©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Establishment of Lipopolysaccharide-dependent Nuclear Factor B Activation in a Cell-free System (*)

(Received for publication, July 18, 1994; and in revised form, October 25, 1994)

Yuji Ishikawa (1) Naofumi Mukaida (1) Kouji Kuno (1) Nancy Rice (2) Shu-ichi Okamoto (1) Kouji Matsushima (1)(§)

From the  (1)Department of Pharmacology, Cancer Research Institute, Kanazawa University, Kanazawa 920, Japan and the (2)ABL-Basic Research Program, NCI-Frederick Cancer Research and Development Center, National Institutes of Health, Frederick, Maryland 21702-1201

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Nuclear factor kappaB (NF-kappaB), consisting of p50 and p65, is bound to a cytoplasmic retention protein, IkappaB, in a resting state, and the stimulation of cells with a variety of inflammatory stimuli induces the dissociation of NF-kappaB from IkappaB and the nuclear translocation of NF-kappaB, thereby activating several genes involved in inflammatory responses, such as interleukin (IL)-6, IL-8, and tumor necrosis factor alpha. In order to elucidate the precise mechanism of NF-kappaB activation, we have established lipopolysaccharide (LPS)-dependent NF-kappaB 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-kappaB in a LPS/CD14-dependent manner only in the presence of ATP as judged by the binding of NF-kappaB to the IL-8 gene kappaB site on an electrophoretic mobility shift assay. LPS-dependent NF-kappaB 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-kappaB activation. In addition, LPS induced the phosphorylation of IkappaB-alpha, 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-kappaB in a cell-free system will facilitate identification of protein kinase(s) and its substrate(s) involved in LPS-mediated NF-kappaB activation.


INTRODUCTION

Several lines of evidence indicate that interleukin 8 (IL-8), (^1)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) alpha(4, 5) . Moreover, the production is regulated at the transcriptional level through the activation of NF-kappaB complexes in conjunction with NF-IL-6 or AP-1 complexes(6, 7, 8) .

NF-kappaB, originally identified as a transcription factor necessary for the Igkappa gene(9) , is a pleiotropic transcription factor that regulates the activation of various inflammatory genes(10, 11, 12) . In quiescent cells, NF-kappaB is ordinarily present in the cytoplasm in association with its inhibitor, IkappaB(13, 14) . Activation of cells can induce the phosphorylation of IkappaB and its dissociation from the complexes with subsequent nuclear translocation of NF-kappaB(13) . No definitive proof, however, has yet been presented on the relationship between the phosphorylation of IkappaB and the activation of NF-kappaB. Alternatively, several independent groups claimed that the activation of NF-kappaB required degradation of IkappaB by a chymotrypsin-like protease (15) and/or the phosphorylation of serine residues of p65 and p50, both of which are components of NF-kappaB(16) .

Interaction of LPS with its receptors on monocytic cells activates the NF-kappaB complexes, inducing a rapid but transient expression of a defined set of genes such as IL-1, IL-6, IL-8, and TNFalpha(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, TNFalpha, and IL-6(25, 29) . Moreover, herbimycin A inhibited LPS-induced NF-kappaB 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-kappaB complexes in a cell-free system using the NF-kappaB 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).


EXPERIMENTAL PROCEDURES

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(2), 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 IkappaB-alpha (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-kappaB 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 times 10^6 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 times 10^5 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-kappaB 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 (10^4 cpm/reaction) and 0.5 µg/ml poly(dI-dC)bulletpoly(dI-dC) in 20 µl of binding buffer (20 mM HEPES, pH 7.9, 60 mM KCl, 4 mM MgCl(2), 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)bulletpoly(dI-dC) were added. Samples were loaded onto 6% polyacrylamide gel (acrylamide/N,N`-methylene bisacrylamide, 30:1) with 0.25times 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 (<10^9 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 times g. After removal of nuclei, the low speed supernatants were centrifuged for 5 min at 300 times g. The supernatant postnuclear fraction was adjusted to a final concentration of 120 mM NaCl and centrifuged for 70 min at 105,000 times 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 times 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(2), 10 mM MnCl(2)) 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)bulletpoly(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, IgG2bkappa) and a control antibody (anti-human monocyte chemotactic and activating factor (MCAF), IgG2bkappa) 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 IkappaB-alpha

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 MgCl(2), 10 mM MnCl(2), 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 IkappaB-alpha 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 1times SDS sample buffer.

Each immunoprecipitate was processed to SDS-PAGE analysis. The gels were dried and visualized by an image analyzer.


RESULTS

NF-kappaB Binding Site Is an Indispensable cis-Element for LPS-induced IL-8 Gene Expression in a Human Monocytic Cell Line

Since we previously observed that IL-8 transcription required the combination of NF-kappaB 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-kappaB 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-kappaB binding was an indispensable cis-element for conferring the responsiveness to LPS.


Figure 1: NF-kappaB 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-kappaB Complex Formation in Intact THP-1 Cells

To confirm that IL-8 NF-kappaB 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-kappaB 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-kappaB complexes as observed on IL-1-induced NF-kappaB 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).


Figure 2: EMSA using the NF-kappaB 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-kappaB 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-kappaB Complex Formation Induced by LPS in a Cell-free System

To analyze biochemically the signaling pathway of LPS-induced activation of IL-8 NF-kappaB proteins, an LPSdependent cell-free activation system for IL-8 NF-kappaB 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-kappaB 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-kappaB 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-kappaB protein. No NF-kappaB complexes were detected using the plasma membrane-enriched fraction alone, even if LPS and ATP were added (Fig. 3B, lanes 1-4). NF-kappaB 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-kappaB 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-kappaB complexes appeared rapidly within 1 min, reaching a maximum by 10 min after the addition of both ATP and LPS (Fig. 4D). NF-kappaB 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-kappaB 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-kappaB complexes of the same components even in a cell-free system as in an intact cell.


Figure 3: IL-8 NF-kappaB 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-kappaB 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-kappaB 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-kappaB oligomer (lane2), a mutated NF-kappaB 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-kappaB

To investigate whether the signaling pathway of LPS-induced activation of IL-8 NF-kappaB 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-kappaB 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-kappaB was largely mediated by CD14.


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.''



The Effects on the Activation of NF-kappaB by Protein Kinase Inhibitors

The activation of NF-kappaB 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-kappaB 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-kappaB 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-kappaB activation.


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.



LPS-dependent Cellular IkappaB-alpha Phosphorylation

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 IkappaB-alpha is phosphorylated in response to LPS in a cell-free system, the reaction mixtures were immunoprecipitated with a specific anti-IkappaB-alpha antiserum. No significant phosphorylation of IkappaB-alpha was observed without LPS stimulation, whereas IkappaB-alpha was strongly phosphorylated at 5 min after LPS stimulation (Fig. 8C, lanes 1-6). Moreover, phosphorylated IkappaB-alpha 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 IkappaB-alpha occurred in response to LPS. In addition, the phosphorylation of IkappaB-alpha 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 IkappaB-alpha phosphorylation.


Figure 8: LPS-dependent cellular IkappaB-alpha 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 IkappaB-alpha 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-IkappaB-alpha 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 IkappaB-alpha 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-IkappaB-alpha antiserum as described under ``Experimental Procedures.'' Then, eluted proteins were analyzed by SDS-PAGE. The position of IkappaB-alpha is indicated by an arrow.




DISCUSSION

Accumulating evidence indicates that the activation of NF-kappaB is crucial for gene expression of several essential inflammatory cytokines and proteins such as IL-6 (10) and TNFalpha(11) . In the case of the IL-8 gene, the NF-kappaB 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-beta (41) suppressed IL-8 gene expression through the inhibition of NF-kappaB activation. These findings suggest that control of NF-kappaB 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-kappaB activation in a cell-free system using NF-kappaB binding sites in the IL-8 gene to explore the precise mechanism of NF-kappaB activation. As revealed by immunochemical analysis, NF-kappaB complexes observed in a cell-free system were identical with those observed in intact cells stimulated with LPS, indicating that LPSdependent NF-kappaB activation could be reconstituted in this system. NF-kappaB 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-kappaB complex. Moreover, LPS induced phosphorylation of several proteins prior to NF-kappaB 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-kappaB 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-kappaB remains to be investigated. Herein, NF-kappaB 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-kappaB complex formation in LPS-simulated THP-1 cells. Staurosporine and several tyrosine kinase inhibitors, to a lesser degree, inhibited NF-kappaB complex formation, suggesting that staurosporine-sensitive kinase(s) and tyrosine kinase(s) are involved in NF-kappaB activation.

Geng et al.(25) reported that herbimycin A inhibited LPS-induced NF-kappaB activation in human blood monocytes. We observed that two tyrosine kinase inhibitors, herbimycin A and tyrphostin, inhibited NF-kappaB activation in a cell-free system, suggesting the involvement of tyrosine kinase(s) in NF-kappaB activation. Moreover, these two tyrosine kinase inhibitors inhibited phosphorylation of IkappaB-alpha in a cell-free system. Since phosphorylation of IkappaB-alpha is presumed to precede the dissociation of and nuclear translocation of NF-kappaB proteins(13, 14) , these results raised the possibility that tyrosine kinase(s) was involved in NF-kappaB complex activation through phosphorylation of IkappaB-alpha. 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 IkappaB-alpha.

In contrast, staurosporine completely inhibited NF-kappaB complex formation in a cell-free system without affecting the phosphorylation of IkappaB-alpha. These results suggest that NF-kappaB complex formation requires activation of additional staurosporine-sensitive kinase(s), in addition to the phosphorylation of IkappaB-alpha. These results raise the possibility that staurosporine-sensitive protein kinase(s) and tyrosine kinase(s) induce NF-kappaB complex formation in a cascade manner. However, staurosporine could not inhibit phosphorylation of IkappaB-alpha, negating the possibility that the target of staurosporine is upstream of that of tyrosine kinase inhibitors. Staurosporine inhibited NF-kappaB 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-kappaB 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 IkappaB.

Identification and purification of IkappaB-alpha kinase(s), p65, p50, p105, and c-Rel kinases are necessary to clarify the mechanism involved in activation of NF-kappaB, 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.


FOOTNOTES

*
This work was supported in part by grants from the Ministry of Education, Science, and Culture of the Japanese Government and Uehara Memorial Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
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The abbreviations used are: IL, interleukin; AP-1, activator protein-1; CaMPKII, calmodulin-dependent protein kinase II; EMSA, electrophoretic mobility shift assay; MAPK, mitogen-activated protein kinase; NF, nuclear factor; PKA, cAMP-dependent protein kinase; PKC, protein kinase C; PKG, cGMP-dependent protein kinase; LPS, lipopolysaccharide; TNF, tumor necrosis factor; PAGE, polyacrylamide gel electrophoresis.


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