Identification of NCF2/p67phox as a novel p53 target gene

Analysis of microarrays performed in p53-, TAp63α- and ΔNp63α-inducible SaOs-2 cell lines allowed the identification of NCF2 mRNA upregulation in response to p53 induction. NCF2 gene encodes for p67phox, the cytosolic subunit of the NADPH oxidase enzyme complex. The recruitment of p67phox to the cell membrane causes the activation of the NADPH oxidase complex followed by the generation of NADP+ and superoxide from molecular oxygen. The presence of three putative p53 binding sites on the NCF2 promoter was predicted, and the subsequent luciferase and chromatin immunoprecipitation assays showed the activation of NCF2 promoter by p53 and its direct binding in vivo to at least one of the sites, thus confirming the hypothesis. NCF2 upregulation was also confirmed by real-time PCR in several cell lines after p53 activation. NCF2 knockdown by siRNA results in a significant reduction of ROS production and stimulates cell death, suggesting a protective function of Nox2-generated ROS in cells against apoptosis. These results provide insight into the redox-sensitive signaling mechanism that mediates cell survival involving p53 and its novel target NCF2/p67phox.     

Annexin Peptide Ac2-26 Suppresses TNFα-Induced Inflammatory Responses via Inhibition of Rac1-Dependent NADPH Oxidase in Human Endothelial Cells

The anti-inflammatory peptide annexin-1 binds to formyl peptide receptors (FPR) but little is known about its mechanism of action in the vasculature. Here we investigate the effect of annexin peptide Ac2-26 on NADPH oxidase activity induced by tumour necrosis factor alpha (TNFα) in human endothelial cells. Superoxide release and intracellular reactive oxygen species (ROS) production from NADPH oxidase was measured with lucigenin-enhanced chemiluminescence and 2′,7′-dichlorodihydrofluorescein diacetate, respectively. Expression of NADPH oxidase subunits and intracellular cell adhesion molecule (ICAM-1) and vascular cell adhesion molecule (VCAM-1) were determined by real-time PCR and Western blot analysis. Promoter activity of nuclear factor kappa B (NFκB) was measured by luciferase activity assay. TNFα stimulated NADPH-dependent superoxide release, total ROS formation and expression of ICAM-1and VCAM-1. Pre-treatment with N-terminal peptide of annexin-1 (Ac2-26, 0.5–1.5 µM) reduced all these effects, and the inhibition was blocked by the FPRL-1 antagonist WRW4. Furthermore, TNFα-induced NFκB promoter activity was attenuated by both Ac2-26 and NADPH oxidase inhibitor diphenyliodonium (DPI). Surprisingly, Nox4 gene expression was reduced by TNFα whilst expression of Nox2, p22phox and p67phox remained unchanged. Inhibition of NADPH oxidase activity by either dominant negative Rac1 (N17Rac1) or DPI significantly attenuated TNFα-induced ICAM-1and VCAM-1 expression. Ac2-26 failed to suppress further TNFα-induced expression of ICAM-1 and VCAM-1 in N17Rac1-transfected cells. Thus, Ac2-26 peptide inhibits TNFα-activated, Rac1-dependent NADPH oxidase derived ROS formation, attenuates NFκB pathways and ICAM-1 and VCAM-1 expression in endothelial cells. This suggests that Ac2-26 peptide blocks NADPH oxidase activity and has anti-inflammatory properties in the vasculature which contributes to modulate in reperfusion injury inflammation and vascular disease.     

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.     

OCT4 as a target of miR-34a stimulates p63 but inhibits p53 to promote human cell transformation

Human cell transformation is a key step for oncogenic development, which involves multiple pathways; however, the mechanism remains unclear. To test our hypothesis whether cell oncogenic transformation shares some mechanisms with the process of reprogramming non-stem cells to induced pluripotent stem cells (iPSC), we studied the relationship among the key factors for promoting or inhibiting iPSC in radiation-transformed human epithelial cell lines derived from different tissues (lung, breast and colon). We unexpectedly found that p63 and OCT4 were highly expressed (accompanied by low expressed p53 and miR-34a) in all transformed cell lines examined when compared with their non-transformed counterparts. We further elucidated the relationship of these factors: the 3p strand of miR-34a directly targeted OCT4 by binding to the 3′ untranslated region (3′-UTR) of OCT4 and, OCT4, in turn, stimulated p63 but inhibited p53 expression by binding to a specific region of the p63 or p53 promoter. Moreover, we revealed that the effects of OCT4 on promoting cell oncogenic transformation were by affecting p63 and p53. These results support that a positive loop exists in human cells: OCT4 upregulation as a consequence of inhibition of miR-34a, promotes p63 but suppresses p53 expression, which further stimulates OCT4 upregulation by downregulating miR-34a. This functional loop contributes significantly to cell transformation and, most likely, also to the iPSC process.     

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.     

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.     

Phosphatidylinositol 3-phosphate-dependent and -independent functions of p40phox in activation of the neutrophil NADPH oxidase

In response to bacterial infection, the neutrophil NADPH oxidase assembles on phagolysosomes to catalyze the transfer of electrons from NADPH to oxygen, forming superoxide and downstream reactive oxygen species (ROS). The active oxidase is composed of a membrane-bound cytochrome together with three cytosolic phox proteins, p40(phox), p47(phox), and p67(phox), and the small GTPase Rac2, and is regulated through a process involving protein kinase C, MAPK, and phosphatidylinositol 3-kinase. The role of p40(phox) remains less well defined than those of p47(phox) and p67(phox). We investigated the biological role of p40(phox) in differentiated PLB-985 neutrophils, and we show that depletion of endogenous p40(phox) using lentiviral short hairpin RNA reduces ROS production and impairs bacterial killing under conditions where p67(phox) levels remain constant. Biochemical studies using a cytosol-reconstituted permeabilized human neutrophil cores system that recapitulates intracellular oxidase activation revealed that depletion of p40(phox) reduces both the maximal rate and total amount of ROS produced without altering the K(M) value of the oxidase for NADPH. Using a series of mutants, p47PX-p40(phox) chimeras, and deletion constructs, we found that the p40(phox) PX domain has phosphatidylinositol 3-phosphate (PtdIns(3)P)-dependent and -independent functions. Translocation of p67(phox) requires the PX domain but not 3-phosphoinositide binding. Activation of the oxidase by p40(phox), however, requires both PtdIns(3)P binding and an Src homology 3 (SH3) domain competent to bind to poly-Pro ligands. Mutations that disrupt the closed auto-inhibited form of full-length p40(phox) can increase oxidase activity approximately 2.5-fold above that of wild-type p40(phox) but maintain the requirement for PX and SH3 domain function. We present a model where p40(phox) translocates p67(phox) to the region of the cytochrome and subsequently switches the oxidase to an activated state dependent upon PtdIns(3)P and SH3 domain engagement.     

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.     

Genes for two autosomal recessive forms of chronic granulomatous disease assigned to 1q25 (NCF2) and 7q11.23 (NCF1).

Chronic granulomatous disease (CGD) is a heterogeneous group of inherited disorders of impaired superoxide production in phagocytes. The most common X-linked recessive form involves the CYBB locus in band Xp21.1 that encodes the membrane-bound beta subunit of the cytochrome b558 complex. Two autosomal recessive forms of CGD result from defects in cytosolic components of the phagocyte NADPH oxidase system, p47phox (NCF1) and p67phox (NCF2). By using human cDNA probes we have mapped the genes for these proteins to chromosomal sites. The combined data from Southern analysis of somatic cell hybrid lines and chromosomal in situ hybridization localize NCF1 to 7q11.23 and NCF2 to band 1q25. The NCF1 localization corrects an erroneous preliminary assignment to chromosome 10. In the mouse, the locus corresponding to NCF2 (Ncf-2) was mapped with somatic cell hybrid panels and recombinant inbred strains to mouse chromosome 1 near Xmv-21 within a region of conserved homology with human chromosome 1 region q21-q32. A second site, probably a processed pseudogene, was identified on mouse chromosome 13.     

The expression of the NADPH oxidase subunit p22phox is regulated by a redox-sensitive pathway in endothelial cells

Endothelial dysfunction is characterized by increased levels of reactive oxygen species (ROS) and a prothrombotic state. The mechanisms linking thrombosis to ROS production in the endothelium are not well understood. We investigated the role of thrombin in regulating NADPH oxidase-dependent ROS production and expression of its subunit p22phox in the endothelial cell line EaHy926. Thrombin elicited a biphasic increase in ROS generation peaking within 15 min, but also at 3 h. The delayed response was accompanied by increased p22phox mRNA and protein expression. Two-photon confocal laser microscopy showed colocalization between p22phox and ROS production. Antioxidant treatment with vitamin C or diphenyleneiodonium abrogated thrombin-induced ROS production and p22phox expression, whereas H2O2 elevated ROS production and p22phox levels. Both responses were dependent on p38 MAP kinase and phosphatidylinositol-3-kinase (PI3 kinase)/Akt. Finally, p22phox was required for thrombin- or H2O2-stimulated proliferation. These data show that thrombin rapidly increases ROS production in endothelial cells, resulting, via activation of p38 MAP kinase and PI3 kinase/Akt, in upregulation of p22phox accompanied by a delayed increase in ROS generation and enhanced proliferation. These findings suggest a positive feedback mechanism whereby ROS, possibly generated by the NADPH oxidase, lead to elevated levels of p22phox and, thus, sustained ROS generation as is observed in endothelial dysfunction.     

p40phox as an alternative organizer to p47phox in Nox2 activation: a new mechanism involving an interaction with p22phox

p40(phox) activated phagocyte NADPH oxidase without p47(phox) in a cell-free system consisting of p67(phox), Rac and cytochrome b(558) relipidated with phosphatidylinositol 3-phosphate. The activation reached to 70% of that by p47(phox). Addition of p47(phox) slightly increased the activation, but not additively. p40(phox) improved the efficiency of p67(phox) in the activation. The C-terminus-truncated p67(phox), p40(phox)(D289A), p40(phox)(R58A), or p40(phox)(W207R) showed an impaired activation. A peptide corresponding to the p22(phox) Pro-rich region suppressed the activation, and far-western blotting revealed its interaction with p40(phox) SH3 domain. Thus, p40(phox) can substitute for p47(phox) in the activation, interacting with p22(phox) and p67(phox) through their specific regions.     

Inhibition of nonsense-mediated decay rescues p53β/γ isoform expression and activates the p53 pathway in MDM2-overexpressing and select p53-mutant cancers

Inactivation of p53 is present in almost every tumor, and hence, p53-reactivation strategies are an important aspect of cancer therapy. Common mechanisms for p53 loss in cancer include expression of p53-negative regulators such as MDM2, which mediate the degradation of wildtype p53 (p53α), and inactivating mutations in the TP53 gene. Currently, approaches to overcome p53 deficiency in these cancers are limited. Here, using non–small cell lung cancer and glioblastoma multiforme cell line models, we show that two alternatively spliced, functional truncated isoforms of p53 (p53β and p53γ, comprising exons 1 to 9β or 9γ, respectively) and that lack the C-terminal MDM2-binding domain have markedly reduced susceptibility to MDM2-mediated degradation but are highly susceptible to nonsense-mediated decay (NMD), a regulator of aberrant mRNA stability. In cancer cells harboring MDM2 overexpression or TP53 mutations downstream of exon 9, NMD inhibition markedly upregulates p53β and p53γ and restores activation of the p53 pathway. Consistent with p53 pathway activation, NMD inhibition induces tumor suppressive activities such as apoptosis, reduced cell viability, and enhanced tumor radiosensitivity, in a relatively p53-dependent manner. In addition, NMD inhibition also inhibits tumor growth in a MDM2-overexpressing xenograft tumor model. These results identify NMD inhibition as a novel therapeutic strategy for restoration of p53 function in p53-deficient tumors bearing MDM2 overexpression or p53 mutations downstream of exon 9, subgroups that comprise approximately 6% of all cancers.     

Inhibition of NADPH oxidase subunits translocation by tea catechin EGCG in mast cell

The inhibitory mechanism of tea catechins for allergy remains undefined. We studied the effect of catechins, mainly EGCG, on the activation of mast cell line canine cutaneous mastocytoma cells (CM-MC). Compound 48/80 induced the degranulation in CM-MC dose dependently, whereas its release of beta-hexosaminidase was inhibited by EGCG and O-methylated EGCG (EGCG-Me). Both catechins were found to inhibit intracellular ROS generation dose dependently together with DPI. Intracellular ROS generation in human polymorphonuclear leukocytes was also inhibited by EGCG. Neither L-NAME, ebeselen nor NAC inhibited ROS generation. From the Western blot analysis of the subunits components of NADPH oxidase, we detected cytosolic subunits; p47(phox), p67(phox), p40(phox), rac2 and membrane subunits; gp91(phox), p22(phox) in CM-MC. Cytosolic subunits were translocated from cytosol to membrane time dependently after stimulation with compound 48/80. EGCG and DPI inhibited cytosolic subunits from translocating into membrane. These data suggest that EGCG inhibits the activation of NADPH oxidase in CM-MC.     

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.