1 Department of Internal Medicine, Cannon Research Center, Carolinas Medical Center, Charlotte 28232; 4 Division of Pulmonary Diseases, Department of Pediatrics, Duke University Medical Center, Durham, North Carolina 27710; 2 Division of Respiratory, Critical Care and Occupational (Pulmonary) Medicine, University of Utah, Salt Lake City, Utah 84132; and 3 Department of Veterinary Molecular Biology, Montana State University, Bozeman, Montana 59717
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ABSTRACT |
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Evidence is rapidly accumulating
that low-activity-reduced nicotinamide adenine dinucleotide phosphate
(NADPH) oxidases homologous to that in phagocytic cells generate
reactive oxygen species as signaling intermediates in both endothelium
and vascular smooth muscle. We therefore explored the possibility of
such an oxidase regulating growth of airway smooth muscle (AWSM).
Proliferation of human AWSM cells in culture was inhibited by the
antioxidants catalase and N-acetylcysteine, and by the
flavoprotein inhibitor diphenylene iodonium (DPI). Membranes prepared
from human AWSM cells generated superoxide anion (OB (NF-
B),
and overexpression of a superrepressor form of the NF-
B inhibitor
I
B
significantly reduced human AWSM growth. These findings
suggest that an NADPH oxidase containing p22phox
regulates growth-factor responsive human AWSM proliferation, and that
the oxidase signals in part through activation of the prototypical
redox-regulated transcription factor NF-
B.
superoxide anion; diphenylene iodonium; p22phox
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INTRODUCTION |
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EVIDENCE IS RAPIDLY ACCUMULATING that low-activity-reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidases homologous to that in phagocytic cells generate reactive oxygen species (ROS) as signaling intermediates in both endothelium and vascular smooth muscle (23). In the arterial circulation, ROS from these oxidases play important roles in regulation of blood pressure and in arterial wall inflammation and vascular smooth muscle proliferation characteristic of atherosclerosis (23). Recently, a C242 to T transition of the p22phox component of the oxidase, resulting in a histidine (H)72 to tyrosine (Y) mutation, has even been associated with accelerated progression of coronary disease (15).
In phagocytes, the NADPH oxidase consists of two membrane proteins,
gp91phox and p22phox,
that bind a flavin adenine nucleotide and form a unique cytochrome with
a redox midpoint potential of 245 mV (4). At least two and possibly three cytosolic peptides (p47phox,
p67phox, and p40phox) are
also essential, and several other cytosolic components participate, including the small GTPases Rac1 or Rac2. The cytochrome appears to
contain all the factors necessary for transporting electrons from the
donor substrate NADPH via flavin adenine nucleotide and then heme to
generate superoxide anion (O
The oxidase in nonphagocytic cells appears to share some of the components with its phagocyte counterpart, but there are important differences, including a delayed time course for activation and low output. In pulmonary artery, the vascular oxidase is reported to exhibit a preference for reduced nicotinamide adenine dinucleotide (NADH) rather than NADPH (38), but recent spin-trap studies suggest that the aortic vascular smooth muscle oxidase either employs NADH or NADPH equivalently (52) or prefers NADPH as substrate (51). Endothelial cells appear to express all the phagocyte oxidase components, including gp91phox, p22phox, p47phox, and p67phox (22, 29). In contrast, aortic smooth muscle cells express p22phox (22, 23, 54), and p47phox is important for oxidase function (27, 45). However, gp91phox appears to be replaced in aortic smooth muscle cells by the unique homolog Mox-1 (54), reported by others as NADPH oxidase homolog-1 (NOH-1) (7), as the heme-containing membrane partner for p22phox. Yet another gp91phox homolog renox has been recently found in renal epithelial cells where it is postulated to serve as an oxygen sensor regulating renal erythropoietin production (20, 48). A consensus terminology has been proposed for the growing family of NADPH oxidases: NOX1 for Mox-1/NOH-1; NOX2 for gp91phox; and NOX4 for renox (48).
Evidence is beginning to accumulate that a phagocyte-like NADPH oxidase is also present in airway smooth muscle (AWSM). Antioxidants and the flavoprotein inhibitor diphenylene iodonium (DPI) inhibit proliferation of rat and bovine AWSM in culture (13, 42). Fetal bovine serum (FBS) and platelet-derived growth factor (PDGF) stimulate generation of ROS by rat (13) and bovine (42) AWSM cells in vitro, and ROS release by cell cultures is inhibited by DPI (13). Treatment of bovine AWSM cells with PDGF or transfection with a constitutively active Rac1 (pEXV-Myc-V12Rac1) increases expression of the cell cycle regulator cyclin D1 in an antioxidant and DPI-inhibitable manner (42), and PDGF-induced expression of cyclin D1 is reduced by inhibition of the oxidase via overexpression of a dysfunctional NH2-terminal fragment of p67phox (42). Thus, a flavoprotein containing oxidase, presumably an NADPH oxidase, also appears to play a role in proliferative signaling of AWSM. However, the components of the oxidase and its functional activity in AWSM have not been determined. We therefore initiated studies to characterize the NADPH oxidase in cultured human AWSM and to better understand its contribution to signaling of growth and proliferation.
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METHODS |
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Culture of human AWSM.
Human AWSM cells were harvested and cultured using methods previously
reported for rat and mouse AWSM (13). With approval of the
Institutional Review Board, human lung was obtained from lobes resected
during thoracotomy for lung cancer. Segmental or subsegmental bronchus
was dissected free from vessels and lung parenchyma. Adventitia and
bronchial epithelium were removed under a dissecting microscope, and
the remaining airway was minced and digested twice for 30 min at 37°C
in Hanks' balanced salt solution (HBSS) containing 0.2% type IV
collagenase and 0.05% type IV elastase. Enzyme digests were
centrifuged at 500 g, and the pellet was resuspended and cultured in Dulbecco's modified Eagle's medium (DMEM)
supplemented with 10% FBS, nonessential amino acids, penicillin (100 U/ml), streptomycin (100 µg/ml), and amphotericin (250 ng/ml) in a
humidified atmosphere of 5% CO2-95% air at 37°C. On
reaching confluence, cells were passed with 0.25% trypsin-0.002%
EDTA. Immunostaining was performed using a polyclonal antibody against
-smooth muscle actin (Sigma) and visualized using an
avidin-biotin-immunoperoxidase technique. Smooth muscle cultures
demonstrated the typical "hill and valley" appearance under
phase-contrast microscopy and stained avidly for
-smooth muscle
actin. Preliminary studies demonstrated that culture of cells in the
presence of 10% FBS resulted in a linear growth phase up to 120 h. Cultures from passages 2-9 were used for experiments.
Measurement of cultured AWSM proliferation. Proliferation of cultured AWSM was quantitated using a previously reported colorimetric method based on metabolic reduction of the soluble yellow tetrazolium dye 3-[4,5-dimethylthiazol]-2yl-2,5-diphenyl tetrazolium bromide (MTT) to its insoluble purple formazan by the action of mitochondrial succinyl dehydrogenase (13). This assay empirically distinguishes between dead and living cells. For proliferation studies, cells were seeded into 24-well uncoated plastic plates (Costar) at 15,000-50,000 cells/well and cultured with DMEM and mitogens. After 24-96 h, medium was removed, cells were washed twice with 1 ml of sterile Dulbecco's modified PBS without Ca2+ or Mg2+ (DPBS), the medium was replaced with 1 ml/well fresh DMEM containing 100 µg/ml MTT and 0.5% FBS, and plates were incubated an additional 1 h. MTT-containing medium was removed, 0.5 ml of dimethyl sulfoxide was added to each well, and the absorbance of the solubilized purple formazan dye was measured at 540 nm. A total of four to six wells were studied at each treatment condition. Preliminary studies were performed to optimize the concentration of MTT and incubation time, and to confirm that the absorbance of the MTT formazan reduction product correlated with direct counts of stained cells within the range employed. For antioxidant interventions and in studies with a final cell density of 40,000 cells/well, cells were fixed in ice-cold buffered formalin (5% in DPBS), permeabilized by two 30-min treatments of ice-cold methanol, stained with Wright's-modified Geimsa, and counterstained with eosin. Direct cell counts were performed on five random fields well viewed at a magnification of ×100 using a 1-mm2 ocular grid.
Cell culture treatments.
Cell proliferation was studied in cultures stimulated with 10% FBS. To
explore the role of oxidants in signaling mitogenesis, we tested the
effects on cell proliferation of supplementing media with the
O-nitro-L-arginine (100 µmol/l), and the flavoprotein inhibitor diphenyleneiodonium (DPI)
(5-100 µmol/l). To probe for expression of NADPH oxidase
components, monolayers of human AWSM cells were grown on 25-mm dishes
to near confluence in 10% FBS, and RNA was harvested for analysis by
the reverse transcriptase-polymerase chain reaction at this point or
after an additional 48 h of growth arrest in serum-free DMEM. To
study the effect of flavoprotein inhibition on signal transduction
pathways, confluent human AWSM cells in 25-mm dishes were growth
arrested for 48 h in serum-free DMEM and stimulated with 10% FBS
and DMEM, in the presence and absence of 50 µmol/l DPI. After
0-6 h, cells were lysed and immunoassayed for the dually
phosphorylated active forms of the mitogen-protein kinase/extracellular
signal-regulated kinases ERK1 and ERK2 and p38 stress-activated kinase,
and for total and phosphorylated inhibitory factor
B
(I
B
),
as detailed below. To study the effect of flavoprotein inhibition on
activation of the redox-regulated transcription factor nuclear factor
(NF)-
B, confluent human AWSM cells in 100-mm dishes were growth
arrested for 48 h in serum-free DMEM and stimulated with 10% FBS
and DMEM, in the presence or absence of 50 µmol/l DPI at the time of
stimulation. In some experiments, growth-arrested monolayers were
also preincubated with DPI 30 min before stimulation. After 30, 60, or
90 min, total cell lysate or nuclear protein was isolated for
immunoblot assay of phosphorylated I
B
or electrophoretic mobility
shift assay of NF-
B DNA binding, as described below.
Measurement of ROS generation.
Generation of ROS by intact monolayers of FBS-stimulated airway smooth
muscle cells was studied by reduction of ferricytochrome c,
employing a modification allowing absorbance reading with an automatic
enzyme immunoassay reader, as previously reported (13). Human AWSM cells grown on 24-well plates in the presence of 10% FBS to
near confluence were washed with DPBS and incubated in serum-free
medium at 37°C with 160 µmol/l ferricytochrome c in a
total volume of 400-µl HBSS (bicarbonate containing, phenol red
free), with and without copper-zinc SOD (300 U/ml). The absorbance of
each well was measured at 550 nm initially and again after 120 min of
incubation at 37°C using an ELx800UV automated microplate reader
(Biotek Instruments, Highland Park, VT). Monolayers were then washed
with DPBS and cell protein was measured using the bicinchoninic protein
assay (Pierce). OEM = 2.1 × 104
M
1/cm
1) and a measured light path length of
1 mm.
Reverse transcriptase-polymerase chain reaction detection of NADPH oxidase components. To probe for presence of components of the neutrophil NADPH oxidase and NOX1, semiquantitatiave reverse transcriptase-polymerase chain reaction (RT-PCR) was performed as recently described (13). Cell monolayers were washed twice with DPBS and lysed with 4 mol/l guanidine thiocyanate, 25 mmol/l sodium citrate, and 0.5% sarkosyl. After being scraped, lysates were sheared with four passes through a pipette. RNA was extracted by the phenol-chloroform method (16) and quantitated spectrophotometrically at 260 and 280 nm. RNA (2 µg) was reverse transcribed using 200 units of Moloney murine leukemia virus reverse transcriptase (Promega) in a reaction mixture containing 1 mmol/l dATP, dCTP, dGTP, and dTTP; 40 units of RNase inhibitor; 25 µmol/l random hexamers; 5 mmol/l MgCl2; 500 mmol/l KCl; and 100 mmol/l Tris · HCl (pH 8.3), in a total volume of 50 µl. The resultant cDNA was PCR amplified for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), p22phox, gp91phox, p47phox, p67phox, and NOX1 using human gene-specific sense and antisense primers based on sequences published in GenBank: GAPDH: 5'-ACCACCATGGAGAAGGCTGG, 3'-CTCAGTGTAGCCCAGGATGC; p22: 5'-ATGGAGCGCTGGGGACAGAAGCACATG, 3'-GATGGTGCCTCCGATCTGCGGCCG; gp91phox: 5'-TCAATAATTCTGATCCTTATTCAG, 3'-TGTTCACAAACTGTTATATTATGC; NOX-1: 5'-CTGGGTGGTTAACCACTGGTTT, 3'-GAATCCCTAAGTGCCGTAACCA; p47phox: 5'-ACCCAGCCAGCACTATGGGT, 3'-AGTAGCCTGTGACGTCGTCT; p67phox: 5'-CGAGGGAACCAGCTGATAGA, 3'-CATGTGAACACTGAGCTTCA. PCR was carried out on a Perkin-Elmer DNA thermal cycler 480. Amplification was carried out for 32 cycles for GADPH and p22phox and 36 cycles for all other primers at 95°C for 1 min, 58°C for 1 min, and 72°C for 2 min, followed by an extension step at 72°C for 10 min. PCR-amplified DNA was separated on 1.2% agarose gel, stained with ethidium bromide, and visualized and photographed under ultraviolet light. PCR products from defined bands were purified with QIAquick gel extraction kits (Qiagen, Chatsworth, CA) and sequenced automatically by an ABI Prism 310 Genetic Analyzer (Applied Biosystems, Foster City, CA) using the same respective primers for sequencing as for PCR. Results of RT-PCR from human AWSM cells were always compared with positive controls (human PMNs for p22phox, gp91phox, p47phox, and p67phox; and Caco colon carcinoma cells for NOX1).
Immunoassay for proteins.
To study activation of signal transduction cascades, cells were lysed
and proteins were isolated and quantitated by immunoassay as previously
detailed (13) using antitobodies against IB
and
phospho-specific antibodies (New England Biolabs, Beverly, MA) against
the dually phosphorylated active forms of ERK1 and ERK2 (phosphorylated
at Thr202/Tyr204) and p38 stress-activated
kinase (phosphorylated at Thr180/Tyr182), and
against phosphorylated I
B
(phosphorylated at Ser32),
and peroxidase-labeled donkey polyclonal anti-rabbit IgG. Cells were
placed on ice, washed twice with cold DPBS, scraped into 0.5-ml boiling
buffer [10% vol/vol glycerol and 2% wt/vol sodium dodecyl sulfate
(SDS) in 83 mM Tris, pH 6.8] and sheared by four passages through a
pipette. Aliquots were removed for protein determination, using the
bicinchoninic protein assay (Pierce). After 10%
-mercaptoethanol
and 0.05% bromphenol blue were added, lysates were boiled for 5 min
and stored at
80°C until immunoblotting was performed. Proteins in
defrosted samples were separated by SDS-PAGE on 12% polyacrylamide
gels (15 µg protein/lane) and electrotransferred to 0.45 µm
Hybond-enhanced chemiluminescence nitrocellulose membranes (Amersham
Life Sciences) using the wet transblot method in transfer buffer (0.025 mol/l Tris, 0.192 mol/l glycine, 2.6 mmol/l SDS, and 20% vol/vol
methanol; pH 8.8) at 100 V for 1 h. Blots were blocked overnight
at 4°C with blocking buffer (PBS with 0.1% Tween 20) containing 5%
fat-free milk powder (Carnation, Glendale, CA). After being rinsed five
times for 5 min each in PBS containing 0.1% Tween 20, blots were
incubated for 1 h at room temperature with primary antiserum
diluted 1:2,000. After being rinsed again as above, blots were
incubated for 1 h at room temperature with horseradish
peroxidase-conjugated secondary antibody diluted 1:2,000 in blocking
buffer. Immunoblots were rinsed again as above and detected using an
enhanced chemiluminescence method (ECL Western blotting detection
system; Amersham Life Science, Buckinghamshire, UK) and autoradiography.
Electrophoretic mobility shift assays.
Nuclear protein was isolated as described by Ogata et al.
(39). Human AWSM cells on 100-mm dishes were scraped in
800 µl of buffer A composed of (in mmol/l) 10 HEPES (pH
7.9), 10 KCl, 0.1 EDTA, 0.1 EGTA, 1.0 DTT, and 1.0 PMSF (plus protease
inhibitor cocktail, 10 µg/ml) at 4°C and kept on ice for 15 min,
followed by the addition of 50 µl of 10% Nonidet P-40, and cells
were homogenized by vortexing for 10 s. The homogenate was
centrifuged at 3,000 g at 4°C for 10 min to prepare
nuclei, and the pelleted nuclei were resuspended in 25 µl of ice-cold
buffer B composed of (in mmol/l) 20 HEPES (pH 7.9), 400 NaCl, 1.0 EDTA, 1.0 EGTA, 1.0 DTT, 1.0 PMSF, and protease inhibitor
cocktail (10 µg/ml). Nuclear proteins were extracted by incubation on
ice for 30 min, and the supernatant containing nuclear protein was
collected after centrifugation at 8,000 g at 4°C for 10 min. Protein concentration was determined on an aliquot by the Pierce
method and the remainder of nuclear protein was frozen at 80°C
until use.
Transduction protocols for IB
gene transfer.
To repress activation of NF-
B, cells were transduced with adenoviral
(Ad serotype 5; Ad5) vectors that were E1a/E1b-deleted and expressed a
superrepressor of NF-
B (Ad-I
B
SR, 2 × 1011
plaque-forming U/ml) under the regulation of the cytomegalovirus (CMV)
immediate-early promoter region (8) or expressed the CMV
immediate-early promoter region alone (AdCMV-3, 2.05 × 1011 plaque-forming U/ml, control vector). These Ad vectors
were constructed in the Vector Core Laboratory at the Gene Therapy
Center of the University of North Carolina School of Medicine and were
generous gifts, respectively, from Dr. Albert S. Baldwin of the
Lineberger Cancer Center and Dr. Andrew Ghio of the Environmental
Protection Agency Human Health Effects Center (Chapel Hill, NC).
Transduction was performed using previously published protocols
(8). Human AWSM cells were seeded onto 24-well plates at a
density of 20,000 cells/well and grown for 6 h in DMEM with 10%
FBS. Media was removed and replaced with 200 µl complete medium
containing ~2.5 × 106 colony- forming units of
Ad-I
B
SR or AdCMV-3. After overnight incubation, the vector
containing media was removed, and cells were washed once with warm DPBS
and reincubated with fresh complete media. After an additional 24 h, cells were washed twice with ice-cold DPBS, fixed twice for 30 min
in ice-cold 5% buffered formalin in DPBS, permeabilized twice with
ice-cold methanol for 30 min, stained with Wright's-modified Giemsa,
and counterstained with eosin before direct counting of cells using an
ocular grid, as outlined earlier.
Transfection protocol for p22phox antisense treatment of human AWSM. To transfect antisense oligonucleotides for p22phox, human AWSM cells were cultured in six-well plates at a density of 20,000 cells/well and grown in DMEM containing 10% FBS. After 24 h, wells were washed once with DPBS and 800 µl of DMEM (serum and antibiotic free) was added to each well. Previously reported (34) p22phox sense (5'-GGTCCTCACCATGGGGCAGATC-3') or antisense (5'-GATCTGCCCCATGGTGAGGACC-3') oligonuleotides (2 µg) were mixed with 5 µl Lipofectace Reagent (Life Technologies) and 200 µl serum- and antibiotic-free DMEM at room temperature for 15 min. This mixture was then added to each well and cells were incubated at 37°C. After 6 h, the transfection mixture was gently removed and replaced with 2.5 ml of DMEM containing 10% FBS. Cells were incubated an additional 48 h before photographs were taken and growth was quantitated with the MTT assay.
Statistical analysis. Data are expressed as means ± SE for a minimum number of four observations, unless indicated. Differences between two groups were compared using Student's t-test. Two-tailed tests of significance were employed. Differences between multiple groups were compared using one-way ANOVA. The post hoc test used was the Newman-Keuls multiple comparison test. Significance was assumed at P < 0.05.
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RESULTS |
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Antioxidants and flavoprotein inhibitors suppress human AWSM cell
proliferation.
Analogous to what we observed in cultured rat AWSM cells
(13), antioxidants also reduced proliferation of cultured
human AWSM (Fig. 1, A and
B). Whereas catalase produced
substantial inhibition of growth, SOD inhibited proliferation only to a
slight, albeit statistically significant, degree. These results suggest that H2O2, and not O-nitro-L-arginine (data not
shown), indicating that a flavoprotein-dependent oxidase other than
xanthine oxidase or nitric oxide synthase mediates an important but not
exclusive pathway for proliferative signaling.
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Human AWSM cell membranes produce ROS.
Confluent human AWSM cell monolayers incubated for 3 h in HBSS
progressively reduced ferricytochrome c in an
SOD-inhibitable manner, generating 4.2 ± 0.4 pmol of
O
|
Proliferating human AWSM cells express the p22phox
component of the NADPH oxidase.
To begin to determine expression of membrane components of the putative
NADPH oxidase, we performed RT-PCR on RNA extracted from human AWSM
cells under two conditions: cells grown to near confluence in the
presence of 10% FBS; and cells grown to near confluence, then growth
arrested in serum-free media for 48 h. Proliferating nearly
confluent human AWSM cells strongly expressed the -subunit of
cytochrome b558, p22phox (Fig.
3A, lanes
1-3), but expression was more variable in
growth-arrested, serum-starved cells (lanes 4-6).
The 252-base pair PCR product obtained is identical to bases
221-372 of the reported human mRNA sequence (26),
except for exhibiting the C242-to-T transition in
p22phox recently reported to be associated with
accelerated coronary disease (15). Protein product for
p22phox was also clearly detectable by
immunoassay in membranes from humans AWSM cells (Fig. 3B).
The cytochrome and flavin-bearing
-subunit of the cytochrome,
gp91phox, and the p67phox
cytosolic component were also detected but expression was variable and
required 36 cycles of amplification for detection (data not shown). No
evidence was found for the NOX1 homolog of
gp91phox or the p47phox
cytosolic component of the leukocyte NADPH oxidase (data also not
shown), but clear expression for both components was seen in controls
(human PMN for p47phox and Caco colon carcinoma
cells for NOX1).
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p22phox is necessary for human AWSM proliferation.
Transfection of sense oligonucleotides for
p22phox had little effect on growth of human
AWSM [Fig. 4B (sense) vs.
4A (control), and Fig. 4D]. However, antisense
oligonucleotides for p22phox reduced expression
of p22phox protein (Fig. 4E), and
significantly impaired AWSM proliferation [Fig. 4C
(antisense) vs. 4B (sense), and Fig. 4D]. This
suggests that the p22phox is important and
necessary for signaling serum-induced proliferation.
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NADPH oxidase influences growth-signaling transduction cascades.
ROS from growth factor-responsive NADPH oxidase might affect AWSM
proliferation by acting on a number of signal transduction cascades.
The flavoprotein inhibitor DPI prevents activation of cyclin D1
expression in bovine AWSM cells by a constitutively active
phosphatidylinositol 3-kinase (PI3K) (43), suggesting that
the PI3K pathway is upstream from the putative Rac1-activated oxidase.
Also, whereas activation of ERK1 and ERK2 is unaffected by ROS
scavengers or flavoprotein inhibition, cyclin D1 expression induced in
bovine AWSM cells by PDGF or a constitutively active Rac1 is
nonetheless prevented by antioxidants or DPI (42). Thus the putative NADPH oxidase must affect proliferation by influencing other signal transduction pathways. Using antibodies specific for their
respective phosphorylated active forms or phosphorylated products, we
studied the effect of flavoprotein inhibition on activity of the IB
kinase, the ERK1/ERK2 mitogen-activated kinases, and the p38
stress-activated kinase. Serum promptly activated the I
B kinase,
resulting in phosphorylation of I
B
(Fig.
5A), and stimulated the
ERK1/ERK2 kinase (Fig. 5B) and p38 stress-activated kinase
cascades (Fig. 5C). Treatment of cells with DPI (50 µmol/l) did not reduce phosphorylation of ERK1/ERK2 (Fig.
6A) or p38 mitogen-activated protein (MAP) kinase (Fig. 6B). However, DPI did reduce
phosphorylation of I
B
, shown in immunoassays using a
phospho-specific antibody (Fig. 6C), suggesting that
phosphorylation of I
B
in human AWSM cells may be in part redox
regulated by the putative NADPH oxidase. Phosphorylation of I
B
is
the initiating event in activation of NF-
B, the prototypical
redox-regulated transcription factor (5, 6, 46, 47). We
therefore performed electrophoretic mobility shift assays to determine
whether serum stimulation activates NF-
B. Serum stimulation for 90 min induced a dramatic increase in DNA binding activity for NF-
B
(Fig. 7A,
lane 2). Supershift experiments with antibodies specific for
potential NF-
B components identified the top band (arrowhead) as the
relevant p50 (lane 3) and p65 (lane 5) dimer.
Consistent with its reduction of I
B
phosphorylation, DPI
treatment also significantly and substantially inhibited serum
stimulation of DNA binding activity for NF-
B in nuclear protein of
human AWSM cells (Fig. 7, B and C). These results
imply that the putative NADPH oxidase influences AWSM proliferation in
part through redox regulation of NF-
B transcriptional activity.
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Transcription factor NF-B is important for human AWSM
proliferation.
The transcription factor NF-
B plays a well-recognized role in
inflammation but has recently (25, 30) also been found important in regulating G0/G1 to S-phase
transition in cell proliferation through increasing expression of
cyclin D1. In vascular smooth muscle, NF-
B is essential for
proliferation. When nuclear activation of NF-
B is interrupted by
microinjection of I
B
or NF-
B consensus oligonucleotides,
growth is disrupted (11). However, the importance of
NF-
B for AWSM proliferation has not been established. We therefore studied the effect on human AWSM proliferation of retarding NF-
B activation by transduction of cells with an adenovirus linked superrepressor form of I
B
(Ad-I
B
SR). Transduction of cells with the adenoviral vector linked to the CMV promoter (Ad-CMV) as a
control had no inhibitory effect on proliferation. However, transduction with Ad-I
B
SR resulted in prominent I
B
SR
expression (Fig. 8C), which
substantially impaired growth of human AWSM cells (Fig. 8, A
and B). Thus interruption of NF-
B activation by DPI might
explain in part the reduction in human AWSM proliferation by this
flavoprotein inhibitor.
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DISCUSSION |
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Signal transducing oxidases have now been reported in a number of
cell types from the blood vessel wall, including vascular endothelium
(22, 29), vascular smooth muscle (7, 15, 23, 27, 38,
45, 54, 56), and blood vessel adventitial fibroblasts (40,
57). In endothelium (22) and adventitial fibroblasts (41), the membrane oxidase components appear
identical to the respective - and
-subunits,
p22phox and gp91phox,
present in leukocytes. In vascular smooth muscle cells,
gp91phox appears to be replaced by a homolog
NOX1 (22, 41, 54). Functionally, these oxidases have been
implicated in ANG II-induced cellular hypertrophy (23,
56), mitogenic signaling (1, 23, 28, 55),
ANG-dependent hypertension (23, 62), impaired endothelial-dependent vascular relaxation (37), and
progression of atherosclerosis (15, 23, 60). In the airway
mucosa, a p22phox- and
gp91phox-containing NADPH oxidase regulating
K+ channel activity has been reported by one group to serve
as an O2 sensor in pulmonary neuroepithelial bodies
(19). In airway smooth muscle, ANG II induces hypertrophy
in cell cultures (36), and a constitutively active form of
Rac1 stimulates cyclin D1 expression in a DPI-inhibitable manner
(42), suggesting that a functionally important NADPH
oxidase analogous to that in vascular smooth muscle exists. However,
its components and relationship to growth signaling cascades have not
been explored.
In this report, we demonstrate evidence for a growth-regulatory oxidase
activity in human AWSM membranes. Serum-induced proliferation of human
AWSM cells in culture was strongly inhibited by the antioxidants catalase and N-acetylcysteine (Fig. 1A),
indicating the importance of redox signaling in growth factor-dependent
cell cycling. Human AWSM membranes (Fig. 2) supported generation of
lucigenin chemiluminescence in a SOD-inhibitable manner, indicating
that the initial product of the growth regulating oxidase activity is
likely O-nitro-L-arginine had an
inhibitory effect on growth. Therefore, neither of these
flavoprotein-containing enzymes is a likely source of growth regulating
ROS in human AWSM cells. Similar to what has been reported for vascular
smooth muscle cells (33, 51), SOD-inhibitable lucigenin
chemiluminescence stimulated by human AWSM cell membranes was
approximately fivefold higher with NADPH rather than NADH as the
electron donating substrate (Fig. 2). Thus the putative NADPH growth
regulating oxidase of AWSM cells shares some of the functional
properties reported for its vascular smooth muscle cell counterpart,
but with important differences.
The vascular smooth muscle oxidase contains a distinct membrane p22phox component that is critical for mediation of oxidase activity and ANG II-induced hypertrophy (55). Human AWSM cells also demonstrate clear expression of p22phox mRNA that, when sequenced, is identical to the sequence reported for the leukocyte analog (Fig. 3A). Furthermore, immunoassays show conspicuous evidence of p22phox protein in human AWSM cell membranes (Fig. 3B). When antisense oligonucleotides for p22phox are transfected into AWSM cells (Fig. 4), growth is significantly impaired. Thus the p22phox NADPH oxidase subunit is expressed in human AWSM cells and is critically important for their proliferation. The structure of the remaining components of the oxidase is less certain. It has been suggested that vascular smooth muscle cells express the gp91phox homolog NOX1 as the partner for p22phox in forming the membrane components of cytochrome b558 (7, 22, 54). We detected no mRNA for NOX1 and only weak expression of gp91phox. Thus, it is not presently clear whether the partner for p22phox in AWSM is gp91phox, expressed at an extremely low level, or a unique tissue-specific protein, such as the recently reported thyroid THOX1 and THOX2 oxidases (17, 18) or NOX4, recently reported in renal tubular epithelium (20, 48) and bone osteoclasts (61). Vascular smooth muscle cells express the p47phox cytosolic component of the leukocyte oxidase (27, 45). Also, overexpression of a dysfunctional NH2-terminal fragment of p67phox disrupts growth factor-stimulated cyclin D1 promoter activity in bovine AWSM cells (42), suggesting that this cytosolic component is present in bovine airway smooth muscle. We detected PCR product for p67phox in human AWSM cells only after 36 cycles of amplification, and were unable to demonstrate any product for p47phox. Thus the two cytosolic components of the leukocyte oxidase are either absent or expressed at low levels. Either circumstance might account for the low rate of ROS production in human AWSM cells compared with the oxidase of leukocytes (32).
In vascular smooth muscle, the putative NADPH oxidase appears to signal
through certain isoforms of MAP kinases (1) and p38
stress-activated protein kinase (56). In AWSM cells, the situation appears different. The AWSM oxidase seems to be part of the
PI3K cascade activated by PDGF in signaling cell proliferation (43). Catalase and DPI block stimulation of the cyclin D1
promoter by expression of either a constitutively active Rac1 (V12Rac1) or a PI3K catalytic subunit (p110PI 3-KCAAX). However, whereas the PI3K
inhibitor wortmannin blocks cyclin D1 promoter activation by PDGF, it
does not prevent stimulation of the promoter by V12Rac1. This would
suggest that the putative NADPH oxidase is downstream from and possibly
activated by phosphorylation of one or more of its components by the
PI3K, similar to the situation in human polymorphonuclear neutrophils,
where receptor-stimulated Rac2 activation is downstream from and
mediated by PI3K (2). Also, stimulation of the NADPH
oxidase with V12Rac1 does not activate the MAP kinases ERK1 and ERK2,
and neither antioxidants nor DPI inhibit ERK1 and ERK2 activation by
PDGF (42). In our studies, we confirmed that serum growth
factor-induced activation of ERK1 and ERK2 is not inhibited by DPI, and
also found that DPI does not prevent serum activation of the p38
stress-activated MAP kinase. Flavoprotein inhibition with DPI did,
however, prevent serum-induced phosphorylation of IB
(Fig.
6C), the first step toward initiating cytosol to nuclear
transport of the transcription factor NF-
B. DPI also inhibited
serum-induced activation of p65/p50-containing NF-
B DNA binding
activity (Fig. 7, B and C). Therefore, in human AWSM, redox regulation of NF-
B appears to be a major pathway mediating the influence of NADPH oxidase activity on cell proliferation.
In unstimulated normal cells NF-B resides in the cytoplasm as a
dimeric protein complex bound to an inhibitor protein, designated I
B
(49). Agonist stimulation activates I
B kinase,
which phosphorylates Ser32 and Ser36 near the
NH2-terminus of I
B
(6, 53), targeting
the inhibitor for ubiquitination and proteolytic degradation by the 26S
proteasome (44). The removal of I
B
unmasks the
nuclear localization signal (10), allowing the NF-
B
complex to translocate to the nucleus, where it binds to its respective
nucleotide sequence and transcriptionally regulates expression or
repression of target genes. The initial upstream response to agonist
activation of I
B kinase and subsequent degradation of I
B
in
normal cells has been proposed to be production of
O
B also induce generation of ROS (5, 6, 46,
47). In human aortic smooth muscle cells, PDGF stimulates
generation of O
B DNA binding activity that is blocked by the
O
B in human umbilical vein endothelial
cells through a pathway involving phosphorylation and activation of the
I
B kinase by O
Activation of NF-B by ROS generated from a putative NADPH oxidase
would be expected to facilitate human AWSM cellular proliferation by
promoting cell cycle G0/G1 to S phase
transition through enhanced expression of cyclin D1 (25,
30). NF-
B activation might also enhance resistance of
vulnerable proliferating cells to apoptosis through increasing
expression of antiapoptotic Bcl-2 family proteins (9, 57,
59). In vascular smooth muscle, NF-
B is essential for
proliferation. When nuclear activation of NF-
B is interrupted by
microinjection of I
B
or NF-
B consensus oligonucleotides, growth is disrupted (11). NF-
B appears to be equally
important for proliferation of human AWSM. When activation of NF-
B
is disrupted through expression of the superrepressor form of I
B
(Fig. 8C), proliferation of human AWSM is substantially
impaired (Fig. 8, A and B). Conversely,
activation of NF-
B might therefore be expected to promote cellular
AWSM growth. This has significant implications for the pathogenesis of
airway wall remodeling in the inflamed, asthmatic airway, where in
mucosal biopsies NF-
B has been shown to be universally activated
(24).
Given the important contribution of the putative NADPH oxidase to
regulation of airway smooth muscle proliferation (13, 42,
43), much additional work is needed to fully characterize the
components of this oxidase and better understand its regulation. It is
unclear whether the regulatory cytosolic component
p47phox is actually missing or simply expressed
at such low levels as to be undetectable by our PCR methods. It has
been postulated that the growth regulatory NADPH oxidase in AWSM and
other mesenchymal tissues is the more primitive form of a signaling
enzyme system that has subsequently evolved into a host defense
function in leukocytes (4). Consistent with this
hypothesis is the demonstration that purified cytochrome
b558 alone can be activated by an anionic amphiphile to
catalyze OS, and at a much higher rate simply with addition of
p67phox (32). Thus not all the
specialized leukocyte cytosolic regulatory components are necessary for
basic enzymatic oxidase function at the low rate of
O
Understanding which oxidase components are necessary, their molecular structure, and the nature of their interactions present compelling tasks for delineating the potentially important role of an AWSM NADPH signaling oxidase in the remodeling of the asthmatic airway wall (12). Airway wall remodeling, caused in large part by hypertrophy and hyperplasia of AWSM within the medial layer, leads to the phenomenon of fixed airways obstruction characteristic of severe asthma (12). Strategies that disrupt the function of a growth regulatory oxidase might retard development of this incapacitating condition. Furthermore, if there is analogy between the condition in the airway and in vascular smooth muscle, polymorphisms in oxidase components predisposing subjects to coronary disease, such as the C242-to-T transition of p22phox (15), might also pose a risk for development of airways remodeling in severe asthma.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. Mark Reames and Edward Lipford for assistance in procuring the human airway specimens from which AWSM was cultured.
![]() |
FOOTNOTES |
---|
This work was funded by grants from the Charlotte-Mecklenberg Health Care Foundation and by National Institutes of Health Grants HL-50153-07 (Dr. Hoidal), RO1-AR-42426, and HL-66767 (Dr. Quinn).
Address for reprint requests and other correspondence: T. Kennedy, Rm. 410, Cannon Research Center, Carolinas Medical Center, PO Box 32861, Charlotte, NC 28232 (E-mail: tkennedy{at}carolinas.org).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajplung.00206.2001
Received 6 June 2001; accepted in final form 10 November 2001.
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