Induction of 15-Lipoxygenase Expression by IL-13 Requires Tyrosine Phosphorylation of Jak2 and Tyk2 in Human Monocytes
1998; Elsevier BV; Volume: 273; Issue: 48 Linguagem: Inglês
10.1074/jbc.273.48.32023
ISSN1083-351X
AutoresBiswajit Roy, Martha K. Cathcart,
Tópico(s)Immune Response and Inflammation
ResumoThe enzyme 15-lipoxygenase (15-LO) participates in the dioxygenation of polyenoic fatty acids. This activity leads to the degradation of mitochondrial membranes during reticulocyte differentiation, the production of pro- and anti-inflammatory mediators by a variety of cell types, and the oxidation of lipids in atherosclerotic lesions. The cytokines, IL-4 and IL-13, are reported to induce the expression of 15-LO in human peripheral blood monocytes. In this report we explore the signaling mechanisms involved in the IL-13-mediated induction of 15-LO expression. First we demonstrate that the delayed induction of 15-LO requires continuous stimulation of monocytes for a minimum period of 12 h. We also found that tyrosine kinase inhibitors blocked the induction of 15-LO in a dose-dependent manner. By immunoprecipitation and antiphosphotyrosine blotting experiments, IL-13 was shown to induce tyrosine phosphorylation of Jak2 and Tyk2, but not Jak1 or Jak3, within 5 min of treatment in human monocytes. To investigate whether the early induction of tyrosine phosphorylation of both Jak2 and Tyk2 was ultimately involved in 15-LO expression, we generated antisense oligodeoxyribonucleotides (ODNs) against Tyk2 and Jak2. We employed a cationic lipid-mediated delivery technique to transfect the monocytes and found that both antisense ODNs inhibited expression of their target proteins by 75–85%. The treatments were specific and did not affect the expression of each other. Furthermore, the antisense ODNs to Jak2 and Tyk2 both inhibited the induction of expression of 15-LO in monocytes treated with IL-13. Parallel experiments with sense ODNs to Jak2 and Tyk2 did not affect their protein levels or the induction of 15-LO by IL-13, and down-regulation of Jak1 also did not affect expression of 15-LO. Our results suggest the novel finding that IL-13 can induce tyrosine phosphorylation of both Jak2 and Tyk2 in primary human monocytes. This occurs as an early and essential signal transduction event for the IL-13-mediated induction of 15-LO expression. These data represent the first characterization of upstream kinases involved in the induced expression of 15-LO. The enzyme 15-lipoxygenase (15-LO) participates in the dioxygenation of polyenoic fatty acids. This activity leads to the degradation of mitochondrial membranes during reticulocyte differentiation, the production of pro- and anti-inflammatory mediators by a variety of cell types, and the oxidation of lipids in atherosclerotic lesions. The cytokines, IL-4 and IL-13, are reported to induce the expression of 15-LO in human peripheral blood monocytes. In this report we explore the signaling mechanisms involved in the IL-13-mediated induction of 15-LO expression. First we demonstrate that the delayed induction of 15-LO requires continuous stimulation of monocytes for a minimum period of 12 h. We also found that tyrosine kinase inhibitors blocked the induction of 15-LO in a dose-dependent manner. By immunoprecipitation and antiphosphotyrosine blotting experiments, IL-13 was shown to induce tyrosine phosphorylation of Jak2 and Tyk2, but not Jak1 or Jak3, within 5 min of treatment in human monocytes. To investigate whether the early induction of tyrosine phosphorylation of both Jak2 and Tyk2 was ultimately involved in 15-LO expression, we generated antisense oligodeoxyribonucleotides (ODNs) against Tyk2 and Jak2. We employed a cationic lipid-mediated delivery technique to transfect the monocytes and found that both antisense ODNs inhibited expression of their target proteins by 75–85%. The treatments were specific and did not affect the expression of each other. Furthermore, the antisense ODNs to Jak2 and Tyk2 both inhibited the induction of expression of 15-LO in monocytes treated with IL-13. Parallel experiments with sense ODNs to Jak2 and Tyk2 did not affect their protein levels or the induction of 15-LO by IL-13, and down-regulation of Jak1 also did not affect expression of 15-LO. Our results suggest the novel finding that IL-13 can induce tyrosine phosphorylation of both Jak2 and Tyk2 in primary human monocytes. This occurs as an early and essential signal transduction event for the IL-13-mediated induction of 15-LO expression. These data represent the first characterization of upstream kinases involved in the induced expression of 15-LO. lipoxygenase oligodeoxyribonucleotide signal transducers and activators of transcription polyacrylamide gel electrophoresis bovine calf serum Dulbecco's modified Eagle's medium dimethyl dioctadecyl ammonium bromide dioleoylphosphatidylethanolamine interleukin polyvinylidene difluoride high performance liquid chromatography. Among the members of the lipoxygenase family (EC 1.13.11.12), 15-lipoxygenase and the closely related leukocyte type 12 lipoxygenases are of special interest because of their ability to oxidize unsaturated fatty acids (1Yamamoto S. Biochim. Biophys. Acta. 1992; 1128: 117-131Crossref PubMed Scopus (570) Google Scholar, 2Takahashi Y. Glasgow W.C. Suzuki H. Taketani Y. Yamamoto S. Anton M. Kuhn H. Brash A.R. Eur. J. 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In human leukocytes, catalysis of arachidonic acid oxidation by 15-LO1 leads to the formation of 15-(S)-hydroperoxyeicosatetraenoic acid, 15-(S)-hydroxyeicosatetraenoic acid, and lipoxins A4 and B4 (7Samuelsson B. Dahlen S.E. Lindgren J.A. Rouzer C.A. Serhan C.N. Science. 1987; 237: 1171-1176Crossref PubMed Scopus (2039) Google Scholar, 8Ford-Hutchinson A. Eicosanoids. 1991; 4: 65-74PubMed Google Scholar, 9Badr K.F. Kidney Int. 1992; 42: S101-S108Google Scholar, 10Serhan C.N. Biochim. Biophys. Acta. 1994; 1212: 1-25Crossref PubMed Scopus (244) Google Scholar). These compounds possess a wide range of biological activities. Both 15-(S)-hydroxyeicosatetraenoic acid and lipoxins A4 are potent endogenous anti-inflammatory molecules in view of their capacities to suppress white cell chemotaxis, adherence, activation, and to specifically antagonize the functional responses of pro-inflammatory 5-LO derivatives, the leukotrienes (11Brezinski M.E. Serhan C.N. Proc. Natl. Acad. Sci. U. S. 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Invest. 1995; 96: 504-510Crossref PubMed Scopus (219) Google Scholar, 15Yla-Herttuala S. Rosenfeld M.E. Parthasarathy S. Glass C.K. Sigal E. Witztum J.T. Steinberg D. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6959-6963Crossref PubMed Scopus (414) Google Scholar, 16Kuhn H. Belkner J. Zaiss S. Fahrenklemper T. Wohlfeil S. J. Exp. Med. 1994; 179: 1903-1911Crossref PubMed Scopus (178) Google Scholar) and to a lesser extent in subtypes of arterial smooth muscle cells (17Hugou I. Blin P. Henri J. Daret D. Larrue J. Atherosclerosis. 1995; 113: 189-195Abstract Full Text PDF PubMed Scopus (23) Google Scholar); however, the role of this enzyme in the pathogenesis of cardiovascular disease is far from clear. Because circulating blood monocytes of normal individuals do not express 15-LO (18Conrad D.J. Kuhn H. Mulkins M. Highland E. Sigal E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 217-221Crossref PubMed Scopus (352) Google Scholar), the enzyme must be induced during activation or macrophage differentiation in the tissue. Numerous lymphokines are known to modulate the inflammatory response through their actions on monocytes (19McKenzie A.N.J. Culpepper J.A. De Waal Malefyt R. Briere F. Punnonen J. Aversa G. Sato A. Dang W. Cocks B.G. Menon S. De Vris J.E. Banchereau J. Zurawski G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3735-3739Crossref PubMed Scopus (545) Google Scholar, 20De Waal Malefyt R. Figdor C.G. Hujibens R. Mohan-Peterson S. Bennett B. Culpepper J.A. Dang W. Zurawski G. De Vris J.E. J. Immunol. 1993; 151: 6370-6381PubMed Google Scholar). The involvement of the local environment of the monocyte in influencing 15-LO expression is supported by studies showing that IL-4 and IL-13 are specific inducers of the enzyme (18Conrad D.J. Kuhn H. Mulkins M. Highland E. Sigal E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 217-221Crossref PubMed Scopus (352) Google Scholar, 21Nassar G.M. Morrows J.D. Roberts L.J., II Fadi F.G. Badr K.F. J. Biol. Chem. 1994; 269: 27631-27634Abstract Full Text PDF PubMed Google Scholar). Attempts to study the molecular signaling mechanisms using monocytic cell lines have not been successful, as the cell lines failed to show similar induction of 15-LO when stimulated with interleukins (22Brinckman R. Topp M.S. Zalan I. Heydeck D. Ludwig P. Kuhn H. Berdel W.E. Habenicht A.J.R. Biochem. J. 1996; 318: 305-312Crossref PubMed Scopus (83) Google Scholar). Of several permanent human hematopoietic and epithelial cell lines, only the lung epithelial cell line, A549, showed induction of 15-LO, even though all the cell lines tested were positive for the presence of cell surface receptors for both IL-4 and IL-13 (22Brinckman R. Topp M.S. Zalan I. Heydeck D. Ludwig P. Kuhn H. Berdel W.E. Habenicht A.J.R. Biochem. 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Although IL-4Rα (140 kDa) was shown to be a substrate for both IL-4- and IL-13-dependent tyrosine phosphorylation, the other constituents of the receptor complex in different cells and cell lines are not yet clear (27Smerz-Bertling C. Duschl A. J. Biol. Chem. 1995; 270: 966-970Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Two different IL-13 receptors are reported, one with lower affinity toward IL-13 (K D = 3–10 nm) having a molecular mass of 56–68 kDa (28Hilton D.J. Zhang J-G. Metcalf D. Alexander W.S. Nicola N.A. Willson T.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 497-501Crossref PubMed Scopus (390) Google Scholar), termed IL-13Rα, and the other with higher affinity toward IL-13 (K D = 20–90 pm) having a molecular mass of 45–50 kDa (29Zhang J-G. Hilton D.J. Willson T.A. McFarlane C. Roberts B.A. Moritz R.L. Simpson R.J. Alexander W.S. Metcalf D. Nicola N.A. J. Biol. 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Cross-competition by IL-4 and IL-13 for receptors was also observed in certain cell types (35Mosley B. Beckmann M.P. March C.J. Idzerda R.L. Gimpel S.D. VandenBos T. Friend D. Alpert A. Anderson D. Jackson J. Wignall J.M. Smith C. Gallis B. Sims J.E. Urdal D. Cosman D. Park L.S. Cell. 1989; 89: 335-348Abstract Full Text PDF Scopus (540) Google Scholar, 36Zurawski S.M. Vega F. Huyghe B. Zurawski G. EMBO J. 1993; 12: 2663-2670Crossref PubMed Scopus (453) Google Scholar, 37Obiri N.I. Debinski W. Leonard W.J. Puri R.K. J. Biol. Chem. 1995; 270: 8797-8804Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 38Vita N. Lefort S. Laurent P. Caput D. Ferrara P. J. Biol. Chem. 1995; 270: 3512-3517Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Although IL-2Rγc is required for IL-4-mediated signal transduction in some cells, other IL-4-responsive cell lines (e.g. plasmacytoma B9 and renal cell lines) exist that do not express IL-2Rγc (39Welham M.J. Learmonth L. Bone H. Schrader J.W. J. Biol. Chem. 1995; 270: 12286-12296Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). Expression of IL-2Rγc on human monocytes has been shown to be very low (40Bosco M.C. Espinoza-Delgado I. Schwabe M. Russell S.M. Leonard W.J. Longo D.L. Varesio L. Blood. 1994; 83: 3462-3467Crossref PubMed Google Scholar) or absent (24de Wit H. Hendriks D.W. Halie M.R. Vellenga E. Blood. 1994; 84: 608-615Crossref PubMed Google Scholar), thus leading to the suggestion that the functional composition of IL-4 and IL-13 receptor complexes may vary from cell type to cell type and that IL-2Rγc is not absolutely necessary for receptor signaling. Different groups have utilized antisense oligodeoxyribonucleotides (ODNs) to inhibit the endogenous level of expression of different enzymes, including the Jaks and STATs, in various cells and cell lines (41Weiler S.R. Mou S. DeBerry C.S. Keller J.R. Ruscetti F.W. Ferris D.K. Longo D.L. Linnekin D. Blood. 1996; 87: 3688-3693Crossref PubMed Google Scholar, 42Lee Y.J. Benveniste E.N. J. Immunol. 1996; 157: 1559-1568PubMed Google Scholar, 43Tilbrook P.A. Bittorf T. Callus B.A. Busfield S.J. Ingley E. Klinken S.P. Cell Growth Differ. 1996; 7: 511-520PubMed Google Scholar). Jak2 antisense ODN treatment resulted in a reduction of expression of up to 46% in human and murine cell lines and normal human progenitor cells (41Weiler S.R. Mou S. DeBerry C.S. Keller J.R. Ruscetti F.W. Ferris D.K. Longo D.L. Linnekin D. Blood. 1996; 87: 3688-3693Crossref PubMed Google Scholar). The level of expression of STAT1α was reduced in the astroglioma cell line (CH235-MG) as well as in glomerular mesangial cells using antisense ODN specific to STAT1α (42Lee Y.J. Benveniste E.N. J. Immunol. 1996; 157: 1559-1568PubMed Google Scholar). Our attempts to inhibit the activity and expression of classical protein kinase C (44Li Q. Cathcart M.K. J. Biol. Chem. 1994; 269: 17508-17515Abstract Full Text PDF PubMed Google Scholar) and cytosolic phospholipase A2 (45Li Q. Cathcart M.K. J. Biol. Chem. 1997; 272: 2404-2411Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar) using antisense ODNs met with an even higher level of inhibition (up to 80%) as this approach worked more efficiently in nonproliferating, rapidly pinocytosing monocytes. In the current study, we examined the signaling intermediates triggered by IL-13 in human monocytes and assessed their involvement in the induction of expression of 15-LO. Our data indicate that the induction of 15-LO was delayed and dependent on tyrosine kinases. Using immunoprecipitation studies, we demonstrated that IL-13 induced tyrosine phosphorylation of Jak2 and Tyk2. The induction of expression of 15-LO could be inhibited by using antisense ODNs against Jak2 and Tyk2, suggesting their involvement in the IL-13-signaling pathway in human monocytes. Genistein, daidzein, tyrphostin 23, and tyrphostin 1 were purchased from Biomol Research Laboratories (Plymouth, PA). All the drugs were dissolved in Me2SO. All the reagents were made as 1000-fold concentrated stock solutions and stored at −20 °C before use. Recombinant human IL-13 was purchased from Upstate Biotechnology, Lake Placid, NY. Antibody against rabbit reticulocyte 15-LO cross-reacting with human 15-LO was raised in sheep and was a kind gift of Dr. Joseph Cornicelli, Parke-Davis. Rabbit antisera against Jak1, Jak2, Tyk2, and Jak3 were purchased from either Upstate Biotechnology or Santa Cruz Biotechnology, Inc., CA. Each one of the Jak antibodies was essentially noncross-reactive with the other members of the Jak family and recognized corresponding antigens under native (in 1% Triton X-100 extracts) as well as denaturing conditions. For detection of tyrosine-phosphorylated proteins by immunoblotting, a mixture (1:1) of anti-phosphotyrosine antibodies PY-20 (Santa Cruz Biotechnology, Inc., CA) and 4G-10 (UBI, Lake Placid, NY) were used, both at dilutions of 1:1000. Antibody to phosphotyrosine, PY-99 (Santa Cruz Biotechnology, Inc.), was used to immunoprecipitate tyrosine-phosphorylated proteins. Human blood monocytes were isolated from heparinized whole blood by sequential centrifugation over a Ficoll-Paque solution and adherence to serum-coated tissue culture flasks as described previously (46Cathcart M.K. McNally A.K. Chisolm G.M. J. Immunol. 1989; 142: 1963-1969PubMed Google Scholar). Nonadherent cells were removed by gentle washing, and adherent cells were collected after releasing with 5 mm EDTA and plated in tissue culture plates (Costar, Cambridge, MA) at 1.0 × 106 cells/ml. The cell population typically contained more than 95% monocytes and was maintained in Dulbecco's modified Eagle's medium (DMEM) (Life Technologies, Inc.) supplemented with 10% bovine calf serum (BCS) (HyClone, Logan, UT) at 37 °C in the presence of 10% CO2. IL-13 (250 pm) was added in tissue culture plates (100 mm) containing 10.0 × 106 cells/plate with 1.0 × 106 cells/ml of DMEM-BCS medium and incubated for up to 36 h. Subsequently, the cells were washed three times with PBS to remove the traces of DMEM, 10% BCS. The plates were placed on ice, and the cells were lysed using 200 μl of lysis buffer (1% Triton X-100, 150 mm NaCl, 50 mm Tris-HCl, pH 7.4, 1 mm phenylmethylsulfonyl fluoride and 10 μl of protease inhibitor mixture (Sigma)/1 ml of lysis buffer). After 30 min, the lysate was centrifuged for 15 min at 9300 × g. The supernatant was collected, and the protein concentration was determined using the Bio-Rad protein assay kit and loaded on a 7.5% SDS-PAGE gel (50 μg of lysate/well). The proteins were transferred to a PVDF membrane (0.2 μm) (Bio-Rad) using a Trans-Blot SD electrophoretic transfer cell (Bio-Rad). The membrane was blocked in 3% nonfat milk in 20 mm Tris-HCl, pH 7.4, 150 mm NaCl, and 0.1% Tween 20 for one h at room temperature and was probed with antibody to rabbit reticulocyte 15-LO (diluted 1:2000 in 20 mm Tris-HCl, pH 7.4, 150 mm NaCl, and 0.1% Tween 20 for 1 h at room temperature. This antibody was shown to be specific for 15-LO and does not cross-react with 5-LO (47Cornicelli J.A. Welch K Auerbach B. Feinmark S.J. Daugherty A. Arterioscler. Thromb. Vasc. Biol. 1996; 16: 1488-1494Crossref PubMed Scopus (19) Google Scholar). A horseradish peroxidase-labeled secondary antibody (ICN Biochemicals, Cleveland, OH) diluted 1:5000 in 20 mm Tris-HCl, pH 7.4, 150 mm NaCl and 0.1% Tween 20 was added for 1 h, and the hybridization signal was detected using Enhanced Chemiluminescence (ECL) detection reagents (Pierce) according to the manufacturer's guide and followed by autoradiography. Freshly isolated monocytes were pretreated with activated sodium vanadate solution (5 mm final concentration) for 30 min followed by treatment with IL-13 (250 pm) for 5 min. The treated cells were immediately lysed at 50 × 106/ml in lysis buffer (1% Triton X-100, 150 mm NaCl, 50 mm NaF, 50 mm Tris, pH 7.4, 5 mm sodium pyrophosphate, 1 mm sodium orthovanadate, 500 μmphenylmethylsulfonyl fluoride, and 1:100 diluted protease inhibitor mixture). After 30 min on ice, the extracts were centrifuged at 9300 × g for 15 min at 4 °C, and the post-nuclear extract (500 μg/ml) was incubated with the relevant antibody for 2 h at 4 °C with constant rotation. Immune complexes were collected using Sepharose-protein A beads (20 μl packed volume). After washing 3 times (10 min each) with lysis buffer, the immune complexes were released in SDS sample buffer and analyzed by SDS-PAGE followed by electrophoretic transfer to PVDF membranes (Bio-Rad). The membranes were then probed with either antibody to phosphotyrosine or individual antibodies to Jak/Tyk kinases and developed using ECL. For immunoprecipitation experiments using anti-phosphotyrosine, PY-99 (Santa Cruz, CA), no sodium pyrophosphate was included in the lysis buffer. Fresh monocytes were isolated and adhered to 6-well plates (5 × 106cells/well) in the presence of BCS (10%) and DMEM for 2 h. The plates were washed once with fresh DMEM (without methionine) containing no BCS and subsequently replaced with DMEM (also without methionine) containing radioactive methionine ([35S]methionine, 100 μCi/ml and 2 ml/well). The plates were incubated for 4 h and washed with fresh DMEM containing 10% BCS, and subsequently, the cells were lysed after 0, 4, 8, 12, and 24 h of further incubations. Each one of the Jak/Tyk kinases were immunoprecipitated from the lysates (10 × 106/group), run on a 7.5% SDS-PAGE gel, transferred onto PVDF membranes, and exposed on a PhosphorImager screen (Molecular Dynamics). The half-life of the protein was calculated as the time necessary for the dpm of incorporated, radioactive methionine in that protein to decrease by 50%. The antisense sequences for human Jak2, Tyk2, and Jak1 were selected after studying the predicted secondary structural conformations of their mRNAs using the software program Mulfold©. The regions lacking major predicted secondary structures, e.g. loops, were mainly targeted to generate the antisense ODNs. Before final selection of the target region, the sequences were screened for uniqueness in all nonredundant GenBank CDS translations + PDB + SwissProt + PIR using Blast© and also were tested for lack of internal secondary structure or pairing using Mulfold© (48Jaeger J.A. Turner D.H. Zuker M. Methods Enzymol. 1989; 183: 281-306Crossref Scopus (387) Google Scholar). The antisense oligomer selected against Jak2 was complementary to nucleotides 59–78 of the human Jak2 sequence (accession numberAA453345), whereas the antisense oligomer against Tyk2 was complementary to nucleotides 481–500 of the human Tyk2 sequence (accession number X54637). The antisense ODN to Jak1 was complementary to bases 462–480 of the human Jak1 sequence (accession numberM64174). Control ODN for Jak2 and Tyk2 consisted of sense ODN. The sense ODN sequence for Jak1 was predicted to possibly serve as an antisense for human γ adaptin mRNA. We therefore chose to use a scrambled ODN as a control for the Jak1 antisense. The scrambled Jak1 ODN did not display internal secondary structure, did not pair with itself, and was unique in the nucleic acid data bases listed above. All ODN contained phosphorothioate-modified oligonucleotides to limit DNA degradation, and all were HPLC-purified before use (Genosys Biotechnology Inc., Woodlands, TX). The sequences of the ODN are as follows. Jak2 antisense: 5′-TCT TAA CTC TGT TCT CGT TC-3′. Jak2 sense: 5′-GAA CGA GAA CAG AGT TAA GA-3′. Tyk2 antisense: 5′-CCA ACT TTA TGT GCA ATG TG-3′. Tyk2 sense: 5′-CAC ATT GCA CAT AAA GTT GG-3′. Jak1 antisense: 5′-GGT TGC ATC TGG AAT CTT T-3′. Jak1 scrambled: 5′-TTG TGA ACT GCC TGT GAT T-3′. Cationic lipids were used to aid the delivery of oligonucleotides (49Wielbo D. Shi N. Sernia C. Bochem. Biophys. Res. Commun. 1997; 232: 794-799Crossref PubMed Scopus (10) Google Scholar). Lipids were prepared from chloroform stock solutions of DDAB and DOPE (250 mg/ml). DDAB and DOPE were added to yield a ratio of 2:5 (w/w), DDAB:DOPE. This mixture was dried under nitrogen and then sonicated in sterile deionized water until clear to yield a concentration of 5 mm DDAB. The lipid preparation was used as a 100× stock solution. Before treating the monocytes with ODNs, the cationic lipid preparation was diluted 100-fold in DMEM without serum or antibiotics, and ODNs were added to yield a final concentration of 1.0 μm sense or antisense ODN. This mixture was incubated for 30 min to form lipid:ODN complexes. Medium was removed from the monocytes, they were rinsed with DMEM without serum or antibiotics, and the lipid:ODN mixture was added. After 1 h of incubation, BCS was added to 10% (v/v), and the incubation was continued for 24 h. After this treatment, monocytes were either lysed, and the level of target proteins was analyzed, or the monocytes were exposed to IL-13 for another 24 h to study the expression of 15-lipoxygenase. Nassar et al. (21Nassar G.M. Morrows J.D. Roberts L.J., II Fadi F.G. Badr K.F. J. Biol. Chem. 1994; 269: 27631-27634Abstract Full Text PDF PubMed Google Scholar) studied the induction of 15-LO in response to constant exposure to IL-13 and found that the expression of 15-LO was not immediate and was detectable by Western blot after 36 h of incubation. To determine whether continuous exposure to IL-13 was required for 15-LO induction, we exposed monocytes to IL-13 (250 pm) for various times, removed the IL-13 by washing, and then detected 15-LO expression at 36 h. The results (Fig. 1) revealed that only after 12 h of incubation with IL-13 was there detectable expression of 15-LO. The level of 15-LO expression was up-regulated when incubations were continued for 24 and 36 h. No 15-LO band was detected in the lysate of cells treated with IL-13 for less than 12 h or in the untreated cell lysate. Numerous studies have reported the involvement of Jak/Tyk kinases and the STAT molecules in interleukin-mediated signaling pathways (23Debinski W. Miner R. Leland P. Obiri N.I. Puri R.K. J. Biol. Chem. 1996; 271: 22428-22433Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 24de Wit H. Hendriks D.W. Halie M.R. Vellenga E. Blood. 1994; 84: 608-615Crossref PubMed Google Scholar, 25Bosco M.C. Espinoza-Delgado I. Schwabe M. Gusella G.L. Longo D.L. Sugamura K. Varesio L. Blood. 1994; 83: 2995-3002Crossref PubMed Google Scholar, 26Espinoza-Delgado I. Bosco M.C. Musso T. Mood K. Ruscetti F.W. Longo D.L. Varesio L. Blood. 1994; 83: 3332-3338Crossref PubMed Google Scholar). At the same time, the reports indicate that these activation and tyrosine phosphorylation events take place immediately, within a few min of treatment. But in our case, we found that for the expression of 15-LO, the cells needed to be stimulated with the ligand (IL-13) for a minimum of 12 h. We were therefore interested in examining the role of Jak/Tyk involvement in 15-LO expression. Previous studies have shown IL-13-mediated tyrosine phosphorylation of numerous proteins in human monocytes (50Adunyah S.E. Pegram M.L. Cooper R.S. Biochem. Biophys. Res. Commun. 1995; 206: 103-111Crossref PubMed Scopus (11) Google Scholar). To examine whether IL-13-mediated induction of 15-LO expression involved signaling by tyrosine phosphorylation events, we examined the effects of potent tyrosine kinase inhibitors on this pathway. First, the monocytes were pretreated with tyrosine kinase inhibitors like genistein and tyrphostin 23 for 30 min at different dosages followed by treatment with IL-13 for 24 h, and the level of 15-LO was studied by Western blotting (Fig. 2). Whereas 10 μg of tyrphostin 23/ml of medium caused only slight inhibition, 25 μg/ml had a more profound effect (≈50% inhibition). At 50 μg/ml, tyrphostin 23 inhibited detectable 15-LO expression by approximately 98%. Genistein also substantially inhibited the induction of 15-LO when applied at 25 μg/ml. In contrast, the negative structural analog controls for both genistein (daidzein) and tyrphostin 23 (tyrphostin 1) had little effect (<10% inhibition) on the expression of 15-LO when tested at identical concentrations. These results indicate that both of the tyrosine kinase inhibitors were able to substantially inhibit IL-13-mediated induction of 15-LO. To determine whether the Jak family of kinases was involved in IL-13-mediated signaling pathways, cell lysates from both untreated and IL-13-treated fresh human monocytes were immunoprecipitated with antibodies to Jak1, Jak2, Tyk2, and Jak3, electrophoresed, blotted on PVDF membranes, and immunoblot
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