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From www.bloodjournal.org by guest on August 1, 2017. For personal use only. Arachidonic Acid Mediates Interleukin-1 and Tumor Necrosis Factor-cuInduced Activation of the c-jun Amino-Terminal Kinases in Stromal Cells By Maria Teresa Rizzo and Carmelo Carlo-Stella We have previously shown that arachidonic acid mediates interleukin-1 (IL-1) and tumor necrosis factor-a (TNF-a)-induced transcription of c-jun. The signaling pathway of arachidonic acid-induced c-jun transcription was independent of protein kinase C activation and involved a tyrosine kinasedependent process. The present study was undertaken t o further elucidate the signal transduction pathway of arachidonate-induced c-jun transcription. We used a glutathioneS-transferase-c-jun fusion protein containing the aminoterminal domain of c-jun (residues 5 t o 89) t o explore the hypothesis that arachidonic acid stimulates c-jun amino-terminal kinase (JNK) activity in the murine stromal cell line +/+.1 LDA 11. Extracts from arachidonic acid-treated cells catalyzed phosphorylation of the c-jun fusion protein, indicating stimulation of JNK activity. Similar results were obtained when cells were challenged with IL-1 and TNF-a. The effect of arachidonic acid was specific, because extracts from stimulated cells failed t o phosphorylate a mutated fusion protein in which serine 63 and 73 of e-jun were each substituted with leucine. Arachidonic acid induced JNK activation in a time- and dose-dependent manner that was not mimicked by saturated fatty acids such as palmitic acid or other unsaturated fatty acids from the n-3, n-6, or n-9 series. Furthermore, other lipids, such as diacylglycerol, phosphatidic acid, and C,-ceramide, failed t o induce a significant increase in JNK activity. Treatment of stromal cells with propyl gallate, a dual inhibitor of lipoxygenase and cyclooxygenase enzymes, did not affect the ability of arachidonic acid t o induce JNK activation. Moreover, ETVA (5,8,11,1Ceicosatetraynoic acid), a nonmetabolizable arachidonate analogue, also induced JNK activation. These results are consistent with the hypothesis that the signal transduction pathway by which arachidonate stimulates c-jun transcription involves activation of the JNK cascade. Furthermore, arachidonic acid itself and not its cyclooxygenase or lipoxygenase metabolites is involved in stimulating JNK activity. Thus, arachidonic acid may act as a second messenger in mediating the effects of IL-I and TNF-a in the activation of c-jun. 0 1996 b y The American Society of Hematology. A acid may act as a second messenger promoting the transfer of signals from the cell surface to the nucleus by a pathway involving a protein tyrosine kinase cascade." Downstream intermediates in this activation cascade have not, as yet, been identified. A novel group of protein kinases, closely related to MAP kinases, has been recently identified.I4 These protein kinases known as stress-activated protein kinases (SAPKs) or c-jun amino-terminal kinases (JNKs) bind to the amino-terminal domain of c-jun and phosphorylate it specifically on Ser 63 and Ser 73, thereby increasing c-jun transcriptional activity and, thus, its ability to induce transcription of AP- 1 -containing genes, including c-jun itself. 15-2n The JNK protein kinases are proline-directed kinases that are activated by phosphorylation at conserved tyrosine and threonine residues by dual specificity protein kinases that have been recently Upon phosphorylation, JNK protein kinases translocate to the nucleus. where they participate in the activation of transcription factors such as c-jun and ATF~,lh.2U.25.26 Induction of JNK activity is detected upon cell stimulation with a variety of agonists, including IL-1 and T N F - ~16.27-29 , Some of the steps of the signaling pathway that lead to activation of JNK by IL-1 and TNF-a have been recently e l u ~ i d a t e d . ~ ' -However, ~ ~ . ~ " the intracellular mediators involved in stimulation of JNK activity by IL-l and TNF-(Uare not well defined. Based on these considerations, we undertook this study to further investigate the signal transduction pathway by which arachidonic acid mediates IL-I and TNF-a-induced c-jun transcription. We have explored the hypothesis that arachidonic acid, released upon stimulation of stromal cells with IL-1 and TNF-a, triggers a signal transduction pathway that involves activation of JNK leading to c-jun phosphorylation and thus to c-jun transcription. RACHIDONIC ACID plays a pivotal role in a wide array of cellular responses.',2 Although metabolites of the lipid exert important effects by influencing many cellular functions, a direct role for arachidonic acid has been implicated in the induction of certain cellular responses.'.'' Studies by us and other investigators have shown that arachidonate may be an important mediator of growth factor-induced gene Thus, we showed that interleukin-1 (IL-1) and tumor necrosis factor-a (TNF-a)-induced granulocytemacrophage colony-stimulating factor (GM-CSF) gene expression involves activation of phospholipase A2 and was mediated by arachidonic acid-induced activation of the transcription factor, c-jun.' Arachidonic acid enhanced c-jun transcription by a signaling pathway that did not require activation of protein kinase C (PKC), but instead resulted from a cellular process that was blunted by the phosphotyrosine kinase inhibitor genistein and potentiated by the tyrosine phosphatase inhibitor vanadate. l o Therefore, arachidonic From the Bone Marrow Transplantation Laboratory, Methodist Cancer Center, Methodist Hospital, Indianapolis, IN; and the Division of Hematology and Bone Marrow Transplantation, Pclrma Universify, P a m a , Italy. Submitted March 25, 1996; accepted July 18, 1996. Supported in part by funds from Methodist Cancer Center, by the Showalter Research Foundation to M.T.R., and by a grant from Consiglio Nazionale delle Ricerche (Progetto Finalizzato A. C.R.0.) f0 c.c.-s. Address reprint requests to Maria Teresa Riuo, MD, Bone Marrow Transplantation Laboratory, MPC 141 7, Methodist Research Institute, Methodist Hospital, I701 N Senate Bhd, Indianapolis 46202, IN. The publication costs of this article were defrayed in part by page charge paymenr. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. section 1734 solely to indicate this fact. 0 1996 by The American Society of Hematology. 0006-4971/96/8810-0032$3.00/0 3192 MATERIALS AND METHODS Materials. Human recombinant IL- lp and murine recombinant TNF-a were purchased from Genzyme (Cambrige, MA). McCoy's Blood, Vol 88, No 10 (November 15). 1996: pp 3792-3800 From www.bloodjournal.org by guest on August 1, 2017. For personal use only. 3793 ACTIVATION OF JNK BY ARACHIDONIC ACID 5A modified medium and HL-I medium were purchased from GIBCO BRL (Grand Island, NY) and from Ventrex Laboratories, Inc (Portland, ME), respectively. Fetal bovine serum was obtained from Hyclone (Logan, UT). [y-”P] adenosine triphosphate (ATP) was purchased from Du Pont New England Nuclear (Boston, MA). Hyperfilm was purchased from Amersham Life Science (Arlington Heights, IL). Arachidonic acid, palmitic acid, oleic acid, linoleic acid, linolenic acid, eicosatetraynoic acid (ETYA), propyl gallate, indomethacin, nordihydroguaiaretic acid (NDGA), melittin, quinacrine, genistein, glutathione, glutathione agarose, and the lactate dehydrogenase (LDH) kit were obtained from Sigma Chemical Co (St Louis, MO). Phosphatidic acid and diacylglycerol were from Avanti Polar Lipids (Alabaster, AL). Ceramide was purchased from Biomol Research Laboratories (Plymouth Meeting, PA). Anti-JNK1 antibodies were purchased from Santa CNZ Biotechnology, Inc (Santa Cruz, CA). Protein G plus/protein A agarose was obtained from Oncogene Science (Uniondale, NY).The glutathione-S-transferase (GST)-cjun fusion proteins (5-89) and (A 63/73) were a generous gift of Dr A. Kraft (University of Alabama, Birmingham, AL). Cell culture. The characteristics of the stromal cell line +/+.1 LDA 11 have been previously de~cribed.~’ Cells were grown in McCoy’s 5A modified medium supplemented with 10% fetal bovine serum and containing 50 U/mL penicillin and 50 pg/mL streptomycin. Cells were maintained in humidified 5% CO, at 37°C. Solid-phase kinase assay. We used a GST-C-jun fusion protein coupled to glutathione agarose beads to assay binding and substrate phosphorylation by jun kinase in cell extracts. Serum-deprived stromal cells were stimulated under serum-free conditions with the indicated stimuli and for the indicated length of time. Arachidonic acid was dissolved in 100% ethanol before use and was presented to cells so that the final concentration of ethanol was less than 0.1 %. Control cells received the same amount of ethanol as did treated cells. After stimulation, cells were washed with cold phosphate-buffered saline (PBS) containing 5 mmoVL NaF and 1 mmoVL Na3V04.Cells were lysed in cold lysis buffer (20 mmoVL HEPES, pH 7.6, 300 mmoV L NaCI, 1.5 mmoVL MgCl,, 1 mmoVL EDTA, 0.5 mmoVL dithiothreitol (DTT), 1 mmoVL Na3V04,0.1% sodium deoxycholate, 1% Triton X-100 with 1 mmoVL phenylmethylsulfonylfluoride,10 pg/ mL aprotinine, 10 pg/mL leupeptin, and 10 pg/mL antipain) and clarified by centrifugation at 10,000g for 10 minutes. C-jun kinase activity was determined as described by Hibi et al,27using recombinant GST-c-jun fusion protein purified by glutathione affinity chrom a t o g r a p h ~Briefly, .~~ cell lysates (200 pg) were mixed with 5 pg of immobilized GST-c-jun (5-89) or, as a negative control, GST protein alone and gently shaken for 3 hours at 4°C. Beads were then collected by centrifugation and washed three times in washing buffer containing 20 “OIL HEPES (pH 7.6), 50 mmom NaCI, 2.5 mmoV L MgCl,, 0.1 mmoVL EDTA, and 0.05% Triton X-100. The activity of JNK bound to GST-c-jun was determined by resuspending beads in 20 pL of kinase assay buffer (20 mmom HEPES, pH 7.6, 20 mmoVL MgCl,, 20 mmol/L P-glycerophosphate, 5 mmoVL NaF, 0.1 mmoVL Na3V04, and 2 mmoVL DTT), and kinase reactions were started by the addition of 50 pmoVL ATP and 10 pCi of [ y ”P-ATP]. Reactions were performed at 30°C and terminated after 20 minutes by the addition of 2X sodium dodecyl sulfate (SDS) sample buffer. Samples were denaturated at 100°C by boiling for 5 minutes and eluted proteins were resolved in 12% SDS-polyacrylamide gel, followed by autoradiography. Phosphorylated GST-cjun bands were excised from the gel and quantitated by liquid scintillation counting. Immune complex kinase assay. For JNK immune complex kinase assays, 500 pg of whole cell extracts from unstimulated and stimulated cells was precleared by incubation with 1 pg/mL rabbit antimouse IgG for 1 hour at 4°C and then for an additional 30 minutes with 10 pL of protein G plus/protein A. After preclearing cell lysates, supematants were incubated overnight at 4°C with 1 pg of a polyclonal anti-JNK1 antibody. A nonimmune rabbit IgG was used as the negative control. Immune complexes were recovered by the addition of 20 pL protein G pludprotein A agarose. The protein complexes were extensively washed with washing buffer followed by a washing with kinase assay buffer and resuspended in kinase assay buffer containing 1 pg of GST-c-jun (5-89), GST-c-jun (A 63/73), or GST protein alone as substrates in the presence of 50 pmoVL ATP and 10 pCi [Y-~~PIATP. The reaction mixture was incubated for 20 minutes at 30”C, terminated by the addition of 2X SDS sample buffer, and boiled for 5 minutes. Phosphorylated proteins were resolved by 12% SDS-polyacrylamide gel and subsequently detected by autoradiography. 32P-Piincorporation into GSTc-jun was quantitated as described above. In-gel kinase assay. Assays of proteins renaturated in polyacrylamide gels were performed as described by Kameshita and Fuji~ a w a . ’Briefly, ~ lysates from control and stimulated cells were separated on a 10% SDS-polyacrylamide gel that was polymerized in the absence or in the presence of GST-c-jun (40 pg/mL) added as a substrate directly to the gel before polymerization. After electrophoresis, the gel was washed several times before denaturation in 6 m o m guanidine-HCI. The gel was then renaturated with five changes of a buffer containing 50 mmoVL Tris-HCI (pH 8.0), 5 mmoVL 2P-mercaptoethano1, and 0.04% Tween 40 at 4°C for 16 hours. After renaturation, the gel was incubated for 1 hour at 25°C in kinase assay buffer containing 50 p m o m ATP and 20 pCi/mL of [y-”P]ATP. After incubation, the gel was washed with 5% trichloroacetic acid containing 1% sodium pyrophosphate. The gel was dried and exposed to x-ray film at -80°C. Protein determination. Protein concentrations of cell lysates were determined by the method of Bradford using bovine serum albumin as a standard.” Cyroroxicity assay. LDH activity was determined according to the manufacturer’s instructions from supematants after treatment of cells with arachidonic acid (10 to 100 pmoVL) for 30 minutes. RESULTS Effect of arachidonic acid on JNK protein kinases. Our recent studies suggested that a signal transduction pathway involving a tyrosine kinase cascade mediated arachidonic acid-induced c-jun transcription.” To further elucidate the components of the signaling route to c-jun transcription by arachidonic acid, we performed experiments to explore the potential involvement of the JNWSAPK cascade. JNK activity was measured in cell lysates by a solid-phase in vitro kinase assay using as a substrate the GST-c-jun fusion protein containing the amino-terminal domain of c-jun from amino acid 5 to 89 immobilized on glutathione-agarose beads.35Stromal cells were serum-deprived and subsequently stimulated with arachidonic acid or IL-1 plus TNF-a. Cell extracts were prepared and assayed for their ability to induce phosphorylation of the GST-c-jun fusion protein. As shown in Fig lA, JNK activity present in unstimulated cells substantially increased upon exposure of cells to 25 pmol/L arachidonic acid for 30 minutes. Exposure of stromal cells to IL1 (500 U/mL) plus TNF-a (500 U/mL) for 15 minutes also potentiated JNK activity. Quantitation of radioactivity within excised gel slices showed a 3.5- and 4.3-fold increase in cjun phosphorylation in cells treated with arachidonic acid or with E-1 plus TNF-a, respectively. Similar results were obtained when IL-1 and TNF-a were used individually (data not shown). No phosphorylation was detected when the ki- From www.bloodjournal.org by guest on August 1, 2017. For personal use only. RlZZO AND CARLO-STELLA 3794 A om-cjun b C oslr-cjun w Fig 1. (A) Activation of JNK by arachidonic acid or IL-1 and TNF-a. Lysates from control cells or from cells stimulated with arachidonic acid (25 pmollLl for 30 minutes or IL-1 (500 UlmL) plus TNF-a (500 UlmL) for 15 minutes were incubated with 5 p g of immobilized GST-c-jun or GST protein alone as a negative control. The beads were washed and incubated in kinase assay buffer containing 50 pmollL ATP and 10 pCi [y-BZPIATP for 20 minutes at 30°C. Reactions were terminated by the addition of 2 x SDS sample buffer. Phosphorylated proteins were resolved by 1296 SDS-polyacrylamide gel electrophoresis, dried, and analyzed by autoradiography. (B) Dose-response of JNK activation by arachidonic acid. Cells were stimulated with different concentrations of arachidonic acid for 30 minutes and cell lysates were prepared. JNK activity was measured by the solid-phase kinase assay, as described in the Materials and Methods. (C) Time course of JNK activation by arachidonic acid. Cells were treated with arachidonic acid (25 pmollL) for varying times. JNK activity was assayed by the solid-phase kinase assay, as described above. Results are from a representative experiment performed twice with similar results. nase reaction was performed using agarose beads coated with GST protein only, indicating that phosphorylation was substrate (c-jun) dependent (Fig 1 A). Arachidonic acid induced activation of JNK in a doseand time-dependent manner (Fig 1B and C). A 2.5-fold increase of GST-c-jun phosphorylation was observed with a concentration of I O pmol/L arachidonic acid as compared with vehicle-treated cell (Fig IB). A 3.2- and 4.5-fold increase of JNK activity was detected at 25 pmol/L and 50 pmoI/L arachidonic acid, respectively (Fig IB). Further increases in the concentration of arachidonic acid did not further potentiate JNK activity (data not shown). The effect of arachidonic acid did not appear to be a result of generalized cytotoxicity, because LDH was not detectable in supematants from cells stimulated with different concentrations of arachidonic acid ( I O to 100 pmol/L) for 30 minutes (data not shown). Phosphorylation of GST-c-jun was observed within 15 minutes of stimulation with arachidonic acid (Fig 1C). Arachidonic acid-induced phosphorylation of GST-cjun increased over 30 minutes and started to decline thereafter at 60 minutes (Fig IC). The JNK group of protein kinases include the 46-kD JNKl and the 55-kD JNK2 isoforms, both of which are activated by IL-I and TNF-a in different cell sy~tems.’~.’’~’‘ To characterize the JNK activity further, we performed in-gel kinase assays. These experiments showed that stimulation of stromal cells with 25 pmol/L arachidonic acid for 30 minutes or with IL-I (500 U/mL) plus TNF-a (500 U/mL) for 15 minutes induced activation of both the 46-kD and 55-kD isoforms of JNKs. although the 46-kD form of JNKl was the major c-jun kinase activated in response to arachidonic acid or IL-I plus TNF-a (Fig 2). No activation was detected when proteins were separated in gel polymerized in the absence of GST-c-jun (data not shown). The ability of arachidonic acid to induce JNK activation was further examined by immune complex kinase assays. JNK activity was immunoprecipitated from lysates of arachidonic acid-treated cells using a polyclonal antibody that recognizes the 46-kD JNKl isoform. As shown in Fig 3, anti-JNKI immunoprecipitates of arachidonic acid-stimulated cells induced phosphorylation of GST-c-jun (5-89). A similar effect was observed when immunoprecipitates from lysates of IL- 1 plus TNF-atreated cells were used (Fig 3). On the contrary, no phosphorylation was detected when immunoprecipitates prepared From www.bloodjournal.org by guest on August 1, 2017. For personal use only. ACTIVATION OF JNK BY ARACHIDONIC ACID Fig 2. Preferential activation of JNKl by arachidonic acid or IL-1 and TNF-a. Lysates from cells treated either with vehicle (Co) or with 25 pmol/L arachidonic acid (AA) for 30 minutes or with 11-1 (500 U / mL) plus TNF-a (500 U/mL) for 15 minutes were subjected to 10% SDS-polyacrylamide gel electrophoresis. GST-c-jun (40 pg/mL) was added to the gel before polymerizationas a substrate. Protein renaturation and kinase assays were performed as described in the Materials and Methods. Shown by the arrows are both isoforms of the JNK protein kinases. This experiment was repeated twice with similar results. with a nonreactive antibody were used. Phosphoamino acid analysis confirmed that GST-c-jun (5-89) was phosphorylated on serine by JNKl immunoprecipitated from arachidonate-treated cells (data not shown). Furthermore, when immune complex kinase assays were performed using a GSTc-jun fusion protein in which Ser 63 and Ser 73 were each replaced with leucine (GST-c-jun A63/73), a marked decrease of phosphorylation was observed (Fig 3). Spec$cio of arachidonate-induced JNK activation. To determine whether the effect of arachidonic acid on JNK activation was specific, we tested the ability of several saturated and unsaturated long-chain fatty acids to activate JNK (Fig 4A). Stimulation of cells with 25 pmol/L oleic acid (n9) for 30 minutes caused a mild increase of JNK activity as compared with that induced by arachidonic acid. Similarly, 25 pmol/L linoleic acid (n-6) and linolenic acid (n-3) induced a modest activation of JNK (Fig 4A). Palmitic acid (25 pmoVL) had a marginal effect on JNK activation as compared with the effect of arachidonic acid (Fig 4A). We next undertook experiments to determine whether other lipid second messengers were able to stimulate JNK activation (Fig 4B and C). Stimulation of stromal cells with 50 ymol/L phosphatidic acid for 30 minutes failed to induce JNK activation (Fig 4B). Diacylglycerol, a known activator of PKC, also failed to induce JNK activation under the conditions used (Fig 4B). Because ceramide has been proposed to mediate the effects of TNF-a in many cellular functions, including activation of JNK,36.37we undertook experiments to determine its effect on induction of JNK activity in stromal cells. As shown in Fig 4C, stimulation with 5 pmol/L Czceramide for 30 minutes induced a weak increase (0.8-fold) of JNK activity as compared with that induced by stimulation with 25 pmol/L arachidonate (3.0-fold) for the same length of time. An equimolar concentration (25 pmol/L) of cera- 3795 mide also failed to induce an increase of JNK activity as compared with that induced by arachidonic acid (data not shown). Thus, in the stromal cell line used, arachidonic acid appears to be the preferential candidate in mediating activation of JNK by IL-I and TNF-a. Role of arachidonic acid metabolites on JNK activation. Arachidonic acid is rapidly metabolized through the cyclooxygenase and the lipoxygenase pathways leading to formation of several biologically active metabolites, including prostaglandins and leukotrienes.’.’ To determine whether JNK activation is a direct effect of arachidonic acid or one of its metabolites, we assessed the effects of arachidonic acid metabolism inhibitors on JNK activation. As shown in Fig 5A, pretreatment of cells with 100 pmolk propyl gallate, an antioxidant that inhibits both lipoxygenase and cyclooxygenase enzymes,” before stimulation did not effect the ability of arachidonic acid to induce JNK activation. The inhibitor had no detectable effect, by itself, on activation of JNK (Fig 5A). Indomethacin (IO pmol/L), a cyclooxygenase inhibitor, and nordihydroguaiaretic acid or NDGA (1 0 pmoVL), a lipoxygenase inhibitor, similarly failed to block JNK stimulation by arachidonic acid (data not shown). We next examined the effect of ETYA on arachidonateinduced JNK activation. ETYA is a structural analogue of arachidonic acid in which four alkyne bonds replace the four alkene bonds present in arachidonic acid.99ETYA competitively inhibits the uptake of arachidonic acid into intracellular membranes and inhibits both cyclooxygenase and lipoxygenase enzymes by acting as a false s u b ~ t r a t e Exposure .~~ of cells to arachidonic acid or ETYA alone for 30 minutes GST-cjun(5-89) C I ..-- GST-cjnn(A63D3) c Fig 3. Specificity of GST-c-jun phosphorylation by arachidonic acid or IL-1 and TNF-a. Lysates from control and arachidonic acidtreated (25 pmol/L; 30 minutes) or IL-1 (500 UlmL; 15 minutes) plus TNF-a (500 UlmL; 15 minutes) -treated cells were subjected to immunoprecipitationwith an antiJNKl antibody or anti-lgG antibody. The kinase activity of the immune complexes was determined by phosphorylationof GST-c-jun (5-89) or the mutated GST-c-jun (A 63/73) in the presence of [y3‘P1ATP. Proteins were analyzed by 12% SDS-gelelectrophoresis and autoradiography. Resultsare from a representative experiment. From www.bloodjournal.org by guest on August 1, 2017. For personal use only. R l U O AND CARLO-STELLA 3796 A 500 B - 400 h S E V 0 S $ Y O C -a'I E I- ul u 100 om-cjpn F Co m o o * AA OA LA LnA PA Fig 4. (A) Effect of fatty acids on JNK activation. Cells were stimulated for 30 minutes with 25 pmol/L each of the following fatty acids: arachidonic acid (AA), oleic acid (OA), linoleic acid (LA), linolenic acid (LnA), and palmitic acid (PA). Cell lysates were prepared and used for the solid-phase kinase assay as described. Data represent the mean k SEM of three independent experiments, expressed as an increase with respect t o unstimulated cells (Co). An autoradiograph corresponding t o the labeled substrate from a representative determination is shown. (6) Effect of lipids on JNK activation. Stromal cells were stimulated with 25 pmol/L arachidonic acid (AA), 50 pmollL phosphatidic acid (PA), or 50 pmol/L diacylglycerol (DAG) for 30 minutes. Cell lysates were assayed using the solid-phase kinase assay with GST-c-jun as a substrate, as described under the Materials and Methods. Results are from a single experiment that was repeated twice. (C) Effect of ceramide on JNK activation. Cells were stimulated with C,-ceramide (5 pmol/L; 30 minutes) or arachidonic acid (25 pmollL; 30 minutes), and lysates were prepared and assayed in the solid-phase kinase assay as described above. induced a 3.7- and 2.9-fold increase of JNK activity, respectively, as determined by scintillation counting of the phosphorylated GST-c-jun proteins excised from the gel (Fig SB). When cells were stimulated with arachidonic acid in the presence of ETYA, a 6.0-fold increase of GST-c-jun phosphorylation was detected (Fig SB). Finally, the effects of propyl gallate and ETYA were examined on IL-I plus TNF-a-induced activation of JNK. As shown in Fig 5C, pretreatment with propyl gallate did not inhibit JNK activity induced by IL-l plus TNF-a. Similar results were obtained when cells were stimulated with IL-l plus TNF-a in the presence of ETYA (Fig SC). Role of arachidonic acid on IL-l and TNF-a-induced JNK activation. We have previously shown that c-jun gene expression induced by IL-l and TNF-a was decreased by treatment of cells with the PLA2 inhibitor, quinacrine.' Moreover, c-jun gene expression induced by IL- l plus TNF-a or by arachidonic acid was sensitive to inhibition by the tyrosine kinase inhibitor, genistein."' To further determine the potential role of arachidonic acid as an intracellular mediator of IL-l plus TNF-a-induced JNK activation, we examined the effect of quinacrine on cytokine-induced JNK activity. Cells were stimulated with IL-I (500 U h L ) plus TNF-a (500 U h L ) for 15 minutes in the presence of different concentrations of quinacrine. As shown in Fig 6, treatment of cells with 1 pmol/L and 5 pmol/L quinacrine caused a 39% and 54% decrease on IL-I plus TNF-a-induced JNK activity, respectively, consistent with the hypothesis that arachidonic acid mediates IL-1 plus TNF-a stimulation of JNK activity. We next examined the effect of genistein on JNK activation induced by exposure of cells to either IL-I plus TNF-a or arachidonic acid. Stromal cells were preincubated for 2 hours with 10 pmol/L genistein after stimulation with IL-I (500 U/mL) plus TNF-a (500 U h L ) for 15 minutes or with 25 pmol/L arachidonic acid for 30 minutes. As shown in Fig 7, treatment with genistein resulted in a 46% and 64% inhibition of JNK activity induced by IL-I plus TNF-a or by arachidonic acid, respectively. DISCUSSION In recent years, a role for arachidonic acid in the regulation of gene expression has been proposed."-'3 Our previous studies have identified arachidonic acid as a potential intracellular mediator of the effect of IL-I and TNF-a on c-jun gene From www.bloodjournal.org by guest on August 1, 2017. For personal use only. ACTIVATION OF JNK BY ARACHIDONIC ACID 3797 expres~ion.~~'" Stimulation of the murine stromal cell line +/ +.1 LDA 11 with arachidonic acid or with IL-I and TNFLY led to enhanced c-jun transcription by a mechanism independent of PKC activation." In addition, increased tyrosine kinase activity was detected upon stimulation with arachidonic acid and treatment of cells with tyrosine kinase inhibitors markedly decreased arachidonic acid-induced c-jun transcription." These observations led us to postulate that a tyrosine kinase-dependent process was involved in the signaling route of c-jun transcription by arachidonic acid. In the present study, we have further clarified a portion of the signal transduction pathway that leads to c-jun transcription when cells are exposed to arachidonic acid. Spe- 700 I 0 I+T Q=*c A co U eL Fig 6. + mL) in the absence or in the presence of 1 pmolIL and 5 pmolIL quinacrine. Lysates were prepared and analyzed for JNK activity as described. Data represent the mean ? SEM of an experiment performed in triplicate. Autoradiography corresponding t o the labeled substrate from a representative determination is shown. GST-cjunb cifically, we have presented evidence that regulation of cjun transcription by arachidonic acid involves activation of the JNK signaling pathway. Consistent with this hypothesis. extracts from cells stimulated with arachidonic acid induced phosphorylation of a c-jun fusion protein containing Ser 63 and Ser 73, indicating the presence of activated JNK proteins. Similar effects were observed with extracts from cells treated with IL-l and TNF-a. The results are consistent with the hypothesis that these growth factors exert their effects by inducing the release of arachidonate, which mediates activation of intracellular c-jun kinases. We used in-gel kinase assays as well as kinase assays of immunoprecipitates to identify the isoform of JNK activated in response to arachidonic acid. In-gel kinase assays have been previously shown to be able to detect both the JNKl and JNK2 isoforms of JNK protein kinases." The major < B t GSTcjunb C st; LL z c + LL Z + c 8 A A r I GST-cjun b Effect of quinacrine on IL-1 plus TNF-a-induced JNK activ- ity. Cells were stimulated with IL-1 (500 UImL) plus TNF-a (500 U I + r I Fig 5. Effect of arachidonic acid metabolism on JNK activation. (A) Cells were pretreated with 100 pmolIL propyl gallate for 10 minutes before stimulation with vehicle (PG) or with 25 pmolIL arachidonic acid for 30 minutes (AA PG). Control cells ICo) were exposed t o vehicle alone. Lysates were prepared and incubated with immobilized GST-e-jun. Reactions were performed as described in the Materials and Methods. Phosphorylated proteins were separated by SDSgel electrophoresis and analyzed by autoradiography. IB) Cells were stimulated with 25 pmolIL arachidonic acid (AA) or 50 pmolIL ETYA alone or in combination with arachidonate IAA + ETYAI. Phosphorylation of GST-c-jun. was analyzed as described above. Bands corresponding t o phosphorylated c-jun were excised from gels and counted by scintillation counting. (CI Cells were stimulated either with vehicle (Co) or with IL-1 (500 UImL) plus TNF-a (500 UImLI in the absence or in the presence of propyl gallate (100 pmol/L) or ETYA 150 pmolIL). Lysates were prepared and analyzed by the solid-phase kinase assay using the immobilized GST-c-jun as described. An autoradiogram from a representative experiment is shown. + From www.bloodjournal.org by guest on August 1, 2017. For personal use only. R I U O AND CARLO-STELLA 3798 800 I 700 L C 0 0 .-6m - 600 ; 500 L 6 400 c n x 300 p. .C 3 200 m OST-cjuu co I+T I+T+Q 0 AA AA+O Fig 7. Effact of genistein on 11-1 plus TNF-a- and arachidonic acidinduced JNK activity. Cells were preincubated for 2 hours with 10 pmol/L genistein (GI in serum-free conditions. Cells were either left unstimulated or stimulated with 11-1 (500 UlmLI plus TNF-a (500 U/ mLJ for 15 minutes or with arachidonic acid (25 pmol/L; 30 minutest. Lysates were made and assayed in the solid-phase kinase assay as described. Shown is a representative experiment performed in triplicate (mean f SEMI. Autoradiography of phosphorylatedGST-c-jun is from a representative determination. JNK protein kinase activated in response to arachidonic acid or IL-I and TNF-a appeared to be the 46-kD JNKl isoform. Additional evidence that JNKl activated in response to arachidonic acid was involved in phosphorylating Ser 63 and Ser 73 of the amino-terminal domain of c-jun was provided by the fact that anti-JNKI immune complexes from arachidonate-treated cell were able to phosphorylate the GST-cjun fusion protein. Conversely, when the mutated GST-cjun fusion protein was used as a substrate, a marked decrease in phosphorylation was detected. Taken together, these results suggest that JNKl or an immunologically related kinase is induced after stimulation of stromal cells with arachidonic acid. Thus, activation of the JNK proteins by arachidonic acid may mediate IL-l and TNF-a-induced phosphorylation of c-jun on Ser 63 and Ser 73. The effect of arachidonic acid on JNK activation was specific. Whereas other fatty acids induced JNK activation, their effect on JNK activity was much smaller that the effect observed after stimulation with arachidonic acid. Moreover, several other lipid second messengers, including phosphatidic acid, diacylglycerol, and ceramide, failed to stimulate JNK activity in these cells. Several reports have implied ceramide as a mediator of TNF-a signalling in other systems?’ A recent study by Verheij et al” showed that, in the human monocytic leukemia cell line, U937, and in bovine aortic endothelial cells, ceramide induced apoptosis by activating the SAPWJNK cascade. Other lipids, including arachidonic acid, which failed to induce apoptosis in these cells, also failed to stimulate the SAPWJNK cascade. The explanation for these divergent results is not presently clear. Signal- ling mediators for IL-1 and TNF-a may differ in different types of cells. Thus, in human umbilical vein endothelial cells, TNF-a-induced activation of the JNK cascade was not mediated by ceramide?’ Although other factors may be involved, it seems clear in our system that arachidonic acid is a relevant messenger mediating the IL- 1 and TNF-a effect on JNK activation. Arachidonic acid rather than ceramide appears to mediate other responses as well. Thus, Jaattella et aI4’ found that, in a TNF-sensitive subclone of MCF7 breast carcinoma cells, TNF-a-induced apoptosis was mediated by PLAz activation, but not by ceramide. More recently, a role for arachidonic acid metabolism in mediating apoptosis it has been postulated?’.44 However, whether this effect is mediated by activation of the JNK cascade or by other effectors it is not yet known. Further investigations are warranted to better define the role of ceramide and arachidonic acid in mediating the effects of IL-I and TNF-a on cell function. Inhibition of arachidonic acid metabolism by propyl gallate did not affect the ability of arachidonate to induce JNK activation. Furthermore, the nonmetabolizable arachidonate analogue, ETYA, stimulated JNK activity at levels similar to that stimulated by arachidonic acid. We also observed that stimulation of stromal cells with arachidonic acid in the presence of ETYA enhanced the ability of arachidonic acid to induce JNK activation. This effect may be ascribed to increased intracellular availability of arachidonic acid due to inhibition of its metabolism. In addition, we have observed that ETYA stimulates c-jun gene expression in the stromal cell line used (unpublished observation). Taken together, these results suggest that metabolites of the cyclooxygenase or lipoxygenase pathway do not mediate the effect of arachidonic acid on JNK activation. Although the involvement of an alternative metabolic pathway cannot be excluded, the results presented herein suggest that arachidonate itself is involved in regulation of JNK activity. In the stromal cell line used, JNK activation induced by IL 1 and TNF-a parallels that induced by arachidonic acid in many ways. Neither response is affected by pretreatment of cells with propyl gallate or ETYA. Moreover, genistein similarly affected IL-f plus TNF-a-induced and arachidonate-induced JNK activation. Furthermore, quinacrine partially inhibited JNK activation by IL-1 plus TNF-a, suggesting that the effect of these cytokines on JNK activity is mediated, in part, by the release of arachidonic acid upon PLA2activation. Together with our previous observations?’’ these results are consistent with the hypothesis that arachidonic acid mimics the effects of IL-1 and TNF-a on c-jun activation. Consistent with this possibility, stimulation of stromal cells with IL-I and TNF-cy induced activation of PLA2,the inhibition of which blunted growth factor-induced, but not arachidonate-induced c-jun gene expression? These findings strengthen our contention that arachidonic acid acts as a second messenger of L-1and TNF-cy signaling network. The precise mechanism by which arachidonic acid induces JNK activation remains to be identified. Arachidonic acid may exert its effect on JNK through other, as yet unidentified, kinases. Experiments by others have shown that induction of JNK kinase (JNKK) precedes JNK activation in growth factor-stimulated cells?’ Moreover, MEK kinase 1 (MEKK) is able to induce SAPKs From www.bloodjournal.org by guest on August 1, 2017. For personal use only. ACTNATION OF JNK BY ARACHIDONIC ACID activation by phosphorylating SAPK activator, SEKl?l Whether these kinases are involved in activation of JNK by arachidonic acid remains to be determined. ACKNOWLEDGMENT We are grateful to Dr A. Kraft for the generous gift of the GSTc-jun fusion proteins. We thank Dr B. Morimoto for helpful discussions and comments, Dr L. Williamson for her advice on the solidkinase assay, and Prof V. Rizzoli for his encouragement and support. REFERENCES 1. Axelrod J: Receptor-mediated activation of phospholipase Az and arachidonic acid release in signal transduction. Biochem SOC Trans 18:503, 1990 2. Piomelli D: Arachidonic acid in cell signaling. Curr Opin Cell Biol 5:274, 1993 3. Fafeur V, Jiang ZP, Bohlen P: Signal transduction by bFGF but not TGFP1, involves arachidonic acid metabolism in endothelial cells. J Cell Physiol 149:277, 1991 4. Peppelenbosch MP, Qiu RG, de Vries-Smits AMM, Tertoolen LGJ, de Laat SW, McCormick F, Hall A, Symons MH, Bos JL: Rac mediates growth factor-induced arachidonic acid release. Cell 81:849, 1995 5. Lennartz MR, Brown EJ: Arachidonic acid is essential for IgG Fc receptor-mediated phagocytosis by human monocytes. 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Modur V, Zimmerman GA, Prescott MS, McInyre MT: Endothelial cell inflammatory responses to tumor necrosis factor-a. Ceramide-dependent and -independent mitogen-activated protein kinase cascade. J Biol Chem 271:13094, 1996 42. Jaattella M, Benedict M, Tewari M, Shayman JA, Dixit VM: Bcl-x and bcl-2 inhibit apoptosis and activation of phospholipase Az in breast carcinoma cells. Oncogene 10:2297, 1995 43. Tang DG, Chen YQ, Honn KV: Arachidonate lipoxygenases as essential regulators of cell survival and apoptosis. Proc Natl Acad Sci USA 93:5241, 1996 44. Ballif BA, Mincek NV, Barrat JT, Wilson ML, Simmons DL: Interaction of cyclooxigenase with an apoptosis and autoimmunityassociated protein. Proc Natl Acad Sci USA 93:5544, 1996 From www.bloodjournal.org by guest on August 1, 2017. For personal use only. 1996 88: 3792-3800 Arachidonic acid mediates interleukin-1 and tumor necrosis factor-alpha- induced activation of the c-jun amino-terminal kinases in stromal cells MT Rizzo and C Carlo-Stella Updated information and services can be found at: http://www.bloodjournal.org/content/88/10/3792.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. 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