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PLENARY PAPER
From the Herman B Wells Center for Pediatric Research,
Department of Pediatrics (Hematology/Oncology) and Medical and
Molecular Genetics, James Whitcomb Riley Hospital for Children, Indiana
University Medical Center, Indianapolis; Department of Biochemistry,
Wake Forest University School of Medicine, Winston-Salem, NC;
Department of Biochemistry, Emory University Medical School, Atlanta,
GA; Department of Immunology, The Scripps Research Institute, La Jolla,
CA.
The phagocyte nicotinamide adenine dinucleotide phosphate (reduced
form) (NADPH) oxidase was functionally reconstituted in monkey kidney
COS-7 cells by transfection of essential
subunits, gp91phox,
p22phox, p47phox, and
p67phox. COS-7 cells express the essential
small guanosine 5'-triphosphatase, Rac1. Transgenic
COS-phox cells were capable of arachidonic acid-induced NADPH oxidase activity up to 80% of that of human neutrophils, and of
phorbol myristate acetate (PMA)-induced activity up to 20% of that of
neutrophils. Expression of all 4 phox components was
required for enzyme activity, and enzyme activation was associated with
membrane translocation of p47phox,
p67phox, and Rac1. Expression of
p47phox Ser303Ala/Ser304Ala
or Ser379Ala phosphorylation-deficient mutants resulted in
significantly impaired NAPDH oxidase activity, compared with expression
of wild-type p47phox or the
p47phox Ser303Glu/Ser304Glu
phosphorylation mimic, suggesting that p47phox
phosphorylation contributes to enzyme activity in the COS system, as is
the case in neutrophils. Hence, COS-phox cells should be useful as a new whole-cell model that is both capable of high-level superoxide production and readily amenable to genetic manipulation for
investigation of NADPH oxidase function. PMA-elicited superoxide production in COS-phox cells was regulated by activation of
protein kinase C (PKC) and Rac. Although COS-7 cells differ from human neutrophils in PKC isoform expression, transient expression of major
neutrophil isoforms in COS-phox cells did not increase
PMA-induced superoxide production, suggesting that endogenous isoforms
were not rate limiting. Val204 in p67phox,
previously shown to be required for NADPH oxidase activity under cell-free conditions, was found to be essential for superoxide production by intact COS-phox cells, on the basis of
transfection studies using a p67phox
(Val204Ala) mutant.
(Blood. 2002;99:2653-2661) The phagocyte nicotinamide adenine dinucleotide
phosphate (reduced form) (NADPH) oxidase plays a central role in host
defense and in the inflammatory response by catalyzing the transfer of electrons from NADPH to molecular oxygen, producing superoxide radicals.1,2 Superoxide is directly microbicidal and is
also converted into reactive oxygen derivatives, which synergize with toxic granule proteins to facilitate microbial killing. Genetic deficiency of the NADPH oxidase results in chronic granulomatous disease, a syndrome characterized by recurrent, often
life-threatening, infections and chronic granulomas.3
However, although the NADPH oxidase is essential for host defense,
excessive or inappropriate release of reactive oxidants has been
implicated as a mediator of inflammatory tissue
injury.4
The NADPH oxidase is a multicomponent enzyme whose activity requires
assembly of cytosolic subunits p47phox,
p67phox, and Rac with membrane-associated
flavocytochrome b558, a heterodimer of
gp91phox and
p22phox.5 In resting neutrophils,
p47phox and p67phox are
present in a high-molecular-weight complex along with
p40phox,6 which interacts with
phosphorylated lipids7,8 but is not required for NADPH
oxidase function. Membrane translocation in intact cells is dependent
on phosphorylation of multiple serine residues in
p47phox.1 This induces a
conformational change in p47phox,9
which reveals binding sites for the flavocytochrome
subunits.10-14 Gene expression of
gp91phox, p47phox, and
p67phox is largely restricted to mature
phagocytes. Genetic defects in any 1 of the 4 essential phox
components, gp91phox,
p22phox, p47phox, or
p67phox, result in chronic granulomatous
disease.3 Rac activation and membrane translocation occur
independently of
p47phox/p67phox
translocation15 and involve release from cytosolic-binding protein RhoGDI16 and exchange of bound guanosine
5'-diphosphate (GDP) for guanosine 5'-triphosphate (GTP).
Rac-GTP interacts with numerous effector proteins, including
p67phox.17 Hematopoietic-specific
Rac2 is the predominant Rac isoform in human neutrophils, whereas Rac1
and Rac3 isoforms are widely expressed.18-20
Decreased neutrophil NADPH oxidase activity has been reported in
Rac2 How the cytosolic components regulate flavocytochrome activation is not
completely understood, but evidence suggests that p47phox functions as a regulated adaptor
protein, which enhances the binding of p67phox
to the flavocytochrome by 100-fold.26 The
p47phox subunit is also essential for
translocation of p67phox in whole cells, on the
basis of studies of p47phox-deficient chronic
granulomatous disease neutrophils.12,27 Association of
p47phox and p67phox is
mediated via reciprocal src homology-3 (SH-3) domain/proline-rich region interactions1,2 Cellular agonists induce
phosphorylation of multiple serine residues along the C terminal
quarter of p47phox inducing an activating
conformational change.1 In particular, phosphorylation of
residues 303, 304, and 328 is sufficient to disrupt an intramolecular
SH-3 domain interaction, thereby allowing p47phox to interact with
p22phox.13,14 Phosphorylation of
Ser379 is also necessary for p47phox membrane
translocation and NADPH oxidase activity in whole cells.28 Evidence suggests that the oxidase can be activated through multiple signaling pathways, which are differentially regulated by various stimuli. Protein kinase C (PKC) has been implicated in virtually all of
the signaling pathways that activate neutrophils.29 PKC can phosphorylate p47phox in
vitro,30 and in cell-free oxidase assays phosphorylation by PKC alone is sufficient to induce an activating conformational change in p47phox, enabling membrane
translocation and low-level superoxide production.31
Only the N-terminal 210 amino acids of p67phox
are necessary for flavocytochrome activation under cell-free
conditions.32 The N-terminal 150 amino acids of
p67phox contain 4 Previous studies of NADPH oxidase assembly and activation in intact
cells have used primary human or murine hematopoietic cells, or
immortalized cell lines of hematopoietic lineage. Primary phagocytic
cells, such as neutrophils, are not easily amenable to genetic
modification. Expression of transgenes in immortalized cells of
hematopoietic lineage35-40 have provided many useful
insights. However, these cell lines suffer variously from limitations
on the ready manipulability of different oxidase subunits, the ease of
transfection, long-term expression of transfected proteins, and level
of superoxide production. Cell-free assays of NADPH oxidase activity
offer flexibility in the use of purified recombinant components.
However, there are clear differences between oxidase activation in
reconstituted cell-free assays compared with the physiologic
environment of whole cells.1,41 For example, in the
cell-free assay, in which oxidase assembly is triggered by the
addition of anionic amphiphile, p47phox
phosphorylation is not required,42,43 nor are the SH-3
domains of p47phox and
p67phox necessary.44,45
The aim of the current study was to develop a readily transfectable
whole-cell system for investigating the NADPH oxidase by genetic
approaches. Transgenic expression of recombinant oxidase subunits in
monkey kidney COS-7 cells conferred high-level superoxide production
that was activated by either phorbol ester or arachidonic acid
(AA). Phorbol myristate acetate (PMA)-activated superoxide production in these cells appeared to be regulated by PKC and Rac, and
was dependent on serine residues in p47phox
previously implicated as critical phosphorylation sites in intact cells. We also established that the p67phox
activation domain, which is essential for superoxide production under
cell-free conditions, was required for assembly of a functional NADPH
oxidase in whole cells.
Reagents and antibodies
Mouse monoclonal antibodies (Abs) for gp91phox
and p22phox were kindly provided by D. Roos and
A. Verhoeven (Central Laboratory of the Netherlands Blood Transfusion
Service, Amsterdam). Polyclonal rabbit serum raised against
p67phox was provided by P. Heyworth (Scripps
Research Institute, La Jolla, CA), and polyclonal rabbit serum against
Rap1A was provided by M. Quinn (Montana State University,
Bozeman). Polyclonal rabbit serum was raised against
recombinant human Rac2, RhoGDI, and p47phox. The
following antibodies were purchased: mouse monoclonal Ab against Rac1
(Upstate, Lake Placid, NY); isoform-specific rabbit polyclonal Abs for
PKC isoforms Expression vectors
Cell culture COS-7 cells were obtained from American Type Culture Collection (Manassas, VA) (CRL-1651) and grown in DMEM with 10% heat-inactivated fetal calf serum (FCS) (Hyclone, Logan, UT), 50 U/mL penicillin, and 50 µg/mL streptomycin. Adherent COS-7 cells were harvested by incubating with trypsin/EDTA for 5 minutes at 37°C. Following addition of DMEM/10%FCS to neutralize the trypsin, cells were pelleted by centrifugation at 4°C, washed in ice-cold PBS, repelleted, and rapidly resuspended in ice-cold PBS.Transfection of COS-7 cell lines COS-7 cells had previously been stably transfected with gp91phox cDNA in pEF-PGKpac and p22phox cDNA in pEF-PGKneo.47 The gp91phox/p22phox-transfected COS-7 cells were either transfected with p47phox/pEF-PGKhygro, transfected with p67phox/pEF-PGKhygro, or cotransfected at a 1:10 DNA ratio with p47phox/pEF-PGKhygro and p67phox/pEF-PGKpac. DOTAP (Boehringer Mannheim, Indianapolis, IN) was used to transfect 11 µg DNA per 100-mm plate of cells per the manufacturer's instructions. Clones were selected by limiting dilution in 0.2 mg/mL hygromycin (Calbiochem, La Jolla, CA), 1.8 mg/mL neomycin, and 1 µg/mL puromycin. Of 50 cotransfected clones screened by immunoblotting, 3 expressed all 4 phox components. The clone with the highest expression of p47phox and p67phox also had the highest rate of superoxide production and was selected for detailed analysis.Lipofectamine Plus was used to transiently transfect 2 µg DNA per 60-mm plate of COS-7-derived cell lines. Cells were analyzed 21 to 24 hours after transfection. Transient transfection efficiency assayed by immunohistochemisty to detect expression of a Myc-epitope tag averaged 35%. Neutrophil isolation Neutrophils were isolated from heparinized venous blood donated by healthy adult volunteers, as described.48 Briefly, whole blood was subjected to dextran sedimentation, centrifugation over Isolymph (Gallard-Schlesinger Industries, Carle Place, NY), or Ficoll/Paque (Amersham Pharmacia, Piscataway, NJ), and to hypotonic lysis of contaminating red cells.Measurement of NADPH oxidase activity Superoxide production by whole COS-7 cell lines, harvested by brief trypsinization as described above, was measured in a quantitative kinetic assay on the basis of the superoxide dismutase-inhibitable reduction of cytochrome c. Assays were performed at 37°C by means of a Thermomax microplate reader and associated SOFTMAX Version 2.02 software (Molecular Devices, Sunnyvale, CA) as described previously.46 Briefly, cells were suspended (2.5 × 105 cells per well) in 250 µL PBS plus 0.5 mM MgCl2, 0.9 mM CaCl2, and 7.5 mM dextrose (PBSG) containing 75µM cytochrome c. Cells were activated by the addition of 0.4 µg/mL PMA, 100 µM AA, or a combination of 0.4 µg/mL PMA plus 60 µM AA. Samples containing 250 U superoxide dismutase, in addition to the reagents listed, were run in parallel. Superoxide production was quantified by means of an extinction coefficient of 21.1 mM 1cm1 for cytochrome
c. The maximum rate of superoxide generation over a 3-minute
interval was calculated by means of SOFTMAX software.
For detection of nitroblue tetrazolium (NBT) reduction, cells were incubated for 30 minutes at 37°C in PBSG containing NBT plus 0.4 µg/mL PMA. Cells were fixed with methanol and stained with 0.2% safrinin. A blinded observer randomly counted 200 cells at × 400 magnification to determine the percentage of NBT+ cells. Analysis of protein expression Cells were pelleted and resuspended in lysis buffer (20 mM Tris-HCl pH 8, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 20 µg/mL chymostatin, 2 mM phenylmethylsulfonyl fluoride [PMSF], 1 mM 4-(2-aminoethyl) benzenesulfonyl fluoride [AEBSF], and 10 µM leupeptin). Lysate was incubated on ice for 30 minutes and cleared by microcentrifuging (14 000 rpm, 2.5 minutes, 4°C). Protein concentration was quantified by means of the BCA Protein Assay (Pierce, Rockford, IL). With the use of the buffer system of Laemmli, samples were electrophoresed in sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) followed by transfer to nitrocellulose membranes. Membranes were blocked in Tris-buffered saline with 0.5% Tween-20 (TBST) containing 6% powdered milk, exposed to primary antibodies diluted in TBST, exposed to horseradish peroxidase-conjugated secondary anti-immunoglobulin G, washed thoroughly, and visualized by enhanced chemiluminescence (ECL) (Amersham Pharmacia). Densitometric analysis of films was performed by means of Stratagene Eagle Eye II Still Video System and associated software (Stratagene, La Jolla, CA). Serial protein dilutions along with multiple exposures were analyzed to ensure that this analysis was performed in the linear range.Measurement of translocation of cytosolic NADPH oxidase components Cells were suspended at 4 × 107/mL in PBSG, warmed for 5 minutes at 37°C, and then stimulated with 0.4 µg/mL PMA for 10 minutes at 37°C, or with 100 µM AA for 5 minutes at 37°C. Control cells were incubated (10 minutes at 37°C) without stimulus. Cells were returned to ice and immediately diluted 10 × with ice-cold PBS, then centrifuged (1200 rpm, 6 minutes, 4°C). Cells were resuspended in 2 mL relaxation buffer (100 mM KCl, 10 mM Hepes, 3.5 mM MgCl2, 3 mM NaCl, 1.2 mM ethyleneglycotetraacetic acid [EGTA], 25 mM NaF, 5 mM Na3VO4, 1 mM p-nitrophenyl phosphate, 0.5 µM microcystin, 20 µg/mL chymostatin, 2 mM PMSF, 10 µM leupeptin, and 1 mM AEBSF), disrupted in a Dounce homogenizer (Kontes Glass, Vineland, NJ), then centrifuged (1300 rpm, 10 minutes, 4°C) to remove unbroken cells and nuclei. Supernatant was layered on a discontinuous 20%/38% sucrose gradient and centrifuged at 204 000g for 30 minutes (SW55 rotor; Beckman Instruments, Palo Alto, CA). The cytosol was removed from the top of the gradient, and the membrane fraction was collected at the 20%/38% interface. Fractions were electrophoresed, and the proteins analyzed as described above.Analysis of PKC isoform expression Cell fractionation was essentially as described.48 Briefly, neutrophils or COS-7 cells were treated with 1 mM diisopropyl fluorophosphate for 5 minutes on ice, washed once with 10 vol ice-cold Hanks balanced salt solution, and resuspended at 2 × 108 cells per milliliter in extraction buffer (50 mM Tris, pH 7.5, containing 2 mM EGTA, 1 mM PMSF, 1 µg/mL leupeptin, 10 µM benzamidine, 10 µM pepstatin, and 0.2 µg/mL aprotinin). Cells were disrupted by sonication on ice, and the resulting homogenate was centrifuged (800g, 10 minutes, 4°C). Neutrophil cytosolic, membrane and granule fractions were obtained from the 800g supernatant after centrifugation over a 15%/40% discontinuous sucrose gradient at 4°C for 30 minutes at 150 000g (SW55 rotor). Neutrophil cytosol was removed from the top of the gradient, and the membrane fraction was collected at the 15%/40% interface. COS-7 cell cytosolic and membrane fractions were obtained from the supernatant and pellet, respectively, following centrifugation at 368 000g for 30 minutes at 4°C. Samples were electrophoresed in 7% SDS-polyacrylamide gels and transferred electrophoretically to nitrocellulose. Blots were processed as previously described.48Statistical analysis Results were analyzed by means of InStat (GraphPad software, San Diego, CA). Treatments were compared with control by means of the 2-tailed paired Student t test or analysis of variance followed by the Student-Newman-Keuls multiple comparisons test, as indicated. P < .05 was considered significant. Data are expressed as mean ± SD.
The phagocyte NADPH oxidase can be functionally reconstituted in heterologous COS-7 cells We previously generated a COS-7 cell line that coexpressed gp91phox and p22phox from stable transgenes.47 The 7D5 monoclonal antibody, which is directed to an extracellular epitope of gp91phox,49-51 stained unpermeabilized COS-7 gp91phox/p22phox cells, and membranes prepared from these cells supported superoxide production in the presence of neutrophil cytosol.52 The p47phox and p67phox cDNAs were cotransfected into COS-7 gp91phox/p22phox cells to determine if superoxide production could be elicited in whole cells in this heterologous system. Of 50 clones screened by immunoblot analysis, 3 expressed all 4 phox components. One of the 3 clones exhibited 25% higher p47phox expression,
50% greater p67phox expression, and a higher
rate of superoxide production (see below) than the other 2 clones, so
it was selected for detailed analysis. The level of phox
protein expression in this clone was estimated by densitometric
analysis of immunoblots relative to serial dilutions of human
neutrophil extract (Table 1). The
transgenic COS-phox cells expressed the 4 phox
proteins at a level equivalent to or higher than human
neutrophils on a per-cell-equivalent basis. However, the
flavocytochrome subunits were less concentrated on the basis of per
milligrams total protein in the COS-phox cells as compared
with human neutrophils because the larger COS-7 cells contained
approximately 8 times as much total protein per cell. Immunoblot
analysis also showed that the COS-phox cells expressed endogenous Rac1 and Rap1A, but not hematopoietic-specific Rac2, as
expected (Table 1).
The transgenic COS-phox cells were capable of
high-level NADPH oxidase activity upon activation by either the phorbol
ester PMA (phorbol 12-myristate 13-acetate), or AA, 2 soluble stimuli commonly used to activate the neutrophil NADPH oxidase (Figure 1). The PMA-elicited rate of superoxide
production by the COS-phox cells was 11 ± 3.3 (n = 43)
nmol/107 cells per minute (maximum velocity
[Vmax]), which was constant over a PMA concentration
range of 100 to 1000 ng/mL. This compared favorably both to a
PMA-elicited Vmax of 47 ± 7.7 nmol/107 cells
per minute (n = 5 from 4 different donors) for human
neutrophils and to a Vmax of 35 ± 18
nmol/107 cells per minute (n = 29) reported for PLB-985
cells differentiated to a neutrophil phenotype.35 Reported
rates of PMA-elicited superoxide production in human neutrophils have
ranged as high as 117 nmol/107 cells per minute in various
studies.53-55 The AA-elicited rate of superoxide
production by the COS-phox cells was 35 ± 12
nmol/107 cells per minute (n = 11) (Vmax) at
an optimal concentration of 100 µM. This was comparable to the rate
of superoxide production by human neutrophils of 41 ± 9.7
nmol/107 cells per minute elicited by 100 µM AA, or
34 ± 11 nmol/107 cells per minute elicited by 80 µM AA
(for both, n = 5 from 4 different donors). AA-elicited
superoxide production of up to 105 ± 24 nmol/107
cells per minute (Vmax) has previously been reported
for human neutrophils with the use of 82 µM AA.54
Wild-type COS-7 cells have been reported to express an endogenous oxidase, genetically distinct from the phagocyte NADPH oxidase, which is capable of low-level superoxide production that may play a role in mitogenic signaling.56,57 However, as shown in Figure 1, any endogenous superoxide production in wild-type COS-7 cells was below the threshold of detection in the cytochrome c reduction assay. NADPH oxidase activity measurable by cytochrome c reduction required expression of the 2 flavocytochrome subunits plus both cytosolic phox components, as COS-7 cell lines expressing only gp91phox/p22phox, only gp91phox/p22phox/p47phox, or only gp91phox/p22phox/p67phox did not produce significant levels of superoxide after either PMA or AA stimulation (data not shown). The p47phox serine residues at 303, 304, and 379 are necessary for optimal NADPH oxidase activation in COS-phox cells To ascertain whether phosphorylation of key p47phox serine residues 303, 304, and 379 contributed to NADPH oxidase activation, COS-7 gp91phox and p22phox cells were transiently transfected with wild-type p67phox plus either empty vector, wild-type p47phox, or p47phox mutants Ser303Ala/Ser304Ala, Ser303Glu/Ser304Glu, or Ser379Ala. The alanine substitutions are kinase insensitive, whereas the glutamate substitutions mimic phosphorylated residues. NADPH oxidase activity was significantly lower in COS cells expressing the kinase-insensitive p47phox Ser303Ala/Ser304Ala or Ser379Ala mutants compared with cells expressing wild-type p47phox or the Ser303Glu/Ser304Glu phosphorylation mimic (Figure 2), suggesting that phosphorylation of p47phox serine residues 303, 304, and 379 contributes to optimal NADPH oxidase activation in the COS cell system.
Both PMA and AA elicit membrane translocation of p47phox, p67phox, and Rac in COS-phox cells To determine whether COS-phox cells resemble neutrophils, in which cytosolic NADPH oxidase components translocate to the membrane in response to soluble agonists, membrane was extracted from cells following activation with 0.4 µg/mL PMA (10 minutes, 37°C), 100 µM AA (5 minutes, 37°C), or no stimulus (10 minutes, 37°C). Membrane fractions were subjected to SDS-PAGE and immunoblotting. Activation of COS-phox cells with either PMA or AA induced membrane translocation of Rac1, p47phox, and p67phox (Figure 3).
Endogenous PKC isoforms are not rate limiting for PMA-induced NADPH oxidase activity in COS-phox cells Preincubation of COS-phox cells with the PKC-selective inhibitor GF109203x (1 µM, 30 minutes, 37°C) reduced PMA-induced superoxide production by 89% ± 12% (n = 3), suggesting that PMA-elicited NADPH oxidase activation was mediated predominately through PKC signaling pathways. A variety of studies have suggested that conventional PKC isoforms or  mediate PMA-induced NADPH
oxidase assembly in neutrophils and monocytes.4,58-60 We
compared PKC isoform expression in COS-7 cells to that in human
neutrophils by using immunoblotting to examine expression of PKC
isoforms , I, II,  ,  ,  ,  ,  ,  , and  . The
most substantial differences were found with PKC isoforms , II,
, , and (Figure 4). COS-7 cells
expressed much more PKC-, PKC-, and PKC- than human
neutrophils, but had undetectable levels of PKC-II and low levels of
PKC-, both of which were expressed at much higher levels in
neutrophils. Both cell types expressed very low levels of PKC-I;
COS-7 cells also expressed low levels of PKC- (not shown). Isoforms
, , and did not appear to be significantly expressed in
either cell type (not shown).
To determine whether PKC isoforms that are enriched in
neutrophils would enhance NADPH oxidase activation in
COS-phox cells, PKC- Rac activation contributes to agonist-elicited superoxide production in COS-phox cells To investigate whether Rac activation plays a role in the regulation of NADPH oxidase activity in transgenic COS-phox cells, we transiently expressed either dominant-negative RacN17 mutants or RhoGDI, a ubiquitously expressed Rho-family binding protein that sequesters Rac in the cytosol.16 Transient transfection efficiency under our conditions was approximately 35%, as assessed by immunohistochemistry to detect expression of Myc epitope-tagged transgenic protein. Relative to endogenous RhoGDI and Rac1, recombinant RhoGDI and, particularly, Myc epitope-tagged Rac1N17 were overexpressed (Figure 5A). Transgenic expression of Rac1N17, Rac2N17, or RhoGDI reduced PMA-elicited superoxide production by 34% to 41% as compared with empty vector-transfected control cells in the cytochrome c reduction assay (Figure 5B). AA-elicited superoxide production was similarly inhibited (data not shown). Using NBT to detect PMA-elicited superoxide production by individual cells, we found that 69% ± 1.7% of empty vector-transfected control cells were NBT+, compared with 52% ± 1.9% of Rac1N17-transfected cells, 48% ± 15% of Rac2N17-transfected cells, and 48% ± 14% of RhoGDI-transfected cells (all P < .05 compared with empty vector-transfected control cells; n = 5). Thus, there were 25% to 31% fewer NBT+ cells in cell populations transfected with Rac1N17, Rac2N17, or RhoGDI compared with empty vector-transfected control cells; this is consistent with complete inhibition of superoxide production in most cells expressing dominant-negative Rac or RhoGDI. Immunohistochemistry confirmed that the majority of cells expressing the Myc-epitope tag were NBT. A few Myc+ cells were NBT+,
which may reflect cell-to-cell variability in expression of the
dominant-negative Rac. Taken together, these results suggest that
inhibition of Rac activation impaired agonist-elicited NADPH oxidase
activity in transgenic COS-phox cells.
A p67phox activation domain is necessary for NADPH oxidase activation in whole cells A p67phox derivative truncated at amino acid 210 and containing a Val204Ala substitution in a putative flavocytochrome "activation" domain was previously found to be completely inactive when added as a recombinant protein to a cell-free superoxide assay.32 The truncated p67phox Val204Ala mutant translocated to the membrane under these conditions and was a potent inhibitor of superoxide production in the presence of recombinant wild-type p67phox.32 To examine the function of full-length p67phox Val204Ala in whole cells, wild-type p67phox or the mutant Val204Ala derivative were transiently expressed in COS-phox cells. In COS-7 cells stably expressing gp91phox, p22phox, and p47phox, transient expression of wild-type p67phox resulted in significant superoxide production upon PMA activation, whereas superoxide production was not detected with transient expression of p67phox Val204Ala (Figure 6A). In the "complete" COS-phox cells, which stably expressed wild-type p67phox in addition to gp91phox, p22phox, and p47phox, transient expression of additional wild-type p67phox did not significantly affect the rate of superoxide production (Figure 6B). In contrast, transient expression of p67phox Val204Ala significantly reduced superoxide production by an average of 30%, as compared with either mock-transfected control cells or wild-type p67phox-transfected cells (Figure 6B). This reduction was similar to the transfection efficiency, suggesting that expression of the Val204Ala mutant was a potent inhibitor of superoxide production in the transfected cell population. These results are consistent with those obtained in cell-free NADPH oxidase activation assays, which suggest that incorporation of p67phox Val204Ala into the oxidase complex results in an inactive enzyme.32
In this study, we show that expression of all 4 essential phox subunits can reconstitute high-level NADPH oxidase activity in a nonhematopoietic, easily transfectable cell line derived from monkey COS-7 cells. These COS-phox cells are a new whole-cell model that is readily amenable to genetic manipulation, providing the ability to easily express oxidase subunits and regulatory proteins, either transiently or stably, to examine the impact on NADPH oxidase function in intact cells. Many features of oxidase assembly cannot be recapitulated in cell-free oxidase assays, including the requirement for the SH-3 domains of p67phox or for p47phox phosphorylation, both of which are important for translocation to the membrane and oxidase activity in whole cells.1,41 Primary phagocytes are not readily transfectable, and functional analysis of mutant phox proteins in cell lines with NADPH oxidase expression, such as PLB-985 or Epstein-Barr virus (EBV)-transformed B cells, are limited to genetically deficient cells that are also not efficiently transfected and, for B-cell lines, have low levels of superoxide production. The NADPH oxidase has also been reconstituted by transfection of hematopoietic but nonphagocytic K562 leukemia cells, which can then produce superoxide at 5% to 15% the rate of human neutrophils.40,61 The COS-phox cells represent the first functional reconstitution of the phagocyte NADPH oxidase in a nonhematopoietic cell line and can produce superoxide at up to 80% the rate of human neutrophils upon activation by either the phorbol ester PMA or AA. Hence, COS-7 cells contain upstream signaling components that can couple to transgenically expressed phox subunits to activate high levels of NADPH oxidase activity. Whereas in human neutrophils, PMA and AA can induce similar rates of superoxide production, in COS-phox cells AA was a more potent stimulus, inducing more than 3-fold higher rates of superoxide production compared with PMA. This difference in agonist efficacy suggests some differences in signaling pathways that lead to oxidase activation in COS cells compared with human neutrophils. Nevertheless, the final assembled state of the oxidase in the COS-phox system seems to simulate the native system because a large amount of superoxide is generated, expression of all 4 essential phox components is required, and oxidase activation is associated with membrane translocation of p47phox, p67phox, and Rac. Furthermore, p47phox kinase-insensitive mutants Ser303Ala/Ser304Ala or Ser379Ala are significantly deficient in NADPH oxidase activity in the COS-phox cells compared with wild-type p47phox or the Ser303Glu/Ser304Glu phosphorylation-mimic mutant. The Ser303Ala/Ser304Ala and Ser379Ala mutants are similarly deficient in supporting whole-cell NADPH oxidase activity in the EBV-transformed B cell or K562 heterologous systems, whereas they are fully active in cell-free assays.13,28,62 The COS-phox system therefore should be a useful model for studying functional domains in NADPH oxidase subunits necessary for assembly of an active enzyme in intact cells. In COS-phox cells, we found that the main effect of PMA on
NAPDH oxidase activation was exerted through PKC, similar to phagocytic cells, where PKC inhibitors effectively abolish PMA-induced superoxide production.63,64 We investigated whether differences
between endogenous PKC isoforms in COS-phox cells compared
with neutrophils accounted for the lower rate of COS-phox
superoxide production elicited by PMA compared with arachidonate. COS-7
cells expressed much more PKC- Stimulation of COS-phox cells with either PMA or AA induced Rac translocation to the membrane. Furthermore, expression of transgenic RacN17 or RhoGDI significantly inhibited both PMA- and AA-induced superoxide production in COS-phox cells. These results suggest that Rac activation and membrane translocation are important for assembly of the active NADPH oxidase complex in whole COS cells. This is consistent with the requirement for Rac-GTP to achieve high-level superoxide-generating activity in cell-free systems derived from neutrophils or using highly purified proteins.65,66 Rac-GTP binds directly to p67phox,17,33 and to a second site in the NADPH oxidase complex, possibly the flavocytochrome.67 Our results also support the finding that expression of D57N dominant-negative Rac2 is associated with reduced NADPH oxidase activity in human neutrophils.23,24,68 Capitalizing on the ease with which the phox components can be manipulated in COS-phox cells, we used this system to assess the importance of a putative activation domain in p67phox in a whole-cell environment. This p67phox activation domain (residues 199 through 210) was originally identified by testing the activity of recombinant p67phox C-terminal truncation mutants in cell-free oxidase assays. Whereas p67phox truncated at amino acid 210 supported NADPH oxidase activity at levels comparable to wild-type p67phox, p67phox truncated at amino acid 204 was inactive.32 Amino acid substitutions within the activation domain of truncated p67phox (1 through 210) revealed that a p67phox Val204Ala mutant was completely inactive in the cell-free oxidase assay.32 The p67phox Val204Ala mutant associated with the membrane fraction after cell-free activation and competed with wild-type p67phox for assembly into the NADPH oxidase complex.32 The p67phox activation domain was not involved in binding interactions with the other cytosolic factors and was postulated to bind directly to flavocytochrome b558.32 In vitro flavin reduction assays suggest that the p67phox activation domain regulates hydride/electron flow from NADPH to FAD in the flavocytochrome.34 Our results in the COS-phox system establish that substitution of alanine for valine at residue 204 in the p67phox activation domain also abrogates superoxide production when NADPH oxidase assembly is triggered in whole cells. Furthermore, coexpression of full-length p67phox Val204Ala mutant with wild-type p67phox inhibited superoxide production in the COS-phox cells. Similar results were obtained in cell-free NADPH oxidase activation assays, which suggest that p67phox Val204Ala is incorporated into the oxidase complex but results in an inactive enzyme.32 In conclusion, we functionally reconstituted the phagocyte NADPH oxidase in nonhematopoietic COS-7 cells by stable transfection of 4 essential phagocyte oxidase components, gp91phox, p22phox, p47phox, and p67phox. This whole-cell model system was capable of high-level superoxide production in response to phorbol ester or AA. The COS-phox system resembled neutrophils in that superoxide production was dependent on Rac activation and the presence of all 4 phox subunits and was associated with membrane translocation of the cytosolic subunits. Serines 303, 304, and 379 in p47phox, previously shown to be important for activation of the NADPH oxidase in whole cells, were also required for optimal superoxide production by COS-phox cells. Using this model system, we established that the p67phox activation domain, which is essential for superoxide production under cell-free conditions, was required for assembly of a functional NADPH oxidase in whole cells. Thus the COS-phox cells represent a new, readily transfectable whole-cell system for studying the phagocyte NADPH oxidase by genetic approaches.
We thank Nancy Pech for assistance with NBT assays, Dianne Greene for assistance with identification of PKC isoforms, Teri Mallory and Shari Upchurch for expert help in manuscript preparation, and David Skalnik for helpful discussions.
Submitted March 30, 2001; accepted December 3, 2001.
Supported by National Institutes of Health grants RO1HL45635 (M.C.D.), 2RO1AI-22564 (L.C.M.), CA84138 (J.D.L.), AI35947 (U.G.K.), and GM37696 (U.G.K.). The Wells Center for Pediatric Research is a Center for Excellence in Molecular Hematology funded by National Institutes of Health grant P50DK49218.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Mary C. Dinauer, Cancer Research Institute R4, Indiana University School of Medicine, 1044 W Walnut St, Rm 402A, Indianapolis, IN 46202; e-mail: mdinauer{at}iupui.edu.
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Y. Gu, Y. C. Xu, R. F. Wu, F. E. Nwariaku, R. F. Souza, S. C. Flores, and L. S. Terada p47phox Participates in Activation of RelA in Endothelial Cells J. Biol. Chem., May 2, 2003; 278(19): 17210 - 17217. [Abstract] [Full Text] [PDF]
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 T. E. Decoursey Voltage-Gated Proton Channels and Other Proton Transfer Pathways Physiol Rev, April 1, 2003; 83(2): 475 - 579. [Abstract] [Full Text] [PDF]
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B. Banfi, R. A. Clark, K. Steger, and K.-H. Krause Two Novel Proteins Activate Superoxide Generation by the NADPH Oxidase NOX1 J. Biol. Chem., January 31, 2003; 278(6): 3510 - 3513. [Abstract] [Full Text] [PDF]
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 N. Touret and S. Grinstein Voltage-gated Proton "Channels": a Spectator's Viewpoint J. Gen. Physiol., November 25, 2002; 120(6): 767 - 771. [Full Text] [PDF]
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A. Maturana, K.-H. Krause, and N. Demaurex NOX Family NADPH Oxidases: Do They Have Built-in Proton Channels? J. Gen. Physiol., November 25, 2002; 120(6): 781 - 786. [Full Text] [PDF]
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K. J. Biberstine-Kinkade, L. Yu, N. Stull, B. LeRoy, S. Bennett, A. Cross, and M. C. Dinauer Mutagenesis of p22phox Histidine 94. A HISTIDINE IN THIS POSITION IS NOT REQUIRED FOR FLAVOCYTOCHROME b558 FUNCTION J. Biol. Chem., August 9, 2002; 277(33): 30368 - 30374. [Abstract] [Full Text] [PDF]
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D. Morgan, V. V. Cherny, M. O. Price, M. C. Dinauer, and T. E. DeCoursey Absence of Proton Channels in COS-7 Cells Expressing Functional NADPH Oxidase Components J. Gen. Physiol., June 1, 2002; 119(6): 571 - 580. [Abstract] [Full Text] [PDF]
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M. O. Price, S. J. Atkinson, U. G. Knaus, and M. C. Dinauer Rac Activation Induces NADPH Oxidase Activity in Transgenic COSphox Cells, and the Level of Superoxide Production Is Exchange Factor-dependent J. Biol. Chem., May 17, 2002; 277(21): 19220 - 19228. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
) or
transgenic COS-phox cells (
), following detachment from
tissue-culture dishes and stimulation with either 0.4 µg/mL PMA or
100 µM AA, was determined by reduction of ferricytochrome
c. Data are expressed as mean ± SD;
n 
3.
