阿托伐他汀通过抑制PKC的激活对抗高糖诱导的人脐静脉内皮细胞氧化应激反应

目的:探讨阿托伐他汀对高糖诱导的人脐静脉血管内皮细胞(HUVECs)产生氧化应激的影响及其作用机制。方法:体外培养HUVECs,以25 mmol/L葡萄糖干预,模拟糖尿病患者体内环境,通过流式细胞术和共聚焦显微镜检测细胞内的活性氧(ROS)水平,采用Lucigenin分析方法测定还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶活性,分别应用实时荧光定量PCR和免疫印迹杂交的方法检测 NADPH氧化酶亚基Nox4和Nox2/gp91^phox的表达水平,用免疫印迹杂交方法检测蛋白激酶C(PKC)蛋白的磷酸化水平。结果:(1)在高糖环境(终浓度为25 mmol/L)下,HUVECs内ROS生成显著增加,NADPH氧化酶的活性显著增强,NADPH 氧化酶Nox4和Nox2/gp91^phox亚基的mRNA和蛋白表达水平显著上调;(2)阿托伐他汀可显著抑制高糖诱导的ROS 生成、NADPH氧化酶活性的增强及NADPH 氧化酶Nox4和Nox2/gp91^phox亚基表达水平的增加幅度,且具有浓度依赖性;(3)PKC抑制剂(PKC inhibitor peptide, 20 μmol/L)可显著抑制高糖环境下ROS的生成、NADPH氧化酶活性的增强及NADPH 氧化酶Nox4和Nox2/gp91^phox亚基表达水平的增加幅度;(4)阿托伐他汀可抑制高糖诱导的PKC蛋白的磷酸化。结论:PKC的活化参与了高糖诱导的HUVECs产生的氧化应激反应。阿托伐他汀通过抑制PKC蛋白的活化对抗高糖诱导的内皮细胞产生的氧化应激反应。     

NADPH oxidases and MMP-9/TIMP-1 balance in human glomeruli: putative links to diabetic nephropathy

Study aim Glomerular basement membrane thickening, the hallmark of diabetic nephropathy, is thought to be related to an enhanced oxidative stress and reduced matrix proteolysis. Our study concerned the mRNA and protein expression of NADPH oxidase (NOX) components, MMP‐2, MMP‐9 and TIMP‐1 in freshly isolated human glomeruli as well as enzymatic activities and their modulation by glucose, H₂O₂ and angiotensin‐2. Material and methods NOX, cytosolic and membrane‐bound associated proteins and mRNA were analysed by RT‐PCR and Western blotting after glomerular extraction. Oxidase activity was identified by cytochrome c reduction and chemiluminescence. Gelatinases and inhibitors were semiquantitatively assessed by RT‐PCR, gelatin zymography and ELISA in a model of glomerular conditioned survival. Results NOX‐2, NOX‐4 and membrane‐bound and cytosolic factors could be observed in freshly extracted glomeruli (RNA + protein). p40^phox, p67^phox and p47^phox molecular weights were increased compared to their phagocytic counterparts advocating for specific glomerular analogues, and a slight specific oxidase activity was retrieved in isolated glomeruli. Also, mRNA coding for MMP‐2, ‐9 and TIMP‐1, ‐2 were detected. High glucose concentrations (25 mm) reduced TIMP‐1 release in glomerular survival media and MMP‐2 activity in glomerular extracts. On the opposite, angiotensin‐2 significantly induced MMP‐2 and ‐9 activities in the survival media as well as H₂O₂ in glomerular extracts, while addition of 25 mm glucose blunted these findings. Conclusion Glomerular matrix remodelling, the backbone of renal fibrosis in diabetic patients, could be induced by H₂O₂ from specific glomerular NADPH oxidases under the influence of extra‐cellular glucose and angiotensin‐2 and could participate in the control of MMP activities.     

Superoxide production and NADPH oxidase expression in human rheumatoid synovial cells: regulation by interleukin-1β and tumour necrosis factor-α

Objectives to evaluate the rheumatoid synovial cell capacity to produce superoxide anion in response to interleukin-1β (IL-1β) and tumour necrosis factor-α (TNF-α), and to study the NADPH oxidase involvement in this production. Material and Methods Synovial cells obtained from 7 rheumatoid arthritis (RA), 5 osteoarthritic (OA) patients, and dermal fibroblasts, were stimulated (i) with IL-1β and TNF-α, or (ii) with specific oxidase activators and inhibitors, before studying superoxide production; we also studied NADPH oxidase mRNAs and protein expression, and p47-phox phosphorylation. Results Constitutive superoxide production by RA cells was increased in comparison to OA cells and dermal fibroblasts, and was stimulated by PMA and ionomycin. This production was increased after cytokine treatment of RA synovial cells. Cytokine-induced superoxide production by RA cells was inhibited by iodonium diphenyl or apocynin, suggesting the involvement of NADPH oxidase. RT-PCR and western blot analysis revealed the presence of p47-phox, gp91-phox and Nox4 in RA and OA cells, and in dermal fibroblasts. P47-phox phosphorylation was enhanced after cytokine-treatment in RA and OA cells, suggesting a PKC-mediated up-regulation of NADPH oxidase. Conclusions NADPH oxidase is involved in the superoxide release by RA synovial cells, constitutively and after cytokine up-regulation. These cells express two different homologues (gp91-phox and Nox4). Received 2 August 2005; returned for revision 12 January 2006; returned for final revision 22 May 2006; accepted by J. Di Battista 9 June 2006     

Assembly of NADPH Oxidase in Human Neutrophils Is Modulated by the Opacity-Associated Protein Expression State of Neisseria gonorrhoeae

Neisseria gonorrhoeae (the gonococcus, Gc) triggers a potent inflammatory response and recruitment of neutrophils to the site of infection. Gc survives exposure to neutrophils despite these cells'' antimicrobial products, such as reactive oxygen species (ROS). ROS production in neutrophils is initiated by NADPH oxidase, which converts oxygen into superoxide. The subunits of NADPH oxidase are spatially separated between granules (gp91^phox/p22^phox) and the cytoplasm (p47^phox, p67^phox, and p40^phox). Activation of neutrophils promotes the coassembly of NADPH oxidase subunits at phagosome and/or plasma membranes. While Gc-expressing opacity-associated (Opa) proteins can induce neutrophils to produce ROS, Opa-negative (Opa⁻) Gc does not stimulate neutrophil ROS production. Using constitutively Opa⁻ and OpaD-positive (OpaD⁺) Gc bacteria in strain FA1090, we now show that the difference in ROS production levels in primary human neutrophils between these backgrounds can be attributed to differential assembly of NADPH oxidase. Neutrophils infected with Opa⁻ Gc showed limited translocation of NADPH oxidase cytoplasmic subunits to cellular membranes, including the bacterial phagosome. In contrast, these subunits rapidly translocated to neutrophil membranes following infection with OpaD⁺ Gc. gp91^phox and p22^phox were recruited to Gc phagosomes regardless of bacterial Opa expression. These results suggest that Opa⁻ Gc interferes with the recruitment of neutrophil NADPH oxidase cytoplasmic subunits to membranes, in particular, the p47^phox “organizing” subunit, to prevent assembly of the holoenzyme, resulting in an absence of the oxidative burst.     

Exercise training raises expression of the cytosolic components of NADPH oxidase in rat neutrophils

Exercise activates neutrophil burst and this effect is dependent on training status and exercise intensity. In this study, the chronic effect of treadmill exercise on phagocytosis, production of reactive oxygen metabolites and expression of NADPH oxidase components in rat neutrophils was investigated. Neutrophils were obtained by intraperitoneal lavage with PBS. After 11 weeks of training the exercised group showed increased phagocytosis capacity (49%) and production of reactive oxygen metabolites (6.6-fold) when compared with neutrophils from the sedentary group. Exercised had no effect on expression of the membrane components of NADPH oxidase (p22 ^phox , gp91 ^phox ). In contrast, there was an increase of the p47 ^phox mRNA levels (by 126%), the cytosolic component of the enzyme. In addition, exercise increased the protein content of p47 ^phox (by 22%) and of p67 ^phox (by 2.8-fold) in neutrophils. Evidence is then presented that training to moderate exercise increases phagocytosis and production of reactive oxygen metabolites and the expression of p47 ^phox and p67 ^phox in neutrophils. Therefore, moderate exercise might enable neutrophils to respond more efficiently when exposed to pathogens.     

Up-regulation of a hydrogen peroxide–responsive pre-mRNA binding protein in atherosclerosis and intimal hyperplasia

BackgroundMultiple lines of investigation have implicated hydrogen peroxide (H₂O₂) as an important endogenous mediator of cell proliferation in the vessel wall. Heterogeneous nuclear ribonucleoprotein C (hnRNP-C), a nuclear pre-mRNA binding protein that plays roles in vertebrate cell proliferation and differentiation, has been identified as a component of a vascular cell signaling pathway activated by low physiologic levels of H₂O₂. The expression of hnRNP-C in human arteries has not previously been assessed.MethodsSegments of human proximal internal carotid arteries were evaluated for the expression of hnRNP-C by immunohistochemistry.ResultsIn normal proximal internal carotid arteries, hnRNP-C is expressed predominantly by the endothelium, with significantly lower expression by medial smooth muscle. In preatherosclerotic intimal hyperplasia, hnRNP-C is up-regulated in the artery wall, due to the robust expression by the intimal smooth muscle cells, without up-regulation in the medial smooth muscle cells. In arteries with atherosclerotic lesions, there is strong expression of hnRNP-C not only by intimal cells but also by medial smooth muscle cells.ConclusionsThe H₂O₂ responsive pre-mRNA binding protein hnRNP-C is up-regulated in atherosclerosis and in preatherosclerotic intimal hyperplasia in humans, supporting the hypothesis that H₂O₂ is a regulator of vascular cell proliferation in these conditions. These data also suggest that hnRNP-C may be useful as a marker of vascular cell activation.     

Regulation of NADPH oxidase in vascular endothelium: the role of phospholipases, protein kinases, and cytoskeletal proteins

The generation of reactive oxygen species (ROS) in the vasculature plays a major role in the genesis of endothelial cell (EC) activation and barrier function. Of the several potential sources of ROS in the vasculature, the endothelial NADPH oxidase family of proteins is a major contributor of ROS associated with lung inflammation, ischemia/reperfusion injury, sepsis, hyperoxia, and ventilator-associated lung injury. The NADPH oxidase in lung ECs has most of the components found in phagocytic oxidase, and recent studies show the expression of several homologues of Nox proteins in vascular cells. Activation of NADPH oxidase of nonphagocytic vascular cells is complex and involves assembly of the cytosolic (p47(phox), p67(phox), and Rac1) and membrane-associated components (Noxes and p22(phox)). Signaling pathways leading to NADPH oxidase activation are not completely defined; however, they do appear to involve the cytoskeleton and posttranslation modification of the components regulated by protein kinases, protein phosphatases, and phospholipases. Furthermore, several key components regulating NADPH oxidase recruitment, assembly, and activation are enriched in lipid microdomains to form a functional signaling platform. Future studies on temporal and spatial localization of Nox isoforms will provide new insights into the role of NADPH oxidase-derived ROS in the pathobiology of lung diseases.     

Baculovirus mediated expression of human phagocytic cell oxidase cytochrome b558 in sf9 insect cells

Chronic granulomatous disease (CGD) results from deficient production of components of the phagocyte NADPH oxidase. Most commonly affected is cytochrome b₅₅₈, a heterodimer composed of a 22-kDa protein (p22^phox noncovalently bound to a 91-kDa transmembrane glycoprotein (gp91^phox). CGD phagocytes lack both p22^phox and gp91^phox peptides when either gene is affected, suggesting that both peptides must be produced for individual subunit stability. Both genes have been cloned, but eukaryotic expression of recombinant gp91^phox has not been reported. To investigate the stability and interaction of cytochrome b₅₅₈ subunits, we introduced p22^phox and gp91^phox cDNA into recombinant baculoviruses. Recombinant gp91^phox (rgp91^phox) and p22^phox (rp22^phox) were detected individually and together in the same cells by in situ immunofluorescence and by SDS-PAGE immunoblotting of membranes from sf9 cells infected with baculovirus constructs. Formation of rp22^phox/ rgp91^phox complexes was demonstrated by coprecipitation using subunit-specific antibodies. This study demonstrates for the first time that cDNA encoding either subunit is capable of initiating production of stable recombinant cytochrome b₅₅₈ subunits in eukaryotic cells.     

NADPH oxidase inhibition improves neurological outcomes in surgically-induced brain injury

Neurosurgical procedures can result in brain injury by various means including direct trauma, hemorrhage, retractor stretch, and electrocautery. This surgically-induced brain injury (SBI) can cause post-operative complications such as brain edema. By creating a mouse model of SBI, we tested whether NADPH oxidase, an important reactive oxygen species producing enzyme, is involved in SBI using transgenic mice lacking gp91^phox subunit of NADPH oxidase (gp91^phox KO) and apocynin, a specific inhibitor of NADPH oxidase. Neurological function and brain edema were evaluated at 24 h post-SBI in gp91^phox KO and wild-type littermates grouped into SBI and sham-surgery groups. Alternatively, mice were grouped into vehicle- and apocynin-treated (5 mg/kg, i.p. 30 min before SBI) groups. Oxidative stress indicated by lipid peroxidation (LPO) was measured at 3 and 24 h post-SBI. The gp91^phox KO mice, but not the apocynin-treated mice showed significantly improved neurological scores. Brain edema was observed in both gp91^phox KO and wild-type groups after SBI; however, there was no significant difference between these two groups. Brain edema was also not affected by apocynin-pretreatment. LPO levels were significantly higher in SBI group in both gp91^phox KO and wild-type groups as compared to sham group. A trend, although without statistical significance, was noted towards attenuation of LPO in the gp91^phox KO animals as compared to wild-type group. LPO levels were significantly attenuated at 3 h post-SBI by apocynin-pretreatment but not at 24 h post-SBI. These results suggest that chronic and acute inhibition of NADPH oxidase activity does not reduce brain edema after SBI. Long-term inhibition of NADPH oxidase, however improves neurological functions after SBI.     

Two X-Linked Chronic Granulomatous Disease Patients with Unusual NADPH Oxidase Properties

Background

Chronic granulomatous disease (CGD) is an immune deficiency syndrome caused by defects in the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, the enzyme that generates reactive oxygen species (ROS) in phagocytizing leukocytes. This study evaluates the NADPH oxidase capacity in two X-linked CGD patients with mutations in gp91^phox that alter the regions in this membrane-bound NADPH oxidase component involved in docking of the cytosolic component p47^phox.

Materials and Methods

Hydrogen peroxide and superoxide generation, bactericidal activity, and NADPH oxidase protein expression by the patients?? neutrophils were measured, and genetic analysis was performed.

Results

We report two patients, each with a novel missense mutation in CYBB, the gene that encodes gp91^phox. Surprisingly, neutrophils from these patients showed total absence of superoxide production, although they retained 13?C30% of the hydrogen peroxide production capability. We speculate that this is due to direct electron transfer from flavin adenine dinucleotide (FAD) in gp91^phox to oxygen, leading to inefficient hydrogen peroxide formation instead of efficient superoxide production.

Conclusions

X-linked CGD patients with mutations that alter the gp91^phox protein in regions involved in docking of the cytosolic NADPH oxidase component p47^phox may have higher than expected hydrogen peroxide generation capability.     

Src kinase participates in LPS-induced activation of NADPH oxidase

The production of superoxide from NADPH oxidase by macrophages in response to endotoxin (LPS) is an important innate immune response, yet it is not clear how LPS signals the activation of NADPH oxidase. The hypothesis is that LPS-induced src kinase and PI3 kinase (PI3K) facilitates the activation of p47^phox, the regulatory subunit of NADPH oxidase. In mouse macrophage RAW264.7 cells, inhibition of src tyrosine family kinases inhibited LPS-induced activation of NADPH oxidase, phosphorylation of p47^phox, activation of PI3K and phosphorylation of the TLR4. Moreover, inhibition of LPS-induced increases in intracellular calcium blunted src kinase activation, PI3K association with TLR4, as well as PI3 kinase activation. These data suggest that both src kinase and PI3 kinase are involved in LPS-induced NADPH oxidase activation. Importantly, these data suggest that LPS-induced src kinase activation is critical for PI3 kinase activation as well as TLR4 phosphorylation and is dependent upon LPS-induced increase in intracellular calcium. These signaling events fill critical gaps in our understanding of LPS-induced free radical production as well as may potentially responsible for the mechanism of innate immune tolerance or desensitization caused by steroids or ethanol.     

Protein Kinase CK1αLS Promotes Vascular Cell Proliferation and Intimal Hyperplasia

Protein kinase CK1α regulates several fundamental cellular processes including proliferation and differentiation. Up to four forms of this kinase are expressed in vertebrates resulting from alternative splicing of exons; these exons encode either the L-insert located within the catalytic domain or the S-insert located at the C terminus of the protein. Whereas the L-insert is known to target the kinase to the nucleus, the functional significance of nuclear CK1αLS has been unclear. Here we demonstrate that selective L-insert-targeted short hairpin small interfering RNA-mediated knockdown of CK1αLS in human vascular endothelial cells and vascular smooth muscle cells impairs proliferation and abolishes hydrogen peroxide-stimulated proliferation of vascular smooth muscle cells, with the cells accumulating in G₀/G₁. In addition, selective knockdown of CK1αLS in cultured human arteries inhibits vascular activation, preventing smooth muscle cell proliferation, intimal hyperplasia, and proteoglycan deposition. Knockdown of CK1αLS results in the harmonious down-regulation of its target substrate heterogeneous nuclear ribonucleoprotein C and results in the altered expression or alternative splicing of key genes involved in cellular activation including CXCR4, MMP3, CSF2, and SMURF1. Our results indicate that the nuclear form of CK1α in humans, CK1αLS, plays a critical role in vascular cell proliferation, cellular activation, and hydrogen peroxide-mediated mitogenic signal transduction.A key morphological distinction between vertebrates and invertebrates is the presence of a closed endothelial-lined vascular system in the vertebrates.¹ Activation of the cells comprising the vertebrate vasculature results in cellular proliferation, enhanced proteoglycan deposition, and secretion of growth factors and cytokines.^2,3,4 Such vascular activation is an important process in both vascular development and in vascular diseases such as atherosclerosis and postangioplasty restenosis. Thus, an understanding of the vertebrate-specific signaling pathways regulating vascular cell activation is of high importance.Protein kinase CK1α regulates several fundamental cellular processes including proliferation and differentiation.⁵ Up to four different forms of the kinase exist owing to the alternative splicing of exons encoding either the L-insert located within the catalytic domain or the S-insert located at the C terminus of the protein.^6,7,8,9 Protein kinase CK1α itself is highly conserved among all metazoans. However, the exon encoding the nuclear localizing L-insert is restricted to vertebrates.¹⁰ Whereas vertebrates may contain up to four different splice forms of CK1α, humans are thought to only express three forms: CK1α, CK1αS, and CK1αLS, which are also referred to as CK1α1, CK1α2, and CK1α3, respectively. In the nucleus, CK1αLS probably plays a role in pre-mRNA processing and alternative splicing based on its ability to phosphorylate the highly abundant vertebrate-specific pre-mRNA binding protein heterogeneous nuclear ribonucleoprotein C (hnRNP-C)^10,11,12 and its localization to nuclear speckles,⁶ sites of accumulation of pre-mRNA processing factors.Within the vessel wall, hydrogen peroxide (H₂O₂) plays important roles in mediating vascular activation resulting from diverse stimuli including altered flow, growth factors, cytokines, and vascular injury.^13,14 In fact, vertebrate cells are known to proliferate in response to low concentrations of H₂O₂.¹⁵ Low levels of H₂O₂ are generated by vertebrate cells in response to growth factor-mediated signaling, and this mitogenic H₂O₂ activates CK1αLS, which then phosphorylates hnRNP-C.^10,11 It is known that hnRNP-C modulates the expression of several genes regulating cell growth and survival, including platelet-derived growth factor B chain (PDGF-B),¹⁶ c-myc,¹⁷ p53,¹⁸ the X-chromosome-linked inhibitor of apoptosis,¹⁹ and the urokinase plasminogen activator receptor.²⁰ Thus, CK1αLS phosphorylation of hnRNP-C may promote H₂O₂-stimulated vertebrate cell growth. However, in the cytoplasm, cytosolic forms of CK1α (CK1α and CK1αS) play important roles inhibiting key proliferative signaling pathways involving both wnt/β-catenin and the nuclear factor of activated T-cells.^21,22 Thus, it has been unclear whether H₂O₂-activated CK1αLS in the nucleus is promoting H₂O₂-stimulated growth or is, in fact, a compensatory counter-regulatory pathway. Here we demonstrate by selective and stable knockdown of CK1αLS that the kinase is, in fact, an important positive regulator of vascular activation and H₂O₂ mitogenic signaling.     

Hyper‐responsive Toll‐like receptor 7 and 9 activation in NADPH oxidase‐deficient B lymphoblasts

Chronic granulomatous disease (CGD) is an inherited immunodeficiency linked with mutations in the multi-subunit leucocyte NADPH oxidase. Myeloid-derived phagocytic cells deficient in NADPH oxidase fail to produce sufficient levels of reactive oxygen species to clear engulfed pathogens. In this study we show that oxidase also influences B-cell functions, including responses to single-stranded RNA or unmethylated DNA by endosomal Toll-like receptors (TLRs) 7 and 9. In response to TLR7/9 ligands, B-cell lines derived from patients with CGD with mutations in either the NADPH oxidase p40^phox or p47^phox subunits produced only low levels of reactive oxygen species. Remarkably, cytokine secretion and p38 mitogen-activated protein kinase activation by these oxidase-deficient B cells was significantly increased upon TLR7/9 activation when compared with oxidase-sufficient B cells. Increased TLR responsiveness was also detected in B cells from oxidase-deficient mice. NADPH oxidase-deficient patient-derived B cells also expressed enhanced levels of TLR7 and TLR9 mRNA and protein compared with the same cells reconstituted to restore oxidase activity. These data demonstrate that the loss of oxidase function associated with CGD can significantly impact B-cell TLR signalling in response to nucleic acids with potential repercussions for auto-reactivity in patients.     

The molecular basis of chronic granulomatous disease

Summary and conclusions CGD is a rare inherited immunodeficiency syndrome, caused by the phagocytes' inability to produce (sufficient) reactive oxygen metabolites. This dysfunction is due to a defect in the NADPH oxidase, the enzyme responsible for the production of superoxide. It is composed of several subunits, two of which, gp9l^phox and p22^phox, form the membrane-bound cytochrome b558, while its three cytosolic components, p47^phox p67^phox and p40^phox, have to translocate to the membrane upon activation. This is a tightly and intricately controlled process that involves, among others, several low-molecular weight GTP-binding proteins. Gp91^phox is encoded on the X-chromosome and p22^phox, p47^phox and p67^phox on different autosomal chromosomes, and a defect in one of these components leads to CGD. This explains the variable mode of inheritance seen in this syndrome.Clinically CGD manifests itself typically already at a very young age with recurrent and serious infections, most often caused by catalase-positive pathogens.Modern treatment options, including prophylaxis with trimethoprim-sulfamethoxazole and rIFN- as well as early and aggressive anti-infection therapy, have improved the prognosis of this disease dramatically.CGD, as a very well-characterized inherited affection of the hematopoietic stem cells, is predestined to be among the first diseases to profit from the advances in cutting-edge therapeutics, such as gene therapy and in utero stem cell transplantation.     

The NADPH Oxidase Subunit p22phox Inhibits the Function of the Tumor Suppressor Protein Tuberin

Mutations in the von Hippel-Lindau (VHL) gene give rise to renal cell carcinoma. Reactive oxygen species, generated by Nox oxidases, are involved in tumorigenesis. We have previously demonstrated that in VHL-deficient cells, p22^phox-dependent Nox1 and Nox4 oxidases maintain hypoxia inducible factor-2α (HIF-2α) protein expression through an Akt-dependent translational pathway. Phosphorylation of tuberin, by Akt, results in its inactivation. Here we show that diphenyleneiodonium chloride, an inhibitor of Nox oxidases, and small-interfering RNA-mediated down-regulation of p22^phox inhibit Akt-dependent phosphorylation of tuberin and stabilizes tuberin protein levels in VHL-deficient renal carcinoma cells. p22^phox-mediated inactivation of tuberin is associated with an increase in ribosomal protein S6 kinase 1 and eukaryotic initiation factor 4E-binding protein-1 (4E-BP1) phosphorylation as well as HIF-2α stabilization. Importantly, we find that marked up-regulation of p22^phox in human renal cell carcinoma correlates with increased tuberin phosphorylation, decreased tuberin protein levels, and increased phosphorylation of 4E-BP1. Our data provide the first evidence that p22^phox-based Nox oxidases maintain HIF-2α protein expression through inactivation of tuberin and downstream activation of ribosomal protein S6 kinase 1/4E-BP1 pathway.Epithelial tumors comprise the majority of renal cell carcinomas (RCCs), in which ∼75% are histologically of the clear cell type. Biallelic inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene is linked to the development of hereditary and sporadic RCC. Mutations within the VHL gene maintain the expression of hypoxia inducible factor-2α (HIF-2α) and up-regulates of genes involved in autonomous cell growth,¹ survival,² and metastasis,^3,4 major features of RCC. NADPH oxidases of the Nox family are major sources of reactive oxygen species (ROS) in cancer, including renal cancer.^{5,6,7,8,9,10,11,12} Selected Nox isoforms form a heterodimer with p22^phox, and the dimers facilitate ROS-generating activity of the Nox subunits.^13,14,15,16 We and others have shown that p22^phox, Nox1, and Nox4 contribute to the maintenance of HIF-2α protein expression and are up-regulated in VHL-deficient cells.^10,11 Moreover, Nox-derived ROS maintain HIF-2α protein levels through a phosphoinositide 3-kinase/Akt-dependent translational mechanism.¹¹ Activation of the phosphoinositide 3-kinase/Akt pathway promotes translation through inactivation of tuberin.¹⁷ Inhibition of tuberin function occurs through Akt-dependent phosphorylation at residues S⁹³⁹, Thr¹⁴⁶² leading to the dissociation of the harmatin/tuberin complex and ubiquitination and degradation of the free tuberin.^17,18 Tuberin negatively regulates mRNA translation by inhibiting mammalian target of rapamycin (mTOR) and subsequent phosphorylation of the translational repressor, eukaryotic initiation factor 4E-binding protein-1 (4E-BP1), and the ribosomal protein S6 kinase 1 (S6K). In this study we show for the first time that p22^phox, a key component in the activation of Noxes, results in HIF-2α protein accumulation via inactivation of tuberin and subsequent activation of S6K/4E-BP1 pathway. Furthermore, we report that in human RCC, expression of p22^phox, Nox1, and Nox4 is elevated and correlates with increased tuberin phosphorylation on Thr¹⁴⁶² and decreased tuberin protein levels together with increased phosphorylation of 4E-BP1.     

Critical role of the NADPH oxidase subunit p47phox on vascular TLR expression and neointimal lesion formation in high-fat diet-induced obesity

Reactive oxygen species (ROS) formation is associated with inflammation and vasculature dysfunction. We investigated the potential role of the NADPH oxidase on vascular Toll-like receptor (TLR) expression and carotid neointimal formation in high-fat (HF) diet-induced obesity (DIO) model. Using mice DIO and common carotid artery flow cessation-induced lesion formation models, we examined vascular TLR2 and TLR4 expression and neointimal formation in NADPH oxidase subunit p47(phox)-deficient (p47(phox-/-)) mice. Feeding C57BL/6J mice an HF diet for 22 weeks resulted in significant increases in p47(phox), TLR2 and TLR4 expression in vascular tissues compared with mice fed a low-fat (LF) diet. Minimal changes in TLR2 and TLR4 expression was detected in p47(phox-/-) DIO mice. Furthermore, flow cessation-induced angiogenic and inflammatory response and neointimal formation were significantly attenuated in p47(phox-/-) DIO mice compared with wild-type DIO mice. In addition, exposure of endothelial cells to leptin led to ROS formation; this was accompanied by upregulation of TLR2, TLR4 expression and its downstream signaling. Leptin also increased endothelial cell migration and proliferation. Pharmacological inhibition of NADPH oxidase or genetic deletion of p47(phox) significantly diminished these alterations. Obesity increases neointimal formation via a mechanism involving p47(phox)-TLRs signaling, suggesting that the NADPH oxidase may represent a potential novel therapeutic target for the treatment of obesity-associated vascular inflammation and dysfunction.     

Cardiac oxidative stress and remodeling following infarction: role of NADPH oxidase

BackgroundThere is growing recognition that oxidative stress plays a role in the pathogeneses of myocardial repair/remodeling following myocardial infarction (MI). Nicotinamide adenine denucleotide phosphate (NADPH) oxidase is a major source for cardiac reactive oxygen species production. Herein, we studied the importance of NADPH oxidase in development of cardiac oxidative stress and its induced molecular and cellular changes related to myocardial repair/remodeling.MethodsMI was created by coronary artery ligation in C57/BL (wild type) and NADPH oxidase (gp91^phox) knockout mice. Cardiac oxidative stress, inflammatory/fibrogenic responses, apoptosis, and hypertrophy were detected by in situ hybridization, immunohistochemistry, terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL), picrosirius red staining, and image analysis, respectively, at different stages post MI.ResultsIn wild-type mice with MI, and compared to sham-operated animals, we observed significantly increased gp91^phox and 3-nitrotyrosine, a marker of oxidative stress, in the infarcted myocardium; accumulated macrophages and myofibroblasts at the infarct site; abundant apoptotic myocytes primarily at border zones on Day 3; and numerous apoptotic inflammatory/myofibroblasts in the later stages. In addition, we detected significantly increased transforming growth factor β1, tissue inhibitor of metalloprotease 2, and type 1 collagen gene expression; continuously increasing collagen volume in the infarcted myocardium; and hypertrophy in noninfarcted myocardium. Compared to wild-type mice with MI, we did not observe significant difference in infarct size/thickness, cardiac hypertrophy, myocyte apoptosis, inflammatory/fibrogenic responses, as well as cardiac oxidative stress in gp91^phox knockout mice.ConclusionOur findings indicate that during NADPH oxidase deficiency, superoxide production can be compensated by other sources, which leads to cardiac oxidative stress and its related molecular/cellular events in the infarcted heart.     

Epithelial-to-mesenchymal transition in podocytes mediated by activation of NADPH oxidase in hyperhomocysteinemia

The present study tested the hypothesis that hyperhomocysteinemia (hHcys) induces podocytes to undergo epithelial-to-mesenchymal transition (EMT) through the activation of NADPH oxidase (Nox). It was found that increased homocysteine (Hcys) level suppressed the expression of slit diaphragm-associated proteins, P-cadherin and zonula occludens-1 (ZO-1), in conditionally immortalized mouse podocytes, indicating the loss of their epithelial features. Meanwhile, Hcys remarkably increased the abundance of mesenchymal markers, such as fibroblast specific protein-1 (FSP-1) and α-smooth muscle actin (α-SMA). These phenotype changes in podocytes induced by Hcys were accompanied by enhanced superoxide ( \textO₂ ^·- {\text{O}}_2^{{ \cdot - }} ) production, which was substantially suppressed by inhibition of Nox activity. Functionally, Hcys significantly enhanced the permeability of the podocyte monolayer coupled with increased EMT, and this EMT-related increase in cell permeability could be restored by Nox inhibitors. In mice lacking gp91 ^phox (gp91^−/−), an essential Nox subunit gene, hHcys-enhanced podocyte EMT and consequent glomerular injury were examined. In wild-type (gp91^+/+) mice, hHcys induced by a folate-free diet markedly enhanced expression of mesenchymal markers (FSP-1 and α-SMA) but decreased expression of epithelial markers of podocytes in glomeruli, which were not observed in gp91^−/− mouse glomeruli. Podocyte injury, glomerular sclerotic pathology, and marked albuminuria observed in gp91^+/+ mice with hHcys were all significantly attenuated in gp91^−/− mice. These results suggest that hHcys induces EMT of podocytes through activation of Nox, which represents a novel mechanism of hHcys-associated podocyte injury.     

Inhibition of the neutrophil NADPH oxidase by adenosine is associated with increased movement of flavocytochrome b between subcellular fractions

Adenosine is a potent inhibitor of reactive oxygen species (ROS) production by the NADPH oxidase in fMLF-stimulated neutrophils. Although much is known about the pharamacology and signal transduction of this effect, it is not known how adenosine affects assembly and localization of the NADPH oxidase components within the neutrophil. We report here that adenosine pretreatment of fMLF-stimulated neutrophils results in decreased plasma membrane/secretory granule content of the flavocytochrome b components (p22^phox and gp91^phox) of the NADPH oxidase, which correlates with inhibition of ROS production. Adenosine treatment did not affect upregulation of secretory and specific granule surface markers, confirming that degranulation was not impaired by adenosine. However, adenosine treatment did result in increased movement of cell-surface flavocytochrome b to heavy granule fractions in fMLF-stimulated neutrophils. These data suggest that adenosine-mediated effects on neutrophil ROS production are due, in part to endocytosis and/or redistribution of flavocytochrome b between various subcellular compartments.