Lipopolysaccharide-induced Tumor Necrosis Factor-alpha Promoter Activity Is Inhibitor of Nuclear Factor-kappa B Kinase-dependent*

Jennifer L. Swantek, Lori Christerson, and Melanie H. CobbDagger

From the Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9041

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The adverse effects of lipopolysaccharide (LPS) are primarily mediated by tumor necrosis factor-alpha (TNF-alpha ). TNF-alpha production by LPS-stimulated macrophages is regulated both transcriptionally and post-transcriptionally. Transcriptional regulation of the TNF-alpha gene is dependent on nuclear factor-kappa B (NF-kappa B). We examined the signaling pathways involved in the regulation of NF-kappa B that lead to TNF-alpha promoter activity. We determined a role for one or both of the recently identified inhibitor of NF-kappa B kinases, Ikappa B kinase-1 and Ikappa B kinase-2, in LPS induction of an NF-kappa B reporter and of TNF-alpha promoter activity. Ikappa B kinase activation is one of the earliest signaling events known to be induced by LPS. Furthermore, our results suggest roles for the Ikappa B kinases NF-kappa B-inducing kinase and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 1 in the regulation of Ikappa B kinase-2, as well as in LPS-induced TNF-alpha transcription.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Lipopolysaccharide (LPS)1 is a surface component of Gram-negative bacteria that is released following host infection and causes tissue injury and shock (1). LPS mediates such adverse effects by inducing the production of pro-inflammatory cytokines. One of the most important of these LPS-induced pro-inflammatory cytokine mediators is tumor necrosis factor-alpha (TNF-alpha ). TNF-alpha production in monocytes and macrophages constitutes between 1 and 2% of secreted proteins in response to LPS (2). Purified TNF-alpha induces many of the deleterious effects of LPS in vivo (3); passive immunization against TNF-alpha protects animals from the lethal effects of LPS (4).

The LPS signaling cascade leading to TNF-alpha production bifurcates to control both transcription of the TNF-alpha gene and translation of TNF-alpha mRNA (5). Translational regulation of TNF-alpha mRNA is mediated by a short AU-rich sequence that is conserved among various cytokines and oncogenes and is present within the 3'-untranslated regions of such genes (6). This element confers a repression of translation that must be overcome in order for translation to proceed (7-11). Recent studies also suggest a role for this element in destabilization of TNF-alpha mRNA (12). Signaling molecules shown to play a role in translational regulation of TNF-alpha mRNA include p38 (13) and Jun-N-terminal kinase/stress-activated protein kinase (JNK/SAPK) (14), members of the mitogen-activated protein kinase family, as well as the more proximal signaling molecules Raf and Ras (15). Transcriptional regulation of the TNF-alpha gene is quite complex; differences exist across species as well as among cell types. Regulation of TNF-alpha transcription is conferred by transcription factor binding sites present within the TNF-alpha promoter. The cis-acting elements within the TNF-alpha promoter that are conserved among species include a Y box motif, an SP-1 binding site, as well as multiple nuclear factor-kappa B (NF-kappa B) sites (16, 17). In addition, the human TNF-alpha promoter contains an AP-1 site (18). LPS induction of both murine and human TNF-alpha promoter activity is dependent on NF-kappa B binding sites. Mutation or deletion of such sites results in a loss of LPS responsiveness, whereas multiple copies of the NF-kappa B sites inserted in front of a reporter gene are LPS-responsive (16, 19, 20). Furthermore, compounds that inhibit NF-kappa B block TNF-alpha transcription and TNF-alpha production in human monocytes (21). Evidence arguing against a role for NF-kappa B in regulation of human TNF-alpha gene transcription exists (22, 23). However, the discrepancy may be due to the stimulus used to induce TNF-alpha promoter activity, as well as the cell type.

LPS treatment of macrophages stimulates the nuclear mobilization of NF-kappa B (24, 25). Under normal conditions, NF-kappa B is found in a cytoplasmic complex with an inhibitory protein, inhibitor of NF-kappa B (Ikappa B) (26). Many signals that lead to the nuclear translocation of NF-kappa B result in the phosphorylation and subsequent degradation of Ikappa B. Phosphorylation of Ser-32 and Ser-36 of Ikappa B-alpha targets Ikappa B-alpha for ubiquitination and degradation by the proteosome, allowing NF-kappa B to translocate to the nucleus (27-29). Recently, two kinases have been identified that inducibly phosphorylate Ikappa B: Ikappa B kinase-1 (IKK-1), also called IKK-alpha (30-33), and Ikappa B kinase-2 (IKK-2), also called IKK-beta (31, 33, 34). These kinases are activated by TNF-alpha and interleukin-1beta (30, 33, 34) and are required for cytokine-induced activation of NF-kappa B (30, 32, 33). IKK-1 and IKK-2 show 52% identity at the amino acid level and can exist in a heterodimer that is able to interact with another kinase called NF-kappa B-inducing kinase (NIK). NIK was originally identified based on its ability to bind to TNF-receptor-associated factor 2 and is a member of the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase (MEKK) family (35). Co-expression of NIK with IKK-1 or IKK-2 enhances IKK activity, whereas overexpression of a kinase mutant NIK blocks cytokine-induced NF-kappa B activation (30, 34). Another kinase implicated in the regulation of NF-kappa B activation is MEKK1. MEKK1 activates an Ikappa B-alpha kinase complex (36, 37) and is also implicated in TNF-alpha -induced NF-kappa B activation (38). Furthermore, MEKK1 is required for Tax-induced NF-kappa B activation (39) and is involved in Fcepsilon RI-induced activation of the human TNF-alpha promoter (40).

We set out to determine the signaling events initiated by LPS that lead to activation of the murine TNF-alpha promoter. This promoter lacks elements, such as AP-1, present in the human promoter, thereby allowing a more straightforward analysis of the underlying signaling mechanisms using a physiological promoter. Here, we demonstrate a requirement for one or both IKKs and examine roles of NIK and MEKK1 in LPS-induced TNF-alpha transcription.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

DNA Constructs-- Expression vectors containing the constitutively active version of MEKK-1 (MEKK-C) and dominant-negative MEKK-1 (D1369A) were described previously (41). The FLAG-tagged wild-type IKK-2, dominant-negative IKK-2 (S177A/S181A), FLAG-tagged wild-type IKK-1, and dominant-negative IKK-1 (S176A/S180A) constructs were described previously (31, 39). Wild-type and dominant-negative NIK (K429A/K430A) were also described previously (39). The TNF-alpha transcriptional reporter (TNFpro-CAT) was provided by B. Beutler and was described previously (14). The NF-kappa B reporter (NF-kappa B-luc) consisted of a triple repeat of the human immunodeficiency virus NF-kappa B site driving a luciferase cDNA and was provided by A. Thorburn. The dominant-negative Ikappa B construct (Ikappa B-alpha SS32, 36AA) was provided by R. Gaynor.

Cells and Transfections-- RAW 264.7 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Life Technologies, Inc.), 50 units/ml penicillin, 50 µg/ml streptomycin, and 2-mM L-glutamine at 37 °C in 5% CO2. Transfections were performed using the Profection DNA transfection kit (DEAE-dextran) from Promega and following the protocol provided by the manufacturer. DNA amounts transfected were kept constant by the addition of empty expression vector. Where indicated, cells were stimulated with LPS (1 µg/ml) or diluent (sterile saline). Cells were treated with LPS for 6 h for both luciferase and chloramphenicol acetyltransferase (CAT) assays. CAT assays were performed as described previously (14). CAT activity was quantitated using a PhosphorImager. Data are represented as percent conversion of chloramphenicol to acetylated chloramphenicol. Luciferase assays were performed using the luciferase assay reagent (Promega) and following the manufacturer's protocol.

In Vitro Kinase Assays-- Where indicated, FLAG-IKK-1 and FLAG-IKK-2 were immunoprecipitated from equal amounts of protein (2 mg) from lysates of transfected RAW 264.7 cells using 1 µg of the M-2 anti-FLAG monoclonal antibody (Sigma) for 4 h at 4 °C on a rocking platform. Where indicated, endogenous IKK-1 and IKK-2 were immunoprecipitated from equal amounts of lysates using 1 µg of anti-IKK-1 (M-280; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or anti-IKK-2 (H-470; Santa Cruz Biotechnology, Inc.), respectively, for 4 h at 4 °C on a rocking platform. Protein A-Sepharose was added to each immunoprecipitate for 1.5 h. Immunoprecipitates were washed three times with lysis buffer (50 mM HEPES (pH 7.5), 150 mM NaCl, 1.5 mM MgCl2, 10% glycerol, 1% Triton X-100, 100 mM NaF, 1% phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin) and once with kinase buffer (50 mM Tris (pH 7.4), 10 mM MgCl2, 1 mM dithiothreitol). The pellets were resuspended in kinase buffer with 50 µM ATP, 10 µCi of [gamma -32P]ATP/sample, and 9 µg/sample of glutathione S-transferase-Ikappa B (amino acids, 1-54). The reactions were carried out for 35 min at 30 °C. Samples were centrifuged, supernatants were added to Laemmli buffer, and the mixtures were boiled for 2 min. Aliquots were loaded on SDS-10% polyacrylamide gels for electrophoresis. The gels were dried and exposed to x-ray film.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

LPS Activates the TNF-alpha Promoter and an NF-kappa B Reporter in RAW 264.7 Cells; LPS-induced TNF-alpha Promoter Activity Is NF-kappa B-dependent-- LPS is a potent inducer of TNF-alpha biosynthesis, activating both transcription of the TNF-alpha gene and translation of TNF-alpha mRNA. We previously demonstrated that multiple kinase signaling pathways are stimulated by LPS and that the JNK/SAPK pathway is required for translational induction of TNF-alpha by LPS (14). In addition, we found no requirement for JNK/SAPK in LPS-induced TNF-alpha transcription, despite the previously described effects of JNK/SAPK on TNF-alpha transcription in another system (14, 40). Therefore, we have explored signaling pathways leading to LPS-induced TNF-alpha promoter activity. It is well documented that LPS activates NF-kappa B and that NF-kappa B is crucial for LPS induction of TNF-alpha gene transcription (20, 21, 24, 25). As a starting point for our experiments, we confirmed that LPS could induce NF-kappa B activation, as well as TNF-alpha promoter activity in our system. We transiently transfected RAW 264.7 cells with an NF-kappa B reporter (NF-kappa B-luc) or a TNF-alpha transcriptional reporter consisting of the murine TNF-alpha promoter driving a CAT cDNA (TNFpro-CAT). After 24 h, cells were stimulated with LPS or diluent for 6 h and were harvested for assessment of luciferase or CAT activity. LPS enhanced NF-kappa B and TNF-alpha promoter activity, as demonstrated by an increase in luciferase (Fig. 1A, left) or CAT activity (Fig. 1A, right) in LPS-treated cells, over that in control cells. The stimulation across experiments was variable, ranging from 2- to 20-fold. To determine the extent to which NF-kappa B contributes to LPS-induced TNF-alpha promoter activity, we tested whether overexpression of a dominant-negative Ikappa B could inhibit LPS-induced TNF-alpha promoter activity. As shown in Fig. 1B, overexpression of the dominant-negative Ikappa B completely abolished LPS-induced TNF promoter activity.


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Fig. 1.   LPS activates TNF-alpha promoter activity and an NF-kappa B reporter; TNF-alpha promoter activity is NF-kappa B-dependent. A, RAW 264.7 cells were transfected with either a TNF-alpha transcriptional reporter, TNFpro-CAT (right), or an NF-kappa B reporter, NF-kappa B-luc (left). 24 h after transfection, cells were stimulated with diluent (No LPS) or LPS (1 µg/ml) for 6 h. Cells were harvested for assessment of CAT activity or luciferase activity. B, dominant-negative Ikappa B was co-transfected with the TNF-alpha transcriptional reporter. Cells were treated as described above and were harvested for assessment of CAT activity. Results are shown as -fold increase over diluent for NF-kappa B reporter activity (n = 3) (error bars represent S.E.) or percent conversion of chloramphenicol to acetylated chloramphenicol for TNF-alpha promoter activity (n = 2, range indicated in A; n = 3, error bars represent S.E. in B).

LPS Activates IKK-1 and IKK-2; IKKs Are Required for LPS-induced TNF-alpha Promoter Activity-- Because LPS induction of the TNF-alpha promoter appears strongly linked to NF-kappa B (20, 21, 24, 25), we set out to examine the signaling molecules involved in NF-kappa B activation by LPS. Recently, two kinases, IKK-1 and IKK-2, were identified that phosphorylate Ikappa B, releasing NF-kappa B, leading to its nuclear translocation (30-34). To determine whether LPS is capable of activating IKK-1 or IKK-2, we immunoprecipitated endogenous IKK-1 or IKK-2 from lysates of RAW 264.7 cells that had been treated with LPS for varying amounts of time or from unstimulated cells (control). After immunoprecipitation, IKK-1 or IKK-2 activity was assessed in vitro in the presence of [gamma -32P]ATP using bacterially expressed glutathione S-transferase-Ikappa B (amino acid, 1-54) as a substrate. As shown in Fig. 2A, LPS activates both IKK-1 and IKK-2. Peak activity occurred 15 min poststimulation and rapidly decreased within 30 min. Because LPS activates an NF-kappa B reporter and IKK activity, we wanted to determine whether IKK-1 or IKK-2 is involved in LPS induction of TNF-alpha promoter activity. RAW 264.7 cells were transiently co-transfected with TNFpro-CAT, and either empty vector (control) or inhibitory mutants of IKK-1 (IKK-1 SS/AA) or IKK-2 (IKK-2 SS/AA). These are phosphorylation-defective mutants previously shown to block Ikappa B phosphorylation (39). Cells were treated with diluent or LPS for 6 h and harvested for assessment of CAT activity. As shown in Fig. 2B, both dominant-negative IKK-2 and dominant-negative IKK-1 decreased basal promoter activity and inhibited LPS-induced TNF-alpha promoter activity. The inhibitory effect was greater with IKK-2. Similar results were obtained with the NF-kappa B reporter (data not shown). Thus, one or possibly both IKKs are required for LPS-induced regulation of TNF-alpha transcription. These results further emphasize the predominant role of NF-kappa B in the LPS-mediated transcription of TNF-alpha .


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Fig. 2.   LPS activates IKK activity; IKKs are required for LPS induction of TNF-alpha promoter activity. A, IKK-1 and IKK-2 were immunoprecipitated from RAW 264.7 cells that had been treated with LPS or diluent as indicated using anti-IKK-1 or anti-IKK-2 antibodies, respectively. Kinase activity was assessed in the presence of [gamma -32P]ATP using glutathione S-transferase (GST)-Ikappa B (1-54) as a substrate. Autoradiographs represent phosphorylated Ikappa B. B, RAW 264.7 cells were co-transfected with the TNF-alpha transcriptional reporter and empty expression vector, a dominant-negative IKK-2, or dominant-negative IKK-1. 24 h after transfection, cells were stimulated with diluent or LPS for 6 h. Cells were harvested for assessment of CAT activity as described above. Data are representative of several independent experiments.

LPS-induced Activation of IKK-2 and TNF-alpha Promoter Activity Is Blocked by Kinase-defective Mutants of NIK and MEKK1-- NIK and MEKK1 can activate IKK-1 or IKK-2 in HeLa cells and fibroblasts (34, 37, 39, 42). We examined the roles of NIK and MEKK1 in the regulation of IKK-1 or IKK-2 in macrophages. We transiently co-transfected a FLAG-tagged IKK-1 or IKK-2 with increasing amounts of either NIK or a constitutively active version of MEKK1 (MEKKC). Overexpression of either NIK or MEKKC activated IKK-2 by 50-fold or more, whereas neither activated IKK-1, although equal amounts of IKKs were present (Fig. 3A and data not shown). To determine the possible requirement of NIK or MEKK1 for LPS induction of IKK-2 activity, we co-expressed FLAG-tagged IKK-2 with either empty vector (control), a dominant-negative NIK (NIK KK/AA), or a dominant-negative MEKK1 (MEKK1 D/A) in RAW 264.7 cells. After 29 h, the cells were treated with LPS or diluent for the indicated amounts of time. As shown in Fig. 3B, dominant-negative NIK and dominant-negative MEKK-1 were each able to block LPS-induced IKK-2 activity. These results suggest functions for both NIK and MEKK-1 in LPS induction of IKK-2 activity. However, because each of these proteins may bind to IKK2, it is possible that they could sequester it, preventing activation by other kinases. Because NIK and MEKK1 stimulated IKK-2 activity (Fig. 3A), we examined their effects on LPS-induced TNF-alpha promoter activity. RAW 264.7 cells transiently transfected with the TNF-alpha transcriptional reporter and either a dominant-negative NIK or a dominant-negative MEKK1 were treated with LPS or diluent. As shown in Fig. 3C, expression of either dominant-negative NIK (right) or dominant-negative MEKK1 (left) abolished LPS induction of TNF-alpha promoter activity. Similar results were obtained with the NF-kappa B reporter (Fig. 3D). These results further suggest a role for NIK and MEKK-1 in LPS induction of the TNF-alpha promoter.


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Fig. 3.   NIK and MEKK1 are involved in the regulation of IKK and TNF-alpha promoter activities. A, RAW 264.7 cells were co-transfected with either FLAG-tagged IKK-1 or IKK-2 and increasing amounts of either NIK (1, 5, 10, or 20 µg) or a constitutively active MEKK1 (MEKKC; 1, 5, or 10 µg). IKK-1 or IKK-2 activity was assessed as described above. Autoradiographs display phosphorylated Ikappa B. GST, glutathione S-transferase; IP, immunoprecipitate. B, RAW 264.7 cells were co-transfected with FLAG-tagged IKK-2 and empty expression vector, a dominant-negative NIK (NIK KK/AA), or a dominant-negative MEKK1 (MEKK1 D/A). 29 h after transfection, cells were stimulated with diluent or LPS for the indicated amounts of time. IKK-2 kinase activity was assessed as described above. Autoradiograph represents phosphorylated Ikappa B. Data are representive of several independent experiments. C, RAW 264.7 cells were co-transfected with the TNF-alpha transcriptional reporter and empty expression vector, dominant-negative MEKK1 (left), or dominant-negative NIK (right). 24 h after transfection, cells were treated with diluent or LPS for 6 h and were harvested for assessment of CAT activity (n = 2; error bars indicate range). D, RAW 264.7 cells were co-transfected with the NF-kappa B reporter (NF-kappa B-luc) and empty expression vector, dominant-negative NIK, or dominant-negative MEKK1. Cells were treated with either diluent or LPS for 6 h and were harvested for assessment of luciferase activity. Data are shown as -fold increase over diluent-treated vector control. Data are representive of two separate experiments.

Overexpression of NIK or MEKK1 Activates TNF-alpha Promoter Activity in the Absence of Exogenous Stimuli-- Overexpression of MEKK1 activates the human TNF-alpha promoter (40) and NF-kappa B (37, 39) in other systems. Because kinase-dead NIK and MEKK1 block LPS-induced TNF-alpha promoter activity, we tested whether overexpression of NIK or MEKK1 could induce TNF-alpha promoter activity in the absence of exogenous stimuli. We co-expressed the TNF-alpha transcriptional reporter with either NIK or MEKK1 (MEKKC) in RAW 264.7 cells. 24 h after transfection, the cells were treated with either diluent or LPS and cells were harvested for assessment of CAT activity. As shown in Fig. 4, in the absence of LPS, overexpression of NIK induced TNF-alpha promoter activity to a level above that obtained with LPS stimulation alone. LPS did not appear to enhance the ability of NIK to activate the TNF-alpha promoter. In a similar manner, in the absence of LPS, overexpression of MEKK-1 activated the TNF-alpha promoter. However, the effects of LPS and MEKK1 were nearly additive in inducing the TNF-alpha transcriptional reporter. Similar results were obtained using the NF-kappa B reporter (data not shown). Thus, NIK and MEKK1 are able to induce TNF-alpha promoter activity in the absence of exogenous stimuli. Because LPS further enhances activation of TNF-alpha promoter activity by MEKK1, but not by NIK, it may be that MEKK1 is acting by a distinct mechanism.


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Fig. 4.   NIK and MEKK1 activate TNF-alpha promoter activity in the absence of exogenous stimuli. RAW 264.7 cells were co-transfected with the TNF-alpha transcriptional reporter and either NIK (left) or a constitutively active version of MEKK1 (right). Cells were stimulated with diluent or LPS and were harvested for assessment of CAT activity as described above (n = 2; range indicated).

Overexpression of Dominant-negative IKKs Inhibit NIK- and MEKK1-induced TNF-alpha Promoter Activity-- Because NIK and MEKK1 can stimulate IKK-2, we tested whether IKK-2 was required for NIK- or MEKK1-induced TNF-alpha promoter activity. We transiently transfected the TNF-alpha transcriptional reporter with empty expression vector, MEKK1 (MEKKC), or NIK in the presence or absence of dominant-negative IKK-2 (IKK2 SS/AA). As shown in Fig. 5, overexpression of dominant-negative IKK-2 inhibited NIK- and MEKK1-induced TNF-alpha promoter activity. Similarly, overexpression of a dominant-negative IKK1 (IKK-1 SS/AA) blocked NIK- and MEKK1-induced TNF-alpha promoter activity, despite the fact that neither NIK nor MEKK1 activated IKK1 in these cells (Fig. 5).


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Fig. 5.   IKKs mediate NIK- and MEKK1-induced TNF-alpha transcription. RAW 264.7 cells were transfected with the TNF-alpha transcriptional reporter; empty vector, NIK (A), or MEKK1 (B); and either dominant-negative IKK-2 or dominant-negative IKK-1. Cells were treated with LPS or diluent and were harvested for assessment of CAT activity. Data are representative of two separate experiments. Error bars indicate range (A).


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this study, we investigated the signaling pathways initiated by LPS that are involved in transcriptional regulation of the TNF-alpha gene in macrophages. In confirmation of earlier findings concerning regulation of the TNF-alpha promoter, our work indicates that most or all of the signaling leading to TNF-alpha transcription is mediated by NF-kappa B. This conclusion is based, in part, on the finding that dominant-negative Ikappa B completely inhibits LPS-induced TNF-alpha promoter activity. Dominant-negative IKKs fully block LPS induction of the TNF-alpha promoter, further implicating NF-kappa B as the primary mediator of LPS-induced TNF-alpha promoter activity. In a previous study examining transcriptional regulation of the human TNF-alpha promoter, it was determined that a dominant-negative JNK/SAPK interfered with Fcepsilon RI induction of transcription in mast cells (40). We showed that dominant-negative JNK/SAPK had no effect on LPS induction of the murine TNF-alpha promoter in macrophages (14). Therefore, regulation of TNF-alpha transcription differs among species and/or cell types. Indeed, the human promoter contains elements, such as an AP-1 site, that are not present in the murine promoter (18).

The mechanism by which NF-kappa B becomes activated by LPS has not been examined. We demonstrate that LPS activates the recently identified Ikappa B kinases, IKK-1 and IKK-2. LPS stimulation of RAW 264.7 cells resulted in similar kinetics of activation for both IKK-1 and IKK-2. We established roles for IKK-2 and IKK-1 in LPS induction of TNF-alpha transcription by demonstrating that overexpression of dominant-negative versions of either IKK blocked LPS induction of the murine TNF-alpha promoter. Because IKK-1 and IKK-2 form heterodimers (3, 33, 34), it is not possible to determine whether one or the other is more important using these approaches.

Because LPS induces TNF-alpha production in macrophages, one could argue that the effects on the IKKs are caused by an autocrine mechanism. This, however, is not the case because of the rapid kinetics of IKK activation. We determined that LPS induced activation of other signaling molecules, including extracellular signal-regulated kinases, p38, JNK/SAPK, MEKs 1, 3, 4, and 6, 30-60 min poststimulation in macrophages (14). In contrast, activation of IKKs peaks within 15 min of LPS stimulation. Therefore, IKK activation is among the earliest signaling events known to be induced by LPS in macrophages.

To test possible links between LPS and the IKKs, we examined two IKK kinases, NIK and MEKK1. Both NIK and MEKK1 stimulate IKK-2 activity in RAW 264.7 cells, and both NIK and MEKK1 are sufficient to activate not only an NF-kappa B reporter but also the TNF-alpha promoter in the absence of ligand. Additional findings suggesting that NIK and MEKK1 may both be involved in LPS-mediated transcriptional regulation of the TNF-alpha gene come from studies with kinase-defective mutants. Either kinase-dead NIK or MEKK1 blocks the induction of IKK-2 activity and induction of the TNF-alpha promoter by LPS. These results may indicate that both enzymes are required. Alternatively, they may block NF-kappa B by sequestering IKK-2 or NF-kappa B activation complexes.

Our results suggest roles for IKK-1, IKK-2, or both, in LPS-induced signaling to the TNF-alpha promoter. However, we propose that IKK-2 is the more significant mediator of LPS-induced signaling to the TNF-alpha promoter, since known IKK kinases lead to activation of IKK-2, not IKK-1. LPS-induced IKK activity observed in the IKK-1 immunoprecipitates could be due to the presence of IKK-2. Dominant-negative IKK-1 inhibited LPS-, MEKK1-, and NIK-induced TNF-alpha promoter, and NF-kappa B activity could also be attributed to the fact that the IKKs form heterodimers (3, 33, 34). Thus, overexpression of dominant-negative IKK-1 could interfere with IKK-2 function; more extensive studies are necessary to determine such matters definitively.

It is clear that regulation of NF-kappa B is complex, as multiple kinases and other molecules, such as NF-kappa B essential modulator (43), appear to be involved. Here, we establish a role for IKKs in LPS-induced NF-kappa B activation and, more importantly, in LPS induction of TNF-alpha transcription. Most studies about IKKs merely demonstrate a role for IKKs in the regulation of NF-kappa B, using NF-kappa B reporters consisting of multiple copies of a particular site driving the expression of a reporter gene. Our studies establish a potential mechanism for initiation of TNF-alpha promoter activity, thus providing a better understanding of the regulation of such an important inflammatory mediator.

    ACKNOWLEDGEMENTS

We thank Frank Mercurio and Miguel Barbosa for providing the IKK2 cDNAs. We thank Richard Gaynor and Min-Jean Yin for providing various reagents. We thank Bruce Beutler for the TNF transcriptional reporter. We also thank Barry Conner, Peiqun Wu, and Don Arnette for excellent technical assistance.

    FOOTNOTES

* This work was funded by National Institutes of Health Research Grants DK34128 and GM53032 (to M. H. C.) and National Research Service Award Postdoctoral Fellowship GM18550-01 (to J. L. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Pharmacology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235. Tel.: 214-648-8710; Fax: 214-648-3811; E-mail: mcobb{at}mednet.swmed.edu.

    ABBREVIATIONS

The abbreviations used are: LPS, lipopolysaccharide; TNF, tumor necrosis factor; NF-kappa B, nuclear factor-kappa B; JNK/SAPK, Jun-N-terminal kinase/stress-activated protein kinase; Ikappa B, inhibitor of NF-kappa B; IKK, Ikappa B kinase; NIK, NF-kappa B-inducing kinase; MEKK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase; CAT, chloramphenicol acetyltransferase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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