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(Received for publication, July 18, 1994; and in revised form, October 25, 1994) From the
Nuclear factor
Several lines of evidence indicate that interleukin 8 (IL-8), ( NF- Interaction of LPS with its
receptors on monocytic cells activates the NF-
Each immunoprecipitate
was processed to SDS-PAGE analysis. The gels were dried and visualized
by an image analyzer.
Figure 1:
NF-
Figure 2:
EMSA using the NF-
Figure 3:
IL-8 NF-
Figure 4:
Kinetic analysis of NF-
Figure 5:
Analysis of components of the NF-
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 I
Accumulating evidence indicates that the activation of
NF- Here, we established LPS-dependent
NF- 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- Geng et al.(25) reported that herbimycin A
inhibited LPS-induced NF- In contrast, staurosporine
completely inhibited NF- Identification and purification of I
Volume 270,
Number 8,
Issue of February 24, 1995 pp. 4158-4164
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
B Activation in a
Cell-free System (*)
B (NF-B), consisting of p50 and p65, is
bound to a cytoplasmic retention protein, IB, in a resting state,
and the stimulation of cells with a variety of inflammatory stimuli
induces the dissociation of NF-B from IB 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 IB-, 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.
)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) .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, IB(13, 14) . Activation of cells
can induce the phosphorylation of IB 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 IB
and the activation of NF-B. Alternatively, several independent
groups claimed that the activation of NF-B required degradation of
IB 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) .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).
Cell Line
A human monocytic cell line, THP-1,
was maintained in RPMI 1640 medium (Nissui Pharmaceutical, Tokyo,
Japan) supplemented with 5% heat-inactivated fetal bovine serum
(Hyclone Laboratories, Logan, UT), 2 mM glutamine, 100
units/ml penicillin G, and 100 µg/ml streptomycin.Reagents
LPS (Escherichia coli O55
B5, Difco) was dissolved in PBS(-) and stored at -20
°C. Staurosporine (Sigma), genistein (Sigma), herbimycin A
(generously provided by Nihon Kayaku Co. Ltd., Tokyo, Japan), and
tyrphostin (Sigma) were dissolved in 2.05% dimethyl sulfoxide. MAPK
substrate (APRTPGGRR, generously provided by Chugai Pharmaceutical
(Gotemba, Japan)), PKG (RKRARKE, Sigma), PKA (fragment
6-22-NH
, Sigma), PKC (fragment 19-31, LC
Laboratories, Woburn, MA), and calmodulin-dependent protein kinase II
(CaMPKII) (fragment 290-309, Sigma) inhibitory peptides were
dissolved in 20 mM HEPES (pH 7.4) and stored at -20
°C. ATP (Sigma) was dissolved in 20 mM HEPES (pH 7.4) and
stored at -20 °C. [
-
P]ATP
(<110 TBq/mM) was purchased from Amersham Japan (Tokyo,
Japan). Anti-CD14 monoclonal antibody producing hybridoma, 3C10, was
provided from ATCC. Rabbit antisera against human
c-Rel(32, 33) , p65, p50, p52, RelB(34) , and
IB- (35) were prepared as described previously.Luciferase Assay
The 5`-flanking region
spanning from -133 to +44 base pair(s) of the IL-8 gene was
subcloned into a firefly luciferase expression vector(36) .
Site-directed mutagenesis of the IL-8 AP-1, NF-IL-6, and NF-B site
was carried out using polymerase chain reaction, and sites of the
introduced mutation were essentially the same as in the case of the
chloramphenicol acetyltransferase expression vector previously
described(37) . THP-1 cells (2 
10
cells)
were transfected with 5 µg of plasmid DNA using DEAE-dextran (500
µg, Pharmacia-Biotech, Uppsala, Sweden) as described
previously(36) . Transfected cells were divided into two parts
and incubated for 24 h at a cell density of 2.5 10
cells/ml. After the cells were further cultured in the presence
or absence of LPS (10 µg/ml) for 24 h, cell lysates were prepared
using Pica Gene
(Toyo Ink Co., Tokyo, Japan) according to
the manufacturer's instructions. The light intensity was measured
on 20 µg of cell lysates using a Lumat model LB950 luminometer
(Berthold, Germany).Extraction of Nuclear Proteins and Electrophoretic
Mobility Shift Assay (EMSA)
The oligomer used for the
present study was the NF-B binding site of the IL-8 gene
(-83 to -68 base pairs; CGTGGAATTTCCTCTG). THP-1 cells were
stimulated with or without 1 µg/ml LPS for 30 min. Nuclear proteins
were extracted according to the method described by Dignam et
al.(38) . Four µg of nuclear proteins were incubated
with a P-labeled probe (10
cpm/reaction) and
0.5 µg/ml poly(dI-dC)
poly(dI-dC) in 20 µl of binding
buffer (20 mM HEPES, pH 7.9, 60 mM KCl, 4 mM MgCl, 0.2 mM EDTA, 1 mM dithiothreitol, 10% (v/v) glycerol, 2% (w/v) polyvinyl alcohol)
for 20 min at 25 °C. In some experiments, nuclear extracts were
incubated with either 100-fold molar excess unlabeled oligomers or 1
µl of 10 times diluted antisera for 10 min at 4 °C before
radiolabeled probe and poly(dI-dC)poly(dI-dC) were added. Samples
were loaded onto 6% polyacrylamide gel
(acrylamide/N,N`-methylene bisacrylamide, 30:1) with
0.25 Tris borate buffer. After electrophoresis, gels were dried
and analyzed using an image analyzer (BAS 2000, Fuji Film Co., Tokyo,
Japan).Preparation of Cytoplasmic and Membrane Fractions
from Unstimulated THP-1 Cells
Cytosol and membrane
fractions were prepared according to the method described by Sadowski et al.(39) . Briefly, THP-1 cells (<10
cells) at logarithmic growth phase were washed twice with
ice-cold PBS(-) and once with hypotonic buffer (20 mM HEPES, pH 7.9, 1 mM EGTA, 0.5 mM phenylmethylsulfonyl fluoride, 1 µg/ml each of aprotinin,
pepstatin, and leupeptin). Cells were resuspended in 3-4 volumes
of hypotonic buffer, sonicated four times for 5 s each, and centrifuged
for 5 min at 300 g. After removal of nuclei, the low
speed supernatants were centrifuged for 5 min at 300 g. The supernatant postnuclear fraction was adjusted to a
final concentration of 120 mM NaCl and centrifuged for 70 min
at 105,000 g. Glycerol was added to the resultant
supernatants to a final concentration of 10%, and they were frozen
without dialysis as a cytosol fraction. The pellets obtained after the
ultracentrifugation were resuspended in buffer containing 150 mM NaCl and 8% glycerol. After centrifugation at 17,000 g for 30 min at 4 °C, pellets were resuspended in buffer (20
mM Tris, pH 7.4, 1 mM EGTA, 10% glycerol, 1 µg/ml
each of leupeptin, aprotinin, and pepstatin, and 0.5 mM phenylmethylsulfonyl fluoride) and frozen at -80 °C as a
plasma membrane-enriched fraction. Protein contents were determined
using a protein assay kit (Bio-Rad)(37) .EMSA in a Cell-free System
Postnuclear
fractions (40 µg of protein) or cytosol fraction (20 µg of
protein) with or without plasma membrane-enriched fraction (20 µg
of protein) in kinase buffer (50 mM HEPES, pH 7.4, 20 mM MgCl, 10 mM MnCl) were incubated
for the indicated time intervals at 30 °C in the presence or
absence of either 20 µg/ml LPS or 10 mM ATP. The reactions
were carried out in 10.25 µl. The reactions were terminated by
adding 0.5 M EDTA (pH 8.0) to a final concentration of 13.5
mM, and half of each mixture was analyzed by EMSA essentially
in the same manner as described above except that the concentration of
poly(dI-dC)poly(dI-dC) was changed to 0.25 µg/ml.Effects of Anti-CD14 Monoclonal Antibody (3C10) on
LPS-dependent Cell-free Activation
Anti-CD14 monoclonal
antibody (3C10, IgG2b) and a control antibody (anti-human monocyte
chemotactic and activating factor (MCAF), IgG2b) were fractionated
by protein G-Sepharose (Pharmacia) from hybridoma culture supernatants.
Both cytosol fraction and plasma membrane-enriched fraction in kinase
buffer were incubated for 10 min at 30 °C in the presence of LPS
(20 µg/ml) and with or without either control antibody or 3C10 (25
or 10 µg/ml). The reactions were carried out, terminated, and
analyzed by EMSA in the same manner as described above.In Vitro Kinase Reaction and Immunoprecipitation of
I
The mixtures of both cytosol (20 µg of
protein) and plasma membrane-enriched (20 µg of protein) fractions
in kinase buffer (50 mM HEPES, pH 7.4, 20 mM MgClB-, 10 mM MnCl, 300 µM ATP, and 10 µCi of [-P]ATP) were
incubated at 30 °C for the indicated time intervals in the presence
or absence of 20 µg/ml LPS. The reactions were carried out in 10.25
µl. After the reaction was terminated by adding 0.5 M EDTA
(pH 8.0) to a final concentration of 13.5 mM, 0.5 volume of
the reaction mixtures was analyzed by SDS-PAGE. In some experiments, 1
µl of IB- antiserum was added to each reaction mixture at
the termination of reaction and incubated at 4 °C for an additional
3 h. Then, the reaction mixture was added to 30 µl of protein
G-Sepharose (Pharmacia) diluted 2-fold with PBS(-) containing 10
mM EDTA (pH 8.0) and incubated for an additional 3 h. After
these mixtures were washed five times by PBS(-) containing 1%
Nonidet P-40, the bound proteins were eluted by boiling for 2 min in
the presence of 1 SDS sample buffer.
NF-
Since we previously observed that IL-8 transcription
required the combination of NF-B Binding Site Is an Indispensable cis-Element
for LPS-induced IL-8 Gene Expression in a Human Monocytic Cell
LineB with NF-IL-6 or AP-1 binding
sites in several types of
cells(7, 8, 36, 37) , we examined
the effects of mutation of each cis-element on IL-8
promotor-driven luciferase activity in THP-1 cells. The mutation of
NF-B completely abolished the responsiveness to LPS whereas that
of AP-1 or NF-IL-6 binding site failed to eliminate the induction of
luciferase activities by LPS (Fig. 1). These results indicated
that NF-B binding was an indispensable cis-element for
conferring the responsiveness to LPS.
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.
LPS-stimulated NF-
To confirm that IL-8 NF-B Complex Formation in Intact
THP-1 CellsB binding activity is
inducible by LPS in intact THP-1 cells, EMSA was performed on the
nuclear proteins extracted from these cells by using an IL-8 NF-B
binding site as a probe. A faint band was observed in the nuclear
proteins extracted from resting cells and did not disappear in the
presence of an excess amount of cold probe, indicating that it was not
a specific one (Fig. 2, lanes1 and 3). LPS induced two slower migrating bands, both of which were
competed out by the cold probe (Fig. 2, lanes2 and 4), indicating the specificity of the complexes.
Moreover, antibodies to p65, p50, and c-Rel but not those to p52 and
RelB supershifted these two complexes. In addition, the antibody to p65
decreased the amount of NF-B complexes as observed on IL-1-induced
NF-B complexes in a human glioblastoma cell line,
T98G(37) . These results indicated that these complexes were
immunochemically identified to be composed of both p65, p50, and c-Rel (Fig. 2, lanes5-10).
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).
NF-
To analyze biochemically the signaling pathway of
LPS-induced activation of IL-8 NF-B Complex Formation Induced by LPS in a Cell-free
SystemB proteins, an LPSdependent
cell-free activation system for IL-8 NF-B proteins was
established. At first, EMSA was performed on the postnuclear fractions
prepared from unstimulated THP-1 cells. No complex was observed in the
absence of LPS (Fig. 3A, lanes1 and 2). While LPS alone increased the intensity of the band of
NF-B complex 10.6-fold (Fig. 3A, lane3), the complex formation was enhanced a further 2.2-fold
by the addition of ATP (Fig. 3A, lane4). These results implied that LPS-induced NF-B
complex formation depended on the presence of ATP even in a cell-free
system. Next, we separated the cytosol and plasma membrane-enriched
fractions from the postnuclear fraction in order to determine the
contribution of these fractions to the cell-free activation of IL-8
NF-B protein. No NF-B complexes were detected using the
plasma membrane-enriched fraction alone, even if LPS and ATP were added (Fig. 3B, lanes 1-4). NF-B
complexes were detected in the cytosol fraction in the presence of LPS
and ATP (Fig. 3B, lanes 5-8). However,
when both fractions were combined, NF-B complexes were induced by
addition of LPS and ATP more than 3-fold (Fig. 3B, lanes 9-12). In the absence of LPS or ATP, NF-kB
complexes did not appear until 30 min (Fig. 4, A-C). On the contrary, IL-8 NF-B complexes appeared
rapidly within 1 min, reaching a maximum by 10 min after the addition
of both ATP and LPS (Fig. 4D). NF-B complexes
formed in a cell-free system were also inhibited by a specific oligomer
but not by a mutated one (Fig. 5, lanes 1-3).
Moreover, the antibody to p65 decreased the amount of NF-B
complexes while specific antibody to p50 and c-Rel but not that to p52
or RelB supershifted the complexes in a cell-free system (Fig. 5, lanes 4-9). These results indicate that
LPS induced NF-B complexes of the same components even in a
cell-free system as in an intact cell.
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.
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).
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.
CD14 Is Essentially Involved in LPS-dependent Cell-free
Activation of NF-
To investigate whether the signaling
pathway of LPS-induced activation of IL-8 NF-BB protein is mediated
by CD14 or not, the effects of anti-CD14 monoclonal antibody were
examined(40) . Although 3C10 at the concentration of 10
µg/ml had little effect on the activation (Fig. 6, lanes1, 2, and 5), at the concentration of
25 µg/ml it inhibited NF-B activation by more than 70% (Fig. 6, lanes1, 3, and 6).
Furthermore, when 3C10 was added just before the application to EMSA,
it did not show any effects on the complex formation (Fig. 6, lanes1, 4, and 7), suggesting that
the antibody did not interfere directly with the complex formation.
These data indicated that LPS-dependent cell-free activation of
NF-B was largely mediated by CD14.
The Effects on the Activation of NF-
The activation of NF-B by Protein
Kinase InhibitorsB proteins by LPS
depended on the presence of ATP as shown in Fig. 3and Fig. 4, suggesting the involvement of protein kinase(s) in the
signaling pathway of LPS. Hence, we examined the effects of several
protein kinase inhibitors on NF-B complex formation induced by LPS
in a cell-free system. A 3-4-fold ED
value of
staurosporine, herbimycin A, tyrphostin, genistein, and CaMPKII
inhibitory peptide inhibited the formation of NF-B complexes (Fig. 7, upperpanel), and the potency of
their inhibitory activities was as follows: staurosporine >
tyrphostin > herbimycin A > genistein > CaMPKII inhibitory
peptide (Fig. 7, lowerpanel). However, the
3-4-fold ED value of peptide inhibitors against
several other protein kinases, including MAPK, PKG, PKA, and PKC,
failed to inhibit complex formation (Fig. 7). These results
suggested that staurosporine-sensitive kinase(s) and tyrosine kinase(s)
are probably involved in the signaling pathway of LPS-mediated
NF-B activation.
LPS-dependent Cellular I
In vitro kinase reaction revealed that
several proteins whose molecular masses were estimated at 105, 97, and
67 kDa were phosphorylated even without LPS stimulation. LPS induced
markedly the phosphorylation of additional proteins whose molecular
masses were estimated at 130, 110, 65, 55, 50, 40, 36, 34, and 30 kDa,
in a time-dependent manner, reaching a maximum generally within 5 min (Fig. 8, A and B). These results implied that
kinase reactions actually occurred in a cell-free system in a
LPS-dependent manner. Finally, to examine whether or not IB-
PhosphorylationB-
is phosphorylated in response to LPS in a cell-free system, the
reaction mixtures were immunoprecipitated with a specific
anti-IB- antiserum. No significant phosphorylation of
IB- was observed without LPS stimulation, whereas
IB- was strongly phosphorylated at 5 min after LPS
stimulation (Fig. 8C, lanes 1-6).
Moreover, phosphorylated IB- could not be recovered during
immunoprecipitation in the presence of corresponding peptides in
reaction mixtures (Fig. 8C, lanes 7-9),
indicating that specific phosphorylation of IB- occurred in
response to LPS. In addition, the phosphorylation of IB- was
inhibited by herbimycin A and tyrphostin but not by staurosporine or
MAPK substrate (Fig. 8D). These observations indicate
that tyrosine kinase(s) but not staurosporine-sensitive protein kinase
or MAPK is involved in IB- phosphorylation.
B-
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
IB- 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-IB- 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 IB- 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-IB- antiserum as described under ``Experimental
Procedures.'' Then, eluted proteins were analyzed by SDS-PAGE. The
position of IB- is indicated by an arrow.
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.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. 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. 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 IB- in a cell-free system. Since
phosphorylation of IB- 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 IB-. 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 IB-.B complex formation in a cell-free system
without affecting the phosphorylation of IB-. These results
suggest that NF-B complex formation requires activation of
additional staurosporine-sensitive kinase(s), in addition to the
phosphorylation of IB-. 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 IB-,
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 IB. B- 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.
)
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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