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INTRODUCTION |
Cloning and sequencing of cDNAs for the three isoforms
of nitric oxide synthase
(NOS)1 has revealed products
of distinct genes that share 50-60% homology at the nucleotide and
amino acid levels (1). Two of these products, NOS-1 and NOS-3,
are constitutively expressed enzymes that are calcium-calmodulin-dependent and produce small amounts of
nitric oxide (NO) in response to transient elevations in intracellular calcium (2, 3). The third isoform, NOS-2, is not normally expressed but
can be transcriptionally activated in a variety of cell types in
response to proinflammatory cytokines and bacterial endotoxins (4, 5).
Unlike the constitutive isoforms, NOS-2 has calmodulin bound tightly at
all times, maintaining the enzyme in a tonically active state and
making it capable of producing a large, continuous flux of NO. The
relatively large amount of NO that can be synthesized by NOS-2 over a
sustained period has been implicated in diverse functions associated
with inflammation and injury (6). Expression of NOS-2 in the central
nervous system has been associated with viral, parasitic, and bacterial infections; in multiple sclerosis and neurodegenerative diseases; and
in trauma and ischemia (7). Given the potential involvement of NOS-2 in
such pathologies, a clear understanding of the mechanisms of NOS-2 gene
regulation in vivo is important.
The role of elements in the 5' untranslated region of the NOS-2 gene
and the mechanisms by which NOS-2 is induced and this induction is
suppressed have all been well characterized in vitro. A
1749-bp fragment of the mouse NOS-2 promoter has been cloned and
sequenced (8), and the transcription initiation site and 22 consensus
sequences for the potential binding of transcription factors have been
identified. Given this diversity and multiplicity, it is not surprising
that maximal induction of the gene requires the synergistic effect of
combinations of stimuli. The nuclear factor
B binding site in the
proximal portion of the promoter is considered obligatory for NOS-2
induction by endotoxin in vitro, whereas the more distal
region of the promoter is important for the synergistic effect of
IFN-
(9).
Recently, a NOS-2 gene-deficient mouse was generated (10) with a
targeted deletion of the proximal region of the promoter (585 bp), as
well as exons 1-4 (including the translational start site). This
manipulation was designed to produce a NOS-2 null allele by interfering
with both transcriptional and translational expression of the gene. In
the descriptions and outcomes of studies in a variety of disease models
using this genetic variant, and in a range of different cell types, the
loss of expression appears to hold true (6). However, here we report
that the generation of a cerebral infarct in such mice initiates
transcriptional expression of the disrupted NOS-2 gene. Although there
is translation of a product, this does not function as a NOS. As such,
these mice provide a unique opportunity to explore further the
regulation of the NOS-2 gene in vivo through the analysis of
a promoter deletion introduced by genetic mutagenesis and should prove
useful in the identification of previously undetected activators of
this gene.
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EXPERIMENTAL PROCEDURES |
Mice and Genotyping--
NOS-2
/
mice, generated from a
mixed background of C57BL/6 × 129/SvEv (10), were outcrossed to
C57BL/6 wild-type mice (+/+) to generate NOS-2 heterozygous (+/
) mice
for breeding. A NOS-2 colony was established at the University of Iowa
using heterozygote breeding pairs in order to generate littermates of all three genotypes (+/+, +/
, and
/
). Genomic screening was performed on DNA extracted from mouse tail snips. To detect the presence of the wild-type NOS-2 allele, a pair of PCR primers were
designed that amplified a 328-bp region within exon 1 of the NOS-2
allele and absent in
/
mice (Fig. 1).
The sequences of the NOS-2 primers are as follows: forward, 5'-TGA AGT
GAC TAC GTG CTG CC-3'; reverse, 5'-AGT CCC TTC ACC AAG GTG G-3'. To
identify the presence of the altered NOS-2 allele, another primer pair was constructed that amplifies a 550-bp region of the neo
insert specific to the disrupted NOS-2 allele. The sequences of the
neo primers are as follows: forward, 5'-TGG AGA GGC TAT TCG
GCT ATG AC-3'; reverse, 5'-CAC CAT GAT ATT CGG CAA GCA G-3'.

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Fig. 1.
Schematic comparison of the wild-type and
disrupted NOS-2 alleles. Shown are the deletions in the /
mice, the actual and potential translational start sites, and the
location of the PCR primers used for genotyping. Light shaded
boxes, promoter response elements; dark shaded boxes,
exons; arrows, PCR primers.
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Induction of Focal Cerebral Ischemia--
Adult mice (19-30 g)
were anesthetized with 2% halothane for induction and were maintained
on 1% halothane. A midline incision was made on the ventral surface of
the neck, and the right external carotid and common carotid arteries
were isolated and ligated. The internal carotid artery and the
pterygopalatine artery were temporarily occluded. An 8-0 nylon
monofilament coated with silicone was introduced into the extracranial
internal carotid artery through an incision in the common carotid
arteries and carefully advanced approximately 10 mm distal to the
carotid bifurcation, beyond the origin of the middle cerebral artery.
After suturing the incision, anesthesia was withdrawn and the animals
allowed to recover for up to 72 h.
Immunohistochemistry--
NOS-2 protein was detected in
paraformaldehyde-fixed tissue using a rabbit anti-mouse NOS-2
polyclonal antibody (1:100, Transduction Laboratories) that recognizes
the C-terminal region of the NOS-2 isoform. Immunohistochemistry was
performed with only slight modifications of standard immunoperoxidase
techniques suggested by Oncogene Science Inc. Immunoreactivity was
detected using an avidin-biotin horseradish peroxidase complex kit
(Vectastain ABC kit, Vector Laboratories, Inc.) along with a peroxidase
substrate kit using nickel-enhanced 3,3'diaminobenzidine as substrate
following the manufacturer's recommendations. All sections were
additionally counterstained with toluidine blue (0.5% aqueous,
acidified with 0.2% glacial acetic acid) and eosin (0.5% aqueous) and
coverslipped. To assist in the determination of cell types expressing
NOS-2 protein, adjacent sections were labeled with cell-specific
markers; a mouse monoclonal anti-glial fibrillary acidic protein
antibody (1:200, Sigma) was used to identify astrocytes, and a rat
anti-mouse F4/80 polyclonal antibody (1:100, Serotec Ltd.) was used to
recognize monocytes and macrophages.
RNA Isolation--
Total cellular RNA was isolated from
approximately 100-150 mg (wet weight) of tissue with Trizol reagent
(Life Technologies, Inc.) according to the manufacturer's protocol.
Precipitated RNA was pelleted by centrifugation, washed with 75%
ethanol, and allowed to air dry. RNA was redissolved in RNase-free
water and precipitated with 0.6 volumes of 2 M sodium
acetate and 3 volumes of 100% ethanol for storage at
70 °C. Prior
to reverse transcription, RNA precipitates were recovered by
centrifugation, washed with 75% ethanol, redissolved in RNase-free
water, and quantitated.
RT-PCR--
First strand cDNA was synthesized from total RNA
using SuperscriptTM II RNase H reverse transcriptase (Life
Technologies, Inc.); conditions were optimized for the various
oligonucleotide primers used. Specific PCR primers were designed to
identify portions of the NOS-2 mRNA that persisted in the
gene-deficient mice and therefore span the entire cDNA sequence.
Typically, the primers were known to span at least one intron, and
therefore amplification of genomic DNA would have resulted in a PCR
product much larger than that generated by amplification of cDNA.
Where exon/intron boundaries were unknown, amplification of genomic DNA
was controlled either by treatment with DNase prior to RT, or the PCR
included a control sample in which all components of the RT reaction
were included except for the reverse transcriptase.
Mixed Glial Cell Cultures--
Primary glial cell cultures from
cortices of neonatal NOS-2 +/+ and
/
mice were obtained as
described previously (11).
RNase Protection Assay--
This was performed as described
previously (12). In all reactions, a probe for the transcript for
ribosomal protein L32 was included to control for RNA loading. For
quantification, autoradiographs were scanned (densitometry imager;
Bio-Rad), and band densities were determined using imaging software
(Molecular Analysis; Bio-Rad). Densities of the transcripts are
expressed as a ratio to the transcript for L32.
Immunoprecipitation and Immunoblotting--
This was performed
essentially as described by Kim et al. (13), except that the
lysis buffer contained 1% Nonidet P-40. Cell lysates were first
incubated with a specific monoclonal antibody directed against a
C-terminal peptide fragment of NOS-2 (Transduction Laboratories) and
then with protein G-agarose (Santa Cruz Biotechnology). The
immunoprecipitates were subjected to SDS-PAGE and transferred to
nitrocellulose membrane. The blot was immunostained with the same NOS-2
monoclonal antibody (1:500), using ECL luminography for detection.
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RESULTS |
Expression of a Protein in NOS-2 Gene-deficient Mice Following
Cerebral Ischemia--
In wild-type (+/+) mice, immunohistochemistry
performed with a specific C-terminal antibody revealed NOS-2 expression
in both infiltrating cells and also in resident astrocytes after
permanent middle cerebral artery occlusion (MCAO). In gene-deficient
(
/
) mice, immunoreactivity was detected 24 h postocclusion in
the infarcted hemisphere in infiltrating inflammatory cells (Fig. 2A). Later, at 72 h after
MCAO, immunostaining was still evident, but was additionally seen in
cells surrounding the infarct that morphologically resemble astrocytes
(Fig. 2B). This pattern of expression was identical,
spatially and temporally, to that seen in wild-type mice following
MCAO.2 As no immunolabeling
was observed in the contralateral cerebral hemisphere at any time
point, expression of the altered gene product appeared to be restricted
to the ischemic hemisphere. No immunoreactivity was detected in tissue
from normal
/
mouse brain (data not shown), suggesting that the
altered NOS-2 gene was not constitutively activated.

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Fig. 2.
Immunoreactivity for NOS-2 in ischemic /
mouse brain. Immunohistochemistry using a C-terminal NOS-2
polyclonal antibody revealed protein expression in / mice at
24 h (A) and 72 h (B) after MCAO.
Positive infiltrating cells were seen in the parenchyma (A)
and in cells resembling astrocytes located adjacent to the infarct
(B). Scale bar, 25 µm.
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A Truncated NOS-2 Transcript Is Expressed in Gene-deficient
Mice--
We used RT-PCR both to verify expression of the disrupted
NOS-2 gene following MCAO, and also in an attempt to determine the extent of the resulting transcript. Residual transcription of the NOS-2
gene was sought using multiple pairings of NOS-2-specific PCR primers.
Using primers corresponding to sequences downstream of exon 4, a 692-bp
portion of the NOS-2 mRNA was detected following MCAO in both +/+
and
/
mice (Fig. 3). However, primers
upstream of the exon 4/5 boundary (bases 484/485, GenBankTM
accession no. M84373) generated a 745-bp product in +/+ but not
/
mice. A product of 858 bp from the 3' region of the NOS-2 transcript
was also detected in +/+ and
/
mice. Sequencing verified the
nucleotide identity with NOS-2 of three different PCR products from
/
mice, generated with primer pairs located 5' (bases 489-1181), 3' (bases 3121-3650), and in the middle of the cDNA (bases
2605-2820).

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Fig. 3.
Detection of NOS-2 transcript in ischemic +/+
and / mouse brain. A, using a forward PCR primer
(436F) located in exon 4, transcript was detected in +/+ but not /
mice. NOS-2 mRNA was detected in +/+ and / animals using
primers (489F-1181R) that amplified regions downstream of the exon 4/5
boundary. The predicted product sizes (692 and 745 bp) are indicated by
arrows. B, the NOS-2 transcript was also detected
in +/+ and / mice using primers (2721F-3579R) located in the 3'
region. The predicted 858-bp product is indicated by an
arrow.
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Transcription of the disrupted NOS-2 gene was additionally verified in
ischemic animals by ribonuclease protection assay (RPA) using a
specific 275-bp NOS-2 cRNA probe, complimentary to a region starting
800 bp downstream from the translational start site. Evidence for
transcriptional activation of the NOS-2 gene was found in both
/
and +/+ mice at 6, 24, and 48 h following MCAO, both ipsilateral
and contralateral to the infarct (Fig.
4A). Quantitation of the NOS-2
transcript revealed somewhat similar expression in the ischemic
hemispheres of both mouse genotypes. If anything, loss of the proximal
promoter region in NOS-2
/
mice correlated with faster onset and/or
more extensive expression.

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Fig. 4.
Activation of the NOS-2 gene in tissue and
cells derived from / mouse brain. A, RNA from
infarcted hemispheres was probed for NOS-2 and the transcript for
ribosomal protein L32 by RPA at various times after MCAO. Densitometric
analysis of autoradiograms was performed using Molecular Analysis
imaging software. Individual data points (2-4 animals per group) are
indicated as the ratio of NOS-2:L32. B, RNA isolated from
cultured glial cells was probed for NOS-2 by RPA. Lanes 1 and 3, unstimulated cells; lanes 2 and
5, IFN- (100 units/ml) and desferrioxamine (0.8 mM); lane 4, IFN- and IL-1 (1 ng/ml);
lanes 6 and 11, mixture of IFN- , IL-1 ,
TNF (50 ng/ml), and lipopolysaccharide (1 µg/ml); lanes
7 and 9, TNF ; lanes 8 and 10, hypoxia; lane 12, 8-bromo-cyclic GMP (1 mM).
L32 indicates relative loading. C, glial cultures
were exposed to cytokines for 30 h, lysed, immunoprecipitated with
anti-NOS-2, and then immunoblotted. Lane 1, +/+ cells
stimulated with cytokine mixture; lane 2, unstimulated /
cells; lane 3, cytokine mixture stimulated / cells. The
position of molecular mass markers (kDa) is indicated.
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The Disrupted NOS-2 Gene Can Be Activated in Cultured Glial
Cells--
A period of cerebral ischemia is immediately followed by
the induction of genes encoding for proinflammatory cytokines, such as
IL-1
and TNF
. Assuming that the +/+ and
/
NOS-2 genes are induced by similar mechanisms, we tested the ability of various combinations of proinflammatory cytokines, as well as hypoxia, to
induce the altered gene in mixed glial cultures. Although the known
hypoxia response element in the NOS-2 promoter is deleted in the
genetically altered mice, we thought that another site might exist more
distally. With this in mind, we tested the responsiveness of the
altered gene to hypoxic conditions and to stimuli (IFN-
and the iron
chelator desferrioxamine) shown to induce the NOS-2 gene in macrophages
through a hypoxia response element (14).
The disrupted NOS-2 gene transcript was detected, using RPA, in glial
cells that had been exposed to a mixture of cytokines, but not in
response to other stimuli (Fig. 4B). A protein product could
also be detected in immunoprecipitates from lysates of
cytokine-activated cells (Fig. 4C). This protein was smaller
than that of NOS-2 in wild-type cells (131 kDa) and displayed a
molecular mass (approximately 112 kDa) similar to that predicted if the
first 113 amino acids of NOS-2 were absent (116 kDa). In similar cell
cultures exposed to the cytokine mixture, we determined nitrite
accumulation in the medium, using the Griess reaction. Detectable NO
production was only found in cells derived from wild-type animals,
indicating a loss of NOS-2 function in
/
mice (data not shown).
As neo has its own promoter (PGK), we predicted that it
should be expressed in
/
mice, even though it was inserted into the
NOS-2 construct in the reverse orientation. With the same primer pair
used for genotyping mice, and using RT-PCR, we were able to amplify a
product from DNase-treated RNA derived from
/
mice (Fig.
5). In cells from
/
mice, a mixture
of cytokines also induced expression of neo, as determined
by northern hybridization using a specific probe. Only one transcript
was detected, and it was of a size corresponding to neo mRNA (data
not shown).

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Fig. 5.
Expression of neo in
ischemic NOS-2 / mouse brain. RNA isolated from mouse cerebral
hemispheres was DNase-treated, reverse transcribed, and subjected to
PCR using specific primers. Lane 1, no target; lane
2, control for residual genomic DNA; lane 3, untouched
mouse; lanes 4 and 5, contralateral and ischemic
hemispheres 24 h after ischemia; lanes 6 and
7, contralateral and ischemic hemispheres 48 h after
ischemia. The predicted product size is 550 bp.
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Inflammation-related Gene Expression following Ischemia is Similar
in +/+ and
/
Mice--
The expression of cytokine and other
inflammation-related genes was examined to evaluate possible variations
in the inflammatory reaction in
/
mice. Multiprobe RPA analysis was
performed on RNA isolated from infarcted and contralateral hemispheres
in order to detect transcripts for the cellular response genes ICAM-1, A20, Mac-1, EB22, and glial fibrillary acidic protein and for the
cytokines TNF-
, TNF-
, IL-4, IL-5, IL-1
, IFN-
, IL-2, IL-6, IL-1
, and IL-3. Increased expression of Mac-1 and glial fibrillary acidic protein genes is indicative of activation of
macrophages/microglia and astrocytes, respectively. Expression of EB22,
an acute-phase response gene, was detected as early as 6 h and was
increased at 24 and 48 h postocclusion. Induction of the gene for
the cellular adhesion molecule ICAM-1 was also apparent in NOS-2
/
mice. Quantification revealed similar ischemic induction of ICAM-1 and Mac-1 in both genotypes at all times following permanent MCAO (Fig.
6). Transcript for IL-1
, a potent
inducer of the NOS-2 gene, was markedly elevated in ischemic
hemispheres of wild-type and NOS-2-deficient mice, whereas expression
of IL-6 and TNF-
was increased to a lesser extent (Fig.
7). Minimal or no induction was seen for
the other cytokine genes.

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Fig. 6.
Expression of markers of ischemic
inflammation in NOS-2 +/+ and / mouse brain. RNA from both
infarcted (ipsilateral) and contralateral hemispheres was probed by RPA
for ICAM-1 (A) and MAC-1 (B) at the indicated
times following MCAO. Densitometric analysis of autoradiograms was
performed using Molecular Analysis imaging software. Mean values (as a
ratio to L32) for 2-4 mice are indicated.
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Fig. 7.
Expression of proinflammatory cytokine genes
after MCAO in NOS-2 +/+ and / mice. RNA from both infarcted
(right) and noninfarcted (left) hemispheres was probed by RPA for
IL-1 (A), IL-6 (B), and TNF (C)
at the indicated times following MCAO. Densitometric analysis of
autoradiograms was performed using Molecular Analysis imaging software.
Mean values (as a ratio to L32) for 2-4 mice are indicated.
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DISCUSSION |
We have found that mice with a disruption of the NOS-2 gene can
undergo tissue-specific transcription, and translation of a
NOS-incompetent product, despite the absence of exons 1-4 and proximal
regions of the promoter considered to be essential for activation.
In vitro investigation into the responsiveness of this gene
to endotoxin, proinflammatory cytokines, and hypoxia have identified
the promoter sequences required for induction, many of which are absent
in the gene-deficient mouse. However, the 5' flanking region of the
mouse NOS-2 gene contains sequences of DNA of which the function is not
yet known, and studies using reporter constructs are limited by the
amount upstream that has been sequenced to date. Therefore, the
regulatory mechanisms that occur in vivo might be complex
and may be oversimplified by in vitro representation.
Because they exhibit a truncated but active promoter, these
/
mice
provide a unique opportunity to study NOS-2 gene regulation in
vivo.
Mice with targeted deletions in the NOS-1 (15), NOS-2 (10, 16, 17), and
NOS-3 (18) genes have been produced. Although gene disruption is
designed to result in a null allele, this is not always the case, and
the resulting mice may produce a nonfunctional gene product, a
truncated product, or alternatively spliced products (19). In some
cases, generation of an inactive product is specifically designed, as
this mimics the effects of pharmacological inhibition. Mice deficient
for the NOS-1 gene showed residual NOS activity, indicating the
presence of splice variants (20). In the NOS-2-deficient mice generated
by Wei et al. (16), an attempt to delete the first five
exons of the gene failed, and a genomic rearrangement occurred. From
this genomic alteration there resulted an aberrant transcript larger
than wild-type NOS-2 mRNA, but reportedly, this was not translated
into a functional NOS. The targeting strategy for another
NOS-2-deficient mouse involved replacement, with the neomycin
resistance gene, of exons 12 and 13, which encode the calmodulin-binding domain (17). Low levels of two abnormal transcripts were detected, and both contained sequences of neo. Although
immunoblotting revealed no detectable NOS-2 protein, a low level of
NO-producing activity was observed.
In the mice used here, a gene replacement vector was designed to delete
the proximal 585 bases of the promoter, as well as exons 1-4. Because
the proximal portion of the promoter was previously shown in
vitro to be required for NOS-2 induction in macrophages in
response to lipopolysaccharide, and because exon 2 contains the
translational start site, homologous recombination of the targeting
vector with the NOS-2 gene was predicted to interfere with both
transcription and translation. Screening of NOS-2-deficient mice for
mRNA, protein, and NOS activity indicated that the gene had been
inactivated (10). However, this screening was performed on organ blots
following lipopolysaccharide injection, or on isolated macrophages
stimulated with various combinations of lipopolysaccharide, TNF
,
IFN-
/
, and IFN-
. As activation of the NOS-2 gene via these
stimuli has been shown to be dependent upon the proximal portion of the
NOS-2 promoter (8), induction of the disrupted gene would not be
predicted. We have shown here that glial cells from gene-deficient mice
did not express the aberrant transcript under such conditions. However,
both a transcript and a protein product could be detected when a
cytokine mixture was used, although there was no evidence for a NOS function.
When gene targeting is designed to modify or delete regulatory DNA
sequences, the activity of the marker gene should be considered as it
brings its own promoter and enhancer (21). Although we have shown that
neo is expressed in NOS-2
/
mice following cerebral ischemia, and in cytokine-activated glial cells, we believe it is
activation of the remaining (distal) NOS-2 promoter region that is
responsible. The temporal and spatial expression of the disrupted gene
product following cerebral ischemia is identical to that in the
wild-type, suggesting a similar mechanism of induction. Less than 2 kilobase pairs of the 5' flanking DNA of the murine NOS-2 gene has been
sequenced. It is possible that unidentified promoter response elements
could be involved in the activation of both the disrupted and the
wild-type gene in response to focal ischemia. By comparison, the human
NOS-2 gene has important regulatory elements extending 16 kilobase
pairs upstream (22). It is also possible that the remaining
transcription element consensus sequences mediate expression by some
mechanism(s) previously undetected by in vitro study.
The NOS-2 protein is a dimeric, bidomain enzyme with iron
protoporphyrin IX, flavin adenine dinucleotide, flavin mononucleotide, and tetrahydrobiopterin as bound prosthetic groups (23). The enzyme has
an N-terminal oxygenase domain, containing binding sites for
L-arginine and heme, and a C-terminal reductase domain that
binds FMN, FAD, and NADPH. When NOS-2 is cleaved at the junction of the
oxygenase and reductase domains, the resulting protein fragments remain
catalytically active but unable to generate NO (24). Activity of NOS-2
is dependent upon dimerization, and recent evidence has identified
amino acids 66-114 of the oxygenase domain as required for dimeric
interaction (25). Exons 2-4 of the NOS-2 gene, deleted in the
deficient mice, encode the first 100 amino acid residues of the NOS-2
protein. Although the native translational initiation site is located
in exon 2, an ATG codon at bases 524-526 could potentially act as a
translational start site in the gene-disrupted mice. If so, then the
disrupted gene would be translated in the same open reading frame and,
using the same termination codon as the wild-type gene, generate a
truncated protein lacking the N-terminal 113 amino acid residues. We
have found evidence, using an antibody against the C terminus, for the
translation of a truncated protein both in ischemic NOS-2-deficient mouse brain, and in cytokine-activated glial cultures. Based on the
work of Ghosh et al. (25) and the lack of NO production in
glial cells activated with a cytokine mixture, we conclude that this
truncated protein is NOS-incompetent because of its inability to dimerize.
Although translation of this altered gene product would not result in a
functional NOS, alternate functions of a truncated protein cannot be
ruled out. The reductase domain of NOS is capable of catalyzing the
transfer of electrons from NADPH to a variety of exogenous acceptors,
including cytochrome c and dioxygen. The NOS-1 protein can
reduce molecular oxygen to superoxide (26), and the reductase domain is
both necessary and sufficient for this reaction (27). Although NADPH
oxidation by NOS-1 is not influenced by the substrate arginine, the
rate of oxidation by NOS-2 increases in the presence of substrate (28).
Recent evidence, though, suggests that the NOS reductase domains are
actually poor superoxide generators, as they are slow to transfer
electrons to dissolved O2 (23). It is therefore predicted
that although the truncated protein might catalyze a superoxide
dismutase-insensitive reduction of cytochrome c or similar
artificial electron acceptors, NADPH oxidation leading to superoxide
formation is unlikely (29).
The serendipitous finding that the disrupted NOS-2 gene can be
expressed, despite the absence of four exons and important promoter
response elements, has several implications. This NOS-2-deficient mouse
can contribute important insights into the regulation of the gene and
to structure-function aspects of the NOS enzymes. The residual
expression of the disrupted gene challenges the idea that proximal
promoter sequences are obligatory for induction. These mice provide a
unique opportunity to study the molecular and physiological effects of
both a truncated NOS-2 promoter and protein in intact animals following
neurodegeneration, trauma, and viral infection, conditions in which the
NO from NOS-2 is proposed to play a role (7).