From the Departments of Molecular and Structural
Biology, § Medicine and Clinical Science, and
Urology, Kyushu University Graduate School of Medical Science,
3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, the ¶ Department of
Integrated Biosciences, Graduate School of Frontier Sciences,
University of Tokyo, Tokyo 113-0033, ** RIKEN, Genomic
Sciences Center, Sagamihara 228-8555, and the
Human Genome Center, Institute of Medical
Science, University of Tokyo, Tokyo 108-8639, Japan
Received for publication, August 21, 2000, and in revised form, October 9, 2000
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ABSTRACT |
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During phagocytosis,
gp91phox, the catalytic subunit of the
phagocyte NADPH oxidase, becomes activated to produce superoxide, a
precursor of microbicidal oxidants. Currently increasing evidence suggests that nonphagocytic cells contain similar superoxide-producing oxidases, which are proposed to play crucial roles in various events
such as cell proliferation and oxygen sensing for erythropoiesis. Here
we describe the cloning of human cDNA that encodes a novel NAD(P)H
oxidase, designated NOX4. The NOX4 protein of 578 amino acids exhibits
39% identity to gp91phox with special
conservation in membrane-spanning regions and binding sites for heme,
FAD, and NAD(P)H, indicative of its function as a superoxide-producing
NAD(P)H oxidase. The membrane fraction of kidney-derived human
embryonic kidney (HEK) 293 cells, expressing NOX4, exhibits NADH- and
NADPH-dependent superoxide-producing activities, both of
which are inhibited by diphenylene iodonium, an agent known to block
oxygen sensing, and decreased in cells expressing antisense NOX4
mRNA. The human NOX4 gene, comprising 18 exons,
is located on chromosome 11q14.2-q21, and its expression is almost
exclusively restricted to adult and fetal kidneys. In human renal
cortex, high amounts of the NOX4 protein are present in distal tubular
cells, which reside near erythropoietin-producing cells. In addition,
overexpression of NOX4 in cultured cells leads to increased superoxide
production and decreased rate of growth. The present findings thus
suggest that the novel NAD(P)H oxidase NOX4 may serve as an oxygen
sensor and/or a regulator of cell growth in kidney.
Reactive oxygen species
(ROS)1 are conventionally
viewed as toxic byproducts of cellular metabolism. On the other hand,
organisms possess enzymatic systems that physiologically generate ROS.
Among the systems to be well characterized is the superoxide-producing NADPH oxidase in mammalian professional phagocytes, which plays a
crucial role in host defense against microbial infection (1-4). The
catalytic core of the phagocyte NADPH oxidase is the
membrane-integrated flavocytochrome b558,
comprising the two subunits p22phox and
gp91phox, the latter of which contains a
complete electron-transferring apparatus (from NADPH to molecular
oxygen) with binding sites for heme, FAD, and NADPH (5-8). The
oxidase, dormant in resting cells, becomes activated during
phagocytosis to generate superoxide, a precursor of microbicidal ROS.
The activation requires the two specialized cytosolic proteins
p47phox and p67phox, and
the small GTPase Rac, all of which translocate upon cell stimulation to
the membrane to interact with and activate the flavocytochrome (9-15).
Such a strict regulation of the phagocyte oxidase activity is clearly
important, since uncontrolled production of high amounts of ROS is
injurious not only to phagocytes themselves but also to their
surrounding tissues, and triggers inflammatory reaction (1-4).
A growing body of evidence suggests that similar superoxide-producing
NAD(P)H oxidases exist in nonphagocytic cells of vascular smooth muscle
(16, 17), endothelium (18), carotid body (19), lung (20, 21), and
kidney (22, 23). Although these oxidases are proposed to play a role in
a variety of events such as signaling for cell growth or cell death,
oxygen sensing, and inflammatory processes (16-25), their bona
fide functions as well as molecular compositions are largely
unknown. Suh et al. (26) have recently reported cell
transformation by the superoxide-generating oxidase Mox1, which is
homologous to gp91phox and highly expressed in
colon and to a lesser extent in prostate, uterus, and vascular smooth
muscle. Interestingly, alternative splicing of the oxidase NOH-1,
equivalent to Mox1, generates a short isoform, which functions as a
voltage-gated proton channel (27). Considerable attention has been paid
to an NAD(P)H oxidase in kidney; this organ highly expresses
p22phox, the small subunit of flavocytochrome
b558 (28), is susceptible to oxidative damage,
suggestive of significance of ROS under pathological conditions (29),
and plays a central role in erythropoiesis by synthesizing
erythropoietin (EPO), which is regulated by an oxygen sensor (24, 25).
Molecular nature of the kidney oxidase, however, has remained elusive.
Here we describe the primary structure and function of a novel
superoxide-producing NAD(P)H oxidase whose expression is almost exclusively restricted to adult and fetal human kidneys. We designate the novel oxidase NOX4 to avoid future confusion about the term, because Drs. Krause, Lambeth, and Lovering have recently established a
consensus terminology for the NOX family of NAD(P)H oxidase: NOX1 for
Mox1/NOH-1, NOX2 for gp91phox, and NOX3 for the
one, thus far only known from its genomic sequence (accession no.
AL031773).2
Cloning of Human cDNA Encoding NOX4--
-A search of EST
data bases with the C-terminal region of human
gp91phox yielded a cDNA clone (GenBankTM
accession no. AI742260). Using the EST clone as a template, we obtained
the PCR product of 500 nucleotides with two unique oligonucleotide
primers: 5'-GCTGGATCCCGAATGGTCAAAGAAAATTT-3' (forward
primer) and 5'-GCTGAATTCATAGTCAGGTCTGTTCTCTTG-3' (reverse primer). Here sequences from the EST clone are underlined. The PCR
product was used as a probe for screening a human kidney cDNA library (Stratagene). Twelve independent positive clones were obtained
from about 6 × 105 plaques, and sequenced in both
directions. Among them, six clones encoded the full-length of NOX4.
Chromosomal Localization--
To determine the chromosomal
localization of the human NOX4 gene, fluorescence in
situ hybridization was performed as described previously (30). In
brief, a NOX4 cDNA fragment (corresponding to amino acids 1-578)
was labeled with biotin-14-dATP by nick-translation and hybridized to
R-banded chromosomes prepared from phytohemaggulutinin-stimulated cultured lymphocytes of normal donors. After overnight hybridization at
37 °C, the slides were washed in 50% formamide, 2× SSC at 37 °C
for 10 min, followed by a wash in 1× SSC at room temperature for 15 min. Hybridization signals were amplified using rabbit anti-biotin IgG
(Enzo) and fluorescein labeled goat anti-rabbit IgG (Enzo). The
chromosomes were counterstained with propidium iodide.
Transfection--
Sense and antisense cDNA encoding human
NOX4 (amino acids 1-578) were subcloned into pREP10 (Invitrogen) or
pEF-BOS (31), a generous gift form Dr. Shigekazu Nagata (Osaka
University, Osaka, Japan), for expression of the N-terminal FLAG-tagged
proteins. COS-7, HEK293, HeLa, or NIH3T3 cells were transfected with
pREP10-NOX4, pREP10-antisense-NOX4, or empty pREP10 vector and/or
pREP10-p22phox, or with pEF-BOS-FLAG-tagged-NOX4
or pEF-BOS-antisense-NOX4, using FuGENE (Roche Molecular Biochemicals).
Western Blot Analysis--
The DNA fragments encoding the
C-terminal domains of NOX proteins, NOX1 (amino acids 378-564), NOX2
(), and NOX4 (), were prepared by PCR, and the products
were ligated to pGEX-2T or -4T (both from Amersham Pharmacia Biotech).
Glutathione S-transferase (GST) fusion proteins were
expressed in Escherichia coli strain BL21 and purified by
glutathione-Sepharose-4B (Amersham Pharmacia Biotech), as described
previously (9, 11). An anti-human NOX4 rabbit polyclonal antibody was
raised against the C-terminal 20 amino acid peptide of human NOX4.
Purified proteins or membrane fractions of cells transfected with human
NOX4 cDNA were subjected to SDS-PAGE, transferred to a
polyvinylidene difluoride membrane (Millipore), and probed with the
anti-human NOX4 antibody or anti-FLAG (M2) antibody (Sigma-Aldrich).
The blots were developed using ECL-plus (Amersham Pharmacia Biotech) to
visualize the antibodies.
RT-PCR for NOX1, NOX2, and NOX4--
Total RNAs of HEK293, HeLa,
KPK13 cells (32) were perpared from with Trizol reagent (Life
Technologies, Inc.), and RT-PCR for NOX1, NOX2, and NOX4 was performed
with GeneAmpTM (PerkinElmer Life Sciences) using specific primers:
5'-GATGATCGTGACTCCCAC-3' (forward primer) and
5'-CAACAATATTGCTGTCCC-3' (reverse primer) for NOX1;
5'-GGGAAAAATAAAGGAATGCC-3' (forward primer) and
5'-AGCCAGTGAGGTAGATGTTG-3' (reverse primer) for NOX2;
5'-GTCATAAGTCATCCCTCAGA-3' (forward primer) and
5'-TCAGCTGAAAGACTCTTTAT-3' (reverse primer) for NOX4.
Superoxide Production--
HEK293 cells were lysed by
sonication, and the sonicate was centrifuged for 10 min at 10,000 × g. The resultant supernatant was further centrifuged for
1 h at 100,000 × g. The pellet was used as the
membrane fraction. Superoxide production by the membrane was determined
as superoxide dismutase (SOD)-inhibitable chemiluminescence detected
with an enhancer-containing luminol-based detection system (DiogenesTM; National Diagnostics) as described previously (11, 14).
The membrane (5 µg of protein) was resuspended in 200 µl of 100 mM potassium phosphate, pH 7.0, with 10 µM
FAD, 1 mM NaN3, and 1 mM EGTA.
After preincubation of the membrane solution with the enhanced
luminol-based substrate (200 µl), NADH or NADPH was added at the
final concentration of 0.5 mM. The chemiluminescence was
assayed using a luminometer (Auto Lumat LB953; EG&G Berthold). The
reaction was stopped by the addition of SOD (50 µg/ml).
Cell Growth--
COS-7, HEK293, or NIH3T3 cells were seeded with
5.0 × 103 cells/well in 96-well plates, and
transfected with pREP10-NOX4 or pREP10-antisense-NOX4 by using FuGENE
(Roche Molecular Biochemicals). Transfected cells were maintained in
DMEM containing 10% FCS for 56 h, and the cell number was
determined photometrically using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (Sigma).
Northern Blot Analysis--
Human Multiple Tissue NorthernTM
blots (CLONTECH) were hybridized with a
32P-labeled NOX4 cDNA fragment, encoding the region
that corresponds to amino acids 324-491, under high stringency
conditions using ExpressHybTM (CLONTECH).
Immunohistological Staining--
Localization of the NOX4
protein in human kidney was investigated by immunohistochemical study
using renal tissue samples obtained by renal biopsy from patients, who
were admitted to our hospital because of chance proteinuria or
hematuria. All samples revealed neither glomerular and tubular
abnormalities nor significant deposition in immunofluorescent study.
Biopsy specimens were fixed in Bouin's solution and embedded in
paraffin. The procedures of the use of the samples in the present study
was in accordance with the guidelines of the local Human Subject
Committee at Kyushu University (Fukuoka, Japan).
Immunohistochemical staining was performed as described previously
(33). Briefly, paraffin sections were cut at 3 µm, deparaffinized, and treated with 10 mM citrate, pH 6.0, in 800-watt
microwave oven for 15 min for antigen retrieval before immunostaining.
After inactivation of endogenous peroxidase with 3%
H2O2 in methanol for 30 min at room
temperature, the sections were preincubated for 1 h with 5% dry
milk and 5% bovine serum albumin in phosphate-buffered saline (PBS;
137 mM NaCl, 2.7 mM KCl, 4.3 mM
Na2HPO4, and 1.4 mM
KH2PO4, pH 7.4). The samples were subsequently
incubated with the anti-human NOX4 rabbit polyclonal antibody (1:2000
dilution) for overnight at 4 °C, washed in PBS with 0.1% Triton
X-100, and probed with biotinylated anti-rabbit IgG antibody (Nichirei
Corp., Tokyo, Japan) for 30 min at room temperature. After washing in PBS with 0.1% Triton X-100, the sections were reacted with
HRP-conjugated streptavidin (Nichirei Corp.) for 30 min at room
temperature, and the sites of HRP were visualized with diaminobenzidine
(Nichirei Corp.) and H2O2, and counterstained
with hematoxylin for 2 min.
Human cDNA Encoding a Novel Homologue of
gp91phox--
A search of EST data bases with the
C-terminal region of human NOX2/gp91phox yielded
a cDNA clone (GenBankTM accession no. AI742260). Using a DNA
fragment of the EST clone as a probe, we screened a human kidney
cDNA library and cloned a cDNA of ~2.4 kb. The cDNA clone contains an open reading frame of 1,734 nucleotides with the first methionine codon surrounded by a consensus Kozak sequence, two in-frame
preceding termination codons, and a consensus polyadenylation signal
(AATAAA) present in the 3'-region (GenBankTM accession no. AB041035).
The size of the cloned cDNA is identical to that of the major
transcript of about 2.4 kb (see below), supporting that the obtained
clone indeed contains the full-length cDNA.
The predicted protein consists of 578 amino acids (Fig.
1), with 39% and 35% identity to
NOX2/gp91phox and NOX1/Mox1, respectively.
Regions, that are considered to be binding sites for FAD and pyridine
nucleotide (5-8), are nearly identical among the NOX proteins (Fig.
1). Six putative membrane-spanning segments are also well conserved
with four histidine residues that are proposed to be ligated to heme in
NOX2/gp91phox (8); His105 and
His119 of the third segment, and His194 and
His207 of the fifth one in NOX4. Since these features in
the primary structure indicate the function as a superoxide-producing
NAD(P)H oxidase, we designated the protein NOX4, a novel member of the NAD(P)H oxidase (NOX) family.
Genomic Organization and Chromosomal Localization of the Human NOX4
Gene--
The human NOX2 gene consists of 13 exons, all
with lengths similar to the 13 exons of the NOX1 gene (27).
To know genomic structure of the NOX4 gene, we screened a
pooled human genomic library by PCR and obtained the BAC clone
RP11-745I13 (GenBank accession no. AP002404), that contains exons
2-16 of NOX4. In addition, a data base search revealed that the two
BAC clones RP11-345O16 and RP11-97D10 (GenBank accession nos.
AC012198 and AP001815, respectively) also contain exons 1-7 and 3-18 of NOX4, respectively. The NOX4 gene comprises 18 exons,
covering a minimum of 160 kb, and its organization is similar to those of NOX2 (Fig.
2A).
To determine the chromosomal localization of the human NOX4
gene, we performed fluorescence in situ hybridization
analysis. As shown in Fig. 2B, specific hybridization
signals were observed on chromosome 11q14.2-q21, whereas no other
hybridization sites were detected. Thus, the human NOX4 gene
is located on chromosome 11q14.2-q21, while the human NOX1
and NOX2 genes are located on chromosome Xq22 and Xp21.1,
respectively (27).
Expression of the NOX4 Protein in COS-7 and HeLa Cells Transfected
with Its cDNA--
We prepared an antibody raised against the
C-terminal peptide of human NOX4 as a tool for characterization of the
NOX4 protein. As expected, the antibody interacted with the C-terminal
cytoplasmic domain of NOX4 expressed as a GST fusion protein, but not
with that of NOX1/Mox1 or NOX2/gp91phox (Fig.
3A). We next expressed NOX4 as
a FLAG-tagged protein in COS-7 cells, and detected the protein by
Western blot analysis. As shown in Fig. 3B, an anti-FLAG
antibody bound to a protein with the apparent molecular mass of 66 kDa,
the value which is essentially the same as the calculated molecular
mass of NOX4. Additional two minor bands with much higher molecular
masses were also observed on the blot (Fig. 3B). The bands
likely indicate dimer and/or oligomer(s) of NOX4, since they increased
with concomitant decrease of the 66-kDa band when SDS-PAGE was
performed under oxidized conditions (data not shown). The antibody to
NOX4 only interacted with the same protein as that recognized by the
anti-FLAG antibody, among proteins in COS-7 cells (Fig. 3B).
These observations indicate that the antibody raised to the C terminus
of human NOX4 specifically recognizes NOX4.
We also tested effect of expression of p22phox
on the protein level of NOX4. The protein
p22phox is the heterodimeric partner of
NOX2/gp91phox in the cytochrome
b558 complex of the phagocyte NADPH oxidase, and
is required for stabilization of NOX2: NOX2 is absent in phagocytes lacking p22phox due to defect in the
p22phox gene (1-4). Cotransfection of COS-7
cells with both NOX4 and p22phox cDNAs did
not alter the protein level of NOX4, compared with transfection with
the NOX4 cDNA alone (data not shown). The finding raises a
possibility that NOX4 may not be complexed with
p22phox, but does not exclude another one that
NOX4 alone might be localized in an intracellular membrane compartment,
but in the presence of p22phox it may be
incorporated into the plasma membrane in a heterodimeric form, a
process which has recently been shown to occur in formation of the
NOX2/gp91phox-p22phox
heterodimer (34). It is also possible that COS-7 cells may express an
heretofore unidentified p22phox homologue that
could complex with NOX4.
Using the membrane fraction of HeLa cells transfected with the NOX4
cDNA, we detected a protein band of about 75 kDa, in addition to
the 66-kDa band, on Western blot (Fig. 3C). The larger
protein may be a glycosylated form of NOX4, since there exist four
putative N-glycosylated sites in the second and third
extracellular loops of NOX4: Asn129-Phe-Ser,
Asn133-Tyr-Ser, Asn230-Arg-Thr, and
Asn236-Ile-Ser (Fig. 1). The extent of glycosylation may be
dependent on cell types; such a 75-kDa protein was not detected in
COS-7 cells transfected with the NOX4 cDNA (Fig. 3B). To
obtain experimental evidence that NOX4 is glycosylated, we attempted to
digest the 75-kDa protein by N-glycosidase F, which was
unsuccessful. Since the NOX4 protein appears to have a tendency to
self-aggregate (Fig. 3B), targets for the glycosidase were
possibly inaccessible. The final conclusion that NOX4 is glycosylated
thus waits for further investigation.
Superoxide-producing NAD(P)H Oxidase Activity of NOX4--
We
searched for cultured cells expressing NOX4, and found that the human
renal cell carcinoma KPK13 cells (32) and HEK293 cells express both
mRNA and protein of NOX4 by RT-PCR using specific primers (Fig.
4A) and by Western blot using
the anti-NOX4 antibody (Fig. 4B), respectively. Neither
NOX1/Mox1 mRNA nor NOX2/gp91phox mRNA
was detected in HEK293 cells, while the former mRNA was expressed
in KPK13 cells (Fig. 4A). In contrast with these cells, human neutrophils exclusively expressed NOX2 (Fig. 4A).
Thus, HEK293 cells appear to be suitable for study on endogenous NOX4, since they express NOX4 but not other NOXs.
To investigate function of the NOX4 protein, we prepared the membrane
fraction of HEK293 cells and assayed its superoxide-producing activity.
We initially used the cytochrome c reduction assay (9, 11),
which is the most reliable one for quantitation of amounts of
superoxide produced, but is not highly sensitive to detect the anion.
We could detect only small amounts of superoxide produced by the
membrane fraction in the presence of NADH or NADPH: the rates were
about or less than 0.5 nmol/min/106 cell equivalents of
membrane, respectively, which are several 10-fold lower than the
phagocyte NADPH oxidase activity in human neutrophil membranes (9, 11,
15).
We next assayed superoxide production by the chemiluminescence method,
which is much more sensitive to superoxide than the cytochrome
c assay, but cannot be used for absolute quantitation. As
shown in Fig. 5A, the addition
of NADH to the HEK293 membrane caused enhancement of chemiluminescence.
The response was completely inhibited by SOD, indicating the
involvement of superoxide. Analysis by this method further revealed
that superoxide was also produced by NADPH; the maximal rate with NADH
was approximately 2-3-fold higher than that with NADPH, whereas an
apparent Km value for NADH was rather higher than
that for NADPH (13 µM for NADH and 9 µM for
NADPH) (Fig. 5B). The results suggest that a
superoxide-producing oxidase in HEK293 cells can use either NADH or
NADPH as an electron donor. We also used the chemiluminescence method
to assay superoxide production by human neutrophil membranes under
cell-free activation conditions (9, 11, 15), and found that the
superoxide producing activity is approximately 40- and 100-fold higher
than those of HEK293 cell membranes with NADH and NADPH, respectively.
This observation is consistent with that obtained by the cytochrome c reduction assay as described above.
Both NADH- and NADPH-dependent superoxide-producing
activities of HEK293 cell membranes were blocked by the flavoprotein
inhibitor diphenylene iodonium (Fig. 5D). The oxidase
activities were not altered by the presence of the cytosolic fraction
of HEK293 cells, by the addition of arachidonic acid, an in
vitro activator of the phagocyte NADPH oxidase (15, 35), or by
added recombinant p47phox,
p67phox, and GTP-loaded Rac2, which are
essential for the phagocyte oxidase activation (data not shown).
Transfection of HEK293 cells with a plasmid expressing an antisense
mRNA for NOX4 decreased amounts of the NOX4 protein, as determined
by Western blot analysis (Fig. 5C), and caused a significant decrease in both NADH- and NADPH-supported superoxide producing activities of the membrane (Fig. 5D), confirming the
identity of NOX4 as a superoxide-producing NAD(P)H oxidase. This
observation also indicates that the superoxide-producing NAD(P)H
oxidase activity in the HEK293 membrane is at least partly due to NOX4.
Role of NOX4 in Cell Growth--
Since it has been reported that
overexpression of NOX1/Mox1 increases the growth rate of NIH3T3 cells
(26), we estimated a role of NOX4 in cell proliferation. The cells did
not express endogenous NOX4 as estimated by RT-PCR (data not shown) and
Western blot (Fig. 6A).
Transfection of NIH3T3 cells with the NOX4 cDNA led to expression
of the NOX4 protein (Fig. 6A) and a slight but significant
increase in NADH-dependent superoxide-producing activity in
the membrane (Fig. 6B). Unexpectedly, overexpression of NOX4 resulted in a retarded growth of NIH3T3 cells (Fig. 6B),
with no increase in apoptotic cells (data not shown). We also
overexpressed the NOX4 protein in COS-7 and HEK293 cells, in both cases
of which the cell proliferation rate was decreased to a similar extent (data not shown). On the other hand, the growth rate was not altered when NIH3T3 cells were transfected with the antisense NOX4 cDNA (Fig. 6B). The oxidases NOX4 and NOX1 thus appear to have
the opposite effect on regulation of cell growth.
Expression of Human NOX4--
We finally studied expression of
NOX4 in various human tissues. Northern blot analysis revealed that
human NOX4 mRNA of about 2.4 kb is expressed almost exclusively in
kidney among adult tissues tested, with faint signals in heart,
skeletal muscle, and brain (Fig. 7).
Human fetal kidney also abundantly expressed the message of NOX4 (Fig.
7). Thus, the tissue distribution of the NOX4 mRNA was totally
different from those of NOX1 and NOX2; the former is most abundantly
expressed in colon (26, 27), and the latter in phagocytic leukocytes
(1-4).
To know which types of cells express the NOX4 protein in human kidney,
we performed immunohistochemical analysis of samples of the renal
cortex using antibodies against NOX4. Strong signals were observed in
epithelial cells of distal tubules, whereas only faint signals can be
detected in proximal tubules and glomeruli (Fig.
8). Thus, the NOX4 protein is most
abundantly expressed in distal tubular cells in human kidney
cortex.
In the present study, we describe a novel
gp91phox homologue, designated NOX4, that is
highly expressed in adult and fetal kidneys. As expected from its
primary structure, NOX4 exhibits NADH- and NADPH-dependent
superoxide producing activities. NOX4-expressing HEK293 cells show such
oxidase activities, which are decreased when transfected with antisense
NOX4 cDNA; and overexpression of NOX4 in NIH3T3 cells leads to
increased superoxide production.
Abundant expression of the NOX4 protein in distal tubular cells may
suggest its function as an oxygen sensor for EPO synthesis. A current
model for the oxygen sensing proposes that, dependent on oxygen
tension, an NAD(P)H oxidase generates superoxide and its derivative
ROS, which oxidatively destabilize HIF-1 During the completion of this study, Geiszt et al. (38)
reported the cloning of essentially the same cDNA as NOX4,
designated Renox in their report, and presented that transfection of
NIH3T3 cells with the cDNA leads to an increased production of
superoxide at the cell level and a decreased rate of proliferation.
They have also shown, by in situ hybridization, that Renox
is expressed in proximal tubular cells of mouse kidney, whereas the
present study demonstrates, using an antibody specific to NOX4, that
the protein is predominantly present in distal tubular cells, not in
proximal tubular cells, of human kidney (Fig. 8). The discrepancy may
be due to difference in species or methods used in the two studies. In
addition, Geiszt et al. (38) showed that mRNA of mouse
Renox is exclusively detected in kidney as human NOX4 is (Fig. 8),
whereas mouse NOX4 mRNA is expressed not only in kidney but also in
liver, but to a lesser extent, as estimated by Northern blot
analysis.3 The reason for
this discrepancy is presently unknown.
Also in the fetus, the NOX4 mRNA is abundantly expressed in kidney
(Fig. 7), an organ that is not considered to highly produce EPO at this
stage. In addition to a suggested function as an oxygen sensor, NOX4
possibly plays a role in other cellular events. One possibility is that
NOX4 may participate in regulation of renal cell growth and/or death.
ROS are known to be important mediators of many pathophysiological
processes in renal diseases, probably by causing cell death via
necrosis or apoptosis (29). The present study shows that overexpression
of NOX4 results in decreased cell proliferation, although we did not
detect an increase in apoptotic cells. Future studies should be
directed to clarify the role of NOX4 in kidney under oxidative damage
such as hypoxia/reoxygenation and ischemia/reperfusion injury. It is
also possible that NOX4 may function as a proton channel; an
alternatively spliced form of NOX1/Mox1 and
NOX2/gp91phox in the active phagocyte oxidase
complex are likely involved in a proton channel (27). This possibility
is currently under investigation in our laboratory.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Deduced amino acid sequence of human NOX4 in
comparison with human NOX2/gp91phox and
NOX1/Mox1. Residues identical to NOX4 are boxed.
Overlined residues are presumed membrane-spanning regions,
and binding sites for heme, FAD, and NAD(P)H. Asterisks
indicate conserved histidine residues that are candidates for heme
ligation.
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Fig. 2.
Genomic organization and chromosomal
localization of the human NOX4 gene.
A, genomic organization of the human NOX4
gene in comparison with the human
NOX2/gp91phox gene. Exons and introns
are shown as horizontal and vertical
bars, respectively. NOX4 comprises 18 exons,
whereas NOX2 does 13 exons. Boxed numbers represent those of
coding nucleotides in each exon. B, chromosomal localization
of the human NOX4 gene. Partial metaphase plate showing
doublet signals on human chromosome 11q14.2-q21 (arrow). The
biotinylated cDNA probe for human NOX4 gene was
hybridized to chromosomes with replication bands prepared from cultured
lymphocytes. After hybridization and washing, hybridization signals
were amplified using rabbit anti-biotin and fluorescein-labeled goat
anti-rabbit IgG. The chromosomes were counterstained with propidium
iodide.
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Fig. 3.
Specific recognition of human NOX4 by an
antibody raised against its C-terminal peptide. A,
Western blot analysis with an anti-NOX4 antibody. The C-terminal
cytoplasmic domains of NOX1, NOX2, and NOX4 were expressed as GST
fusion proteins. Purified proteins (0.2 µg) were subjected to 10%
SDS-PAGE, followed by staining with Coomassie Brilliant Blue
(left panel) or by immunoblot with an antibody raised
against the C-terminal peptide of NOX4 (right panel).
B, Western blot analysis of the membrane fraction of COS-7
cells transfected with the plasmid pEF-BOS encoding FLAG-tagged NOX4 or
vector alone. The membrane proteins (0.7 µg) were subjected to 10%
SDS-PAGE, followed by immunoblot with the anti-NOX4 or anti-FLAG
antibody or by silver or Coomassie Brilliant Blue staining. Positions
for marker proteins are indicated in kilodaltons. C, Western
blot analysis of the membrane fraction of HeLa cells transfected with
the plasmid pEF-BOS encoding FLAG-tagged NOX4 or vector alone. The
membrane proteins (7.0 µg) were subjected to 10% SDS-PAGE, followed
by immunoblot with the anti-FLAG antibody. Positions for marker
proteins are indicated in kilodaltons. For details, see "Experimental
Procedures." The experiments have been repeated more than three times
with similar results.
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Fig. 4.
Expression of endogenous NOX4 in human
cells. A, RT-PCR using RNA from KPK13 cells, HEK293
cells, or human neutrophils, with specific primers for NOX1, NOX2, or
NOX4. RT-PCR was performed using RNA (1.0 µg) from KPK13 cells,
HEK293 cells, or human neutrophils, with specific primers for NOX1,
NOX2, or NOX4. The PCR products were subjected to 1% agarose-gel
electrophoresis, and stained with ethidium bromide. B,
Western blot analysis of the membrane fraction of KPK13 cells, HEK293
cells, or human neutrophils, with the anti-NOX4 antibody. The membrane
fraction (5.0 µg) of KPK13 cells, HEK293 cells, or human neutrophils
was subjected to 10% SDS-PAGE, followed by immunoblot with the
anti-NOX4 antibody. Positions for marker proteins are indicated in
kilodaltons. For details, see "Experimental Procedures." The
experiments have been repeated more than three times with similar
results.
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Fig. 5.
Superoxide production by the membrane
fraction of HEK293 cells. A, NADH-dependent
chemiluminescence by the membrane fraction of HEK293 cells. After
preincubation of the membrane fraction of HEK293 cells (12.5 µg/ml)
in the presence (trace b) or absence (trace a) of
SOD (50 µg/ml) for 5 min, 500 µM NADH was added to the
reaction mixture and chemiluminescence change was continuously
monitored with an enhanced luminol-based substrate, DiogenesTM. Where
indicated, SOD was added at the final concentration of 50 µg/ml
(trace a). B, Lineweaver-Burk plot for superoxide
production. The membrane fraction was incubated with various
concentrations of NADH (closed squares) or NADPH
(closed circles), and the rate of superoxide production was determined by SOD-inhibitable
chemiluminescence. C, effect of expression of antisense NOX4
mRNA on its protein level. Membrane fractions were prepared from
HEK293 cells transfected with pREP10-antisense-NOX4 (AS) or
empty pREP10 vector (vector). The membrane proteins (10 µg) were subjected to 10% SDS-PAGE, followed by immunoblot with the
anti-NOX4 antibody. D, superoxide-producing activity of the
membrane fraction of HEK293 cells in the presence of 0.5 mM
NADH (left panel) or NADPH (right panel). NADH-
and NADPH-dependent superoxide-producing activities of the
membrane fraction from HEK293 cells transfected with
pREP10-antisense-NOX4 (AS) or empty pREP10 vector
(vector) were determined as SOD-inhibitable
chemiluminescence. For details, see "Experimental Procedures." When
indicated, the membrane was preincubated with 10 or 20 µM
diphenylene iodonium. Superoxide production is expressed as the
percentage of activity relative to untransfected cells. Each
graph represents the mean of data from six independent
transfections, with bars representing the S.D. for
percentage of activity.
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Fig. 6.
Effect of NOX4 expression on cell
growth. A, expression of human NOX4 in NIH3T3 cells.
NIH3T3 cells were transfected with pREP10-NOX4, pREP10-antisense NOX4,
or empty pREP10 vector. The membrane fraction (10 µg) from
untransfected NIH3T3 or HEK293 cells (left panel) or that
from the transfected NIH3T3 cells (right panel) was
subjected to 10% SDS-PAGE, followed by immunoblot with the anti-NOX4
antibody. B, effect for expression of NOX4 in NIH3T3 cells
on NADH-dependent superoxide producing activity (left
panel) and cell growth (right panel). For details, see
"Experimental Procedures." Both superoxide-producing activity and
cell growth rate of untransfected cells are set as 100%. Each
graph represents the mean of data from four independent
transfections, with bars representing the S.D. for
percentage of activity.
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Fig. 7.
Northern blot analysis for expression of NOX4
mRNA in human tissues. Human Multiple Tissue NorthernTM blots
(CLONTECH) were hybridized with
32P-labeled human NOX4 cDNA fragments (corresponding to
amino acids 324-491) under high stringency conditions using
ExpressHybTM (CLONTECH). The results are
representative of two independent blotting experiments. Positions of
RNA molecular size markers are shown in kilobases.
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Fig. 8.
Expression of the NOX4 protein in human renal
cortex. Immunohistochemical localization of anti-NOX4 antibody in
human renal cortex. The sections were incubated with antisera to NOX4
(B and C) or preimmune sera (A),
probed with biotinylated anti-rabbit IgG antibody, and reacted with
HRP-conjugated streptavidin. The sites of HRP were visualized with
diaminobenzidine and H2O2, and counterstained
with hematoxylin for 2 min. For details, see "Experimental
Procedures." The results are representative of three independent
experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, a dominant transcriptional
activator of the gene for EPO, thereby decreasing expression of EPO
(24, 36). EPO is generally considered to be produced by the peritubular
interstitial cells in the renal cortex (24), that are adjacent to
NOX4-expressing tubular cells and thus easily exposed to ROS produced
by NOX4. Diphenylene iodonium, a flavoprotein inhibitor that blocks the
oxygen sensing for EPO synthesis (24, 25), blocks superoxide production
by NOX4, which is consistent with the idea of NOX4 as an oxygen sensor. In this context, it should be noted that the gene responsible for
familial benign polycythemia (FBP), a rare autosomal recessive condition characterized by erythrocytosis usually with increased EPO
production, is assigned by linkage analysis of affected families to the
region of chromosome 11q23 (37), close to the locus of NOX4 (chromosome
11q14.2-21). It may be possible that the gene for FBP corresponds to
the NOX4 gene. If this is the case, increased levels of EPO
in individuals with FBP can be explicable by defective function of
NOX4, a plausible oxygen sensor that negatively regulates production of
the cytokine in kidney. This possibility is currently under
investigation. On the other hand, the major organ of EPO synthesis in
the fetus, i.e. liver, expresses a negligible amount of the
NOX4 mRNA (Fig. 7). Another oxidase or a different oxygen-sensing system in the regulation of EPO expression might occur in fetal liver.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Yohko Kage (Kyushu University, Fukuoka, Japan) for technical assistance, Dr. Takashi Ito (Kanazawa University, Kanazawa, Japan) for helpful discussion and encouragement, and Dr. Shigekazu Nagata (Osaka University, Osaka, Japan) for providing the plasmid pEF-BOS.
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FOOTNOTES |
---|
* This work was supported in part by grants-in-aid for scientific research from the Ministry of Education, Science, Sports, and Culture of Japan, and grants from Kato Memorial Bioscience Foundation and from CREST (Core Research for Evolutional Science and Technology) of Japan Science and Technology Corporation, and was conducted in part at Kyushu University Station for Collaborative Research.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.
The nucleotide sequence reported in this paper has been submitted to the DDBJ/GenBankTM/EBI Data Bank with accession number AB041035.
§§ To whom correspondence should be addressed. Tel.: 81-92-642-6213; Fax: 81-92-642-6215; E-mail: hsumi@mailserver.med.kyushu-u.ac.jp.
Published, JBC Papers in Press, October 13, 2000, DOI 10.1074/jbc.M007597200
2 K.-H. Krause, personal communication.
3 J. Kuroda, A. Shiose, and H. Sumimoto, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: ROS, reactive oxygen species; NOX, NAD(P)H oxidase; EPO, erythropoietin; PCR, polymerase chain reaction; GST, glutathione S-transferase; SOD, superoxide dismutase; FBP, familial benign polycythemia; EST, expressed sequence tag; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; HRP, horseradish peroxidase; RT, reverse transcription; kb, kilobase pair(s); HEK, human embryonic kidney.
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