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* This work was supported by Korea Science and Engineering Foundation Grant 981-0505-027-2 and Molecular Medical Science Research Grant 02-03-A-05.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.
We investigated the extent to which phosphatidylinositol 3-kinase (PI 3-kinase) and Rac, a member of the Rho family of small GTPases, are involved in the signaling cascade triggered by tumor necrosis factor (TNF)-α leading to activation of c-fos serum response element (SRE) and c-Jun amino-terminal kinase (JNK) in Rat-2 fibroblasts. Inhibition of PI 3-kinase by LY294002 or wortmannin, two specific PI 3-kinase antagonists, or co-transfection with a dominant negative mutant of PI 3-kinase dose-dependently blocked stimulation of c-fos SRE by TNF-α. Similarly, LY294002 significantly diminished TNF-α-induced activation of JNK, suggesting that nuclear signaling triggered by TNF-α is dependent on PI 3-kinase-mediated activation of both c-fos SRE and JNK. We also found nuclear signaling by TNF-α to be Rac-dependent, as demonstrated by the inhibitory effect of transient co-transfection with a dominant negative Rac mutant, RacN17. Our findings suggest that Rac is situated downstream of PI 3-kinase in the TNF-α signaling pathway to the nucleus, and we conclude that PI 3-kinase and Rac each plays a pivotal role in the nuclear signaling cascade triggered by TNF-α.
tumor necrosis factor-α
c-Jun amino-terminal kinase
cytosolic phospholipase A2
Dulbecco's modified Eagle's medium
serum response element
PBS plus Tween 20
tumor necrosis factor receptor-1
fetal bovine serum
Phosphatidylinositol 3-kinase (PI 3-kinase)1 is a lipid kinase involved in mitogenic signal transduction and cellular transformation (
). For example, an inhibition of PI 3-kinase was shown to block growth factor induction of membrane ruffling, while activated PI 3-kinase is sufficient to induce membrane ruffling, acting through Rac (
). Thus, Rac appears to lie downstream of PI 3-kinase within a signaling pathway that controls actin remodeling.
Rac is also crucially involved in the regulation of signal transduction cascades to the nucleus evoked by environmental stresses and proinflammatory cytokines; elements of such cascades include c-Jun amino-terminal kinase (JNK) (
), almost nothing is known about the potential role of PI 3-kinase in Rac-mediated gene regulation in response to environmental stress or proinflammatory cytokines.
Tumor necrosis factor (TNF)-α is one of the most pleiotropic proinflammatory cytokines, signaling a large number of cellular responses, including cytotoxicity, antiviral activity, fibroblast proliferation, and the transcriptional regulation of various genes (
). TNF engagement of TNFR1 leads to the recruitment of TNFR1-associated death domain protein, receptor-interacting protein, and TNFR-associated factor-2 (TRAF2) leads to the formation of a receptor complex (
). Nonetheless, little is known about the intracellular signaling mediating activation of nuclear transcription factors. In particular, the roles of PI 3-kinase and Rac in the nuclear signaling by TNF-α are as yet unclear. In the present study, we investigated the extent to which PI 3-kinase and Rac are involved in the TNF-α-induced activation of c-fos SRE and JNK. Our findings suggest that both PI 3-kinase and Rac have crucial functions within the intracellular signaling cascade triggered by TNF-α in Rat-2 fibroblasts.
Chemicals and Reagents
Lysophosphatidic acid (LPA), mepacrine, and wortmannin (
), a PI 3-kinase antagonist, were purchased from Sigma. LY294002, another PI 3-kinase antagonist, and C2-ceramide were purchased from BioMol (Plymouth Meeting, PA). TNF-α was either purchased from Sigma or was obtained as a gift from Dr. D.-M. Jue (Catholic University Medical College, Seoul, Korea). Fetal bovine serum (FBS), gentamycin, and Dulbecco's modified Eagle's medium (DMEM) were purchased from Life Technologies, Inc. Control (GsTsGCTCCTAAGTTTCTsAsT) and antisense (GsTsGCTGGTAAGGATCTsAsT) cytosolic phospholipase A2 (cPLA2) oligonucleotides were purchased from BioMol. The antisense oligonucleotide is directed against codons 4–9 of human cytosolic, Ca2+-dependent PLA2. Note that the linkages are phosphothioated at both the 5′ and 3′ ends (lowercase “s” in sequences). All other chemicals were from standard sources and were molecular biology grade or higher.
Reporter gene pSRE-Luc contains c-fos SRE oligonucleotide sequences (23-mer) inserted at the −53 position of a truncated basal c-fos promoter fused to the luciferase gene (
). The pEXV, pEXV-RacV12 (Rac1Val12), and pEXV-RhoV14 (RhoAVal14) plasmids were gifts from Dr. A. Hall (University College, London, United Kingdom). All Rac and Rho proteins were expressed as NH2-terminally 9E10 epitope-tagged derivatives driven by SV40 promoter (
), was a gift from Dr. J. Downward (Imperial Cancer Research Fund, London, United Kingdom). Amino acids 478–513 are deleted in the mutant, which, consequently, lacks the binding site for the catalytic subunit.
Cell Culture, Transfections, and Luciferase Assay
Rat-2 fibroblasts were obtained from the American Type Culture Collection (ATCC, CRL 1764). The cells were grown in DMEM supplemented with 0.1 mm nonessential amino acids (Life Technologies, Inc.), 10% FBS, 50 units/ml penicillin, and 50 μg/ml streptomycin at 37 °C under a humidified 95%, 5% (v/v) mixture of air and CO2. The Rat2-RacN17 stable clone expressing a dominant negative Rac1mutant, RacN17, has been described previously (
Transient transfection was carried out by plating approximately 5×105 cells in 100-mm dishes for 24 h and then adding calcium phosphate:DNA precipitates prepared with 20 μg of DNA/dish. The quantities of plasmid used were 3 μg of reporter gene (pSRE-Luc) and 5 μg of small GTPase expression plasmids (e.g. pEXV-RacV12). To control for variations in cell number and transfection efficiency, all clones were co-transfected with 1 μg of pCMV-βGAL, a eukaryotic expression vector in which the Escherichia coliβ-galactosidase (lacZ) structural gene is under the transcriptional control of the cytomegalovirus promoter. In each transfection, the quantity of DNA used was held constant (20 μg) by adding sonicated calf thymus DNA (Sigma). After a 6-h incubation with calcium phosphate:DNA precipitates, cells were rinsed twice with phosphate-buffered saline (PBS) before incubating them in fresh DMEM supplemented with 0.5% FBS for an additional 36 h. Each dish of cells was then rinsed twice with PBS and lysed in 0.2 ml of lysis solution (0.2 m Tris (pH 7.6) + 0.1% Triton X-100), after which lysed cells were scraped and spun for 1 min. Supernatants were assayed for protein concentration as well as luciferase and β-galactosidase activities.
Luciferase activity was assayed using 10 μl of extract according to the manufacturer's protocol (Promega Luciferase Assay System; Promega, Madison, WI) and counted for 10 s in a Beckman liquid scintillation spectrometer using the tritium channel with the coincidence circuit disconnected. Transfection experiments were performed in triplicate with two independently isolated sets of cells, and the results were averaged. β-Galactosidase assays were carried out on 50-μl aliquots of extract (diluted with 100 μl of H2O) using 150 μl of 2× reaction buffer (3 mg/ml O-nitrophenyl-β-galactopyranoside, 2 mmMgCl2, 61 mm Na2HPO4, 39 mm NaH2PO4, 100 mm2-mercaptoethanol). Once a faint yellow color appeared, the reactions were stopped by the addition of 350 μl of 1 mNa2CO3. Optical density at 410 nm was then measured in a spectrophotometer and used to normalize luciferase activity to transfection efficiency. Protein concentrations were determined routinely using the Bradford procedure with Bio-Rad dye reagent and bovine serum albumin as a standard.
Phosphatidylinositol 3-Kinase Assay
PI 3-kinase activity was measured by in vitro phosphorylation of phosphatidylinositol (PI), using essentially the same method as described previously (
). Subconfluent Rat-2 cells were serum-starved in serum-free DMEM for 16 h and then stimulated with TNF-α. Each dish of cells was then washed twice in ice-cold PBS and lysed for 30 min at 4 °C in 1 ml of lysis buffer (20 mm Tris-HCl (pH 7.5), 137 mm NaCl, 1 mm MgCl2, 1 mm CaCl2, 100 μm sodium vanadate, 2 mm EDTA, 1% Nonidet P-40, 10% glycerol, 2 μg/ml aprotonin, 10 μg/ml antipain, 5 μg/ml leupeptin, 0.5 μg/ml pepstatin, and 1.5 μg/ml benzamidine) with 34 μg/ml phenylmethylsulfonyl fluoride. After lysis, the soluble fractions were harvested by centrifugation (15,000 rpm for 15 min) at 4 °C. The harvested fractions (containing 1 mg of protein) were incubated with anti-phosphotyrosine agarose beads to immunoprecipitate PI 3-kinase. The immunoprecipitates were successively washed three times in washing buffer-1 (PBS containing 1% Nonidet P-40 and 100 μmsodium vanadate), three times in washing buffer-2 (100 mmTris-HCl (pH 7.5) containing 500 mm LiCl2 and 100 μm sodium vanadate), and finally two times in washing buffer-3 (25 mm Tris-HCl (pH 7.5) containing 100 mm NaCl, 1 mm EDTA, and 100 μmsodium vanadate). To these immunoprecipitates were added 20 μl of reaction buffer (10 μl of 100 mm MgCl2 + 10 μl of phosphatidylinositol (200 μg/μl) sonicated in 10 mm Tris-HCl (pH 7.5) containing 1 mm EGTA). After adding 10 μl of ATP solution (10 μm) containing 10 μCi of [γ-32P]ATP, the immunoprecipitates were incubated for 20 min at room temperature with constant shaking. The reaction was stopped by the addition of 100 μl of 1 mHCl and 200 μl of CHCl3-methanol (1:1). The samples were then centrifuged, and the lower organic phases were harvested and applied to silica gel TLC plates (Merck Co.) coated with 1% potassium oxalate. The TLC plates were developed in CHCl3-CH3OH-H2O-NH4OH (60:47:11.3:2), dried, and visualized autoradiographically.
JNK/Stress-activated Protein Kinase Assays
To assay JNK activity mediated by TNF-α or C2-ceramide, subconfluent Rat-2 cells were serum-starved for 24 h in DMEM containing 0.5% FBS and then stimulated with TNF-α or C2-ceramide for 30 min. Each dish of cells was then washed with cold PBS, lysed by incubation for 5 min at 4 °C in 0.5 ml of ice-cold lysis buffer (20 mm Tris (pH 7.4) 150 mm NaCl, 1 mmEDTA, 1 mm EGTA, 1% Triton X-100, 2.5 mmsodium pyrophosphate, 1 mm β-glycerophosphate, 1 mm Na3VO4, 1 μg/ml leupeptin) with 1 mm phenylmethylsulfonyl fluoride, scraped into Eppendorf tubes, and triturated by 10 passes through a 21.1-gauge needle on ice. The supernatant (cell lysate) was harvested by microcentrifugation at 14,000 rpm for 10 min. Protein concentrations were equalized by normalizing them to the protein levels (assayed by Bradford procedure with Bio-Rad dye reagent) measured before the JNK assay.
JNK activity was determined using a JNK assay kit according to the manufacturer's protocol (New England Biolabs). Briefly, an amino-terminal c-Jun (amino acid residues 1–89) fusion protein bound to glutathione-Sepharose beads was used to pull down JNK from cell lysates. The kinase reaction (50 μl) was then carried out using the c-Jun fusion protein as a substrate in the presence of cold ATP. Phosphorylation of the c-Jun fusion protein at Ser-63 was measured by Western blot using an anti-phospho-c-Jun rabbit polyclonal antibody that detects only catalytically activated c-Jun phosphorylated at Ser-63. Protein samples were heated to 95 °C for 5 min and subjected to SDS-polyacrylamide gel electrophoresis on 8% acrylamide gels, followed by transfer to polyvinylidene difluoride membranes for 2 h at 100 V using a Novex wet transfer unit. Membranes were then blocked overnight in PBS-T (PBS containing 0.01% (v/v) Tween 20) with 5% (w/v) nonfat dried milk, after which they were incubated for 2 h with primary antibody (anti-phospho-c-Jun) in PBS-T and then for 1 h with horseradish peroxidase-conjugated secondary antibody. The blots were developed using enhanced chemiluminescence kits (ECL, Amersham Pharmacia Biotech). Bands on XAR-5 film (Eastman Kodak Co.) corresponding to phospho-c-Jun were measured by densitometry.
c-fos SRE Is One of the Nuclear Target Sequences of TNF-α
As an initial approach to understanding the role of PI 3-kinase in the signal transduction pathway between TNF-α and the nucleus, we assessed the capacity of TNF-α to stimulate c-fos SRE, which is a primary nuclear target for various extracellular signals (
). TNF-α-induced SRE activation was monitored by measuring luciferase activities normalized to co-transfected β-galactosidase activity. As shown in Fig.1, TNF-α stimulated c-fos SRE-dependent reporter gene activity in a dose- and time-dependent manner. A maximal 5.7-fold increase in the luciferase activity occurred at a TNF-α concentration of 10 ng/ml (Fig. 1, left panel) 1 h after its addition (Fig. 1, right panel). No TNF-α-induced luciferase activity was seen in cells transiently transfected with pO-Luc (vector without SRE; data not shown).
PI 3-Kinase Activity Is Essential for TNF-α Signaling to c-fos SRE
To assess the role of PI 3-kinase, we examined the effects of PI 3-kinase antagonists, LY294002 (Fig.2A) and wortmannin (Fig.2B) on TNF-α-induced c-fos SRE activation. Both compounds dose-dependently inhibited TNF-α-evoked SRE luciferase activity at levels that selectively inhibit PI 3-kinase activity (
). As examples, 25 μm LY294002 reduced TNF-α-evoked SRE luciferase activity by ∼40%, while 100 nm wortmannin reduced the activity by ∼50%. C2-Ceramide-induced SRE activation was similarly attenuated by PI 3-kinase inhibition (Fig. 2, A and B). Further, co-transfection with pSG5-Δp85 encoding a dominant negative PI 3-kinase mutant significantly and dose-dependently diminished TNF-α-induced stimulation of SRE-luciferase activity (Fig.2C). Of the quantities tested, co-transfection with 5 μg of pSG5-Δp85 reduced TNF-α-induced stimulation of SRE-luciferase activity by ∼75%, whereas lysophosphatidic acid (LPA)-induced SRE activation was little affected. Taken together, these results are strongly suggestive of the participation of PI 3-kinase in TNF-α signaling to c-fos SRE.
Encouraged by above results, we next directly assayed TNF-α-evoked PI 3-kinase activity by measuring the levels of the product, phosphatidylinositol phosphate, in serum-starved Rat-2 cells exposed to TNF-α for 10 min (Fig. 3). Consistent with the above results, addition of TNF-α stimulated PI 3-kinase activity significantly. Interestingly, we observed a similar stimulation of PI 3-kinase activity by TNF-α in Rat2-RacN17 cells (
). Therefore, to investigate the potential role of Rac in the TNF-α signaling to c-fos SRE, we tested the effect of transfection with the expression vector encoding RacN17. As shown in Fig.4A, TNF-α-induced SRE activation was dramatically inhibited by co-transfection with 5 μg of pEXV-RacN17 (∼65% reduction in luciferase activity), suggesting that Rac activity is crucial for TNF-α-induced signaling to c-fos SRE. On the other hand, SRE activation induced by 10 μm LPA was unaffected by pEXV-RacN17 transfection.
The role of Rac was further investigated by comparing the SRE-luciferase activities in Rat-2 and Rat2-RacN17 cells. Fig.4B shows TNF-α-induced SRE activation was inhibited by 50% in serum-starved Rat2-RacN17 cells. In contrast, levels of LPA-induced SRE activation were similar in Rat-2 and Rat2-RacN17 cells (Fig. 4B), while epidermal growth factor-evoked activation of SRE was reduced somewhat in Rat2-RacN17 cells. We, therefore, conclude that TNF-α signaling to c-fos SRE is mediated, at least in part, by a Rac-dependent cascade.
Pretreatment with LY294002 Inhibits JNK Activation by TNF-α
The effect of LY294002 on TNF-α-induced JNK activation was assessed to determine the extent to which it is dependent on PI 3-kinase and Rac activities. Serum-starved Rat-2 cells were pretreated with LY294002 (+) or control buffer (−) for 30 min before adding TNF-α (10 ng/ml), C2-ceramide (5 μm), or arachidonic acid (AA; 100 μm), a principal product of Rac-activated phospholipase A2 (
). TNF-α and C2-ceramide each induced a ∼5-fold increase of JNK activity as compared with control buffer, an effect that was dramatically inhibited by LY294002 (Fig.5A). On the other hand, LY294002 had no inhibitory effect on AA-induced JNK activation, which suggests that PI 3-kinase is specifically required for activation of JNK by TNF-α or C2-ceramide and implies a common, essential role for PI 3-kinase in TNF-α-evoked activation of both JNK and c-fos SRE.
To determine the function of Rac in TNF-α signaling to JNK, levels of JNK activation were compared between control cells and cells stably expressing RacN17. As shown in Fig. 5B, TNF-α- and C2-ceramide-induced JNK activation was dramatically reduced in Rat2-RacN17 cells, indicating the importance of Rac activity in those cases. On the other hand, JNK activation induced by 100 μm AA was unaffected by RacN17 expression.
The signaling hierarchy between PI 3-kinase and Rac was investigated further by assessing the LY294002 sensitivity of SRE activation by RacV12, a constitutively activated form of Rac1. LY294002 had no inhibitory effect on SRE activation by RacV12 or RhoV14 (Fig.6), whereas RasV12-induced SRE activation was significantly and dose-dependently inhibited by LY294002. This is consistent with previous reports showing that PI 3-kinase acts as a downstream mediator of H-Ras within the signaling cascades leading to actin remodeling and transformation (
), and is further evidence that Rac is situated downstream of PI 3-kinase in the nuclear signaling cascade leading to activation of c-fos SRE or JNK. In a separate experiment, we observed that LY294002 had no inhibitory effect on RacV12-induced JNK activation in Rat-2 cells (data not shown).
Role of cPLA2 in TNF-α Signaling to SRE Activation
We previously reported that cytosolic phospholipase A2 (cPLA2) plays an essential role in mediating Rac signaling to c-fos SRE and thus acts as an important downstream mediator of Rac (
). Considering the linkage between TNF-α and Rac signaling, it seems reasonable to hypothesize that cPLA2 may be involved in TNF-α signaling to SRE. To test this possibility, we assessed the extent to which mepacrine, a potent PLA2 inhibitor, inhibited TNF-α-induced activation of SRE. Fig. 7A shows that pretreatment with 1 μm mepacrine inhibited TNF-α-induced SRE activation by approximately 50% without affecting LPA-induced activation, suggesting PLA2 is specifically required for TNF-α signaling to c-fos SRE.
To further analyze the role of PLA2 in TNF-α signaling, especially that of cPLA2, we examined the effect of transfecting cells with antisense cPLA2 oligonucleotide on TNF-α-induced SRE activation. Co-transfection with the antisense oligonucleotide but not the control oligonucleotide significantly inhibited TNF-α-induced SRE activation (Fig. 7B). For example, cotransfection with 0.5 μm cPLA2antisense oligomer reduced SRE activation by ∼45%, which suggests that a Rac-cPLA2-linked cascade is involved in TNF-α signaling to c-fos SRE. In contrast, LPA-induced SRE activation was unaffected by transfection of the antisense oligonucleotide, suggesting that the involvement of cPLA2is specific to TNF-α-induced signaling to c-fos SRE.
In the present study, we provide evidence supporting novel roles for PI 3-kinase and Rac in the nuclear signaling cascade triggered by TNF-α in Rat-2 fibroblasts. TNF-α was previously reported to rapidly induce protooncogene c-fos in the adipogenic TA1 cell line, although the exact target promoter sequences by which TNF-α stimulates c-fos transcription remain unknown (
). Our results clearly indicate that SRE is at least one of the nuclear target sequences by which TNF-α stimulates c-fos expression. Consistent with this conclusion, c-fos SRE is also reported to be a nuclear target of ceramide, a putative second messenger for certain stresses (e.g. ultraviolet and x-rays) and inflammatory cytokines such as TNF-α (
) showing that AA and its lipoxygenase-generated metabolite are downstream elements in the TNF-α signaling pathway to c-fos. The function of AA as a downstream mediator of TNF-α signaling was also demonstrated in stromal cells, where AA mediates TNF-α-induced activation of JNK (
The involvement of PI 3-kinase in TNF-α-induced signaling to c-fos SRE was confirmed by the significant inhibitory effects of LT294003 and wortmannin, specific PI 3-kinase antagonists, and of transient transfection with pSG5-Δp85 encoding a dominant negative PI 3-kinase mutant. Consistent with this conclusion, JNK activation by TNF-α was dramatically inhibited by LY294002, implying PI 3-kinase functions broadly as a downstream TNF-α mediator in the signaling pathways leading to SRE and JNK activation. That TNF-α stimulates PI 3-kinase activity in vitro lends additional support to this idea. We do not yet know the TNF-α target molecule(s) that mediates PI 3-kinase activation; nonetheless, since the mode of action of C2-ceramide is quite similar to that of TNF-α, especially with respect to inhibition by LY294002, we postulate that enhanced production of ceramide might be involved. On the other hand, although further characterization is needed for confirmation, our evidence suggests the role of TRAF2 in the TNF-α signaling to SRE or JNK is minimal. For example, a dominant negative mutant of TRAF2 does not inhibit activation of either JNK or SRE in cells exposed to TNF α (data not shown). This finding is in contrast to previous reports (
) in which TRAF2 was shown to be essential for TNF-α-induced JNK activation in lymphocytes, suggesting the function of TRAF2 differs in Rat-2 fibroblasts and lymphocytes. In any event, our present findings make us confident that PI 3-kinase is essential for mediating the nuclear signaling cascades triggered by TNF-α or ceramide, which is consistent with increasing evidence indicating that PI 3-kinase is activated by environmental stresses and growth factors (
We also found evidence for the role of Rac in TNF-α signaling to the nucleus, which is consistent with earlier findings demonstrating an essential role of Rac in the nuclear signaling by C2-ceramide, cytokines and environmental stresses (
). Thus, the present study shows that TNF-α stimulates c-fos SRE and JNK via a signaling cascade involving PI 3-kinase and Rac. Although precise determination of the mechanisms of action of PI 3-kinase and Rac will require further study, we postulate a hierarchical relationship among these proteins (TNF-α → PI 3-kinase → Rac), whereby Rac serves as a PI 3-kinase downstream molecule in a TNF-α-triggered nuclear signaling pathway. Future studies elucidating the linkage between PI 3-kinase and Rac will likely be pivotal to a complete understanding of TNF-α-evoked intracellular signaling.
We thank Dr. D.-M. Jue and Dr. A. Hall for providing recombinant human TNF-α and expression plasmids (pEXV, pEXV-RacV12, and pEXV-RhoV14), respectively. We also thank Dr. J. Downward for providing us pSG5-Δp85 plasmid.