitrite formation mediate ceramide-induced endothelial dysfunction in small coronary arteries.     

Role of PKCζ‐NADPH oxidase signaling axis in PKCα‐mediated Giα2 phosphorylation for inhibition of adenylate cyclase activity by angiotensin II in pulmonary artery smooth muscle cells

We sought to determine the mechanism by which angiotensin II (AngII) inhibits isoproterenol induced increase in adenylate cyclase (AC) activity and cyclic adenosine monophosphate (cAMP) production in bovine pulmonary artery smooth muscle cells (BPASMCs). Treatment with AngII stimulates protein kinase C‐ζ (PKC‐ζ), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, and PKC‐α activities, and also inhibits isoproterenol induced increase in AC activity and cAMP production in the cells. Pertussis toxin pretreatment eliminates AngII caused inhibition of isoproterenol induced increase in AC activity without a discernible change in PKC‐ζ, NADPH oxidase, and PKC‐α activities. Treatment of the cells with AngII increases α2 isoform of Gi (Giα2) phosphorylation; while pretreatment with chemical and genetic inhibitors of PKC‐ζ and NADPH oxidase attenuate AngII induced increase in PKC‐α activity and Giα2 phosphorylation, and also reverse AngII caused inhibition of isoproterenol induced increase in AC activity. Pretreatment of the cells with chemical and genetic inhibitors of PKC‐α attenuate AngII induced increase in Giα2 phosphorylation and inhibits isoproterenol induced increase in AC activity without a discernible change in PKC‐ζ and NADPH oxidase activities. Overall, PKCζ‐NADPH oxidase‐PKCα signaling axis plays a crucial role in Giα2 phosphorylation resulting in AngII‐mediated inhibition of isoproterenol induced increase in AC activity in BPASMCs.     

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.     

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.     

Ascorbate inhibits NADPH oxidase subunit p47phox expression in microvascular endothelial cells

The production of reactive oxygen species (ROS) is central to the etiology of endothelial dysfunction in sepsis. Endothelial cells respond to infection by activating NADPH oxidases that are sources of intracellular ROS and potential targets for therapeutic administration of antioxidants. Ascorbate is an antioxidant that accumulates in these cells and improves capillary blood flow, vascular reactivity, arterial blood pressure, and survival in experimental sepsis. Therefore, the present study tested the hypothesis that ascorbate regulates NADPH oxidases in microvascular endothelial cells exposed to septic insult. We observed that incubation with Escherichia coli lipopolysaccharide (LPS) and interferon-gamma (IFNgamma) increased NADPH oxidase activity and expression of the enzyme subunit p47phox in mouse microvascular endothelial cells of skeletal muscle origin. Pretreatment of the cells with ascorbate prevented these increases. Polyethylene glycol-conjugated catalase and selective inhibitors of Jak2 also abrogated induction of p47phox. Exogenous hydrogen peroxide induced p47phox expression that was prevented by pretreatment of the cells with ascorbate. LPS+IFNgamma or hydrogen peroxide activated the Jak2/Stat1/IRF1 pathway and this effect was also inhibited by ascorbate. In conclusion, ascorbate blocks the stimulation by septic insult of redox-sensitive Jak2/Stat1/IRF1 signaling, p47phox expression, and NADPH oxidase activity in microvascular endothelial cells. Because endothelial NADPH oxidases produce ROS that can cause endothelial dysfunction, their inhibition by ascorbate may represent a new strategy for sepsis therapy.     

Protein Kinase B (AKT) Mediates Phospholipase D Activation via ERK1/2 and Promotes Respiratory Burst Parameters in Formylpeptide-stimulated Neutrophil-like HL-60 Cells

Phospholipase D (PLD), a major source of lipid second messengers (phosphatidic acid, diglycerides) in many cell types, is tightly regulated by protein kinases, but only a few of them have been identified. We show here that protein kinase B (AKT) is a novel major signaling effector of PLD activity induced by the formylpeptide f-Met-Leu-Phe (fMLP) in human neutrophil-like HL-60 cells (dHL-60 cells). AKT inhibition with the selective antagonist AKTib1/2 almost completely prevented fMLP-mediated activity of PLD, its upstream effector ERK1/2, but not p38 MAPK. Immunoprecipitation studies show that phosphorylated AKT, ERK, and PLD2 form a complex induced by fMLP, which can be prevented by AKTib1/2. In cell-free systems, AKT1 stimulated PLD activity via activation of ERK. AKT1 actually phosphorylated ERK2 as a substrate (K_m 1 μm). Blocking AKT activation with AKTib1/2 also prevented fMLP- but not phorbol 12-myristate 13-acetate-mediated NADPH oxidase activation (respiratory burst, RB) of dHL-60 cells. Impaired RB was associated with defective membrane translocation of NADPH oxidase components p67^phox and p47^phox, ERK, AKT1, AKT2, but not AKT3. Depletion of AKT1 or AKT2 with antisense oligonucleotides further indicates a partial contribution of both isoforms in fMLP-induced activation of ERK, PLD, and RB, with a predominant role of AKT1. Thus, formylpeptides induce sequential activation of AKT, ERK1/2, and PLD, which represents a novel signaling pathway. A major primarily role of this AKT signaling pathway also emerges in membrane recruitment of NOX2 components p47^phox, p67^phox, and ERK, which may contribute to assembly and activation of the RB motor system, NADPH oxidase.     

HSCARG Inhibits NADPH Oxidase Activity through Regulation of the Expression of p47phox

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase catalyzes the transfer of electrons from NADPH to O₂, which is the main source of reactive oxygen species (ROS) in nonphagocytic cells. Excess ROS are toxic; therefore, keeping ROS in homeostasis in cells can protect cells from oxidative damage. It is meaningful to further understand the molecular mechanism by which ROS homeostasis is mediated. Human protein HSCARG is a newly identified oxidative sensor and a negative regulator of NF-κB. Here, we find that HSCARG represses the cellular ROS generation through inhibiting mRNA and protein expression of p47phox, a subunit of NADPH oxidase. In contrast, shRNA-mediated HSCARG knockdown increases endogenous p47phox expression level. And HSCARG has no obvious effect on ROS production in p47phox-depleted cells. Furthermore, HSCARG regulates p47phox through inhibition of NF-κB activity. Our findings identify HSCARG as a novel regulator in regulation of the activity of NADPH oxidase and ROS homeostasis.     

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.     

NADPH oxidase is involved in H2O2-induced differentiation of human promyelocytic leukaemia HL-60 cells

The expression and activity of NADPH oxidase increase when HL‐60 cells are induced into terminally differentiated cells. However, the function of NADPH oxidase in differentiation is not well elucidated. With 150–500 μM H₂O₂ inducing differentiation of HL‐60 cells, we measured phagocytosis of latex beads and investigated cell electrophoresis. Two inhibitors of NADPH oxidase, DPI (diphenyleneiodonium) and APO (apocynin), blocked the differentiation potential of cells induced by 200 μM H₂O₂. However, H₂O₂ stimulated the generation of intracellular superoxide (O₂ ^{? ?}), which decreased in the presence of the two inhibitors. DPI also inhibited H₂O₂‐induced ERK (extracellular‐signal‐regulated kinase) activation, as detected by Western blotting. Furthermore, PD98059, the inhibitor of the ERK pathway, inhibited the differentiation of HL‐60 cells induced by H₂O₂. This shows that H₂O₂ can activate NADPH oxidase, leading to O₂ ^{? ?} production, followed by ERK activation and ultimately resulting in the differentiation of HL‐60 cells. The data indicate that NADPH oxidase is an important cell signal regulating cell differentiation.     

Diphenyleneiodonium suppresses apoptosis in cerulein-stimulated pancreatic acinar cells

NADPH oxidase has been considered a major source of reactive oxygen species in phagocytic and non-phagocytic cells. Apoptosis linked to oxidative stress has been implicated in pancreatitis. Recently, we demonstrated that NADPH oxidase subunits Nox1, p27phox, p47phox, and p67phox are constitutively expressed in pancreatic acinar cells, which are activated by cerulein, a cholecystokinin analogue. Cerulein induces an acute and edematous form of pancreatitis. We investigated whether inhibition of NADPH oxidase by diphenyleneiodonium suppresses the production of reactive oxygen species and apoptosis by determining viable cell numbers, DNA fragmentation, TUNEL staining, caspase-3 activity, and the expression of apoptosis-inducing factor in pancreatic acinar AR42J cells stimulated with cerulein. Inhibition on NADPH oxidase by diphenyleneiodonium was assessed by the alterations in NADPH oxidase activity and translocation of the cytosolic subunits p67phox and p47phox to the membrane. Intracellular Ca2+ level was monitored to investigate the relationship between NADPH oxidase and Ca2+ in cells stimulated with cerulein. As a result, cerulein induced the activation of NADPH, increased production of reactive oxygen species, and apoptotic indices determined by the expression of apoptosis-inducing factor, caspase-3 activation, TUNEL staining, DNA fragmentation, and cell viability. Treatment with DPI inhibited cerulein-induced activation of NADPH oxidase, the production of reactive oxygen species, and apoptosis, but not the increase of intracellular Ca2+ levels in pancreatic acinar cells. These results demonstrate that the cerulein-induced increase in intracellular Ca2+ level may be an upstream event of NADPH oxidase activation. Diphenyleneiodonium, an NADPH oxidase inhibitor, inhibits the expression of apoptosis-inducing factor and caspase-3 activation, and thus apoptosis in pancreatic acinar cells.