Janus Kinase-Signal Transducer and Activator of Transcription Mediates Phosphatidic Acid-Induced Interleukin (IL)-1 and IL-6 Production

We have found previously that phosphatidic acid (PA) can induce inflammatory mediators such as cytokines, which implies that PA plays a role in inflammatory response. In the present study, we provide evidence of the PA-mediated activation of the Janus tyrosine kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway, which results in the production of interleukin (IL)-1 and IL-6. PA elicited the rapid phosphorylations of JAK2 and STAT1/3, and the subsequent nuclear translocation. Macrophages that had been transiently transfected with a luciferase reporter construct containing eight consecutive -interferon activating sequence (GAS) elements, a known STAT binding site, exhibited enhanced reporter gene activity in response to PA stimulation, which further supports the involvement of JAK-STAT activation in the PA-induced signaling pathway. Of the inflammatory cytokines, IL-1 , IL-6, and tumor necrosis factor (TNF)were detected in media from macrophages stimulated with PA. Moreover, the JAK2 inhibitor -cyano-(3,4-dihydroxy)-N-benzylcinnamide (AG-490) abolished PA-induced IL-1 and IL-6 release but not TNFproduction, which is consistent with the notion that IL-1 and IL-6 but not TNFcontain a STAT binding element in their promoter region. The knockdown of JAK2 in macrophages by small interfering RNA significantly attenuated PA-induced IL-1 and IL-6 production. In addition, JAK2 inhibitor suppressed PA-induced Akt phosphorylation, and the Akt inhibitor 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002) blocked GAS activation (GAS contains a promoter that responds to PA), suggesting that PA-mediated JAK2 activation leads to phosphatidylinositol 3-kinase/Akt phosphorylation and STAT activation, and the subsequent translocation of STAT to the nucleus. Together, our data demonstrate that PA-activated macrophages produce IL-1 and IL-6 and that these processes require the activation of the JAK2-STAT1/3 or JAK2-Akt-STAT signaling pathways. Phosphatidic acid (PA) is an important metabolite that is involved in phospholipid biosynthesis and membrane remodeling (Lim et al., 2003). PA can be generated by several cellular processes, such as the hydrolysis of phosphatidylcholine by phospholipase D, the phosphorylation of DAG by DAG kinase, and the acylation of lyso-PA by lyso-PA acyltransferase (Koch et al., 2004; van Baal et al., 2005), and can be metabolized to other bioactive lipids, such as lyso-PA and DAG (Nanjundan and Possmayer, 2003). It has been suggested that PA may play a crucial role in the regulation of various biological events. For example, PA is involved in the phosphorylations of many proteins (Chen et al., 2003; Avila-Flores et al., 2005), activation because of oxidative stress (de Jong et al., 2004), modulation of membrane trafficking (Kooijman et al., 2003), and regulation of inflammatory response (Lim et al., 2003; Tou and Gill, 2005). This work was supported by the Basic Research Program Grant R01–2004000-10023-0 (2004) and the Aging-Associated Vascular Disease Research Center at Yeungnam University of Korea Science and Engineering Foundation Grant R13-2005-005-01003-0 (2005). Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.105.018481. ABBREVIATIONS: PA, phosphatidic acid; DAG, diacylglycerol; mTOR, mammalian target of rapamycin; ERK, extracellular signal-regulated kinase; JAK, Janus tyrosine kinase; STAT, signal transducers and activators of transcription; siRNA, small interfering RNA; IL, interleukin; PI3K, phosphatidylinositol 3-kinase; AG-490, -cyano-(3,4-dihydroxy)-N-benzylcinnamide; LY294002, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4one; PD98059, 2 -amino-3 -methoxyflavone; SB203580, 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole; SP600125, 1,9pyrazolo anthrone anthra (1,9-cd) pyrazol-6(2H)-one; MAPK, mitogen-activated protein kinase; GAS, -interferon activating sequence; Ab, antibody; PAGE, polyacrylamide gel electrophoresis; TNF, tumor necrosis factor; ELISA, enzyme-linked immunosorbent assay; PMSF, phenylmethylsulfonyl fluoride; PBS, phosphate-buffered saline; TF, transcription factor; TTBS, Tris-buffered saline/0.05% Tween 20. 0026-895X/06/6903-1041–1047$20.00 MOLECULAR PHARMACOLOGY Vol. 69, No. 3 Copyright © 2006 The American Society for Pharmacology and Experimental Therapeutics 18481/3090187 Mol Pharmacol 69:1041–1047, 2006 Printed in U.S.A. 1041 at A PE T Jornals on A uust 4, 2017 m oharm .aspeurnals.org D ow nladed from We found evidence that the addition of PA to macrophages induces a number of inflammatory responses, such as proinflammatory cytokine production, cyclooxygenase-2 expression, and the up-regulation of inducible nitric-oxide synthase (Lim et al., 2003). Other studies have also found that PA stimulates cardiac ATP-sensitive K channels with novel gating kinetics (Fan et al., 2003), which is also observed in the study of phosphatidylinositol, and promotes hair growth in epithelial cells (Anthony et al., 2004). It is noteworthy that many of these processes are highly selective and specific; thus, it was suggested that like other lipid second messengers, PA seems to function via selected targets in specific cell membrane regions (Ktistakis et al., 2003). It was recently reported that PA causes the activation of the mammalian target of rapamycin (mTOR), and subsequently targets mTOR, S6 kinase, and 4E-binding protein 1 (Avila-Flores et al., 2005). Likewise, PA directly regulates protein kinase activities such as Fgr and protein kinase C, and also regulates the mitogen-activated protein kinase kinase/ERK cascade (Bollag et al., 2005). Moreover, Alderton et al. (2001), who reported PA-mediated ERK activation, speculated that ERK phosphorylation might occur via a specific PA receptor, although its existence was unproven. Another study demonstrated that PA binds to the orphan receptor GPR63 (Niedernberg et al., 2003; Kostenis, 2004), suggesting that PA acts through binding to cell membrane targets. However, the cellular target of PA has not been identified, and the mechanisms involved remain to be elucidated. The JAK pathway may be triggered by diverse ligands, including cytokines and growth factors (Ihle, 1995; Yadav et al., 2005). Moreover, JAK-mediated signals trigger proliferation, migration, inflammatory responses, immune responses, and other cellular events (Hu et al., 2002; Niwa et al., 2005; Smith et al., 2005). The JAK family consists of four members, namely, JAK1, JAK2, JAK3, and TYK2, which are all ubiquitously expressed in cells, whereas in macrophages JAK2 is dominantly expressed (Natarajan et al., 2004; de Jonge et al., 2005). The stimulation of cells with suitable ligands, such as cytokines, induces receptor oligomerization and causes the local aggregation of associated JAKs, which results in their activation by transphosphorylation. JAKs are activated by tyrosine transphosphorylation, which allows them to phosphorylate their major downstream targets (e.g., STATs). When tyrosine is phosphorylated by JAKs, STATs are translocated to the nucleus and turn on target genes (Murray et al., 2005; Niwa et al., 2005). Here, we demonstrate that the JAK2-STAT1/3 pathway has critical and distinctive role in PA-induced cytokine release. Our data show that PA elicits JAK2 activation and phosphorylation and the nuclear translocation of STAT1/3 in Raw 264.7 cells (a murine macrophage cell line). A JAK2 inhibitor or JAK2 siRNA suppressed PA-induced IL-1 and IL-6 production, indicating the involvement of the JAK2 pathway in PA-activated macrophages. We also found that Akt is activated by PA and that an Akt inhibitor abolished promoter activity containing -interferon activating sequence (GAS) element in response to PA, suggesting that STAT might be a downstream target of Akt in PA-induced signaling. Together, our data demonstrate that novel signal transduction pathways are required for cytokine release by PA and that these include the JAK2-STAT1/3 or the JAK2PI3K/Akt-STAT pathway in PA-activated macrophages. Materials and Methods Reagents and Antibodies. Dioctanoyl PA ( 99%) was obtained from Sigma-Aldrich (St. Louis, MO), and its endotoxin content was determined using a chromogenic Limulus polyphemus amebocyte lysate kit (BioWhittaker, Walkersville, MD). AG-490, LY294002, and several MAPK inhibitors—PD98059, SB203580, and SP600125— were purchased from BIOMOL Research Laboratories (Plymouth Meeting, PA), and JAK3 inhibitor was from Calbiochem (San Diego, CA). The GAS-luciferase reporter construct was generously provided by Dr. M. H. Song (Chungnam University, Daejon, Korea). For Western blot analysis, we used Abs against JAK1, JAK2, JAK3 (Chemicon International, Temecula, CA), and TYK2 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and against phosphorylated JAK2 (Tyr1007/1008), STAT1 (Tyr701), STAT3 (Tyr705), and Akt (Thr308) (Cell Signaling Technology, Inc., Beverly, MA). Peroxidase-conjugated anti-rabbit IgG, anti-goat IgG, or anti-mouse IgG (Santa Cruz Biotechnology, Inc.) were used as secondary antibodies. Cell Culture. Raw 264.7 cells were obtained from the American Type Culture Collection (Manassas, VA) and were cultured in RPMI 1640 medium (Gibco BRL, Gaithersburg, MD) containing 10% fetal bovine serum, 2 mM L-glutamine, 10 U/ml penicillin, and 10g/ml streptomycin at 37°C in 5% CO2 in a water-saturated atmosphere. Cells were treated with synthetic PA for the indicated times. Isolation of Murine Peritoneal Macrophages. Resident peritoneal macrophages were obtained by peritoneal cavity lavage using 10 ml of RPMI 1640 medium. Cells from five mice were pooled, pelleted by centrifugation (200g; 10 min), and washed once with RPMI 1640 medium. The cells were then seeded in a 100-mm culture dish to remove any contaminating cells and to enrich adhering macrophages, and after 2 h of incubation, adherent cells were collected and used in experiments. Cytokine Measurements. The conditioned media from RAW 264.7 cells stimulated with PA amounts of TNF, IL-1 , and IL-6 were determined by specific enzyme-linked immunosorbent assay (ELISA), according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN). Kinase Activity Measurement. Raw 264.7 cells were seeded in 35-mm dishes, cultured overnight, and treated with PA for the indicated times. Whole cell lysates were prepared in ice-cold lysis buffer containing 50 mM Tris-HCl, pH 8.0, 5 mM EDTA, 150 mM NaCl, 1% Triton X-100, 50 mM NaF, 1 M sodium orthovanadate, 1 mM PMSF, and protease inhibitor cocktail. To determine JAK activities, 0.5 mg of total protein was incubated with phosphotyrosine Ab (4G10) for 4 h and then with protein A-agarose for 2 h at 4°C. After being washed seven times with lysis buffer, immunocomplexes were resolved by SDS-PAGE and transferred to nitrocellulose for subsequent immunoblot analysis using JAK2 Ab. For kinase phosphorylation, immunoblotting was performed using Abs specific for the phosphorylated, activated forms of proteins. Nuclear Extracts. Raw 264.7 cells were incubated with PA as indicated. Cells were harvested in PBS containing 2% serum, washed twice with ice-cold PBS, and resuspended in 400 l of buffer A (10 mM HEPES, pH 7.9, 5 mM MgCl2, 10 mM KCl, 1 mM ZnCl2, 0.2 mM EGTA, 1 mM Na3VO4, 10 mM NaF, 0.5 mM dithiothreitol, 0.5 mM PMSF, and protease inhibitors). After cells had been incubated on ice for 10 min and lysed by adding 50 l of 10% Nonidet P-40 (to a final concentration of 1.1%), nuclei were harvested by centrifugation. Nuclear pellets were then resuspended in 60 l of extraction buffer (10 mM HEPES, pH 7.9, 5 mM MgCl2, 300 mM NaCl, 1 mM ZnCl2, 0.2 mM EGTA, 25% glycerol, 1 mM Na3VO4, 10 mM NaF, 0.5 mM dithiothreitol, 0.5 mM PMSF, and protease inhibitors) and incubated on ice for 15 min. Nuclear debris was then removed by centrifugation (13,000 rpm 10 min), and nuclear protein extracts were subjected to gel shift analysis. Protein concentrations were determined using the Bradford method. Electrophoretic Mobility Shift Assays. Mobility shift assays were performed with nuclear extracts and biotin-labeled oligonucle1042 Lee et al. at A PE T Jornals on A uust 4, 2017 m oharm .aspeurnals.org D ow nladed from otides containing the consensus sequences of transcription factor (TF) STAT1 or STAT3 using gel shift kits (Panomics, Redwood City, CA). Typical binding reaction mixtures consisted of 5 g of nuclear extract, labeled TF probe, 2 g/ml poly[d(I-C)] in a buffer containing 20 mM HEPES, pH 7.9, 50 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, and 5% glycerol, and these were incubated at room temperature for 30 min. Reaction mixtures were then separated on 6% Tris-glycine nondenaturing polyacrylamide gels in a 2 Tris-glycine buffer system and transferred to nitrocellulose membranes. The shifted bands corresponding to protein/DNA complexes were visualized using a chemiluminescence system. Unlabeled TF probes were used as a competitor to confirm the identities of the protein/DNA complexes. Transfection and Promoter Activity Assays. A promoter-reporter construct containing eight copies of the GAS element was transfected into RAW 264.7 cells using LipofectAMINE 2000 (Gibco BRL). After transfection, cells were incubated in complete media for 24 h at 37°C and stimulated with PA for 8 h at 37°C. In some experiments, cells were preincubated with specific inhibitor for 1 h at 37°C before PA stimulation. Cell lysates were assayed for luciferase activity using a luminometer (Promega, Madison, WI) according to the manufacturer’s instructions. For JAK2 silencing, Raw 264.7 cells were transfected with a 100 nM concentration of a pool of JAK2specific siRNA (SMARTpool kit; Dharmacon, Chicago, IL) using LipofectAMINE2000. As a control, nonspecific siRNA duplexes were transfected in parallel into cells. Protein Extraction and Western Blot Analysis. Raw 264.7 cells were stimulated with PA, washed twice in ice-cold PBS, and lysed on ice using lysis solution (1% Triton X-100, 50 mM Tris, pH 8.0, 150 mM NaCl, 1 mM PMSF, 1 mM Na3VO4, and protease inhibitor cocktail). Protein concentrations were determined using Bio-Rad protein assays. In brief, proteins from cell lysates (50 g) were boiled at 95°C in Laemmli SDS loading buffer, separated on 8% SDS-PAGE, and electrotransferred to nitrocellulose membranes. Membranes were blocked for 30 min at room temperature in Trisbuffered saline/0.05% Tween 20 (TTBS) containing 5% nonfat dry milk and then incubated with TTBS containing a primary Ab for 4 h at room temperature. After five 10-min washes in TTBS, membranes were incubated with peroxidase-conjugated secondary Ab for 1 h. After 5 10 min with TTBS, protein bands of interest were visualized using an enhanced chemiluminescence detection system (Amersham Biosciences Inc., Little Chalfont, Buckinghamshire, UK).