Location of the epitope for 7D5, a monoclonal antibody raised against human flavocytochrome b558, to the extracellular peptide portion of primate gp91phox

Flavocytochrome b558 is the membrane component of the phagocyte NADPH oxidase, and is a heterodimer composed of gp91phox and p22phox subunits. Human flavocytochrome b558 is recognized by monoclonal antibody 7D5 at an unidentified extracellular domain, although our previous study suggested it might recognize p22phox. 7D5 has proven useful in rapid screening of individuals for X-linked chronic granulomatous disease by flow-cytometry. Therefore, we re-evaluated the location of the 7D5 epitope using gene-engineered cell lines expressing hybrid flavocytochromes composed of human and murine subunit homologues. The current study demonstrates that the 7D5 recognizes epitope only of primate gp91phox. Flow-cytometric analyses showed that 7D5 consistently bound to cells expressing human gp91phox. In addition, 7D5 immunoprecipitated the approximately 58 kDa unglycosylated gp91phox protein from solubilized membrane fractions of tunicamycin-treated PLB-985 granulocytes, indicating that glycans were not required for 7D5 binding. Transgenic COS7 cells expressing human gp91phox but not p22phox were recognized by 7D5. These results localized the epitope of 7D5 to an extracellular peptide portion of primate gp91phox and indicate that the antibody will be useful for monitoring the efficiency of gene therapy in patients with flavocytochrome b558-deficient chronic granulomatous disease and for elucidating structural characteristics of flavocytochrome b558.     

Mutagenesis of p22(phox) histidine 94. A histidine in this position is not required for flavocytochrome b558 function

The NADPH oxidase is a multicomponent enzyme that transfers electrons from NADPH to O2 to generate superoxide (O2*-), the precursor of microbicidal oxygen species that play an important role in host defense. Flavocytochrome b558, a heterodimeric oxidoreductase comprised of gp91(phox) and p22(phox) subunits, contains two nonidentical, bis-histidine-ligated heme groups imbedded within the membrane. Four histidine residues that appear to serve as noncovalent axial heme ligands reside within the hydrophobic N terminus of gp91(phox), but the role of p22(phox) in heme binding is unclear. We compared biochemical and functional features of wild type flavocytochrome b558 with those in cells co-expressing gp91(phox) with p22(phox) harboring amino acid substitutions at histidine 94, the only invariant histidine residue within the p22(phox) subunit. Substitution with leucine, tyrosine, or methionine did not affect heterodimer formation or flavocytochrome b558 function. The heme spectrum in purified preparations of flavocytochrome b558 containing the p22(phox) derivative was unaffected. In contrast, substitution of histidine 94 with arginine appeared to disrupt the intrinsic stability of p22(phox) and, secondarily, the stability of mature gp91(phox) and abrogated O2*- production. These findings demonstrate that His94 p22(phox) is not required for heme binding or function of flavocytochrome b558 in the NADPH oxidase.     

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

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

Deletion mutagenesis of p22phox subunit of flavocytochrome b558: identification of regions critical for gp91phox maturation and NADPH oxidase activity

The heterodimeric flavocytochrome b558, comprised of the two integral membrane proteins p22phox and gp91phox, mediates the transfer of electrons from NADPH to molecular oxygen in the phagocyte NADPH oxidase to generate the superoxide precursor of microbicidal oxidants. This study uses deletion mutagenesis to identify regions of p22phox required for maturation of gp91phox and for NADPH oxidase activity. N-terminal, C-terminal, or internal deletions of human p22phox were generated and expressed in Chinese hamster ovary cells with transgenes for gp91phox and two other NADPH oxidase subunits, p47phox, and p67phox. The results demonstrate that p22phox-dependent maturation of gp91phox carbohydrate, cell surface expression of gp91phox, and the enzymatic function of flavocytochrome b558 are closely correlated. Whereas the 5 N-terminal and 25 C-terminal amino acids are dispensable for these functions, the N-terminal 11 amino acids of p22phox are required, as is a hydrophilic region between amino acids 65 and 90. Upon deletion of 54 residues at the C terminus of p22phox (amino acids 142-195), maturation and cell surface expression of gp91phox was still preserved, although NADPH oxidase activity was absent, as expected, due to removal of a proline-rich domain between amino acids 151-160 that is required for recruitment of p47phox. Antibody binding studies indicate that the extreme N terminus of p22phox is inaccessible in the absence of cell permeabilization, supporting a model in which both the N- and C-terminal domains of p22phox extend into the cytoplasm, anchored by two membrane-embedded regions.     

The phagocyte NADPH oxidase depends on cholesterol-enriched membrane microdomains for assembly

The superoxide-producing phagocyte NADPH oxidase consists of a membrane-bound flavocytochrome b558 complex, and cytosolic factors p47phox, p67phox and the small GTPase Rac, which translocate to the membrane to assemble the active complex following cell activation. We here show that insolubility of NADPH oxidase subunits in nonionic detergents TX-100, Brij-58, and Brij-98 is a consequence of inclusion into cholesterol-enriched membrane microdomains (lipid rafts). Thus, flavocytochrome b558, in a cholesterol-dependent manner, segregated to the bouyant low-density detergent-resistant membrane (DRM) fraction, and the cytosolic NADPH oxidase factors associated dynamically with low-density DRM. Further, superoxide production following cholesterol depletion was severely compromised in intact cells or in a cell-free reconstituted system, correlating with a reduced translocation of cytosolic phox subunits to the membrane. In analogy with the widely accepted role of lipid rafts as signaling platforms, our data indicate that cholesterol-enriched microdomains act to recruit and/or organize the cytosolic NADPH oxidase factors in the assembly of the active NADPH oxidase.     

Functional epitope on human neutrophil flavocytochrome b558

mAb NL7 was raised against purified flavocytochrome b(558), important in host defense and inflammation. NL7 recognized the gp91(phox) flavocytochrome b(558) subunit by immunoblot and bound to permeabilized neutrophils and neutrophil membranes. Epitope mapping by phage display analysis indicated that NL7 binds the (498)EKDVITGLK(506) region of gp91(phox). In a cell-free assay, NL7 inhibited in vitro activation of the NADPH oxidase in a concentration-dependent manner, and had marginal effects on the oxidase substrate Michaelis constant (K(m)). mAb NL7 did not inhibit translocation of p47(phox), p67(phox), or Rac to the plasma membrane, and bound its epitope on gp91(phox) independently of cytosolic factor translocation. However, after assembly of the NADPH oxidase complex, mAb NL7 bound the epitope but did not inhibit the generation of superoxide. Three-dimensional modeling of the C-terminal domain of gp91(phox) on a corn nitrate reductase template suggests close proximity of the NL7 epitope to the proposed NADPH binding site, but significant separation from the proposed p47(phox) binding sites. We conclude that the (498)EKDVITGLK(506) segment resides on the cytosolic surface of gp91(phox) and represents a region important for oxidase function, but not substrate or cytosolic component binding.     

Heme-ligating histidines in flavocytochrome b(558): identification of specific histidines in gp91(phox).

The phagocyte NADPH-dependent oxidase generates superoxide (O(2)) by reducing molecular oxygen through flavocytochrome b(558) (flavocytochrome b), a heterodimeric oxidoreductase composed of gp91(phox) and p22(phox) subunits. Although each flavocytochrome b molecule contains two heme groups, their precise distribution within the heterodimer is unknown. Among functionally and/or structurally related oxidoreductases, histidines at codons 101, 111, 115, 119, 209, 210, and 222 of gp91(phox) are conserved and potential candidates to ligate heme. We compared biochemical and functional features of normal flavocytochrome b with those in cells expressing gp91(phox) harboring amino acid substitutions at each of these histidines. Surface expression of flavocytochrome b and heterodimer formation were relatively unaffected in cells expressing gp91(phox) H111L, H119L, or H210L. These mutations also had no effect on the flavocytochrome b heme spectrum, although NADPH oxidase activity was decreased in cells expressing gp91(phox) H119L or H210L. In contrast, gp65 was not processed to gp91(phox), heterodimers did not form, and flavocytochrome b was not expressed on the surface of cells expressing gp91(phox) H101L, H115L, H115D, H209C, H209Y, H222L, H222C, or H222R. Similarly, this subset of mutants lacked detectable O(2)-generating activity, and flavocytochrome b purified from these cells contained little or no heme. These findings demonstrate that His(101), His(115), His(209), and His(222) of gp91(phox) are critical for heme binding and biosynthetic maturation of flavocytochrome b.     

Mutagenesis of an arginine- and lysine-rich domain in the gp91(phox) subunit of the phagocyte NADPH-oxidase flavocytochrome b558

Site-directed mutagenesis was used to generate a series of mutants harboring point or multiple substitutions within the hydrophilic, polybasic domain of gp91(phox) encompassed by residues 86-102, which was previously identified as a site of interaction with p47(phox) during phagocyte NADPH oxidase assembly. Recombinant wild-type or mutant gp91(phox) was expressed in a human myeloid leukemia cell line in which the endogenous gp91(phox) gene was disrupted by gene targeting. NADPH oxidase activity was measured in a cytochrome c reduction assay following granulocytic differentiation of cells that expressed recombinant gp91(phox). Expression of a gp91(phox) mutant in which amino acids 89-97 were replaced with nine alternate amino acids abolished NADPH oxidase activity. Expression of gp91(phox) mutants R89T, D95A, D95R, R96A, R96E, or K102T did not significantly affect NADPH oxidase activity. However, mutations of individual or paired arginine residues at positions 91 and 92 had substantial effects on superoxide generation. The R91E/R92E mutation completely abolished both NADPH oxidase activity and membrane-translocation of the cytosolic oxidase proteins p47(phox), p67(phox), Rac1, and Rac2. The phorbol 12-myristate 13-acetate-induced rate of superoxide production was reduced by approximately 75% in cells expressing R91T/R92A, R91E, or R92E gp91(phox) along with an increased lag time to the maximal rates of superoxide production relative to cells expressing wild-type gp91(phox). Taken together, these results demonstrate that Arg91 and Arg92 of gp91(phox) are essential for flavocytochrome b558 function in granulocytes and suggest that these residues participate in the interaction of gp91(phox) with the cytosolic oxidase proteins.     

Heme histidine ligands within gp91(phox) modulate proton conduction by the phagocyte NADPH oxidase

The membrane subunit of the phagocyte NADPH oxidase, gp91(phox), possesses a H(+) channel motif formed by membrane-spanning histidines postulated to coordinate the two heme groups forming the redox center of the flavocytochrome. To study the role of heme-binding histidines on proton conduction, we stably expressed the gp91(phox) cytochrome in human embryonic kidney 293 cells and measured proton currents with the patch clamp technique. Similar to its shorter homologue, NADPH oxidase homologue 1, which is predicted not to bind heme, gp91(phox) generated voltage-activated, pH-dependent, H(+)-selective currents that were reversibly blocked by Zn(2+). The gp91(phox) currents, however, activated faster, deactivated more slowly, and were markedly affected by the inhibition of heme synthesis. Upon heme removal, the currents had larger amplitude, activated faster and at lower voltages, and became sensitive to the histidine reagent diethylpyrocarbonate. Mutation of the His-115 residue to leucine abolished both the gp91(phox) characteristic 558-nm absorbance peak and voltage-activated currents, indicating that His-115 is involved in both heme ligation and proton conduction. These results indicate that the gp91(phox) proton channel is activated upon release of heme from its His-115 ligand. During activation of the oxidase complex, changes in heme coordination within the cytochrome might increase the mobility of histidine ligands, thereby coupling electron and proton transport.     

The X+ chronic granulomatous disease as a fabulous model to study the NADPH oxidase complex activation

Chronic granulomatous disease (CGD) is a rare inherited disorder in which phagocytes lack NADPH oxidase activity. Patients with CGD suffer from recurrent bacterial and fungal infections because of the absence of superoxide anions (O2- degrees ) generatingsystem. The NADPH oxidase complex is composed of a membranous cytochrome b558, cytosolic proteins p67phox, p47phox, p40phox and two small GTPases Rac2 and Rap1A. Cytochrome b558 consists of two sub-units gp91phox and p22phox. The most common form of CGD is due to mutations in CYBB gene encoding gp91phox. In some rare cases, the mutated gp91phox is normally expressed but is devoided of oxidase activity. These variants called X+ CGD, have provided interesting informations about oxidase activation mechanisms. However modelization of such variants is necessary to obtain enough biological material for studies at the molecular level. A cellular model (knock-out PLB-985 cells) has been developed for expressing recombinant mutated gp91phox for functional analysis of the oxidase complex. Recent works demonstrated that this cell line genetically deficient in gp91phox is a powerful tool for functional analysis of the NADPH oxidase complex activation.     

Expression of functional mammal flavocytochrome b558 in yeast: Comparison with improved insect cell system

Activity of phagocyte NADPH-oxidase relies on the assembly of five proteins, among them the transmembrane flavocytochrome b₅₅₈ (Cytb₅₅₈) which consists of a heterodimer of the gp91^phox and p22^phox subunits. The Cytb₅₅₈ is the catalytic core of the NADPH-oxidase that generates a superoxide anion from oxygen by using a reducing equivalent provided by NADPH via FAD and two hemes. We report a novel strategy to engineer and produce the stable and functional recombinant Cytb₅₅₈ (rCytb₅₅₈). We expressed the gp91^phox and p22^phox subunits using the baculovirus insect cell and, for the first time, the highly inducible Pichia pastoris system. In both hosts, the expression of the full-length proteins reproduced native electrophoretic patterns demonstrating that the two polypeptides are present and, that gp91^phox undergoes co-translational glycosylation. Spectroscopic analyses showed that the rCytb₅₅₈ displayed comparable spectral properties to neutrophil Cytb₅₅₈. In contrast to rCytb₅₅₈ produced in the insect cells with higher yield, the enzyme expressed in yeast displayed a superoxide dismutase-sensitive NADPH-oxidase activity, indicating a superoxide generation activity. It was also blocked by an inhibitor of the respiratory burst oxidase, diphenylene iodonium (DPI). As in neutrophil NADPH-oxidase, activation occurred by the interactions with the soluble regulatory subunits suggesting comparable protein-protein contact patterns. We focus on the stability and function of the protein during solubilisation and reconstitution into liposomes. By comparing oxidase activities in different membrane types, we confirm that the lipid-protein environment plays a key role in the protein function.     

Expression and activity of polymorphisms in the 67-kDa protein of the NADPH oxidase system

The NADPH oxidase system plays a central role in the antimicrobial activity of phagocytes. This system is initiated by the translocation of cytosolic proteins p67phox, p47phox and p40phox to be in close contact with membrane flavocytochrome b558. This event begins the electron transfer from cytosolic NADPH to molecular oxygen to produce superoxide anions. Herein, a functional analysis is presented of p67phox polymorphisms identified from healthy humans. Mutations were generated in the p67phox cDNA by site-directed mutagenesis and then transiently expressed in COS7 cells that also expressed gp91phox, p22phox, and p47phox from stable transgenes. The changes Va1166lle, Pro329Ser and His389Gln correspond to possible polymorphisms identified in healthy individuals revealed a functional activity similar to COSphox cells transiently transfected with WT p67phox; therefore, these modifications are not associated with genetic deficiencies in NADPH oxidase. In conclusion, the COSphox system represents an easily transfectable model for analysis of NADPH oxidase function in intact cells. The analysis of mutant derivatives of p67phox provides insight into molecular mechanisms by which this subunit regulates the NADPH oxidase.     

Processing and maturation of flavocytochrome b558 include incorporation of heme as a prerequisite for heterodimer assembly

The phagocyte NADPH-dependent oxidase generates superoxide by reducing molecular oxygen through a transmembrane heterodimer known as flavocytochrome b(558) (flavocytochrome b). We investigated the biosynthesis of flavocytochrome b subunits gp91(phox) and p22(phox) to elucidate features of flavocytochrome b processing in myeloid cells. Although the gp91(phox) precursor, gp65, was processed to gp91(phox) within 4-8 h of chase, unassembled gp65 and p22(phox) monomers were degraded by the cytosolic proteasome. gp65 associated with p22(phox) post-translationally, within 1-4 h of chase, but prior to its modification in the Golgi complex. Moreover, p22(phox) coprecipitated with unglycosylated gp91(phox) primary translation product made in the presence of tunicamycin, suggesting that heterodimer formation does not require glycosylation. Blocking heme synthesis with succinyl acetone completely inhibited heterodimer formation, although biogenesis of gp65 and p22(phox) was unaffected. In succinyl acetone-treated cells, p22(phox) and gp65 were degraded completely by 8 h of chase, a process mediated by the cytosolic proteasome. Taken together, these data suggest that the formation of the gp65-p22(phox) heterodimer is relatively inefficient and that acquisition of heme by gp65 precedes and is required for its association with p22(phox), a process that requires neither the addition of N-linked oligosaccharides nor modification in the Golgi complex.     

Characterization of six novel mutations in the CYBB gene leading to different sub-types of X-linked chronic granulomatous disease

Chronic granulomatous disease is an inherited disorder in which phagocytes lack a functional NADPH oxidase and so cannot generate superoxide anions (O₂^–). The most common form is caused by mutations in CYBB encoding gp91 phox, the heavy chain of flavocytochrome b₅₅₈ (XCGD). We investigated 11 male patients and their families suspected of suffering from X-linked CGD. These XCGD patients were classified as having different variants (X91⁰, X91^– or X91⁺) according to their cytochrome b₅₅₈ expression and NADPH oxidase activity. Nine patients had X91⁰ CGD, one had X91^– CGD and one had X91⁺ CGD. Six mutations in CYBB were novel. Of the four new X91⁰ CGD cases, three were point mutations: G65A in exon 2, G387T in exon 5 and G970T in exon 9, leading to premature stop codons at positions Try18, Try125 and Glu320, respectively, in gp91 phox. One case of X91⁰ CGD originated from a new 1005G deletion detected in exon 9. Surprisingly, four nonsense mutations in exon 5 led to the generation of two mRNAs, one with a normal size containing the mutation and the other in which exon 5 had been spliced. A novel X91^– CGD case with low gp91 phox expression was diagnosed. It was caused by an 11-bp deletion in the linking region between exon 12 and intron 12, activating a new cryptic site. Finally, a new X91⁺ CGD case was detected, characterized by a missense mutation Leu505Arg in the potential NADPH-binding site of gp91 phox. No clear correlation between the severity of the clinical symptoms and the sub-type of XCGD could be established.     

Functional analysis of two-amino acid substitutions in gp91<Emphasis Type="Italic">phox</Emphasis> in a patient with X-linked flavocytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>558</Subscript>-positive chronic granulomatous disease by means of transgenic PLB-985 cells

Chronic granulomatous disease (CGD) is a rare inherited disorder in which phagocytes lack NADPH oxidase activity. The most common form is caused by mutations in the CYBB gene encoding gp91phox protein, the heavy chain of cytochrome b₅₅₈, which is the redox element of NADPH oxidase. In some rare cases, the mutated gp91phox is normally expressed but no NADPH oxidase can be detected. This type of CGD is called X91⁺ CGD. We have previously reported an X⁺ CGD case with a double-missense mutation in gp91phox. Transgenic PLB-985 cells have now been made to study the impact of each single mutation on oxidase activity and assembly to rule out a possible new polymorphism in the CYBB gene. The His303Asn/Pro304Arg gp91phox transgenic PLB-985 cells exactly mimic the phenotype of the neutrophils of the X⁺ CGD patient. The His303Asn mutation is sufficient to inhibit oxidase activity in intact cells and in a broken cell system, whereas in the Pro304Arg mutant, residual activity suggests that the Pro304Arg substitution is less devastating to oxidase activity than the His303Asn mutation. The study of NADPH oxidase assembly following the in vitro and in vivo translocation of cytosolic factors p47phox and p67phox has demonstrated that, in the double mutant and in the His303Asn mutant, NADPH oxidase assembly is abolished, although the translocation is only attenuated in Pro304Arg mutant cells. Thus, even though the His303Asn mutation has a more severe inhibitory effect on NADPH oxidase activity and assembly than the Pro304Arg mutation, neither mutation can be considered as a polymorphism.Clara Bionda and Xing Jun Li contributed equally to this work     

Reconstituted high-density lipoprotein suppresses leukocyte NADPH oxidase activation by disrupting lipid rafts

Reconstituted discoidal high-density lipoprotein (rHDL) has potent vascular protective actions. Native HDL suppresses cellular generation of reactive oxygen species, whereas this antioxidant effect of rHDL is less clear. This study examined the effects of rHDL on NADPH oxidase, a major source of cellular superoxide generation, in both leukocytes and human umbilical vein endothelial cells. Superoxide was measured with lucigenin-enhanced chemiluminescence. Expression of NADPH oxidase sub-units was determined by real-time PCR. Pre-treatment of HL-60 cells with rHDL (10 and 25 µM) for 1 h significantly reduced phorbol 12-myristate 13-acetate-stimulated superoxide production. Treatment with rHDL for up to 24 h did not change the mRNA expression of NADPH oxidase sub-units. In HL-60 cells, depletion of cholesterol from the plasma membrane by methyl-β-cyclodextrin mimicked the effect of rHDL, whereas cholesterol repletion blunted the effects of rHDL. Treatment with rHDL induced disruption of the lipid raft structures and blunted PMA-induced redistribution of p47phox into lipid rafts. In contrast, treatment of endothelial cells with rHDL for up to 18 h had no effect on either basal or tumour necrosis factor-α-stimulated NADPH oxidase activity, but markedly suppressed the cytokine-induced expression of proinflammatory adhesion molecules. The results suggest that rHDL inhibits NADPH oxidase activation in leukocytes, probably by interrupting the assembly of NADPH oxidase sub-units at the lipid rafts. This effect may contribute to the vascular protective actions of rHDL against inflammation-mediated oxidative damage.     

Analysis of human phagocyte flavocytochrome b(558) by mass spectrometry

The catalytic core of the phagocyte NADPH oxidase is a heterodimeric integral membrane protein (flavocytochrome b (Cyt b)) that generates superoxide and initiates a cascade of reactive oxygen species critical for the host inflammatory response. In order to facilitate structural characterization, the present study reports the first direct analysis of human phagocyte Cyt b by matrix-assisted laser desorption/ionization and nanoelectrospray mass spectrometry. Mass analysis of in-gel tryptic digest samples provided 73% total sequence coverage of the gp91(phox) subunit, including three of the six proposed transmembrane domains. Similar analysis of the p22(phox) subunit provided 72% total sequence coverage, including assignment of the hydrophobic N-terminal region and residues that are polymorphic in the human population. To initiate mass analysis of Cyt b post-translational modifications, the isolated gp91(phox) subunit was subject to sequential in-gel digestion with Flavobacterium meningosepticum peptide N-glycosidase F and trypsin, with matrix-assisted laser desorption/ionization and liquid chromatography-mass spectrometry/mass spectrometry used to demonstrate that Asn-132, -149, and -240 are genuinely modified by N-linked glycans in human neutrophils. Since the PLB-985 cell line represents an important model system for analysis of the NADPH oxidase, methods were developed for the purification of Cyt b from PLB-985 membrane fractions in order to confirm the appropriate modification of N-linked glycosylation sites on the recombinant gp91(phox) subunit. This study reports extensive sequence coverage of the integral membrane protein Cyt b by mass spectrometry and provides analytical methods that will be useful for evaluating posttranslational modifications involved in the regulation of superoxide production.     

Gp91phox contributes to NADPH oxidase activity in aortic fibroblasts but not smooth muscle cells

Reactive oxygen species (ROS) derived from vascular NADPH oxidase are important in normal and pathological regulation of vessel growth and function. Cell-specific differences in expression and function of the catalytic subunit of NADPH oxidase may contribute to differences in vascular cell response to NADPH oxidase activation. We examined the functional expression of gp91phox on NADPH oxidase activity in vascular smooth muscle cells (SMC) and fibroblasts (FB). As measured by dihydroethidium fluorescence in situ, superoxide (O2-*) levels were greater in adventitial cells compared with medial SMC in wild-type aorta. In contrast, there was no difference in O2-* levels between adventitial cells and medial SMC in aorta from gp91phox-deficient (gp91phox KO) mice. Adventitial-derived FB and medial SMC were isolated from the aorta of wild-type and gp91phox KO mice and grown in culture. Consistent with the observations in situ, basal and stimulated ROS levels were reduced in FB isolated from aorta of gp91phox KO compared with FB from wild-type aorta, whereas ROS levels were similar in SMC derived from gp91phox KO and wild-type aorta. There were no differences in expression of superoxide dismutase between gp91phox KO and wild-type FB to account for these observations. Because gp91phox is associated with membranes, we examined NADPH-stimulated O2-. production in membrane-enriched fractions of cell lysate. As measured by chemiluminescence, NADPH oxidase activity was markedly greater in wild-type FB compared with gp91phox KO FB but did not differ among the SMCs. Confirming functional expression of gp91phox in FB, antisense to gp91phox decreased ROS levels in wild-type FB. Finally, deficiency of gp91phox did not alter expression of the gp91phox homolog NOX4 in isolated FB. We conclude that the neutrophil subunit gp91phox contributes to NADPH oxidase function in vascular FB, but not SMC.