(Received for publication, October 20, 1995; and in revised form, March 7, 1996)
From the
We report the first nonmammalian inducible nitric-oxide synthase
(NOS) cDNA obtained from chicken macrophages. It exhibits an open
reading frame encoding 1,136 amino acid residues, predicting a protein
of 129,648-Da molecular mass. The deduced NOS protein sequence showed
66.6%, 70.4%, 54.2%, and 48.7% sequence identity to mouse and human
inducible NOS and to two constitutive NOSs from rat brain and bovine
endothelium. Overall, NOS appears to be a moderately conserved protein.
Northern analysis showed that chicken iNOS mRNA is approximately 4.5
kilobases (kb), a size similar to mammalian inducible NOS. Analysis of
3.2 kb of 5`-flanking sequence of the chicken iNOS gene showed a
putative TATA box at 30 base pairs (bp) upstream of the transcription
initiation site. The functional importance of the upstream region was
determined by transient expression of deletion constructs. An endotoxin
regulatory region was located exclusively within 300 bp upstream of the
transcription initiation site. This is in contrast to the two distinct
sites identified in the mouse macrophage NOS promoter. Transcription
factor binding sites such as NF-B, PEA1, PEA3, and C/EBP were
identified. Using a NF-
B inhibitor, we showed that NF-
B is
indeed involved in the induction of chicken iNOS gene by
lipopolysaccharide. Our results suggest that NF-
B is a common
regulatory component in the expression of both mammalian and
nonmammalian iNOS genes.
The oxidation of L-arginine (1, 2, 3) is now recognized to be an
important biochemical pathway in many organisms. One of the products of
this reaction, nitric oxide (NO), ()performs many diverse
and significant biological
functions(4, 5, 6, 7, 8) .
In the nervous system, NO is a novel neurotransmitter (9, 10, 11) which is synthesized as needed
and not stored in synaptic vesicles. Importantly, nitric oxide does not
interact with receptors on the surface of neurons but targets redox
centers within neighboring neurons. In the vascular system, NO acts as
the endothelium-derived relaxing factor (12, 13) which
is a mediator of blood vessel relaxation and blood pressure. It can
also inhibit platelet aggregation (14) and adhesion. In the
immune system, NO is synthesized by activated macrophages and acts as a
cytotoxic and tumoricidal agent(1, 2, 5) . NO
also mediates important functions in other tissues and organs such as
the gastrointestinal tract(15) , liver (16) ,
pancreas(17) , kidney(18) , and the reproductive
system(19) .
The enzyme, nitric-oxide synthase (NOS), which catalyzes the biosynthesis of NO has been purified(20, 21, 22, 23) , and its cDNA cloned in mammals from the brain(24, 25) , endothelium(26, 27, 28, 29, 30) , macrophage (31, 32) , and hepatocyte(33) . There are, at least, three genetically distinct types of NOS: type 1 (nNOS), a constitutive form which was initially identified in neurons; type 2 (iNOS), an inducible form from macrophages, and type 3 (eNOS), a constitutive form which was initially identified in endothelium. All isoforms utilize the amino acid arginine, molecular oxygen, and NADPH as substrates and require tetrahydrobiopterin, FAD, and FMN as cofactors(3) . The two constitutive forms are activated by and dependent on changes in intracellular calcium(5) , whereas the inducible isoform is calcium independent apparently because calmodulin is a tightly bound subunit of the iNOS(34) .
Although there
has been considerable research on the biological functions of NO and
the regulation of NOS in humans and rodents(35) , little is
known of NOS in any nonmammalian system. It has been reported that
other species are capable of producing NO, such as Limulus
polyphemus(35) , hematophagous insects(36) ,
fish(37) , and chickens(38) . In addition, there is a
single report of a primary structure of the constitutive NOS from Drosophila(39) but there are no known NOS sequences
from other nonmammalian species. This has not only impeded the
understanding the evolution of NOS protein but also made the study of
the regulation of NOS at the molecular level in other species
impossible. Since chickens do not possess the urea cycle (40) and thus can not synthesize the substrate of NOS
(arginine) directly, the chick represents a unique and potentially
important model to study NOS gene expression. In an effort to study NOS
regulation and to evaluate the evolution of NOS, we have cloned the
first nonmammalian inducible NOS cDNA from a chicken macrophage cell
line. In addition, we report the cloning and analysis of chicken iNOS
5`-flanking region. We have identified an upstream region of chicken
iNOS gene responsible for LPS stimulation. In this LPS-responsive
region, several transcription factor binding elements were identified,
but NF-B was shown to be involved in the induction of chicken iNOS
gene expression.
Figure 4: Nucleotide sequence of the chicken iNOS 5`-flanking region. The mRNA initiation site is denoted as nucleotide position +1 which is bold. Intron 1 is enclosed with parentheses located from +86 to +182 (97 nucleotides). The complementary sequence of nucleotides, +52 to +71, was made as a primer (underlined arrow), 20 nucleotides long, for the primer extension experiment. A putative TATA box is located at nucleotide -30 (boxed). The region associated with LPS response was labeled with a vertical line. Potential transcription factor binding sites are underlined and labeled. All the transcription factor binding sites shown are perfect matches with reported consensus sequences.
Figure 1: The deduced amino acid sequence of chicken macrophage iNOS (CKiNOS) aligned with the amino acid sequences of murine macrophage iNOS (MUiNOS)(31, 32) , human chondrocyte iNOS (HUiNOS)(44) , bovine endothelial cNOS (BOeNOS)(28) , and rat neuronal c NOS (RAnNOS)(24) . Gaps introduced in the sequences to optimize alignments are shown as dots. The identical amino acids are shown as asterisks. Putative cofactor binding sites are drawn in boxes as indicated. CM, calmodulin binding site; Heme, heme binding site; FMN, FMN binding site; F, FAD binding site; N, NADPH binding site. The residues used to design the primers for polymerase chain reaction are shown in bold italic.
Table 1shows the percent identity (homology) of NOS protein sequences between different isoforms and species by using ``GAP'' algorithm in the GCG program. The deduced chicken macrophage NOS protein sequence showed 66.6% (79.1%), 70.4% (81.2%), 54.2% (71.7%), and 48.7% (66.1%) sequence identity (similarity) to mouse iNOS(31, 32) , human iNOS(44) , rat brain cNOS(24) , and bovine endothelium cNOS(30) , respectively. Among the three different isoforms of NOS, there is approximately 50% identity within the same species, e.g. identity among iNOS, eNOS, and nNOS in human. However, eNOS or nNOS among different species exhibited 93% identity, whereas the identity of iNOS among different species was about 81%. Thus, it appears that the percent identity among the same isoforms of NOS between species was considerably higher than that of different isoforms within the same species.
Figure 2: Northern blot analysis of total RNA from endotoxin-treated HD11 chicken macrophages. Total RNA was obtained from chicken macrophages treated with LPS for 0, 4, 8, 12, or 24 h respectively (lanes 1-5), and 30 µg of total RNA was loaded in each lane. The results indicate that NOS mRNA accumulated from 0-12 h after cells were treated with LPS; the most NOS mRNA was at the 12-h time point, and, at 24 h, NOS mRNA was decreased.
Figure 3:
Transient expression of chicken iNOS cDNA.
NOS activity in COS-1 cells transfected with chicken iNOS cDNA which
was cloned into an expression vector, pcDNA 3 (Invitrogen), in a sense
direction or antisense direction as assessed 48 h after transfection.
NO was measured as indicated under
``Materials and Methods.'' In COS-1 cells transfected with
sense direction, NOS specific inhibitors, L-NMMA or
aminoguanidine (100 µM), were added in culture medium with
or without L-arginine or D-arginine (1 mM).
Each data point was a mean ± S.E. obtained from four samples.
These data were similar in three independent
experiments.
The mRNA transcription initiation site, designated nucleotide +1, was identified by primer extension analysis using total RNA from LPS-stimulated HD11 cells (Fig. 5). There was no band in the same primer extension analysis by applying total RNA isolated from unstimulated HD11 cells (lane U). A putative TATA box was identified at 30 bp upstream of the transcription initiation site. Comparing the sequence identity of the 300 bases upstream of the transcription start site of chicken iNOS gene to that of human and mouse iNOS genes showed 39.8% and 35% identity, respectively.
Figure 5:
Primer extension analysis to map the
chicken iNOS mRNA initiation site. The oligonucleotides, 20-mers, as
indicated in Fig. 4were used in the study. Total RNA isolated
from HD11 cells with or without LPS treatment was hybridized to the P-end-labeled primer. The primer was extended with avian
myeloblastosis virus reverse transcriptase, and the products were
electrophoresed on a 6% sequencing gel along with a dideoxy sequencing
ladder applying the same primer. The primer extension using RNA from
unstimulated HD11 cells was denoted U; RNA induced by LPS
stimulation was denoted as I.
Figure 6:
Deletion analysis of the 5`-flanking
region of the chicken iNOS gene. Progressive 5` end deletions of
chicken iNOS promoter were ligated in front of the luciferase gene, and
transfected into chicken macrophages, HD11, along with control plasmid,
-galactosidase. Transfected cells were stimulated with medium
alone or in the presence of LPS (100 ng/ml) for 15 h. Activities of
luciferase and
-galactosidase were determined (as described under
``Materials and Methods''). The luciferase activities were
normalized by dividing the activities of
-galactosidase to adjust
the transfection efficiency. The clone containing the longest iNOS
promoter in front of the luciferase gene was assigned as 100 as
relative luciferase. LPS treatment did not affect
-galactosidase
activity (data not shown). Data are means ± S.E. of triplicates
from 2-3 independent experiments.
Figure 7: The effect of pyrrolidine dithiocarbamate (PDTC) on the synthesis of nitrite and the accumulation of iNOS mRNA in chicken macrophages. A, HD11 cells were treated with LPS (100 ng/ml) in the presence of various amounts of PDTC as indicated in the figure. Nitrite concentration was determined 48 h after treatment. Values from cells treated with LPS only were set as 0% inhibition. Data were obtained from duplicate wells. B, Northern blot analysis of HD11 cells treated with or without LPS (100 ng/ml) together with or without PDTC (40 µM) as shown in the figure. Total RNA was isolated 12 h after treatment, and 10 µg of total RNA was loaded in each lane. The ethidium bromide staining showed equal loading of total RNA across samples.
We report herein the first nonmammalian inducible NOS cDNA
isolated from chicken macrophages. Our data show that NOS is moderately
conserved overall, and it is highly conserved with respect to critical
cofactor binding sequences. We also identified approximately 3.2 kb of
the upstream region of the chicken iNOS gene. From the deletion study (Fig. 6), we show that LPS responsive elements are located
exclusively within 300 bp upstream of the transcription initiation
site. Within these 300 bp, several consensus DNA binding elements were
identified for transcriptional factors which may be regulated by LPS.
NF-B was identified as a potentially important transcription
factor since both mouse and human iNOS (31, 32, 44) contain this response element.
The results from the deletion and inhibitor studies ( Fig. 6and Fig. 7) appear to indicate that NF-
B is indeed a required
nuclear factor to activate the chicken iNOS gene expression by LPS.
The chicken iNOS amino acid sequence is approximately 70% identical
with mammalian inducible forms of NOS. Assuming divergence of chickens
and mammals at approximately 220 million years(47) , the rate
of evolution can be calculated to be approximately 12 residues/100
million years. This rate of evolution is comparable to that described
for histone H1 protein (47) and metallothionein(48) ,
greater than some proteins like histone H4, H3, H2A, and H2B (0.25,
0.30, 1.7, and 1.7 residues/100 million years), cytochrome c (6.7 residues/100 million years), and insulin (7.1 residues/100
million years) and lower than other proteins like albumin, hemoglobin
and
, lysozymes, and fibrinopeptide B (20, 27, 30, 40, 91
residues/100 million years, respectively). This analysis suggests that
the rate of evolution of the iNOS is moderate. Furthermore, upon
comparing the different isoforms of NOS among several different
species, the percent identity between the same isoform among the
different species is considerably higher than different isoforms within
the same species. This suggests that the same isoform among different
species was derived from the same ancestral gene and that the different
isoforms of NOS evolved earlier than the separation of species.
Recently, a bidomain structure of rat brain NOS protein was suggested by Sheta et al.(49) . The authors demonstrated that nNOS can be isolated in two functionally intact subunits. We were interested to determine whether there is a difference in the conservation within these two domains and therefore examined the chicken macrophage NOS protein sequence by individual domains. The N-terminal domain of chicken iNOS, the sequence before the putative calmodulin binding site, showed 73% to 61% identity to all published NOS sequences and no sequence similarity to other known proteins. The C-terminal domain of chicken iNOS also showed similar sequence identity to all the published NOS sequences (66% to 49%) and high sequence similarity to P450 reductase (50) and ferrodoxin reductase(51) . Thus, the two domains of iNOS appear to be equally conserved. Since the putative calmodulin (52) and heme (53) binding sites were highly conserved, this suggests that the N-terminal domain of NOS assumes a function unique to NOS with no similarity to other known enzymes. When aligned with the ferridoxin reductase sequence(54, 55) , the published NOS protein sequences were highly conserved at the NADH and FAD cofactor binding sequences (Fig. 1). This reinforces the notion that the catalytic function of the C-terminal domain of NOS is to transfer electrons to heme moiety binding at the N-terminal domain.
The transcription initiation site of chicken iNOS was identified to be a single start site, G nucleotide, by primer extension. There is a putative TATA box, 30 bp upstream of the transcriptional initiation site which is identical with the mouse (30 bp) and human (30 bp) iNOS genes(56, 57, 58) . Furthermore, when total RNA from unstimulated chicken macrophages was used in primer extension analysis, there was no detectable iNOS mRNA. This result confirmed the data seen in the Northern blot analysis and reinforced the observation that the accumulation of chicken macrophage iNOS mRNA is induced by LPS.
The 5`-flanking region, 3,145 bp, of chicken iNOS gene conferred inducibility by LPS. The major finding of the present study is that a 267-bp upstream region contained complete LPS inducibility. The relative luciferase activity of a reporter gene containing 267 bp of the upstream region of the iNOS gene responded fully to LPS stimulation and was not enhanced by the additional 5`-flanking sequence. Thus, the 267 bp upstream of iNOS gene seemed to contain the elements essential for endotoxin-induced transcriptional activation. Moreover, this region can be divided into two subregions: subregion I, -179 bp to -93 bp and subregion II, -267 bp to -179 bp based on the differential activity following LPS activation.. The clone containing minimum 5`-flanking sequences, -93 bp to +259 bp, failed to exhibit any promoter activity as stimulated by LPS. With the addition of subregion I in front of this minimum 5`-flanking region, there was a 34-fold increase in LPS-induced luciferase activity as compared to that of the clone with minimum 5`-flanking sequences. When subregion II was also present, LPS induced luciferase activity by an additional 5-fold which then totaled a 178-fold increase in LPS-induced luciferase activity when compared to the minimum 5`-flanking sequence. This result suggested that only subregions I and II are required for maximum induction of iNOS gene transcription by endotoxin. Again, this result contrasts observations reported for the mouse macrophage NOS where two distinctly separated regions (-1029 to -913 and -209 to -48) were identified(58) .
Analyzing the chicken LPS-sensitive regions
revealed several consensus elements for transcription factors which may
be responsible for LPS stimulation. Subregion I contains NF-B (59) and C/EBP consensus binding elements(60) .
NF-
B has also been shown to exist in the promoters of mouse (56, 58) and human (57) iNOS genes. Moreover,
NF-
B has been shown to be a required nuclear factor for the
induction of mouse iNOS gene by LPS(46) . In the present study,
the NF-
B inhibitor, PDTC, was able to block almost completely both
nitrite accumulation in culture medium and NOS mRNA accumulation in
LPS-stimulated macrophages. This result suggests that NF-
B is also
functionally involved in the transcriptional regulation or activation
of chicken iNOS gene by LPS. From our data and those of the mouse, we
might predict that either the NF-
B consensus element is also
functionally significant in human iNOS promoter. If not, then the human
iNOS gene represents a unique iNOS system relative to mouse and avian.
Subregion II of the chicken promoter contains PEA1 and PEA3 consensus
binding elements(61, 62) . These elements have been
shown in the enhancer region of polyomavirus, and they are important
for polyomavirus DNA replication and RNA
transcription(63, 64) . PEA1 and PEA3 also appear in
the upstream region of collagenase gene(65) , and they have
been demonstrated to modulate
12-O-tetradecanoylphorbol-13-acetate induction of collagenase
gene. However, PEA1 and PEA3 are not present in the promoters of mouse
and human iNOS genes. Further studies are needed to characterize the
promoter sequence of chicken iNOS gene in detail to gain a greater
understanding of the mechanisms that regulate chicken iNOS gene
expression.
The early portion (400 bp) of the 5`-flanking region of
human iNOS gene is approximately 67% identical with the mouse iNOS
gene(57) . Although this upstream sequence of iNOS gene between
human and mouse shares high sequence similarity, there is as yet no
functional analysis of the 5`-flanking region of human iNOS gene. The
5`-flanking region of chicken iNOS gene was shown in the present study
to contain the LPS-responsive region within 300 bp upstream of the
transcription initiation site. When comparing the sequence identity
between this 300 bp of 5`-flanking sequences of chicken iNOS gene with
that of mouse or human iNOS genes, there was a low identity: 35% to
mouse iNOS gene and 39.8% to human iNOS gene. Essentially, the data
suggest that there is little sequence identity in 300 bp of 5`-flanking
sequences of chicken iNOS gene compared with those of mouse or human
iNOS genes. On the other hand, the promoter sequences of the chicken,
mouse, and human iNOS genes show: 1) a TATA box located at 30 bp
upstream of the transcription initiation site, 2) an NF-B response
element located at approximately 100 bp upstream of the transcription
initiation site. Moreover, both chicken and mouse iNOS genes have been
demonstrated to possess functional LPS-responsive activity within 300
bp upstream of the transcription initiation site. This indicates that
the avian iNOS promoter shares both similarities and differences to the
mammalian iNOS promoters. The similarities would suggest that either
the 400-bp upstream region of the human iNOS gene is functionally
important or that the human gene is uniquely different.
In summary,
we have cloned the first nonmammalian iNOS cDNA from a chicken
macrophage cell line. The sequence comparison showed that chicken iNOS
protein sequence is approximately 70% identical with mammalian iNOS.
The rate of evolution of the iNOS protein, overall, appears to be
moderate. The molecular size of chicken macrophage iNOS mRNA (4.5 kb)
is comparable to the mammalian iNOS. Functional analysis of 3.2 kb of
the 5`-flanking sequences of chicken iNOS gene showed a LPS-responsive
region located exclusively within 300 bp upstream of the transcription
initiation site, a result in marked contrast to that of mouse iNOS
gene(56, 58) . Also, when comparing the sequences of
these 300 bp between chicken iNOS gene to that of mouse (or human)
gene, there was low identity (35%). Several consensus sequences for
transcription factors were identified: NF-B, C/EBP, PEA1, and
PEA3. Using a NF-
B inhibitor, PDTC, NF-
B was shown to be
involved in the LPS-induced iNOS gene expression, a result similar to
that of mouse iNOS gene. The similarities and differences observed
between the promoters of chicken iNOS gene and those of human and mouse
iNOS genes may provide insight into the molecular mechanisms that
regulate the expression of iNOS and the factors that influence the
expression patterns of iNOS in different species.
Note Added in Proof-deVera et al.(67) recently reported an analysis of the human iNOS promoter.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U46504 [GenBank](cDNA) and U46503 [GenBank ](5`-flanking genomic).