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J. Biol. Chem., Vol. 275, Issue 27, 20861-20866, July 7, 2000

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Cellular Events Mediated by Lipopolysaccharide-stimulated Toll-like Receptor 4

MD-2 IS REQUIRED FOR ACTIVATION OF MITOGEN-ACTIVATED PROTEIN KINASES AND Elk-1^*

Hua Yang, Donna W. Young, Fabian Gusovsky, and Jesse C. Chow

From the Division of Inflammatory Diseases, Eisai Research Institute, Andover, Massachusetts 01810

Received for publication, April 5, 2000, and in revised form, April 27, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Lipopolysaccharide (LPS) stimulates multiple signaling events, including nuclear factor-B (NF-B) activity and the mitogen-activated protein (MAP) kinases, ERK, JNK, and p38 in LPS-responsive cells, resulting in transcriptional activation and cytokine generation. LPS-induced signaling via toll-like receptor 4 (TLR4) results in the activation of the transcription factor NF-B. Since LPS activates other signaling cascades in responsive cells, the objective of this study was to determine whether such events are mediated by TLR4 in response to LPS. We generated human embryonic kidney cells (HEK293) that stably express TLR4 (HEK-TLR4) and examined their responsiveness to LPS by measuring NF-B activity and production of interleukin-8 (IL-8). A trans-reporting system was used to measure the activity of Elk-1, an ETS-domain transcription factor targeted by MAP kinase pathways. LPS stimulated NF-B reporter activity and IL-8 production but not Elk-1 activity in HEK-TLR4 cells. When MD-2, a protein associated with the extracellular domain of TLR4, was expressed in these cells, there was a marked increase in Elk-1 activity as well as ERK, JNK, and p38 MAP kinase phosphorylation in response to LPS. TLR4-mediated NF-B reporter activity and IL-8 production was enhanced by the expression of MD-2. This study demonstrates that expression of both TLR4 and MD-2 is required for LPS to activate or augment the MAP kinase pathways, Elk-1 stimulation, and IL-8 generation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The pathophysiology of Gram-negative sepsis and septic shock is caused by lipopolysaccharide (LPS),¹ a component of the outer membrane of bacteria that can activate a variety of mammalian cell types, including monocytes and macrophages (1, 2). This, in turn, causes the release of a number of proinflammatory mediators, such as interleukin (IL)-1, IL-6, IL-8, and tumor necrosis factor- (1). Activation of LPS-responsive cells occurs rapidly after LPS interacts with circulating LPS-binding protein and CD14, a glycosylphosphatidylinositol-linked cell surface or soluble protein necessary for efficient responses to LPS (2). Evidence from studies in vitro indicates that expression of TLR2 and TLR4, two members of the toll-like receptor (TLR) family of cell surface proteins, can enable the activation of nuclear factor-B (NF-B) and expression of genes for IL-1, IL-6, and IL-8 (3, 4). TLR4 has been shown to mediate LPS-induced activation of NF-B in human embryonic kidney cells (HEK293) (5, 6), whereas in 293T and Ba/F3 cells, TLR4-mediated responses to LPS requires the presence of MD-2, a cell surface protein that associates with TLR4 (7). Observations from studies with TLR2 and TLR4 knockout mice indicate that TLR4, but not TLR2, is necessary for responses to LPS under physiological conditions; TLR2 knockout mice respond normally to LPS, whereas TLR4 knockout mice or spontaneous TLR4 mutants (C3H/HeJ and C57/10ScCr) are not responsive to LPS (8-10). On the other hand, studies with TLR2 knockout mice suggest that TLR2 has a broader recognition pattern than TLR4, which extends to a number of different bacterial cell wall components (10).

LPS has been shown to initiate multiple intracellular signaling events, including the stimulation of pathways that lead to the activation of NF-B and three distinct mitogen-activated protein (MAP) kinases, the ERK, p38, and JNK proteins (11). TLR4-mediated NF-B activation is thought to occur via a signaling pathway that is also utilized by IL-1 and IL-18 (12). This pathway is activated by the interaction between myeloid differentiation protein (MyD88) with the receptor followed by stimulation of IL-1 receptor-associated kinase (IRAK) and subsequent recruitment of tumor necrosis factor receptor-associated factor-6 (TRAF6). Stimulation of these proximal signaling molecules results in the activation of members of the MAP kinase kinase kinase family that activate inhibitory kappa B (IB) kinases (12). IB kinases can then phosphorylate IB proteins that retain the rel-type transcription factor NF-B in the cytosol, leading to ubiquitination and degradation of IB proteins by the 26 S proteosome, followed by release and nuclear translocation of NF-B (13).

The proximal signaling molecules involved in LPS-induced activation of ERK, p38, and JNK are not well defined. LPS is thought to activate ERK1 and ERK2 proteins via the Ras/Raf-1/MAP kinase kinase (MEK) pathway, JNK proteins via the MEKK-1/MEK-4 pathway, and p38 proteins via activation of MEK-3 (14, 15). These pathways are coupled to the production of certain proinflammatory cytokines, such as IL-8, by phosphorylating and activating the ternary complex Elk-1, resulting in increased levels of c-fos and enhanced formation of the transcription factor AP-1 (16). The IL-8 promoter contains a NF-B binding site that is necessary for activation as well as AP-1 binding sites that, depending on the cell type, contribute to transcriptional activation (17, 18). The aim of this study was to determine whether TLR4, which activates NF-B in response to LPS (5), also mediates LPS-induced activation of the MAP kinase family of proteins in responsive cells.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- The pcDNA3.0 expression vector encoding human TLR4 cDNA was kindly provided by Drs. Ruslan Medzhitov and Charles Janeway, Jr. (Yale University). The pEFBOS expression vector encoding human MD-2 cDNA was kindly provided by Dr. Kensuke Miyake (Saga Medical School, Japan). The Elk-1 reporter system was purchased from Stratagene (La Jolla, CA). The ELAM-1-luciferase reporter construct (pELAM-luc) and human soluble CD14 (sCD14) were generated as described previously (5). Lipopolysaccharide from Escherichia coli 0111:B4, epidermal growth factor, and IL-1 were purchased from List Biological Laboratories (Campbell, CA), Life Technologies, Inc., and PeproTech (Rocky Hill, NJ), respectively. HEK 293 cells and HL-60 cells were from American Type Culture Collection (Manassas, VA). Plasmid DNA was isolated with Qiagen Endo-free^TM Maxi-prep columns (Chatsworth, CA). Leukocyte cDNA was purchased from CLONTECH (Palo Alto, CA). Cycloheximide was purchased from Sigma.

Cell Culture and Transfections-- HEK293 cells were transfected with the pcDNA3.0 expression vector (Invitrogen, Inc) with or without an insert encoding human TLR4. Transfectants (HEK vector or HEK-TLR4) were selected with the antibiotic G418, screened for LPS responsiveness using pELAM-luc, and analyzed for TLR4 mRNA expression by reverse transcription-polymerase chain reaction (RT-PCR). For the ERK, p38, and JNK signaling experiments, TLR4/MD-2 transfectants were generated by transfecting HEK-TLR4 cells with pcDNA3.1/Hygro encoding human MD-2 plus pELAM-luc/Zeocin followed by antibiotic selection with G418, hygromycin, and Zeocin^TM. Transfectants were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Life Technologies, Inc.) and the appropriate antibiotic(s). Multiple clones of stable transfectants as well as stable cell lines that contain a heterogeneous population of transfectants were used in this study.

For transfection experiments, HEK-TLR4 cells were plated in 24-well tissue culture plates (2 × 10⁵ cells/well) and maintained in the above medium without antibiotics overnight. The cells were transfected with MD-2 plasmid or empty vector (pEFBOS) and pELAM-luc or the Elk-1 reporter constructs (pFA2-Elk-1 and pFR-Luc) using a calcium phosphate protocol described by CLONTECH (Palo Alto, CA). All cells were also transfected with pRL-TK plasmid, a Renilla luciferase control reporter vector (Promega, Inc.), to normalize transfection efficiencies. After transfection, cells were maintained in Dulbecco's modified Eagle's medium supplemented with 0.5% fetal bovine serum for 18 h. The following day, cells were either left untreated or incubated with the indicated amount of ligand and/or inhibitor for 6 h or the indicated time. The medium was then removed and stored at 80 °C until further analysis for IL-8 by enzyme-linked immunosorbent assay (PharMingen, Inc). The cells were harvested in lysis buffer and assayed for firefly and Renilla luciferase activity as described by the manufacturer of the Dual-luciferase^TM Reporter System (Promega, Inc). The amount of luciferase activity in each sample was quantified by a Wallac 1450 MicroBetaTrilux counter. The data are shown as mean ± S.E. for one representative experiment in which each transfection was performed in quadruplicate. Each experiment was repeated at least three times in all figures.

RT-PCR Analysis-- Total RNA was isolated with RNeasy mini columns (Qiagen, Inc.), and first-strand synthesis of RNA was carried out with the ProStar RT-PCR kit (Stratagene, Inc.) using 200 ng of total RNA. The resulting cDNA was amplified with primers for MD-2 (5'-GAAGCTCAGAAGCAGTATTGGGTC-3' and 5'-GGTTGGTGTAGGATGACAAACTCC-3') and -actin (CLONTECH, Inc.). PCR products were resolved by electrophoresis on agarose gels containing ethidium bromide and visualized by UV transillumination.

Immunoblotting Experiments-- HEK-TLR4 or HEK-TLR4/MD-2 cells were stimulated with LPS (1 µg/ml) plus sCD14 (10 nM) for 15, 30, 60, or 120 min. Cells were lysed in 1× SDS sample buffer (Invitrogen) containing -mercaptoethanol, sonicated for 5 s, and boiled at 95 °C for 5 min. Proteins were separated by Tris-glycine gels (Invitrogen) and electroblotted on nitrocellulose membranes (Schleicher & Schuell). The blots were subsequently blocked and probed with an antibody specific for phospho-ERK (Promega), phospho-JNK (Promega), phospho-p38 (New England Biolabs), pan-ERK (Transduction Labs), JNK (NEB), or p38 (NEB) as described by the manufacturer of the antibody. Blots were then incubated with an anti-mouse (Transduction Labs) or anti-rabbit (Pierce) IgG coupled to horseradish peroxidase for 1 h at 25 °C. Proteins were visualized using enhanced chemiluminescence (ECL) as described by the manufacturer (Amersham Pharmacia Biotech). Blots that were probed with phospho-specific antibodies were stripped as recommended in the ECL protocol and reblotted with antibodies specific for the particular protein of interest.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

To study the role of TLR4 in LPS-stimulated signaling events, HEK293 cells expressing human TLR4 (HEK-TLR4) were generated and tested for LPS responsiveness after transfection with the NF-B reporter construct, pELAM-luc, or an Elk-1 reporter system. Twenty-four hours post-transfection, cells were left untreated or incubated with 1 µg/ml LPS plus 10 nM sCD14 for an additional 6 h, at which time medium was collected to measure IL-8 production, and the cells were lysed to determine luciferase activity in the supernatants. HEK-TLR4 cells respond to LPS/sCD14 treatment by elevating NF-B reporter activity 10-fold above vector controls (Fig. 1A). We have previously shown that this response to LPS is dependent on the presence of sCD14 (5). The HEK-TLR4 cells also responded to LPS/sCD14 by secreting IL-8 (Fig. 1B), a chemokine that elicits neutrophil chemotaxis and is regulated by NF-B and AP-1 (17). The concentration of IL-8 in the culture medium was elevated by 2-fold in the basal state and 5-6-fold above vector controls after LPS/sCD14 treatment (Fig. 1B). Consistent with this finding, the level of IL-8 mRNA, as determined by RT-PCR was increased by LPS/sCD14 in a time-dependent manner from 3 to 24 h in HEK-TLR4 cells but not in HEK-vector cells (data not shown). Induction of IL-8 mRNA by LPS did not require de novo protein synthesis, since pretreatment with the protein synthesis inhibitor cycloheximide (10 µg/ml) for 15 min before a 6-h LPS or IL-1 stimulus did not inhibit the response (Fig. 2). Cycloheximide caused a superinduction of IL-8 mRNA in the basal and stimulated state, which does not occur with -actin mRNA (Fig. 2), suggesting that new protein synthesis is required for repression of basal transcription of IL-8. Superinduction of IL-8 by cycloheximide has been previously reported in bone marrow-derived mononuclear cells (19).

View larger version (23K): [in this window] [in a new window]   Fig. 1.   TLR4-mediates activation of NF-B and IL-8 production by LPS in HEK-TLR4 cells. HEK293-vector or HEK-TLR4 cells were transfected with pELAM-luc (panel A) as described under "Experimental Procedures." Cells were either left untreated or incubated with 1 µg/ml LPS plus 10 nM sCD14 for 6 h. Cell lysates were assayed for luciferase activity using the Dual-luciferaseTM Reporter Assay System. The cell media was collected and analyzed for IL-8 production by enzyme-linked immunosorbent assay (panel B).

View larger version (45K): [in this window] [in a new window]   Fig. 2.   Induction of IL-8 mRNA by LPS does not require de novo protein synthesis in HEK-TLR4 cells. HEK-TLR4 cells were incubated in the presence or absence of 10 µg/ml cycloheximide (CHX) for 15 min and then either left untreated or incubated with 1 µg/ml LPS plus 10 nM sCD14 (L) or 20 ng/ml IL-1 (I) for 6 h. Total RNA was isolated, reversed-transcribed, and amplified with IL-8- or -actin-specific primers as described under "Experimental Procedures." IL-8 or -actin cDNA was used as a template for the positive control (PC) reaction. Molecular weights of DNA markers in lane 1 are noted to the left of each figure.

In contrast to NF-B-driven gene expression (Fig. 1), Elk-1 reporter activity was not affected by LPS/sCD14 in HEK-TLR4 cells despite a robust increase in Elk-1 activity after epidermal growth factor treatment (Fig. 3). These results suggest either TLR4 does not mediate the LPS signals that result in Elk-1 stimulation or HEK293 cells lack other signaling molecules that are necessary for Elk-1 activation in response to LPS. MD-2 is a recently identified cell surface protein that physically associates with TLR4 and is a requisite for LPS signaling to NF-B in 293T and Ba/F3 cells (7). We found that HEK-TLR4 cells do not express MD-2 as determined by RT-PCR (Fig. 4). In contrast, MD-2 mRNA was detected in LPS-responsive cells such as differentiated HL-60 monocytes and leukocytes (Fig. 4). These data suggest that MD-2 may be the missing protein whose expression is required for LPS to stimulate Elk-1 reporter activity in HEK-TLR4 cells.

View larger version (27K): [in this window] [in a new window]   Fig. 3.   LPS does not stimulate Elk-1 activity in HEK-TLR4 cells. HEK293 vector or HEK-TLR4 cells were transfected with the Elk-1 reporter system as described under "Experimental Procedures." Cells were either left untreated or incubated with 1 µg/ml LPS plus 10 nM sCD14 or 100 ng/ml epidermal growth factor for 6 h. The cell lysates were assayed for Elk-1 reporter activity using the Dual-luciferaseTM Reporter Assay System.

View larger version (86K): [in this window] [in a new window]   Fig. 4.   MD-2 is not expressed in HEK-TLR4 cells. Total RNA from HEK-TLR4 or HL-60 cells was reversed-transcribed and, along with cDNAs from leukocytes, amplified with MD-2- or -actin-specific primers as described under "Experimental Procedures." PCR products were resolved by electrophoresis on a 1% agarose gel and visualized by UV transillumination. Plasmid encoding MD-2 was used as a template for the positive control (MD-2) reaction. Molecular weights of DNA markers in lane 1 are noted to the left of each figure.

We co-transfected HEK-TLR4 cells with an pEFBOS expression vector encoding MD-2 cDNA and either pELAM-luc or an Elk-1 reporter system followed by the indicated treatment (Fig. 5). Expression of MD-2 in HEK-TLR4 cells increased LPS/sCD14-induced NF-B reporter activity 35-fold above unstimulated cells and 7-fold above LPS/sCD14-treated MD-2-negative vector controls (Fig. 5A), consistent with previous observations in HEK293T cells (7). When MD-2 and the Elk-1 reporter system were transfected into HEK-TLR4 cells, Elk-1 activity was elevated 5-6-fold above basal controls after LPS/sCD14 treatment, a response that was not observed in HEK-TLR4 cells transfected with the pEFBOS vector (Fig. 5B). Neither sCD14 nor LPS alone was capable of eliciting such a response in these cells, indicating that this MD-2-mediated event is dependent on the presence of sCD14 in the cell medium. The activation of Elk-1 by LPS/sCD14 was dose-dependent and reached maximal levels at 10 ng/ml LPS (Fig. 6A). Elk-1 activity remained elevated in response to increasing concentrations of LPS up to 10 µg/ml. In contrast, LPS/sCD14 treatment did not induce Elk-1 reporter activity in HEK293 cells transfected with MD-2 alone (data not shown), indicating that expression of both MD-2 and TLR4 is required for LPS/sCD14 to activate Elk-1. Stimulation of Elk-1 activity occurred in a time-dependent fashion and reached maximal levels at 6 h after LPS treatment (Fig. 6B).

View larger version (22K): [in this window] [in a new window]   Fig. 5.   MD-2 augments TLR4-mediated activation of NF-B and confers Elk-1 activation by LPS in HEK-TLR4 cells. HEK-TLR4 cells were transfected with pEFBOS vector encoding human MD-2 or not plus pELAM-luc (panel A) or an Elk-1 reporter system (panel B) as described under "Experimental Procedures." Cells were either left untreated or stimulated with 1 µg/ml LPS plus 10 nM sCD14 for 6 h. Cells transfected with the Elk-1 reporter system were also treated with sCD14 or LPS alone for 6 h. Cell lysates were tested for luciferase activity using the Dual-luciferaseTM Reporter Assay System.

View larger version (11K): [in this window] [in a new window]   Fig. 6.   Dose- and time-dependent stimulation of Elk-1 activity by LPS. HEK-TLR4 cells were transfected with pEFBOS vector encoding human MD-2 or not and the Elk-1 reporter system as described under under "Experimental Procedures." Cells were left untreated or incubated with 10 nM sCD14 and the indicated concentration of LPS for 6 h (panel A) or 1 µg/ml of LPS for 3, 6, 12, or 24 h (panel B). The cells were subsequently lysed, and the amount of luciferase activity in each sample was quantified using the Dual-luciferaseTM Reporter Assay System.

Previous studies have shown that the ERK, p38, and Jnk signaling pathways contribute to Elk-1 activation (16). Cellular lysates from HEK293 cells that stably co-express TLR4 and MD-2 (HEK-TLR4/MD-2) were examined for ERK, p38, and Jnk phosphorylation by immunoblotting analysis using phospho-specific antibodies after stimulation with LPS/sCD14 for 15, 30, 60, or 120 min. LPS/sCD14 activated all three MAP kinase proteins tested in HEK-TLR4/MD-2 cells but not in HEK-TLR4 cells (Fig. 7, upper panels). The stimulation of ERK, p38, and Jnk phosphorylation was time-dependent, reaching maximal levels at 60 min after the addition of LPS/sCD14 to the cell medium. Thus, the presence of MD-2 enables LPS/sCD14 to induce phosphorylation/activation of ERK, p38, and Jnk proteins in HEK293 cells that co-express TLR4.

View larger version (50K): [in this window] [in a new window]   Fig. 7.   MD-2 is required for LPS-induced ERK, Jnk, and p38 phosphorylation. HEK-TLR4 or HEK-TLR4-MD-2 cells were left untreated or stimulated with 1 µg/ml LPS and 10 nM sCD14 for 15, 30, 60, or 120 min and then lysed in 1× SDS sample buffer. Proteins were resolved by polyacrylamide gel electrophoresis, electroblotted, and immunoblotted with an antibody specific for phospho-ERK (panel A), phospho-JNK (panel B), or phospho (p)-p38 (panel C). The same blots were stripped and re-blotted with an antibody specific for the non-phosphorylated form of each respective signaling protein. These are representative blots from three independent experiments.

We next examined the effect of MD-2 expression on LPS-induced IL-8 production in HEK-TLR4 cells. As shown above, cellular production of IL-8 was elevated above basal levels in HEK-TLR4 cells 6 h after LPS/sCD14 treatment (Fig. 8). Expression of MD-2 enhanced IL-8 production by approximately 2.5-fold above LPS-stimulated vector transfected cells and 20-fold above basal levels.

View larger version (21K): [in this window] [in a new window]   Fig. 8.   MD-2 enhances TLR4-mediated production of IL-8 by LPS. HEK-TLR4 cells were transfected with pEFBOS vector encoding human MD-2 or not as described under "Experimental Procedures." Cells were either left untreated or stimulated with 1 µg/ml LPS plus 10 nM sCD14 for 6 h. Cell lysates were tested for luciferase activity using the Dual-luciferaseTM Reporter Assay System. The cells media was collected, frozen at 80 °C, and subsequently analyzed for IL-8 by enzyme-linked immunosorbent assay.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The discovery that TLR4 mediates LPS-induced responses in cells and in vivo (5, 9) prompted us to identify further cellular events that are triggered by TLR4 in response to LPS. The data presented here provide additional evidence that TLR4 mediates LPS-stimulated IL-8 production in HEK293 cells stably expressing TLR4. In support of these findings, expression of a constitutively active mutant of TLR4 has been shown to induce IL-8 gene expression in THP-1 cells (3). Induction of IL-8 message levels in HEK-TLR4 cells is a rapid event that occurs as early as 3 h after LPS stimulation and does not require new protein synthesis or autocrine factors (Fig. 2). The increase in IL-8 mRNA and IL-8 production by LPS-activated TLR4 is in agreement with reports demonstrating that LPS stimulates IL-8 synthesis in macrophages and other cell types (20), a response that is in part dependent upon NF-B activity (11). We have shown that LPS stimulates TLR4-mediated NF-B activity in this and a previous study (5), suggesting that the increase in NF-B activity is responsible for elevated IL-8 transcription and synthesis in HEK-TLR4 cells. Increasing evidence suggests that the proximal signaling components of the IL-1 pathway are involved in coupling the LPS-induced signals that emanate from activated TLR4 to NF-B. For example, constitutively active constructs of TLR4 have implicated the involvement of MyD88, IRAK, TRAF6, and NF-B-inducing kinase in signaling the activation of NF-B from TLR4 (21, 22). In fact, dominant negative constructs of these signaling proteins have been shown to inhibit LPS-induced NF-B activity in human dermal microvessel endothelial cells, and monocytic leukemia cells (23). Most recently, transforming growth factor--activated kinase 1, a MAP kinase kinase kinase upstream of NF-B-inducing kinase, was found to mediate an LPS activation signal from TLR4 to NF-B (6).

We show here that the stimulation of NF-B and IL-8 production in HEK-TLR4 cells is enhanced by co-expression of MD-2, suggesting that MD-2 increases the sensitivity of TLR4-expressing cells to LPS. These data are consistent with a previous report describing the absolute requirement of MD-2 for LPS-induced NF-B activity in HEK293T and Ba/F3 cells (7). In contrast to NF-B and IL-8 stimulation, we did not detect Elk-1 reporter activity or phosphorylation of MAP kinase proteins upon LPS treatment in HEK-TLR4 cells lacking MD-2. When MD-2 was transfected into these cells, Elk-1 and the three MAP kinase proteins were stimulated by LPS, indicating that MD-2 expression is required for the activation of these signaling molecules. Thus, in this cell model system, TLR4 and MD-2 are both responsible for initiating the signaling pathways that lead to ERK, p38, and Jnk activation as well as Elk-1. It seems likely that the activation of these signaling pathways and the enhancement of NF-B activity might contribute to the increase in IL-8 generation in HEK-TLR4/MD-2 cells. Holtmann et al. (24) recently showed that maximal IL-8 gene expression requires the coordinate action of at least three different signal transduction pathways, which cooperate to induce mRNA synthesis and suppress mRNA degradation. Pathways involving the activation of NF-B, p38, and stress-activated protein kinase (SAPK)/JNK were shown to induce IL-8 synthesis and/or transcription from a minimal IL-8 promoter containing NF-B and AP-1 binding sites (24). In addition, the ERK proteins have been shown to activate AP-1 via Elk-1 (25) and modulate IL-8 production in a variety of cells including mast cells and monocytes (26, 27). Likewise, we have shown that when HEK-TLR4 cells are treated with the MAP kinase kinase (upstream regulator of ERK proteins) inhibitor U0126, LPS-stimulated IL-8 production is partially suppressed,² indicating that the ERK signaling pathway plays a role in IL-8 generation in these cells. It remains to be determined whether or not p38 or stress-activated protein kinase (SAPK)/JNK or both pathways participate in the regulation of IL-8 by LPS in HEK-TLR4 cells. It will be of interest to quantify the relative contribution of each one of these signaling pathways that regulate LPS-induced IL-8 synthesis.

To date the identity of the molecules that link TLR4 and MD-2 to the three MAP kinase proteins are not well defined. Previous studies have suggested that tyrosine kinases, small G-proteins, and serine/threonine kinases mediate LPS signals to these MAP kinase pathways (8). It is possible that signals generated by LPS may bifurcate from known signaling molecules that are thought to mediate LPS-induced NF-B activation, such as MyD88, IRAK, TRAF6, TGF--activated kinase 1, NF-B-inducing kinase, and IB kinases or via signal transducers that have yet to be identified. For example, JNK activation caused by expression of TLR4-FLAG is blocked by a dominant-negative version of MyD88 but not TRAF6 (21), and Medzhitov et al. (22) report that TLR4-activated AP-1 reporter activity is inhibited by a dominant negative construct of IRAK or TRAF6. Taken together, these studies suggest that MyD88 and IRAK are involved in TLR4-activated JNK phosphorylation or AP-1 activity, respectively, whereas TRAF6 does not contribute to JNK phosphorylation but is required for AP-1 activation. Thus, it will be necessary to identify which proximal signaling pathways mediate LPS-induced JNK, ERK, and p38 MAP kinase activation in HEK-TLR4/MD-2 cells.

In summary, these data provide new evidence that the expression of MD-2 in cells that express TLR4 is essential for TLR4-mediated activation of the three MAP kinases and a downstream target of these kinases, Elk-1. In addition, TLR4 is capable of mediating the signals initiated by LPS that induce the production of IL-8, which is enhanced further when MD-2 is co-expressed in HEK293 cells. These studies demonstrate that, although TLR4 alone can mediate the activation of NF-B and IL-8 production, co-expression of MD-2 is absolutely necessary for LPS to activate MAP kinase proteins and Elk-1. The cell surface interactions among the components of the LPS signaling pathway are not well defined. However, genetic complementation studies have demonstrated that there is physical contact between LPS and TLR4 in the course of signal transduction (28). Based on the present and a previous study (29), it appears that TLR4·MD-2 complex is capable of recognizing/sensing LPS with sCD14 better than LPS alone, or perhaps MD-2 is able to "lock" LPS on its binding site for a longer period of time. In the future, it will be important to understand the molecular interactions between TLR4 and other LPS signaling molecules that result in a cellular response. Continued identification and characterization of the mechanistic steps by which TLR4 and other TLRs mediate ligand-induced signals within the cell will enable further improvements in the discovery of anti-septic agents.

	ACKNOWLEDGEMENT

We thank Dr. Sally Ishizaka for helpful comments on this manuscript.

	FOOTNOTES

^* 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.

To whom correspondence should be addressed: Eisai Research Institute, 100 Research Dr., Wilmington, MA 01887. Tel.: 978-661-7276; Fax: 978-657-7715; E-mail: Jesse_Chow@eri.eisai.com.

Published, JBC Papers in Press, April 28, 2000, DOI 10.1074/jbc.M002896200

² H. Yang and J. C. Chow, unpublished observations.

	ABBREVIATIONS

The abbreviations used are: LPS, lipopolysaccharide; TLR, toll-like receptor; IL, interleukin; NF-B, nuclear factor-B; IB, inhibitory B; sCD14, soluble CD14; pELAM-luc, ELAM-1-luciferase reporter plasmid; MyD88, myeloid differentiation protein; IRAK, IL-1 receptor-associated kinase; TRAF, tumor necrosis factor-associated factor; RT-PCR, reverse transcription-polymerase chain reaction; HEK cells, human embryonic kidney cells; MAP, mitogen-activated protein; ERK, extracellular signal-regulated kinase; JNK, c-Jun NH₂-terminal kinase; MEK, MAP kinase kinase; MEKK-1, MAP kinase kinase kinase-1.

	REFERENCES

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				P. Li, B. Ho, and J. L. Ding Recombinant Factor C competes against LBP to bind lipopolysaccharide and neutralizes the endotoxicity Innate Immunity, June 1, 2007; 13(3): 150 - 157. [Abstract] [PDF]

				K. A. Shirey, J.-Y. Jung, and J. M. Carlin Up-Regulation of Gamma Interferon Receptor Expression Due to Chlamydia-Toll-Like Receptor Interaction Does Not Enhance Signal Transducer and Activator of Transcription 1 Signaling Infect. Immun., December 1, 2006; 74(12): 6877 - 6884. [Abstract] [Full Text] [PDF]

				A. Visintin, K. A. Halmen, N. Khan, B. G. Monks, D. T. Golenbock, and E. Lien MD-2 expression is not required for cell surface targeting of Toll-like receptor 4 (TLR4) J. Leukoc. Biol., December 1, 2006; 80(6): 1584 - 1592. [Abstract] [Full Text] [PDF]

				A. E. Medvedev, I. Sabroe, J. D. Hasday, and S. N. Vogel Invited review: Tolerance to microbial TLR ligands: molecular mechanisms and relevance to disease Innate Immunity, June 1, 2006; 12(3): 133 - 150. [Abstract] [PDF]

				A. Motegi, M. Kinoshita, K. Sato, N. Shinomiya, S. Ono, S. Nonoyama, H. Hiraide, and S. Seki An in vitro Shwartzman reaction-like response is augmented age-dependently in human peripheral blood mononuclear cells J. Leukoc. Biol., March 1, 2006; 79(3): 463 - 472. [Abstract] [Full Text] [PDF]

				A. Teghanemt, D. Zhang, E. N. Levis, J. P. Weiss, and T. L. Gioannini Molecular Basis of Reduced Potency of Underacylated Endotoxins J. Immunol., October 1, 2005; 175(7): 4669 - 4676. [Abstract] [Full Text] [PDF]

				H.-Y. Cho, A. E. Jedlicka, R. Clarke, and S. R. Kleeberger Role of Toll-like receptor-4 in genetic susceptibility to lung injury induced by residual oil fly ash Physiol Genomics, June 16, 2005; 22(1): 108 - 117. [Abstract] [Full Text] [PDF]

				J. Pugin, S. Stern-Voeffray, B. Daubeuf, M. A. Matthay, G. Elson, and I. Dunn-Siegrist Soluble MD-2 activity in plasma from patients with severe sepsis and septic shock Blood, December 15, 2004; 104(13): 4071 - 4079. [Abstract] [Full Text] [PDF]

				I. H Haralambieva, I. D Iankov, P. V Ivanova, V. Mitev, and I. G Mitov Chlamydophila pneumoniae induces p44/p42 mitogen-activated protein kinase activation in human fibroblasts through Toll-like receptor 4 J. Med. Microbiol., December 1, 2004; 53(12): 1187 - 1193. [Abstract] [Full Text] [PDF]

				P. Vora, A. Youdim, L. S. Thomas, M. Fukata, S. Y. Tesfay, K. Lukasek, K. S. Michelsen, A. Wada, T. Hirayama, M. Arditi, et al. {beta}-Defensin-2 Expression Is Regulated by TLR Signaling in Intestinal Epithelial Cells J. Immunol., November 1, 2004; 173(9): 5398 - 5405. [Abstract] [Full Text] [PDF]

				W. Wu, R. D. Mosteller, and D. Broek Sphingosine Kinase Protects Lipopolysaccharide-Activated Macrophages from Apoptosis Mol. Cell. Biol., September 1, 2004; 24(17): 7359 - 7369. [Abstract] [Full Text] [PDF]

				R Arroyo-Espliguero, P Avanzas, S Jeffery, and J C Kaski CD14 and toll-like receptor 4: a link between infection and acute coronary events? Heart, September 1, 2004; 90(9): 983 - 988. [Abstract] [Full Text] [PDF]

				H. P. Jia, J. N. Kline, A. Penisten, M. A. Apicella, T. L. Gioannini, J. Weiss, and P. B. McCray Jr. Endotoxin responsiveness of human airway epithelia is limited by low expression of MD-2 Am J Physiol Lung Cell Mol Physiol, August 1, 2004; 287(2): L428 - L437. [Abstract] [Full Text] [PDF]

				S. Ishihara, M. A. K. Rumi, Y. Kadowaki, C. F. Ortega-Cava, T. Yuki, N. Yoshino, Y. Miyaoka, H. Kazumori, N. Ishimura, Y. Amano, et al. Essential Role of MD-2 in TLR4-Dependent Signaling during Helicobacter pylori-Associated Gastritis J. Immunol., July 15, 2004; 173(2): 1406 - 1416. [Abstract] [Full Text] [PDF]

				P. Samuelsson, L. Hang, B. Wullt, H. Irjala, and C. Svanborg Toll-Like Receptor 4 Expression and Cytokine Responses in the Human Urinary Tract Mucosa Infect. Immun., June 1, 2004; 72(6): 3179 - 3186. [Abstract] [Full Text] [PDF]

				L. C. Parker, M. K. B. Whyte, S. N. Vogel, S. K. Dower, and I. Sabroe Toll-Like Receptor (TLR)2 and TLR4 Agonists Regulate CCR Expression in Human Monocytic Cells J. Immunol., April 15, 2004; 172(8): 4977 - 4986. [Abstract] [Full Text] [PDF]