ABSTRACT
It has been suggested that the NF-
[kappa]
B transcription factor family may mediate expression of the gene encoding the
cytokine-inducible form of nitric oxide synthase (iNOS). To establish if nitric
oxide (NO) could in turn affect activity of NF-
[kappa]
B, the ability of NO-donor compounds to influence NF-
[kappa]
B DNA binding activity
in vitro
was investigated. NO-donor compounds sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine (SNAP) both inhibited the DNA
binding activity of recombinant NF-
[kappa]
B p50 and p65 homodimers and of p50-p65 heterodimers. Inhibition of NF-
[kappa]
B p50 DNA binding by NO-donor compounds involved modification of the conserved redox-sensitive C62 residue, as a C62S p50 mutant was significantly more
resistant to SNP-mediated inactivation. Non-reducing SDS-polyacrylamide gel electrophoresis demonstrated that SNP
could inhibit p50 DNA binding by mechanisms other than the formation of
intersubunit disulphide bonds involving p50 residue C62. Electrospray
ionization mass spectrometry of a synthetic NF-
[kappa]
B p50 peptide containing the C62 residue suggested that NO gas can modify C62 by
S-nitrosylation. This study indicates that NO-donors can directly inhibit the DNA binding activity of NF-
[kappa]
B family proteins, suggesting that cellular NO provides another control
mechanism for modulating the expression of NF-[kappa] B-responsive genes.
The physiological roles of the short-lived free radical gas nitric oxide (NO) have been the subject of intense
interest since the identification of NO as the diffusible agent mediating the
relaxation of smooth muscle (
1
), reviewed in (
2
-
5
). NO has subsequently been shown to be involved in a wide range of biological
processes including vasodilation, platelet aggregation, macrophage cytotoxicity
and neurotransmission.
It has been demonstrated that treatment of cells with NO-generating compounds such as sodium nitroprusside (SNP), S-nitroso-N-acetylpenicillamine (SNAP) and S-nitrosoglutathione (GSNO) influences activation of
the nuclear factor [kappa]B (NF-[kappa]B)/rel/dorsal family reviewed in (
6
,
7
). An early study in normal human peripheral blood mononuclear cells suggested
that treatment with NO-donors SNP and SNAP resulted in the activation of the DNA binding activity
of NF-[kappa]B family proteins (
8
). In contrast, in more recent studies using human vascular endothelial cells,
activation of NF-[kappa]B DNA binding activity following treatment with tumour necrosis
factor-[alpha] (TNF-[alpha]) could be inhibited by treatment with SNP and GSNO (
9
). It was proposed that the mechanism of inhibition by NO-donors involved both stabilization of I[kappa]B[alpha], and increased transcription of the I[kappa]B[alpha] gene (
10
).
Inducibility of the DNA binding activity and nuclear translocation of NF-[kappa]B, whose classical form is a heterodimer of p50 and p65 subunits,
is mediated by the disruption of complexes of NF-[kappa]B and I[kappa]B inhibitor proteins. In unstimulated cells NF-[kappa]B family proteins are normally held in a non
DNA-binding form in the cytoplasm by I[kappa]B proteins. Stimulation of cells with a wide range of agents
results in proteolytic degradation of I[kappa]B[alpha], allowing the nuclear translocation of NF-[kappa]B, now in a form competent for DNA binding. NF-[kappa]B family proteins recognize DNA
sequences related to the [kappa]B motif (5'-GGGACTTTCC-3'). Similar DNA sequences are present in
transcriptional control regions of cellular genes involved in immune and
inflammatory responses, and are important for transcription of a number of
viral genes, such as the human immunodeficiency virus (HIV) provirus (reviewed
in
6
,
7
).
Members of the NF-[kappa]B transcription factor family include the p50 (NF-[kappa]B1), p52 (NF-[kappa]B2), p65 (Rel A), Rel B, c-Rel, v-Rel, dorsal and Dif proteins.
These NF-[kappa]B family proteins share a conserved N-terminal ~300 amino acid region known as the NF-[kappa]B/rel/dorsal (NRD) homology region which is
responsible for DNA binding, dimerization and nuclear localization (
6
,
7
). Amino acid residues in the N-terminal part of the NRD region contribute to specific DNA recognition and
are responsible for redox modulation of DNA binding activity (
11
-
16
). The [kappa]B motif DNA is recognised in an unusual way, with the NF-[kappa]B making base and backbone contacts with the DNA over one
complete turn of the double helix (
17
,
18
). Structural analysis of p50 homodimer-[kappa]B motif DNA co-crystals (
19
,
20
) has revealed that the p50 monomer contains two domains separated by a
potentially flexible linker. The N-terminal domain is responsible for most of the specific DNA contacts,
while the C-terminal domain forms the dimer interface but also contacts [kappa]B motif DNA.
It has been suggested that reactive oxygen species such as hydroxyl radicals and
superoxide anions act as mediators of NF-[kappa]B activation by I[kappa]B degradation (
21
,
22
) although NF-[kappa]B must be in a reduced state to bind DNA
in vitro
(
23
). This effect is mediated by the redox state of a cysteine residue conserved in
all NF-[kappa]B/rel/dorsal subunit species, corresponding to C62 in the NF-[kappa]B p50 subunit (
11
-
15
). The C62 residue is located within a polypeptide loop used to make many of the
specific contacts with [kappa]B motif DNA, and contacts a phosphate(s) in the DNA backbone (
19
,
20
).
Following activation of NF-[kappa]B DNA binding activity and NF-[kappa]B translocation to the nucleus, a mechanism is
required to shut off expression of NF-[kappa]B-responsive genes to allow the cell to return to its initial
state. This could be achieved by exporting NF-[kappa]B from the nucleus back to the cytoplasm, a process recently shown
to be mediated by I[kappa]B[alpha] (
24
), and/or by inactivating nuclear NF-[kappa]B.
The promoter of the murine inducible nitric oxide synthase (iNOS) gene contains
two [kappa]B motifs, one of which is critical for inducible gene expression (
25
). Similarly, several cytokine-responsive elements are present in the human iNOS gene promoter, including
a [kappa]B motif, three interferon-[gamma] response elements and a tumour necrosis factor response
element (
26
), suggesting an important role for NF-[kappa]B in NO production. NO has also been shown to inactivate several
proteins by modification of reactive thiol groups (
27
) and mass spectroscopic analysis has shown that NO gas can modify cysteine
groups by S-nitrosylation (
28
). As members of the NF-[kappa]B/rel/dorsal family all posess a reactive redox-sensitive cysteine residue towards the N-terminus of their NRD region, this study explored the
possibility of a direct effect of NO on NF-[kappa]B DNA binding activity using recombinant NF-[kappa]B p50 and p65 proteins.
NF-[kappa]B p65 (residues 12-317), NF-[kappa]B p50 (residues 35-381) and a C62S p50 mutant were
expressed in
Escherichia coli
JM101. The wild-type p50 and C62S mutant p50 proteins were purified as described
previously (
12
), p65 protein was purified similarly. Protein fractions were frozen in liquid
nitrogen and stored at -70oC. Protein purity was determined by analysis under reducing
conditions in 10% polyacrylamide gels containing SDS.
Binding of recombinant NF-[kappa]B proteins to
32
P-labelled DNA containing an NF-[kappa]B recognition site was assayed by electrophoresis on 6% non-denaturing polyacrylamide (44:0.8 acrylamide: bisacrylamide) gels. Typically, 0.7 ng (0.018 pmol)
recombinant p50 protein, 0.62 ng (0.018 pmol) recombinant p65 protein or 0.35
ng of recombinant p50 plus 0.31 ng of recombinant p65 (diluted in PBS, 1 mg/ml
BSA) was added without reducing agents to 17 [mu]l of binding buffer (85 mM NaCl, 8.5% v/v glycerol, 22 mM HEPES pH 8.0, 1.3
mg/ml BSA, 0.17% NP-40, 3.6 mM spermidine, 0.85 mM EDTA pH 8.0, 6.1 mM MgCl
2
). If needed, reducing agents were added at this point and the mixture incubated
on ice for 15 min. Where indicated, NO-generating agents or other reagents were then added and the mixture
incubated on ice [15 min for SNP (Sigma) or 1 h for SNAP (Sigma)]. Finally, 5'-
32
P-labelled double-stranded 16mer [kappa]B motif oligonucleotide (5'-CTGGGGACTTTCCAGG-3', typically 0.05 pmol) was added and the
binding mixture (total volume 20 [mu]l) incubated for 15 min at 20oC before electrophoresis at 200 V for 40 min in 0.5* TBE buffer. After electrophoresis, gels were dried on DEAE-cellulose paper (Whatman, DE81) and visualised by
autoradiography.
To detect intersubunit disulphide crosslinks involving C62 thiol groups of p50
homodimers (or other possible covalent intersubunit crosslinks), p50 proteins
were analysed by electrophoresis in the presence of SDS on non-reducing 10% polyacrylamide gels (with 29:1 acrylamide:bisacrylamide). Following electrophoresis, SDS-protein complexes were electroblotted (LKB semi-dry blotter, with 20 mM Tris, 150 mM glycine, 20% methanol
transfer buffer) onto polyvinylidene difluoride (PVDF, Sigma) membrane. PVDF
membranes were blocked for 1 h in PBS, 0.1% Tween-20, 10% non-fat dry milk (PTM) before incubating for 1 h at room temperature
with polyclonal anti-p50 antiserum diluted 1:1000 with PTM. Membranes were again washed with
PTM and incubated with horseradish peroxidase-conjugated donkey anti-rabbit immunoglobulin (Amersham, diluted 1:500 in PTM) for 30 min at
room temperature. Finally, PVDF membranes were washed twice with PTM, once with
PBS, 0.1% Tween-20 before incubating with enhanced chemiluminescence detection reagent
(Amersham) and detection with X-ray film.
NO concentration measurements were performed in 80 ml of DNA binding assay
binding buffer in an argon-blanketed sealed container at room temperature using an isolated nitric
oxide meter and probe (World Precision Instruments Iso-NO, previously calibrated with NO gas) with 3 min intervals between SNP or
SNAP sample additions.
An oligopeptide corresponding to p50 amino acid residues 43-77 and of predicted average molecular mass 3933.47 Da was synthesised and
then purified by reverse phase HPLC. The integrity of the peptide was confirmed
by N-terminal amino acid sequencing. The p50 peptide was analysed by
electrospray ionization mass spectrometry in the positive ion mode using a VG
Quattro instrument (VG Organic, Altrincham, UK) previously calibrated with
myoglobin. Nitric oxide gas was generated by the reaction of ascorbic acid with
sodium nitrite in solution under a nitrogen atmosphere. The gas generated was
passed through a sodium hydroxide scrubbing solution to remove NO
2
before being passed via a Pasteur pipette at ~20 ml/min for ~5 min into 0.15 mg of reduced p50 peptide dissolved in 50 [mu]l of ultrapure water:methanol:acetic acid (1:1:0.01). Samples of
the peptide solution (5 [mu]l) were analysed by electrospray ionization mass spectrometry before and ~20 min after the NO gas treatment.
To investigate direct effects of NO on NF-[kappa]B DNA binding activity, purified recombinant p50 homodimers were
incubated with NO-donor compounds in the presence or absence of DTT and the DNA binding
activity determined in a gel electrophoresis DNA binding assay using a
32
P-labelled DNA oligonucleotide containing the recognition site for NF-[kappa]B. While SNP in the absence of DTT inhibited p50 DNA binding
in a gradual manner, SNP in combination with 1 mM DTT resulted in a dramatic
inhibition of p50 DNA binding activity (Fig.
1
A). SNP in combination with 2 mM [beta]-mercaptoethanol or with the physiological antioxidant reduced
glutathione (at 2 mM) also potentiated the inhibitory effect of SNP on p50,
although again not as effectively as 1 mM DTT (Fig.
1
A). The dramatic inhibition of NF-[kappa]B p50 DNA binding activity by the combination of SNP and DTT was
not due to any inhibitory effect of DTT itself as reducing agents stimulate the
DNA binding activity of oxidised native and recombinant NF-[kappa]B proteins (
11
-
15
,
23
). Comparison of the two control lanes (+-1 mM DTT) in the absence of SNP clearly illustrates this point (Fig.
1
A).
As several studies had previously identified the critical role of C62 (which is
conserved throughout the NF-[kappa]B/rel/dorsal family) in regulating the DNA binding activity of p50,
the effect of SNP on the DNA binding activity of a C62S mutant of p50 (
12
) was examined. While the wild-type p50 DNA binding activity was completely inhibited by 1 mM SNP in the
presence of DTT (Fig.
4
A), the C62S mutant was considerably more resistant to inactivation by SNP
either in the absence or presence of DTT (Fig.
4
B). This result implicates the redox-sensitive C62 residue of p50 as the target of inactivation by SNP.
As the concentrations of both SNP and SNAP needed to inhibit NF-[kappa]B DNA binding activity
in vitro
were relatively high compared with estimated physiological NO concentrations,
the free NO concentration in the gel electrophoresis DNA binding assay binding
buffer after NO-donor addition was measured using an NO-sensitive probe (World Precision Instruments Iso-NO). The free NO concentrations as measured by the probe were
several orders of magnitude lower than the concentrations of SNP and SNAP (Fig.
5
A and B), suggesting that these
in vitro
phenomena are likely to be physiologically relevant.
Of the chemical modification events which could occur during SNP-mediated inhibition of p50 DNA binding, one possibility might be the
formation of an intersubunit disulphide bond involving residue C62. It has been
previously shown that in oxidised p50 homodimers, the inactivation of p50 DNA
binding is mediated by the formation of an intersubunit disulphide bond
involving C62, a residue known to make close contacts with [kappa]B motif DNA (
11
,
12
,
15
,
19
,
20
). Hence, NF-[kappa]B p50 protein was treated with SNP or the sulphydryl oxidising
agent diamide (Azodicarboxylic acid bis [dimethylamide]) in the presence or
absence of 1 mM DTT, the reaction products analysed by non-reducing SDS-PAGE and correlated with their activity in a DNA binding assay.
The polyclonal anti-p50 western blot analysis of the non-reducing SDS-PAGE suggests that in the absence of 1 mM DTT, the majority
of the SNP-treated p50 is non-covalently associated (although a significant amount of covalently
linked p50 dimer and higher molecular weight forms are visible). Whereas
essentially all of the diamide-treated p50 is present as covalently linked dimer and higher molecular
weight forms in the absence of 1 mM DTT (Fig.
6
A). The relatively weak chemiluminescence signals from the covalently linked p50
forms is probably due to poor transfer of these high molecular weight species
from the polyacrylamide gel to the PVDF membrane. Comparison with the
corresponding DNA binding assay reveals that, as expected, treatment of p50
with either SNP or diamide in the absence of DTT causes almost complete loss of
DNA binding activity (Fig.
6
B).
As the redox-sensitive p50 C62 residue has been implicated as the primary target of SNP-mediated inactivation of p50 DNA binding activity, a synthetic
peptide corresponding to p50 residues 43-77 (of predicted average molecular mass 3933.47 Da) was studied by
electrospray ionization mass spectrometry. Initially the mass of the p50
peptide in a water:methanol:acetic acid (1:1:0.01) solution was measured
following transformation from a mass/charge scale to a true molecular mass
scale as 3933.5 Da (Fig.
7
A). Following exposure to the NO gas stream for ~5 min, a significant fraction of the p50 peptide had been modified to yield
a species of mass 3962.6 Da (Fig.
7
B). This increase in mass of 29.1 Da would be consistent with the nitrosylation
of the peptide with the loss of a hydrogen. A similar mass increase was seen
following treatment of the p50 peptide with the NO-donor SNAP (data not shown).
As the common property of SNP and SNAP is their behaviour
in vivo
and
in vitro
as NO-donors, it seems likely that free NO or a closely related species such as
NO
+
is directly involved in the
in vitro
modification of NF-[kappa]B proteins. This behaviour is similar to that seen with AP-1 transcription factor DNA binding activity in cerebellar
granule cell nuclear extracts, in that SNP treatment inactivated AP-1 (
29
). Interestingly, AP-1 transcription factor DNA binding activity is also modulated by the redox
state of a conserved cysteine residue in each subunit of the dimer (
30
).
Although the concentrations of SNP and SNAP needed to inhibit the DNA binding
activity of NF-[kappa]B proteins
in vitro
in these studies were high (in the range of 0.1-1 mM) compared with normal physiological NO concentrations, ~10 nM in the brain (
31
), the concentrations of free NO as measured by the NO probe were much lower (in
the range 20-100 nM) suggesting that the NO-donor inhibitory species could be present at physiologically-relevant concentrations. Although the exact nature(s) of the
inhibitory species is uncertain, for the NO-donor SNP one candidate is NO
+
which, unlike NO, can readily react with thiolate groups to form RS-N=O (
4
,
32
).
Intriguingly, a recent report has shown that NO can regulate the reactivation of
latent Epstein-Barr virus by down-regulation of the redox-sensitive viral transcription factor Zta. Since Zta can
activate the expression of its own gene, it was suggested that NO could inhibit
Zta function by reaction with its reactive cysteine residue(s) either through S-nitrosylation or by accelerating formation of an intersubunit disulphide
bond (
33
).
The role of NO in modulating the activation of NF-[kappa]B
in vivo
remains confused. In normal human peripheral blood mononuclear cells, SNP and
SNAP have been proposed to activate the latent, cytoplasmic form of NF-[kappa]B family proteins and allow their nuclear translocation (
8
) in a process which was suggested to involve the direct activation of guanine
nucleotide-binding proteins by NO (
34
). Whereas more recent studies with human and bovine vascular endothelial cells
have suggested that NO-donor treatment could inhibit NF-[kappa]B-dependent gene transcription (
9
), this inhibition of NF-[kappa]B activation being shown to result from the protection of I[kappa]B[alpha] from proteolytic degradation, and from the
transcriptional upregulation of the I[kappa]B[alpha] gene (
10
). Further recent studies have shown that the NOS inhibitor NMMA can
significantly enhance the activation of NF-[kappa]B DNA binding activity following treatment of a mouse macrophage
cell line with bacterial lipopolysaccharide (LPS). The authors suggested that
endogenous NO could have a negative feedback or modulatory role on the
regulation of the NF-[kappa]B factor, possibly involving modification of the conserved NF-[kappa]B p50 cysteine 62 residue (
35
).
It was recently demonstrated that the promoter/enhancer region of the murine
iNOS gene contains two [kappa]B motif binding sites for NF-[kappa]B family proteins, one of whose function is critical for the
induction of iNOS gene expression in LPS-treated macrophages (
25
), similarly the human iNOS gene has a [kappa]B motif among the cytokine-responsive elements in its 5' flanking region (
26
). These [kappa]B motifs would be consistent with studies on human brain microglial cells
showing that LPS plus tumour necrosis factor-[alpha] induction of iNOS gene expression could be inhibited by pre-treating cells with either SNP or NO gas solution (
36
). This presence of functionally important [kappa]B motifs in iNOS genes has interesting parallels with the transcriptional
regulation of genes encoding I[kappa]B inhibitor proteins which normally function to maintain NF-[kappa]B family proteins in the cytoplasm and to inhibit their DNA
binding activity. Thus, I[kappa]B[alpha] gene promoters have functionally important [kappa]B motifs (
37
-
39
), as have the NF-[kappa]B1 (p50 precursor) and NF-[kappa]B2 (p52 precursor) gene promoters (
40
,
41
).
Early studies of NF-[kappa]B-dependent HIV transcriptional enhancer activity indicated that chronic HIV infection of a human
monocytic cell line resulted in increased NF-[kappa]B DNA-binding activity and activation of HIV proviral transcription
(
42
). Recently, significant levels of iNOS mRNA were shown to be present 6-7 days after infection of normal human monocytes with HIV but to be
undetectable in uninfected monocyte cultures (
43
). This correlated with NO levels, assayed as total nitrite formation, in the
culture medium rising from being undetectable to 2-5 [mu]M. Interestingly while HIV reverse transcriptase activity in the
infected monocyte culture reached a maximum after 6 days, activity fell
dramatically after this, meanwhile NO levels peaked sharply then declined after
7 days post-infection. It seems possible that this behaviour in HIV infected monocytes
might have a periodic nature as an earlier study showed some evidence for long-term oscillations in reverse transcriptase activity in an HIV infected
monocytic cell line (
42
).
Thus one scheme could envisage cell activation leading to the activation of DNA
binding and the nuclear translocation of NF-[kappa]B-in turn causing the transcriptional activation of a range of
genes (including the iNOS, I[kappa]B[alpha] and several NF-[kappa]B family genes). NO produced by newly expressed iNOS
might then act in concert with newly synthesised I[kappa]B to inhibit the DNA binding activity of NF-[kappa]B and shut off the transcription of NF-[kappa]B-responsive genes to allow the cellular NF-[kappa]B/I[kappa]B system to return to
its initial state.
We are very grateful to John Nicholson and Ellis Jaffray for large scale protein
purification, and to Bill Blyth for photographic services. We are also most
grateful to Dr A. R. Butler for kindly supplying SNAP and for much useful
advice, and thank Haitham Al-Sadony for patient instruction on use of the NO meter. This work was
supported by the BBSRC and the EC concerted action project ROCIO.
REFERENCES
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