Toll-like Receptor 4 Signaling Regulates Cytosolic Phospholipase A2 Activation and Lipid Generation in Lipopolysaccharide-stimulated Macrophages*

Inflammatory lipid mediators such as prostaglandins and leukotrienes play crucial roles in the pathogenesis of bacterial lipopolysaccharide (LPS)-induced inflammation. Cytosolic phospholipase A2 (cPLA2) is a key enzyme in the generation of pro-inflammatory lipid mediators. Here, we found that Toll-like receptor 4 (TLR4) is essential for LPS-induced cPLA2 activation and lipid release. Inhibition of TLR4 protein expression by TLR4 small interfering RNA or neutralization of TLR4 by the specific antibody against TLR4/MD2 blocked cPLA2 phosphorylation and cPLA2-hydrolyzed arachidonic acid release. Furthermore, activation of the TLR4 signaling pathway by LPS regulated cPLA2 activation and lipid release. cPLA2 phosphorylation and cPLA2-hydrolyzed lipid release were significantly impaired when TLR4 adaptor protein, either MyD88 or TRIF, was knocked down in LPS-stimulated macrophages. Similarly, LPS-induced arachidonate release was inhibited in cells transfected with a dominant-negative MyD88 or TRIF construct. Subsequently, cPLA2 activation could be suppressed by inhibition of the TLR4 adaptor protein-directed p38 and ERK MAPK pathways. These findings suggest that, in LPS-induced inflammation, the TLR4-mediated MyD88- and TRIF-dependent MAPK pathways result in cPLA2 activation and production of pro-inflammatory lipid mediators.

Since the identification of the Toll-like receptor (TLR) family, the pathways of pro-inflammatory cytokine production in response to LPS and other pathogen-associated molecular patterns from invading microbes have been well demonstrated (9,10). In mammals, the TLR family comprises at least 11 members (TLR1-TLR11) expressed on the surface of macrophages and other innate immune cells (11,12). TLRs act as primary innate immune sensors, each of which specifically recognizes distinct pathogen-associated molecular patterns (10,(12)(13)(14). Stimulation of TLRs by TLR ligands triggers the recruitment of the cytoplasmic adaptor protein MyD88 and accordingly culminates in the activation of two distinct downstream signaling pathways, the transcription factor NF-B and MAPK pathways, which induce the expression of inflammatory cytokines (15). TLR4 is the signaling receptor of LPS (16 -18). After recognizing LPS in the presence of LPS-binding protein, CD14, and MD2 protein, TLR4 activates the common MyD88dependent signaling pathway as well as a MyD88-independent pathway that is unique to TLR3 and TLR4 signaling pathways, leading to interferon-␤ production (15, 19 -21). In the MyD88-independent pathway, TLR4 interacts with the adaptor protein TRIF, instead of MyD88, to activate NF-B and MAPK as well as interferon-␤ induction (20,21). Thus, the LPS signaling pathways show a bipartite nature. Both TRIF and MyD88 are independently capable of initiating signaling events that lead to pro-inflammatory cytokine production (19,21). However, the mechanism by which LPS induces the production of pro-inflammatory lipid mediators in macrophages remains to be further determined. PAF and eicosanoids, including leukotrienes, prostaglandins, and thromboxanes, are the major constituents of pro-inflammatory lipid mediators, which play a critical role in inflammation and host defense (22)(23)(24)(25)(26). They have been implicated in the pathogenesis of asthma, sepsis, and other inflammatory diseases as well (22,(27)(28)(29). When LPS and other inflammatory stimuli activate macrophages, the lipid mediators are synthesized de novo from membrane phospholipid through a cascade of enzymes (22). The initial step of eicosanoid biosynthesis, which is thought to be the rate-limiting step, is arachidonic acid release from membrane phospholipids by activation of cytosolic phospholipase A 2 (cPLA 2 ). The liberated arachidonic acid is the common precursor molecule of all eicosanoids and is in turn converted to prostaglandins and thromboxanes by the cyclooxygenase pathway and to leukotrienes by the 5-lipoxygenase pathway (22,23). Concomitant with the release of arachidonate, lysophospholipid is formed and can be enzymatically converted to PAF (30).
As an important rate-limiting enzyme in the hydrolysis of arachidonic acid release, cPLA 2 plays a key role in initiating and regulating the multistage biosynthetic process of eicosanoid production (22,31). cPLA 2 is a member of a diverse superfamily of PLA 2 enzymes that hydrolyze fatty acid from the sn-2 position of phospholipids (32). However, cPLA 2 is the only well characterized PLA 2 that is highly selective for phospholipids containing arachidonic acid at the sn-2 position (33)(34)(35). cPLA 2 is activated by phosphorylation by MAPKs and translocated to the membrane in response to submicromolar concentrations of Ca 2ϩ (33, 36 -38). cPLA 2 -deficient mice have provided the most definitive evidence for the central role of cPLA 2 in eicosanoid and PAF production (39 -42) as well as in the pathogenesis of several inflammatory diseases such as adult respiratory distress syndrome due to bacterial sepsis (43,44). Peritoneal macrophages derived from cPLA 2 knockout mice have diminished ability to generate leukotrienes, prostaglandins, and PAF (40). Furthermore, the bone marrow-derived mast cells from these mice fail to produce eicosanoids in either immediate or delayed phase responses (42). Animals that lack cPLA 2 are resistant to bronchial hyper-reactivity and have significantly reduced pulmonary edema, polymorphonuclear leukocyte sequestration, and deterioration of gas exchange caused by LPS administration (43). The role of cPLA 2 as an important mediator of inflammatory diseases has made it a therapeutic target (41,(45)(46)(47).
Because cPLA 2 is essential for production of lipid mediators, to address the mechanism by which cPLA 2 is activated and leads to lipid production in macrophage responses to LPS will be a key to understanding the production of pro-inflammatory lipid mediators in LPS-induced inflammation. Here, we report that cPLA 2 activation and lipid release are regulated by TLR4 signaling in LPS-activated macrophages. The regulation is through TLR4-mediated MyD88-and TRIF-dependent MAPK signaling pathways.
Cell Culture-The mouse macrophage cell line RAW264.7 was obtained from American Type Culture Collection (Manassas, VA) and maintained in Dulbecco's modified Eagle's medium (BIOSOURCE, Camarillo, CA) supplemented with 10% fetal calf serum (Invitrogen).
Expression Plasmids and Transient Transfection-pEF-BOS-TRIF⌬N⌬C for dominant-negative TRIF was a gift from Dr. S. Akira (Osaka University, Osaka, Japan). TRIF was a gift from Dr. Douglas Golenbock (University of Massachusetts Medical School). MyD88 was a gift from Dr. David Segal (NCI, National Institutes of Health). Dominant-negative MyD88 and TLR3 were purchased from InvivoGen (San Diego, CA). The expression plasmids were transfected into the mouse macrophage cell line RAW264.7 by Nucleofection electroporating transfection (Amaxa Inc., Gaithersburg, MD) following the manufacturer's directions. [ 3 H]Arachidonic acid release and Western blot analysis were performed after 36 h of transfection.
Western Blot Analysis-RAW264.7 cells and TLR4, MyD88 or TRIF knockdown RAW264.7 cells were grown on 6-well plates with stimuli or other treatment as indicated. Cells were lysed in cell lysis buffer (0.3 ml/well; 20 mM Hepes (pH 7.4), 2 mM EGTA, 50 mM ␤-glycerophosphate, 1 mM Na 3 VO 4 , 1% Triton X-100, and 10% glycerol) containing protease inhibitor mixture (Roche Applied Science, Basel, Switzerland). The cell lysates were centrifuged, and equivalent amounts of lysate protein were loaded onto 8 -16% Tris/glycine/SDS-polyacrylamide gels (Invitrogen). After electrophoresis, the proteins were transferred to nitrocellulose membranes. The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline/Tween (20 mM Tris-HCl (pH 7.4), 137 mM NaCl, and 0.1% Tween 20). The blots were then incubated with primary antibodies overnight at 4°C and washed with Tris-buffered saline/Tween. The blots were incubated with horseradish peroxidaseconjugated secondary antibodies for 1 h. After washing, the blots were developed with the ECL chemiluminescence detection kit (Amersham Biosciences), and the signals were captured on x-ray films or an Image Station 440CF (Eastman Kodak Co.).
[ 3 H]Arachidonic Acid Release-RAW264.7 cells; TLR4, MyD88, or TRIF knockdown RAW264.7 cells; or cells transfected with the plasmid with or without wild-type or dominant-negative mutant TRIF and MyD88 were incubated overnight in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 0.5 Ci of [5,6,8,9,11,12,14, H]arachidonic acid (150 -230 Ci/mmol/ml; Amersham Biosciences). The medium was removed, and the cells were washed thoroughly and incubated in fresh medium with or without the various treatments as indicated. At the time points indicated, the media were collected and centrifuged, and the radioactivity was determined in the supernatants. The data are presented as the mean Ϯ S.D. of -fold stimulation compared with the control in triplicate experiments.
Dual-Luciferase Reporter Assay for NF-B Activation-RAW264.7 cells or TLR4 knockdown RAW264.7 cells were cultured until they reached 70 -85% confluence. The NF-B-luciferase reporter construct (a gift from the laboratory of Dr. Ulrich Siebenlist, NIAID, National Institutes of Health) was cotransfected with a Renilla luciferase reporter gene (Promega Corp., Madison, WI) into the cells by Nucleofection electroporating transfection. The cells were seeded into 12-well plates at a density of 6 ϫ 10 5 cells/well. The following day, the cells were treated TLR4 Regulates cPLA 2 Activation and Lipid Release as indicated. After 6 h of stimulation, the cells were lysed in passive lysis buffer (Promega), and reporter gene activity was measured using the Dual-Luciferase assay reporter system (Promega Corp.) and a Victor2 plate reader luminometer (PerkinElmer Life Sciences). In all cases, the data were normalized for transfection efficiency and are presented as the means Ϯ S.D. of -fold stimulation in triplicate experiments.

RESULTS
LPS Induces cPLA 2 Activation in Mouse RAW264.7 Macrophages-To examine whether TLR4 is required for cPLA 2 activation, we used LPS preparations to stimulate the mouse macrophages for various times. We then tested cPLA 2 activation first by measuring phosphorylation of cPLA 2 by Western blot analysis with the antibody against phospho-cPLA 2 or total cPLA 2 . Fig. 1A shows that phosphorylation of cPLA 2 became evident at 30 min after LPS stimulation, peaked at 2 h, and slowly declined thereafter. However, total cPLA 2 did not show any change in expression at the different time points. As a positive control, LPS-induced IB␣ degradation, which results in the release and activation of NF-B, was observed at 30 min and returned to the base-line level by 2-3 h (Fig. 1A), suggesting that the LPS preparation used to induce the activity of cPLA 2 in our experiments activates the TLR4 signaling pathway. We next determined the activation of cPLA 2 by measuring the release of its catalytic product arachidonic acid from [ 3 H]arachidonic acid-labeled RAW264.7 cells. After stimulation with different types of LPS and lipid A (which is the core component of LPS),   NOVEMBER 25, 2005 • VOLUME 280 • NUMBER 47 the arachidonic acid release was significantly increased in an LPS dosedependent manner (Fig. 1B). Although cPLA 2 is a major enzyme of arachidonic acid release from membrane phospholipids, secretory PLA 2 , another member of the PLA 2 superfamily with a low molecular mass, may also mediate arachidonic acid release (48). To determine whether LPS-induced arachidonic acid release is specifically catalyzed by cPLA 2 , the specific chemical inhibitors of cPLA 2 and secretory PLA 2 were used to treat RAW264.7 cells. As shown in Fig. 1C, the cPLA 2 inhibitor methyl arachidonyl fluorophosphonate markedly blocked arachidonic acid release by LPS. In contrast, the secretory PLA 2 inhibitor thioetheramide-PC did not inhibit the LPS response, suggesting that arachidonic acid release in response to LPS is due mainly to activation of cPLA 2 .

TLR4 Regulates cPLA 2 Activation and Lipid Release
TLR4 Mediates LPS-induced cPLA 2 Activation and Lipid Release-Two lines of evidence suggest that TLR4 is the receptor that mediates the LPS signal to cPLA 2 activation and lipid release. First, RAW264.7 macrophages were treated with the monoclonal antibody to mouse TLR4/MD2 (MTS510), which specifically inhibits cytokine production induced by lipid A (which is the core component of LPS) (49). We confirmed that antibody MTS510 inhibited lipid A-induced TLR4-mediated NF-B activation using a luciferase reporter assay as well (data not shown). Likewise, Fig. 2 (A and B) shows that antibody MTS510 significantly blocked cPLA 2 -hydrolyzed arachidonic acid release after lipid A or LPS stimulation. By contrast, the control monoclonal antibody against TLR3 did not inhibit either lipid A-or LPS-induced arachidonic acid release in macrophages. Second, RAW264.7 cells were stably transfected with a vector expressing TLR4 siRNA or nonspecific siRNA.
Western blot analysis indicated the silencing of TLR4 expression in RAW264.7 cells by TLR4 siRNA (Fig. 2C). To further confirm the knockdown of TLR4 signaling in macrophages, we measured LPS-induced NF-B promoter activity using the luciferase reporter plasmid in TLR4 siRNA-treated RAW264.7 cells. As shown in Fig. 2D, NF-B promoter activity was suppressed after LPS stimulation, suggesting the inhibition of TLR4-mediated NF-B activation by TLR4 siRNA. We next examined LPS-induced arachidonic acid release and cPLA 2 phosphorylation in TLR4 knockdown RAW264.7 cells. The results in Fig. 2E show that the arachidonic acid release induced by LPS was suppressed in TLR4 siRNA-treated cells compared with control siRNA-treated cells. In addition, Western blotting showed reduced cPLA 2 phosphorylation in TLR4 knockdown RAW264.7 cells after LPS stimulation (see Fig. 4).

Both the TRIF and MyD88 Adaptor Proteins of the TLR4 Signaling Pathway Are Involved in LPS-induced cPLA 2 Activation and Lipid
Release-Both MyD88-and TRIF-dependent pathways in TLR4 signaling are capable of activating downstream NF-B and MAPK pathways (20,21). We next asked whether TRIF and MyD88 adaptor proteins are required for cPLA 2 activation and lipid release. We generated MyD88 and TRIF knockdown RAW264.7 cells by transfection of MyD88 and TRIF siRNA constructs into the macrophages, respectively. Western blot analysis using antibodies against MyD88 and TRIF showed that MyD88 protein expression was knocked down by MyD88 siRNA, but not by TRIF siRNA. Likewise, TRIF siRNA inhibited TRIF protein expression, but did not affect MyD88 protein expression (Fig. 3A). Subsequently, cPLA 2 -hydrolyzed arachidonic acid release and cPLA 2 phos- phorylation were assessed in the MyD88 and TRIF knockdown macrophages after LPS stimulation. Arachidonic acid release was suppressed in both the MyD88 and TRIF knockdown macrophages. However, no inhibition of arachidonic acid release was observed in the cells treated with control siRNA (Fig. 3B). cPLA 2 phosphorylation was significantly reduced in both adaptor protein knockdown macrophages as well (Fig.  4). In addition, overexpression of wild-type TRIF and MyD88 in the macrophages slightly induced cPLA 2 -hydrolyzed lipid release (Fig. 3C). In contrast, overexpression of the dominant-negative form of MyD88 or TRIF suppressed LPS-stimulated arachidonic acid release (Fig. 3D). To further study whether TRIF or MyD88 is capable of independently transmitting the TLR ligand signal from the cell surface to the cytoplasm for cPLA 2 activation, we used the TLR3 ligand poly(I⅐C) RNA, which specifically activates the TRIF adaptor protein, and the TLR9 ligand CpG DNA, which activates only the MyD88 adaptor protein, to stimulate macrophages. Because there is a lower expression of endogenous TLR3 compared with TLR4 in the RAW264.7 cell line (64,65), a TLR3expressing construct was transfected into the macrophages. Poly(I⅐C) only slightly induced cPLA 2 -hydrolyzed lipid release before TLR3 transfection (Fig. 3E), but significantly induced arachidonic release and cPLA 2 phosphorylation after TLR3 overexpression in the macrophages (Fig. 3F). Fig. 3 (G and H) shows CpG DNA ligand-induced arachidonic release and cPLA 2 phosphorylation. Therefore, either TRIF or MyD88 is able to independently activate cPLA 2.
TLR4-mediated MAPK Pathways Are Required for LPS-induced cPLA 2 Activation and Lipid Release-We have demonstrated that LPS induced cPLA 2 activity through TLR4 and its adaptor proteins. The MAPK pathway is one of the major downstream pathways in TLR4 signaling. As shown in Fig. 4, knockdown of TLR4 or TLR4 adaptor proteins MyD88 and TRIF diminished LPS-induced phosphorylation of p38 and ERK MAPKs. In addition, it has been shown that cPLA 2 activation requires phosphorylation by MAPKs and that cPLA 2 is a substrate of p38 and ERK MAPKs (36,37). Therefore, we next studied whether the TLR4-mediated MAPK pathway is required for LPS-in-duced cPLA 2 activation and lipid release. Fig. 5A shows that inhibitors of p38 (SB 203580) and ERK (U0126) inhibited LPS-induced phosphorylation of p38 and ERK1/2, respectively, suggesting that these inhibitors block TLR4-mediated MAPK pathways. Similarly, LPS-stimulated cPLA 2 phosphorylation (Fig. 5B) and cPLA 2 -hydrolyzed arachidonic acid release (Fig. 5C) were markedly inhibited by these MAPK inhibitors as well. Thus, the TLR4-mediated MyD88-and TRIF-dependent MAPK pathways are involved in LPS-induced cPLA 2 activation.
The Phosphoinositide 3-Kinase (PI3K)/Akt Pathway Is Not Required for TLR4-mediated cPLA 2 Activation by LPS-It has been reported that the PI3K/Akt pathway participates in LPS-induced TLR4 signaling, leading to NF-B activation (55)(56)(57). To determine whether the PI3K/ Akt pathway affects TLR4-mediated cPLA 2 activation and lipid release by LPS, RAW264.7 macrophages were treated with LPS with or without two specific pharmacological inhibitors (LY-294,002 and wortmannin) that block the activation of PI3K by different mechanisms. PI3K activation was evaluated by immunoblot analysis of phosphorylated Akt. As shown in Fig. 6A, LPS stimulation of the cells resulted in phosphorylation of Akt. Preincubation with either LY-294,002 or wortmannin completely abolished LPS-induced activation of Akt. However, blockade of . TLR4, MyD88, and TRIF siRNAs decrease the phosphorylation of cPLA 2 , p38, and ERK. RAW264.7 cells were stably transfected with the control siRNA, TLR4 siRNA, TRIF siRNA or MyD88 siRNA construct. cPLA 2 , p38, and ERK phosphorylation was examined by Western blotting with antibodies against phosphorylated (p) and total cPLA 2 , p38, and ERK, respectively.  NOVEMBER 25, 2005 • VOLUME 280 • NUMBER 47 PI3K/Akt by the inhibitors failed to inhibit phosphorylation of cPLA 2 (Fig. 6A) and arachidonic acid release (Fig. 6B) induced by LPS, suggesting that the PI3K/Akt pathway is not required for TLR4-mediated cPLA 2 activation and lipid release in response to LPS.

DISCUSSION
In this study, we have examined the role of the TLR4 signaling pathway in LPS-induced cPLA 2 activation and lipid release in macrophages. The evidence shows that LPS induces cPLA 2 activation and lipid release via the TLR4-mediated MyD88-and TRIF-dependent MAPK pathways. The data suggest that the TLR4 signaling pathway plays an important role in activating cPLA 2 -initiated and cPLA 2 -regulated pro-inflammatory lipid production in response to LPS challenge. cPLA 2 is the only known PLA 2 activated by receptor-mediated events (50). A wide variety of extracellular stimuli have been found to induce activation or increase synthesis of cPLA 2 in diverse cell models (34,51,52). However, in LPS-induced inflammation, the phagocyte receptor and pathway required for cPLA 2 activation leading to pro-inflammatory lipid production remain to be defined. To pursue whether TLR4 is the receptor that mediates cPLA 2 activation in LPS-stimulated macrophages, we first used LPS to stimulate the macrophages and confirmed that the LPS preparation in our experiments activated the TLR4 pathway. By assessing cPLA 2 phosphorylation and cPLA 2 -hydrolyzed arachidonic acid release, we showed that cPLA 2 was activated in macrophages in response to LPS, suggesting that LPS, which activates TLR4, induces cPLA 2 activation and lipid release. Furthermore, direct evidence was obtained by treating the macrophages with TLR4 siRNA or antibody MTS510 (raised against TLR4/MD2). Antibody MTS510, which is capable of neutralizing the TLR4-MD2 complex on the cell surface, blocked cPLA 2 -hydrolyzed arachidonate release induced by LPS and lipid A. On the other hand, LPS-induced arachidonate release and cPLA 2 phosphorylation were significantly inhibited in TLR4 knockdown macrophages by TLR4 siRNA. Together, these data strongly support the conclusion that TLR4 is required for cPLA 2 activation and lipid release in response to LPS stimulation. In addition, our data have shown that not only LPS, but also the ligands of TLR3 and TLR9 are capable of inducing cPLA 2 activation, suggesting that cPLA 2 activation can be regulated by different members of the TLR family.
As described above, the LPS-induced signaling pathway has a bipartite nature. Mouse macrophages with defective or missing TRIF or MyD88 protein exhibit severely impaired production of inflammatory cytokines in response to LPS (20,21). MyD88 and TRIF might each be capable of independently initiating the major signaling pathway for production of inflammatory cytokines. The reason that a cell needs two branches of the pathway responsible for signals that emanate from the LPS receptor remains unclear. To determine which branch of the TLR4 signaling pathway is responsible for cPLA 2 activation and lipid release, we examined cPLA 2 phosphorylation and cPLA 2 -hydrolyzed arachidonic acid release in MyD88 or TRIF protein knockdown macrophages by siRNAs and dominant-negative mutants after LPS challenge. LPSinduced arachidonate release and cPLA 2 phosphorylation were impaired in either adaptor protein-deficient macrophages. In addition, cPLA 2 activation and lipid release could be induced by poly(I⅐C) RNA and CpG DNA ligands, which specifically activate the TRIF and MyD88 pathways through TLR3 and TLR9, respectively, indicating that MyD88 and TRIF may independently mediate LPS-induced cPLA 2 activation and pro-inflammatory lipid production. cPLA 2 is essential for both the immediate and delayed phases of eicosanoid generation in the mouse bone marrow-derived mast cell response to stimuli (42). Activation of cPLA 2 for the rapid generation of leukotrienes is thought to be through the post-translational mechanism of cPLA 2 phosphorylation (53), whereas in a more delayed response (such as in response to inflammatory cytokines and certain growth factors), cPLA 2 activity may be regulated by its expression level (34). Thus, cPLA 2 activation can be regulated by both transcriptional and posttranslational mechanisms. Our data show that activation of cPLA 2 was accompanied by its phosphorylation in macrophages exposed to LPS. However, no increased expression of cPLA 2 was observed during 6 h of stimulation by LPS, suggesting that, in immediate response to LPS stimulation, cPLA 2 is activated through the post-translational mechanism leading to rapid generation of pro-inflammatory lipid mediators. cPLA 2 phosphorylation is an important mechanism for cPLA 2 activation (42). Extracellular ligands that activate cPLA 2 cause the phosphorylation of the catalytic domain at conserved serines 505 and 727 (36,48). The mutation of Ser 505 to Ala is known to block cPLA 2 activation by members of the MAPK family (36), but the functional relevance of Ser 727 has not been reported. Previous reports have demonstrated that either one or multiple MAPK members are able to activate cPLA 2 upon extracellular stimulation and the ability of a specific receptor to trigger the different MAPK family members (48,54). TLR4 signaling activates multiple members of the MAPK family, including ERK1/2, p38 kinase, and JNK (c-Jun NH 2 -terminal kinase) (15). We have found that either a p38-specific or an ERK1/2-specific inhibitor was capable of blocking

TLR4 Regulates cPLA 2 Activation and Lipid Release
TLR4-mediated cPLA 2 phosphorylation and cPLA 2 -hydrolyzed arachidonic acid release after LPS stimulation. Thus, both the ERK1/2 and p38 signaling pathways act to activate cPLA 2 in response to LPS challenge. Whether cPLA 2 phosphorylation is sufficient to account for prolonged activation of cPLA 2 is uncertain. cPLA 2 activation may also be enhanced by binding to lipids such as phosphatidylinositol bisphosphate (58) and ceramide 1-phosphate (59). LPS may induce intracellular release of ceramide (60). Ceramide 1-phosphate has been reported to bind to the C2 domain of cPLA 2 and to increase its sensitivity to intracellular calcium.
PI3K and the downstream serine/threonine kinase Akt play important roles in host defense (61). It has been shown that LPS stimulation of inflammatory cells activates the PI3K/Akt pathway (55)(56)(57)62). Although the signaling steps between TLR4 and activation of PI3K have not been characterized, it has been suggested that the activation of PI3K by LPS is TLR4-and MyD88-dependent (56,57). PI3K and Akt participate in the TLR4 signaling pathway to regulate NF-B activation induced by LPS (55,62,63). However it is not clear whether the LPSactivated PI3K/Akt pathway involves TLR4-mediated cPLA 2 activation by LPS. We have found that incubation of macrophages with LPS resulted in phosphorylation of Akt. Addition of the PI3K inhibitors to the macrophage cultures blocked LPS-induced Akt phosphorylation. However, the blockade of the PI3K/Akt pathway did not impair LPSinduced cPLA 2 phosphorylation and arachidonic acid release. Therefore, PI3K/Akt is not necessary for TLR4-dependent cPLA 2 activation and lipid release.
TLR4 studies have focused on the mechanism by which TLR4 mediates the production of pro-inflammatory cytokines. Our data now suggest that TLR4 may also be involved in the generation of potent proinflammatory lipid mediators by regulating cPLA 2 activation. This TLR4-cPLA 2 -lipid mediator mechanism may provide insights into novel therapies for LPS-induced inflammation.