(Received for publication, July 18, 1994; and in revised form, October 25, 1994)
From the
Nuclear factor B (NF-
B), consisting of p50 and p65, is
bound to a cytoplasmic retention protein, I
B, in a resting state,
and the stimulation of cells with a variety of inflammatory stimuli
induces the dissociation of NF-
B from I
B and the nuclear
translocation of NF-
B, thereby activating several genes involved
in inflammatory responses, such as interleukin (IL)-6, IL-8, and tumor
necrosis factor
. In order to elucidate the precise mechanism of
NF-
B activation, we have established lipopolysaccharide
(LPS)-dependent NF-
B activation in a cell-free system using plasma
membrane-enriched, cytosol, and nuclear fractions extracted from a
human monocytic cell line, THP-1, by disruption with sonication
followed by a differential centrifugation. The combination of plasma
membrane-enriched fraction and cytosol was sufficient to activate
NF-
B in a LPS/CD14-dependent manner only in the presence of ATP as
judged by the binding of NF-
B to the IL-8 gene
B site on an
electrophoretic mobility shift assay. LPS-dependent NF-
B
activation was inhibited by protein kinase inhibitors, such as
staurosporine, herbimycin A, tyrphostin, and genistein, but not
mitogen-activated protein kinase substrate, cGMP-dependent protein
kinase, cAMP-dependent protein kinase, protein kinase C, and
calmodulin-dependent protein kinase II inhibitory peptides, suggesting
that staurosporine-sensitive kinase(s) as well as tyrosine kinase(s)
are involved in LPS-mediated NF-
B activation. In addition, LPS
induced the phosphorylation of I
B-
, starting at 5 min after
the stimulation in a cell-free system. Moreover, the phosphorylation
was inhibited by herbimycin A and tyrphostin, but not staurosporine,
suggesting that these protein kinase inhibitors act at distinct steps
of signal transmission. Establishment of ligand-dependent activation of
NF-
B in a cell-free system will facilitate identification of
protein kinase(s) and its substrate(s) involved in LPS-mediated
NF-
B activation.
Several lines of evidence indicate that interleukin 8 (IL-8), ()a member of the leukocyte chemotactic cytokine (chemokine)
family, is essentially involved in neutrophil-dependent tissue damage
in acute inflammatory reactions(1, 2, 3) .
Most types of cells can produce IL-8 massively and rapidly only in
response to various inflammatory stimuli, including lipopolysaccharide
(LPS), IL-1, and tumor necrosis factor (TNF)
(4, 5) . Moreover, the production is regulated at
the transcriptional level through the activation of NF-
B complexes
in conjunction with NF-IL-6 or AP-1
complexes(6, 7, 8) .
NF-B, originally
identified as a transcription factor necessary for the Ig
gene(9) , is a pleiotropic transcription factor that regulates
the activation of various inflammatory
genes(10, 11, 12) . In quiescent cells,
NF-
B is ordinarily present in the cytoplasm in association with
its inhibitor, I
B(13, 14) . Activation of cells
can induce the phosphorylation of I
B and its dissociation from the
complexes with subsequent nuclear translocation of
NF-
B(13) . No definitive proof, however, has yet been
presented on the relationship between the phosphorylation of I
B
and the activation of NF-
B. Alternatively, several independent
groups claimed that the activation of NF-
B required degradation of
I
B by a chymotrypsin-like protease (15) and/or the
phosphorylation of serine residues of p65 and p50, both of which are
components of NF-
B(16) .
Interaction of LPS with its
receptors on monocytic cells activates the NF-B complexes,
inducing a rapid but transient expression of a defined set of genes
such as IL-1, IL-6, IL-8, and
TNF
(17, 18, 19, 20, 21, 25) ,
although the precise mechanisms remain to be investigated. Recently,
several independent groups documented that LPS induced activation of
several protein kinases, such as mitogen-activated protein kinases
(MAPK)(22, 23, 24) , protein kinase C
(PKC)(25, 26, 27, 28) , and
cAMP-dependent protein kinase (PKA)(25) . In addition, a
tyrosine kinase inhibitor could inhibit LPS-induced production and mRNA
expression of IL-1, TNF
, and IL-6(25, 29) .
Moreover, herbimycin A inhibited LPS-induced NF-
B complex
formation in intact cells(25) . These results raised the
possibility of the involvement of tyrosine kinase(s) in LPS-induced
signal transmission (25, 26, 27, 28, 29, 30, 31) .
In these previous reports, however, the signal transduction pathway has
been examined by adding synthetic protein kinase inhibitors to intact
cells. Thus, permeability and specificity of these protein kinase
inhibitors could hinder the identification of the protein kinase(s)
essentially involved in LPS-induced signal transmission. Hence, we
examined the activation of NF-
B complexes in a cell-free system
using the NF-
B binding site in the IL-8 gene. Several protein
kinase inhibitors were used in this system to explore the LPS-signaling
pathway, particularly with a reference to the role of MAPK and tyrosine
kinase(s).
Each immunoprecipitate was processed to SDS-PAGE analysis. The gels were dried and visualized by an image analyzer.
Figure 1:
NF-B
binding site is indispensable for IL-8 gene expression. The effects of
point mutation on inducibility of luciferase activity by LPS are shown.
The cells were transfected with the indicated luciferase expression
vectors. Intracellular luciferase activities were determined on cells
stimulated with (closedbar) or without LPS (10
µg/ml) (openbar) for an additional 24 h. Mean
± 1 S.D. is calculated on the results from three independent
experiments.
Figure 2:
EMSA using the NF-B binding site in
the IL-8 gene as probe. Nuclear proteins were extracted from THP-1
cells stimulated for 30 min with medium (lanes1 and 3) or LPS (1 µg/ml, lanes2 and 4-8). EMSA was performed on nuclear extracts
preincubated with no reagents (lanes1 and 2), NF-
B oligomer (lanes3 and 4), anti-c-Rel against the C-terminal 15 peptides of human
c-Rel (lane5), anti-c-Rel against residues
304-321 of human c-Rel (lane6), anti-p65 (lane7), anti-p50 (lane8),
anti-p52 (lane9), and anti-RelB (lane10).
Figure 3:
IL-8 NF-B complex formation induced
by LPS in a cell-free system. A, EMSA on postnuclear fraction
prepared from unstimulated THP-1 cells. EMSA was performed on the
postnuclear fraction in the absence (lanes1 and 2) or presence (lanes3 and 4) of
LPS (20 µg/ml) and in the absence (lanes1 and 3) or presence (lanes2 and 4) of
ATP (10 mM) as described under ``Experimental
Procedures.'' The lowerpanel showed relative
binding activity by quantitation. B, EMSA on cytosol and
plasma membrane-enriched fractions prepared from unstimulated THP-1
cells. EMSA was performed on plasma membrane-enriched fraction alone (lanes 1-4), cytosol fraction alone (lanes
5-8), or both fractions (lanes 9-12), in the
absence (lanes 1, 3, 5, 7, 9, and 11) or presence (lanes 2, 4, 6, 8, 10, and 12) of ATP (10
mM) and in the absence (lanes 1, 2, 5, 6, 9, and 10) or presence (lanes 3, 4, 7, 8, 11, and 12) of LPS (20 µg/ml) as described under
``Experimental Procedures.'' The lowerpanel showed relative binding activity by quantitation. C,
experimental procedure is shown here
schematically.
Figure 4:
Kinetic analysis of NF-B complex
formation. EMSA was performed on the combined plasma membrane-enriched
and cytosol fractions incubated for 0 (lane1), 1 (lane2), 2 (lane3), 5 (lane4), 10 (lane5), and 30 min (lane6) in the absence (panelsA and C) or presence (panelsB and D) of
ATP (10 mM) and the absence (panelsA and B) or presence (panelsC and D) of
LPS (20 µg/ml).
Figure 5:
Analysis of components of the NF-B
complex induced by a cell-free activation. The plasma membrane-enriched
and cytosol fractions were incubated at 30 °C for 10 min in the
presence of ATP and LPS. Then, EMSA was performed after preincubation
with no reagents (lane1), an NF-
B oligomer (lane2), a mutated NF-
B oligomer (lane3), anti-c-Rel against the C-terminal 15 peptides of
human c-Rel (lane4), anti-c-Rel against residues
304-321 of human c-Rel (lane5), anti-p65 (lane6), anti-p50 (lane7),
anti-p52 (lane8), and anti-RelB (lane9) antibody.
Figure 6: The effects of anti-CD14 monoclonal antibody on LPS-dependent cell-free activation of NF-kB IL-8 protein. The plasma membrane-enriched and cytosol fractions were incubated at 30 °C for 10 min without (lanes1, 4, and 7) or with a control antibody (lanes2 and 3) or 3C10 (lanes5 and 6) at the concentration of 10 µg/ml (lanes2 and 5) or 25 µg/ml (lanes3 and 6) in the presence of 20 µg/ml LPS and ATP (10 mM). 25 µg/ml control antibody (lane4) or 3C10 (lane7) were added just before the application to EMSA. Then, EMSA was performed as described under ``Experimental Procedures.''
Figure 7: The effects of several protein kinase inhibitors on NF-kB complex formation. Plasma membrane-enriched and cytosol fractions were incubated at 30 °C for 10 min in the presence of ATP (10 mM) and LPS (20 µg/ml) as well as several protein kinase inhibitors as follows: lane1, no inhibitor; lane2, 100 nM staurosporine; lane3, 50 µg/ml genistein; lane4, 30 µg/ml herbimycin A; lane5, 25 µM tyrphostin; lane6, 50 µg/ml MAPK substrate; lane7, 24.4 µM PKG inhibitory peptide; lane8, 312.5 nM PKA inhibitory peptide; lane9, 400 nM PKC inhibitory peptide; lane10, 300 nM CaMPKII inhibitory peptide. Then, EMSA was performed as described under ``Experimental Procedures.'' The lowerpanel showed relative binding activity by quantitation.
Figure 8:
LPS-dependent cellular IB-
phosphorylation. A, patterns of protein phosphorylation in a
cell-free system after LPS stimulation 0 (lane1), 1 (lane2), 2 (lane3), 5 (lane4), 10 (lane5), and 30 min (lane6) are shown. Significantly phosphorylated proteins and
molecular standards (Bio-Rad) are indicated by arrows. B, kinase reaction was performed in a cell-free system
composed of plasma membraneenriched and cytosol fractions for 10 min
with (lanes 2-7) or without (lane1)
LPS (20 µg/ml) in the presence of protein kinase inhibitors as
follows: lanes1 and 2, no inhibitor; lane3, staurosporine; lane4,
genistein; lane5, tyrphostin; lane6, herbimycin A; lane7, MAPK
substrate. The reaction mixtures were analyzed by SDS-PAGE as described
under ``Experimental Procedures.'' C, time-dependent
I
B-
phosphorylation. After kinase reaction in a cell-free
system for 0 (lane1), 0.5 (lane2), 1 (lane3), 2 (lane4), 5 (lanes5 and 7-9),
and 10 min (lane6) in the presence (lanes
1-6, 8, and 9) or absence (lane7) of LPS (20 µg/ml), immunoprecipitation was
performed using anti-I
B-
antiserum (lanes 1-7 and 9) or preimmune serum (lane8) in
the presence (lane9) or absence of corresponding
peptide (1 µg) (lanes 1-8) as described under
``Experimental Procedures.'' Then, eluted proteins were
analyzed by SDS-PAGE. The position of I
B-
is shown by an arrow. D, kinase reaction were performed in a
cell-free system for 10 min with LPS (20 µg/ml) in the presence of
protein kinase inhibitors as follows: lane1, no
inhibitor; lane2, staurosporine; lane3, genistein; lane4, MAPK substrate; lane5, herbimycin A; lane6,
tyrphostin. Immunoprecipitation was then performed using
anti-I
B-
antiserum as described under ``Experimental
Procedures.'' Then, eluted proteins were analyzed by SDS-PAGE. The
position of I
B-
is indicated by an arrow.
Accumulating evidence indicates that the activation of
NF-B is crucial for gene expression of several essential
inflammatory cytokines and proteins such as IL-6 (10) and
TNF
(11) . In the case of the IL-8 gene, the NF-
B
binding site is indispensable for gene expression in any type of cell
so far examined(6, 7, 8) . Moreover, several
agents including FK506(36) , glucocorticoid(37) , and
interferon-
(41) suppressed IL-8 gene expression through
the inhibition of NF-
B activation. These findings suggest that
control of NF-
B activation may be beneficial for various types of
inflammatory diseases by controlling the activation of genes encoding
pro-inflammatory cytokines.
Here, we established LPS-dependent
NF-B activation in a cell-free system using NF-
B binding
sites in the IL-8 gene to explore the precise mechanism of NF-
B
activation. As revealed by immunochemical analysis, NF-
B complexes
observed in a cell-free system were identical with those observed in
intact cells stimulated with LPS, indicating that LPSdependent
NF-
B activation could be reconstituted in this system. NF-
B
complexes were observed only when both cytosol and plasma
membrane-enriched fractions were combined in the presence of LPS and
ATP, suggesting the essential involvement of the interaction between
plasma membrane-associated receptor complex containing CD14 and
cytosolic NF-
B complex. Moreover, LPS induced phosphorylation of
several proteins prior to NF-
B complex formation in this system,
implying that this system can be employed for the analysis of the
signaling pathway involved in a ligand-dependent NF-
B activation.
This system has additional advantages over an intact cell system,
since it can avoid the problem about permeability of synthetic protein
kinase inhibitors. Moreover, highly specific peptide protein kinase
inhibitors or antibodies can be directly added in this system. Several
independent groups claimed that LPS could activate
MAPK(22, 23, 24) , tyrosine kinase (25, 26, 27, 28, 29, 30, 31) ,
PKA(25) , or PKC (25, 26, 27, 28) using different
cell lines. However, due to a lack of specific and permeable protein
kinase inhibitors, the relationship between activation of these kinases
and that of NF-B remains to be investigated. Herein, NF-
B
complex formation was not affected by the addition of highly specific
inhibitory peptides or substrates against MAPK, PKA, PKC, PKG, or
CaMPKII, making it unlikely that these kinases are involved in
NF-
B complex formation in LPS-simulated THP-1 cells. Staurosporine
and several tyrosine kinase inhibitors, to a lesser degree, inhibited
NF-
B complex formation, suggesting that staurosporine-sensitive
kinase(s) and tyrosine kinase(s) are involved in NF-
B activation.
Geng et al.(25) reported that herbimycin A
inhibited LPS-induced NF-B activation in human blood monocytes. We
observed that two tyrosine kinase inhibitors, herbimycin A and
tyrphostin, inhibited NF-
B activation in a cell-free system,
suggesting the involvement of tyrosine kinase(s) in NF-
B
activation. Moreover, these two tyrosine kinase inhibitors inhibited
phosphorylation of I
B-
in a cell-free system. Since
phosphorylation of I
B-
is presumed to precede the
dissociation of and nuclear translocation of NF-
B
proteins(13, 14) , these results raised the
possibility that tyrosine kinase(s) was involved in NF-
B complex
activation through phosphorylation of I
B-
. Recently,
Stefanova et al.(42) reported that LPS induced
activation of CD14-associated p53/p56 lyn, one of the src family tyrosine kinases. Hence, it is tempting to speculate that
one of the src family tyrosine kinases such as lyn was involved in LPS signal transmission, particularly in
phosphorylation of I
B-
.
In contrast, staurosporine
completely inhibited NF-B complex formation in a cell-free system
without affecting the phosphorylation of I
B-
. These results
suggest that NF-
B complex formation requires activation of
additional staurosporine-sensitive kinase(s), in addition to the
phosphorylation of I
B-
. These results raise the possibility
that staurosporine-sensitive protein kinase(s) and tyrosine kinase(s)
induce NF-
B complex formation in a cascade manner. However,
staurosporine could not inhibit phosphorylation of I
B-
,
negating the possibility that the target of staurosporine is upstream
of that of tyrosine kinase inhibitors. Staurosporine inhibited
NF-
B complex formation more strongly than tyrosine kinase
inhibitors, making it unlikely that staurosporine inhibits the
activities of kinase(s) downstream of tyrosine kinase inhibitors.
Several lines of evidence indicate that phosphorylation of both p65 and
p50 of NF-
B at serine residues is required for the factors to bind
to their cognate cis-element(16) . Since staurosporine
can inhibit the activities of a wide variety of serine/threonine
protein kinases, the target of staurosporine may be a kinase that
phosphorylates p65/p50, whereas tyrosine kinase(s) may be involved in
the pathway leading to the phosphorylation of I
B.
Identification and purification of IB-
kinase(s), p65,
p50, p105, and c-Rel kinases are necessary to clarify the mechanism
involved in activation of NF-
B, which is essentially involved in
the gene transcription of a wide variety of inflammatory proteins. A
cell-free system that we described herein will facilitate
identification of these related protein kinases and their substrates.