From the Biology of Ageing Laboratory, Department of
Geriatrics, Geneva University Hospitals, 1225 Geneva, Switzerland, the
¶ Department of Medicine, University of Texas Health Science
Center and South Texas Veterans Health Care System, San Antonio,
Texas 78229-3900, and the
Institute of Veterinary Anatomy,
D-35392 Giessen, Germany
Received for publication, November 4, 2002, and in revised form, December 2, 2002
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ABSTRACT |
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NOX1, an NADPH oxidase expressed predominantly in
colon epithelium, shows a high degree of similarity to the phagocyte
NADPH oxidase. However, superoxide generation by NOX1 has been
difficult to demonstrate. Here we show that NOX1 generates superoxide
when co-expressed with the p47phox and
p67phox subunits of the phagocyte NADPH oxidase
but not when expressed by itself. Since p47phox
and p67phox are restricted mainly to myeloid
cells, we searched for their homologues and identified two novel
cDNAs. The mRNAs of both homologues were found predominantly in
colon epithelium. Differences between the homologues and the phagocyte
NADPH oxidase subunits included the lack of the autoinhibitory domain
and the protein kinase C phosphorylation sites in the
p47phox homologue as well as the absence of the
first Src homology 3 domain and the presence of a hydrophobic
stretch in the p67phox homologue. Co-expression
of NOX1 with the two novel proteins led to stimulus-independent high
level superoxide generation. Stimulus dependence of NOX1 was restored
when p47phox was used to replace its homologue.
In conclusion, NOX1 is a superoxide-generating enzyme that is
activated by two novel proteins, which we propose to name NOXO1
(NOX organizer 1) and NOXA1 (NOX activator 1).
Superoxide generation by phagocytes plays a crucial role in the
elimination of invading microorganisms. It is catalyzed by the
phagocyte NADPH oxidase, an enzyme consisting of two transmembrane subunits, p22phox and
gp91phox, and at least three cytosolic subunits,
p47phox, p67phox, and
Rac2 (1). Upon activation, the NADPH oxidase subunits assemble, and
electrons are transported from intracellular NADPH to extracellular
oxygen by the flavo-heme gp91phox subunit
(2).
Recently six gp91phox homologues have been
described in mammals: NOX1 1
(3, 4), NOX3 (5, 6), and NOX4 (7, 8) with an overall structure similar
to gp91phox (alias NOX2), NOX5 with an
N-terminal EF hand-containing extension (9), and DUOX1 and DUOX2 with
an additional peroxidase homology domain (10-12). NOX1 is found mainly
in colon epithelium (3, 4); NOX3 in embryonic kidney (5, 6), NOX4 in
the kidney cortex (7, 8), NOX5 in lymphoid organs and testis (9), DUOX1
in thyroid and lung, and DUOX2 in thyroid and colon (10-12).
Based on their primary structure all members of the NOX/DUOX family
should be flavo-heme electron transporters. However, it is not
established whether all NOX enzymes transfer electrons to oxygen or
whether some of them may use other electron acceptors as has been shown
for a yeast homologue of gp91phox that functions
as a ferric reductase (13). Among NOX enzymes, only
gp91phox and NOX5 have appeared capable of
generating large amounts of superoxide, both of them in a
stimulus-dependent manner (1, 9).
Based on data gained with NOX1-transfected NIH 3T3 cell
clones NOX1 has been suggested to be a subunit-independent, low
capacity superoxide-generating enzyme involved in the regulation of
mitogenesis (4, 14). However, we have not been able to measure any
superoxide generation in NOX1-transfected cells, and the question arose
whether NOX1 is a subunit-dependent enzyme or possibly not
a superoxide-generating enzyme at all.
Cloning of Mouse p47phox and
p67phox Homologues--
Translated BLAST nucleotide
searches were conducted in the mouse genome data base
(www.ensembl.org/Mus_musculus/blastview) with the mouse
p47phox and p67phox
sequences. The exons of the genes identified were predicted with the
GENSCAN software (genes.mit.edu/GENSCAN.html), and primers were
designed to amplify the coding regions of both predicted transcripts
(5'-catggcaagcccaagacacccagta-3' and 5'-ctcaacggggaacccgagtccctt-3' for
the p47phox homologue and
5'-ccatgagctctctaggggatcag-3' and 5'-tgctagttctggtctcctggct-3' for the
p67phox homologue). Total RNA from mouse colon
was purified with the TRIzol® reagent (Invitrogen), and
cDNA was synthesized with Superscript® reverse
transcriptase (Invitrogen) using random primers followed by PCR with
Taq DNA polymerase using "Q buffer" (Qiagen).
Analysis of mRNA--
Northern blot analysis and in
situ hybridization were performed as described previously (9)
using the entire coding region of NOXO1 and NOXA1 for probe generation
in Northern blot experiments and using the region containing base pairs
1-360 of NOXO1 and the region containing base pairs 171-490 of
NOXA1 cDNA for probe generation in the in situ
hybridization experiments.
Cell Culture and Transfection--
Mouse NOX1 cDNA, mouse
NOXO1 cDNA, and mouse NOXA1 cDNA with inserted Kozak sequences
were subcloned into pcDNA3.1 (Invitrogen). HEK293, Chinese hamster
ovary, COS-7, and HeLa cells were cultured and transfected as
described previously (9).
Superoxide Measurements--
Luminol-enhanced chemiluminescence
and SOD-inhibitable cytochrome c reduction were
measured as described previously (9). For the microscopic NBT test,
cells were seeded in 96-well plates and incubated at 37 °C for 15 min in Hanks' balanced salt solution containing 0.5 mg/ml NBT.
Activation of NOX1 by Cytoplasmic Subunits of the Phagocyte NADPH
Oxidase--
As NOX1 expression by itself did not lead to superoxide
generation in transfected cells (Fig. 1),
we considered that NOX1, like gp91phox, might be
a subunit-dependent enzyme. We therefore co-transfected HEK293 cells with NOX1 and the cytoplasmic subunits of the phagocyte NADPH oxidase, p47phox and
p67phox. Cells transfected with NOX1 alone (Fig.
1) or with p47phox and
p67phox alone (not shown) did not generate
superoxide either with or without stimulation by the phorbol ester PMA.
In contrast, HEK293 cells co-transfected with NOX1,
p47phox, and p67phox
generated superoxide but only after addition of PMA. These results suggested that NOX1 is a subunit-dependent enzyme. However,
p47phox and p67phox are
expressed mainly in myeloid cells. We therefore searched for homologues
that may activate NOX1 in the colon.
Molecular Cloning of Putative NOX1 Cytoplasmic Subunits--
A
search for p47phox homologues yielded a
full-length human sequence deposited as colon cancer antigen NY-CO-31
(GenBankTM accession number AAC18046). Subsequently, we
found the open reading frame of the mouse
p47phox homologue in chromosome 17 and
cloned the cDNA of the coding region by reverse
transcription PCR from mouse colon RNA (GenBankTM
accession number AF539797). The homologue had an expected molecular
mass of 39 kDa and displayed 25% sequence identity with p47phox (Fig.
2A). A search for motifs
located a phox homology domain, which targets proteins to
the phosphoinositide groups of membranes (15), two Src homology
SH3 domains, and a C-terminal proline-rich region that is crucial in
p47phox for interaction with
p67phox (16) (Fig. 2A).
Interestingly, the homologue was missing the proline-proline-arginine-arginine-containing region of
p47phox, which is involved in autoinhibition
(17) through binding to the N-terminal SH3 domain, as well as the
adjacent serine phosphorylation sites (18), which relieve
autoinhibition when phosphorylated (19). This suggested a
stimulus-independent activity of the homologue.
A search of the mouse genome for p67phox
homologues yielded an open reading frame on chromosome 2. Based on the
genomic sequence we cloned the mouse p67phox
homologue by reverse transcription PCR (GenBankTM accession
number AF539798). The predicted protein had a molecular mass of 49 kDa
and displayed 30% sequence identity with
p67phox (Fig. 2B). A computer search
for motifs revealed tetratricopeptide repeats, which are important for
Rac binding in p67phox (20) (Fig.
2B). The so-called activator domain of
p67phox, which interacts with
gp91phox, was also found in the homologue (21)
and it conserved the C-terminal but not the N-terminal SH3 domain of
p67phox. The
p40phox-binding PB1 domain of
p67phox was weakly conserved (22). In addition
the homologue had a C-terminal hydrophobic stretch, which may represent
a transmembrane tail (Fig. 2B).
After discussions with Dr. Ruth Lovering from the Human Genome
Organization Nomenclature Committee and with colleagues working in the
field, the novel factors have been named NOXO1 (NOX organizer 1) and
NOXA1 (NOX activator 1). NOXO1 is the p47phox
homologue, and NOXA1 is the p67phox homologue.
The terms NOXO2 and NOXA2 have been introduced as aliases for
p47phox and
p67phox, respectively.
NOXO1 and NOXA1 mRNAs Are Expressed Predominantly in Colon
Epithelium--
We next investigated the tissue distribution of NOXO1
and NOXA1 by Northern blotting (Fig.
3A). The probe derived from
NOXO1 labeled a ~1.5-kb mRNA, while the NOXA1 probe labeled a
~1.7-kb mRNA. Interestingly, both mRNAs showed a relatively
similar tissue distribution with a predominant expression in the colon.
Low level expression was seen in uterus, small intestine, and stomach
for both transcripts. Testis expressed NOXO1 but not NOXA1 (the
increased length of the testis NOXO1 mRNA is due to differences in
the non-coding regions, not shown). In situ hybridization
with NOXO1 and NOXA1 antisense probes labeled colon epithelial cells
strongly, while neither the NOXA1 sense probe (Fig. 3B,
control) nor the NOXO1 sense probe (not shown) hybridized.
Thus, NOXO1 and NOXA1 were expressed in the same tissue and same cell
type as NOX1.
NOXO1 and NOXA1 Enable Superoxide Generation by NOX1--
To
investigate the function of NOXO1 and NOXA1, we transfected HEK293
cells with NOX1, NOXO1, and NOXA1, or with the empty expression vector
(control). The cells transfected with all three constructs generated
superoxide, while control-transfected cells did not (Fig.
4, A and B).
Addition of a flavoprotein inhibitor, diphenylene iodonium (DPI),
inhibited superoxide production (Fig. 4A). The block of NOX1
activity by DPI was slower than that observed for other NOX proteins.
The half-time of DPI inhibition of superoxide generation was 239 ± 16 s in NOX1-transfected HEK293 cells, while it was 40 ± 1.5 s in NOX5-transfected HEK293 cells (not shown). This is, to
our knowledge, the first clear indication of pharmacological variability among different NOX enzymes.
Individual transfection of NOX1, NOXO1, or NOXA1 did not lead to any
detectable superoxide generation (Fig. 4B). Similarly, no
superoxide generation was observed upon co-transfection of NOX1 with
only one of the novel homologues or co-transfection of NOXO1 and NOXA1
without NOX1 (Fig. 4B). The co-transfection of NOX1, NOXO1,
and NOXA1 led to superoxide generation not only in HEK293
cells but also in Chinese hamster ovary, HeLa, and COS-7 cells (not
shown), although we detected the highest activity in HEK293 cells.
Next we verified the NOX1-dependent superoxide generation
using the SOD-inhibitable cytochrome c reduction assay. As
shown in Fig. 4C, cells transfected with NOX1, NOXO1, and
NOXA1 reduced cytochrome c in an SOD-inhibitable manner. The
observed activity was continuous and did not require any stimulus. The
rate of superoxide generation was 0.69 ± 0.04 nmol of
superoxide/min/107 cells.
Microscopic analysis by the NBT reduction assay showed that ~10% of
the transiently NOX1 + NOXO1 + NOXA1-transfected cells but none of the
control-transfected cells stained NBT-positive (Fig. 4D). We
infer that the superoxide generation per cell expressing an active
NADPH oxidase was ~10 times higher than the mean value given above,
or ~7 nmol of superoxide/min/107 cells. This is in the
range reported for phagocyte NADPH oxidase-expressing COS-7 cells (23),
and NOX5-expressing HEK293 cells (9).
Stimulus Independence of NOX1 Activation by Cytoplasmic
Factors--
The superoxide generation by NOX1 + NOXO1 + NOXA1-transfected cells shown in Fig. 4 occurred in the absence of
external stimuli. In contrast the phagocyte NADPH oxidase
requires external stimuli even in reconstituted systems (23, 24). To
investigate whether external stimuli are able to enhance the NOX1
respiratory burst, we treated the transfected cells with agents known
to activate the phagocyte NADPH oxidase (1). However, none of the
following treatments increased significantly the superoxide production
compared with non-stimulated cells (100%): 100 nM PMA
(84 ± 2%), 100 µM arachidonic acid (108 ± 2%), and 1 µM ionomycin (107 ± 18%).
Cofactor Requirement for Stimulus-dependent and
Stimulus-independent NOX1 Activation--
To understand further the
activation of NOX1 by cofactors, we co-transfected NOX1 with
the following combinations of cofactors: NOXO1 + NOXA1,
p47phox + p67phox, p47phox + NOXA1,
and NOXO1 + p67phox (Fig. 4E).
Stimulus-independent activation of NOX1 occurred exclusively with the
NOXO1 + NOXA1 combination. When NOXO1 was replaced by p47phox, stimulus-independent activation
disappeared, but the system could be activated by PMA. Under this
condition, the superoxide generation was lower than that observed with
NOXO1 + NOXA1 but similar to that observed with
p47phox + p67phox. Only
minimal NOX1 activity was detected with the combination of NOXO1 + p67phox. These results suggested that the
differences between p47phox and NOXO1 are
important for stimulus-dependent versus
stimulus-independent NOX1 activity. This is substantiated by the
different primary structures of the two proteins, i.e. the
lack of the autoinhibitory region and the adjacent phosphorylation
sites in NOXO1. However, as the combination of NOXO1 + p67phox did not display any
stimulus-independent activation, some NOXA1-specific element might also
be involved in the stimulus-independent activity.
In summary, we have described two novel proteins, NOXO1 and NOXA1, that
support superoxide generation by NOX1. As is the case for NOX1, these
proteins are expressed predominantly in colon epithelium and are thus
likely to be physiologically relevant partners of NOX1. It is an
intriguing question whether other subunits are involved in NOX1
function. In particular p22phox and Rac1 are of
interest as both of them have wide tissue distribution and both of them
were reported to be expressed in HEK293 cells (25, 26).
The activation of NOX1 by NOXO1 and NOXA1 is stimulus-independent in
the reconstituted system and probably also in colon. However, we cannot
exclude that there are some distinct mechanisms of NOXO1 inhibition in
the colon, which are not reproduced in the HEK293 system. Is the
stimulus-dependent activation of NOX1 by
p47phox and p67phox of
physiological relevance? Superoxide generation by vascular smooth
muscle, which contains NOX1, is stimulus-dependent and involves p47phox (27). Thus, an interaction of
NOX1 with phagocyte NADPH oxidase subunits may occur in some tissues
and would make NOX1 a versatile enzyme that could change activation
mechanisms depending on the subunits present in the cell type in which
it is expressed.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
View larger version (15K):
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Fig. 1.
Activation of NOX1 by phagocyte
NADPH oxidase subunits. HEK293 cells were transfected with mouse
NOX1 either alone or together with human p47phox
and p67phox, and superoxide generation was
measured by the peroxidase-dependent luminol-amplified
chemiluminescence technique. The filled symbols indicate the
presence of 100 nM PMA. The signal was measured with
relative light units (RLU). Data are from a single
experiment representative of three independent studies.
View larger version (45K):
[in a new window]
Fig. 2.
Comparison of amino acid sequence and domain
structure of NOXO1 and NOXA1 with p47phox
and p67phox. A,
alignment of mouse NOXO1 with mouse p47phox. The
phox homology domain (light shaded box), the SH3
domains (bold characters), the autoinhibitory domain of
p47phox (underlined), the
proline-rich region (dark shaded box), and the serine
residues phosphorylated in p47phox
(asterisks) are indicated. B, comparison of mouse
NOXA1 with mouse p67phox. The four
tetratricopeptide repeat domains (light shaded boxes), the
activator domain (dark shaded box), the proline-rich region
(in italics), the potential p21-activated protein
kinase phosphorylation sites (asterisks), the SH3
domains (bold characters), the PB1 domain
(underlined), and the hydrophobic tail of mNOXA1
(double underlined sequence) are indicated.
View larger version (69K):
[in a new window]
Fig. 3.
Tissue distribution and cellular localization
of NOXO1 and NOXA1 mRNA. A, Northern blot analysis
of tissue distribution of NOXO1 (upper panel) and NOXA1
(middle panel) mRNA. The bottom panel shows
the 28 S and 18 S rRNA as a loading control. B,
in situ hybridization of mouse colon with NOXO1 sense probe
(left panel, control), NOXO1 antisense probe
(middle panel), and NOXA1 antisense probe (right
panel).
View larger version (34K):
[in a new window]
Fig. 4.
NOXO1 and NOXA1 are cofactors for
NOX1-dependent superoxide generation. A,
NOX1, NOXO1, and NOXA1 or vector only (control) were
transfected into HEK293 cells, and superoxide generation was detected
as in Fig. 1. 5 µM DPI was added at the indicated time.
B, statistical analysis of peak superoxide production by
HEK293 cells transfected with the indicated constructs. Superoxide was
detected as in Fig. 1. C, measurement of superoxide
generation with the SOD-inhibitable cytochrome c assay by
HEK293 cells transfected with NOX1, NOXO1, and NOXA1 or with vector
(control). Note that the seemingly different kinetics shown
in A and C are caused by the two different types
of assays, e.g. cytochrome c reduction
measurement yields cumulative values, while the chemiluminescence
measurement yields instantaneous ones. D, NBT reduction was
assessed on HEK293 cells transfected with NOX1, NOXO1, and NOXA1 or
with vector (control) after incubation with 0.5 mg/ml NBT at
37 °C for 15 min. E, statistical analysis of peak
superoxide production by HEK293 cells co-transfected with NOX1 plus the
indicated combinations of NOXO1, NOXA1, p47phox,
and p67phox either without stimulus (black
bars) or in the presence of 100 nM PMA (white
bars). Data shown in A, C, and D
are from a single experiment representative of three or more
independent studies. Data shown in B and E
represent the mean ± S.E. of three or more independent
experiments. RLU, relative light units.
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ACKNOWLEDGEMENTS |
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We thank Lasta Kocjancic Curty for skillful technical assistance; Péter Kiss for providing some cDNA samples; Drs. Anthony Valente, Terry Kay Epperson, Lena Serrander, and Ildikó Szántó for helpful discussions; and Drs. Miklós Geiszt and Thomas L. Leto for having first pointed out the existence of a p47phox homologue data bank entry.
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FOOTNOTES |
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* This research was supported by Swiss National Foundation Grants 31-55805.98 and by United States Public Health Service, National Institutes of Health Grants AI20866 and AG19519.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(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF539799 (mouse NOX1), AF539797 (mouse NOXO1), AF539798 (mouse NOXA1), and AF539796 (human NOXO1).
§ To whom correspondence should be addressed: Dept. of Geriatrics, Geneva University Hospitals, 2, Ch. du Petit-Bel-Air, CH-1225 Geneva, Switzerland. Tel.: 41-22-305-5450; Fax: 41-22-305-5455; E-mail: Botond.Banfi@hcuge.ch.
Published, JBC Papers in Press, December 6, 2002, DOI 10.1074/jbc.C200613200
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ABBREVIATIONS |
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The abbreviations used are: NOX, NADPH oxidase; DPI, diphenylene iodonium; DUOX, dual domain oxidase; NBT, nitro blue tetrazolium; NOXA1, -2, NOX activator 1, 2; NOXO1, -2, NOX organizer 1, 2; PMA, phorbol 12-myristate 13-acetate; SOD, superoxide dismutase; SH3, Src homology 3.
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