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• W2039883000 abstract "Interleukin-4 (IL-4) induces expression of reticulocyte-type 15-lipoxygenase-1 (15-LOX-1) in various mammalian cells via the Janus kinase/signal transducer and activator of transcription 6 (STAT6) signaling system. We studied the mechanism of 15-LOX-1 induction in A549 lung epithelial cells and found that genistein, a potent tyrosine kinase inhibitor, prevented phopsphorylation of STAT6, its binding to the 15-LOX-1 promoter, and the expression of catalytically active enzyme. In contrast, cycloheximide did not prevent 15-LOX-1 induction. Surprisingly, we found that IL-4 up-regulated the histone acetyltransferase activity of CREB-binding protein (CBP)/p300, which is responsible for acetylation of nuclear histones and STAT6. The acetylation of both proteins appears to be essential for the IL-4-induced signal transduction cascade, because inhibition of CBP/p300 by the viral wild-type E1A oncoprotein abrogated acetylation of both histones and STAT6 and strongly suppressed transcriptional activation of the 15-LOX-1 gene. Moreover, we found that the inhibition by sodium butyrate of histone deacetylases, which apparently suppress 15-LOX-1 gene transcription, synergistically enhanced the IL-4-stimulated 15-LOX-1 expression. These data suggest that both phosphorylation and acetylation of STAT6 as well as acetylation of nuclear histones are involved in transcriptional activation of the 15-LOX-1 gene, although these reactions follow differential kinetics. STAT6 phosphorylation proceeds within the first hour of IL-4 stimulation. In contrast, CBP/p300-mediated acetylation requires 9–11 h, and similar kinetics were observed for the expression of the active enzyme. Thus, our results suggest that in the absence of IL-4, nuclear histones may be bound to regulatory elements of the 15-LOX-1 gene, preventing its transcription. IL-4 stimulation causes rapid phosphorylation of STAT6, but its binding to the promoter appears to be prevented by nonacetylated histones. After 9–11 h, when histones become acetylated, STAT6 binding sites may be demasked so that the phosphorylated and acetylated transcription factor can bind to activate gene transcription. Interleukin-4 (IL-4) induces expression of reticulocyte-type 15-lipoxygenase-1 (15-LOX-1) in various mammalian cells via the Janus kinase/signal transducer and activator of transcription 6 (STAT6) signaling system. We studied the mechanism of 15-LOX-1 induction in A549 lung epithelial cells and found that genistein, a potent tyrosine kinase inhibitor, prevented phopsphorylation of STAT6, its binding to the 15-LOX-1 promoter, and the expression of catalytically active enzyme. In contrast, cycloheximide did not prevent 15-LOX-1 induction. Surprisingly, we found that IL-4 up-regulated the histone acetyltransferase activity of CREB-binding protein (CBP)/p300, which is responsible for acetylation of nuclear histones and STAT6. The acetylation of both proteins appears to be essential for the IL-4-induced signal transduction cascade, because inhibition of CBP/p300 by the viral wild-type E1A oncoprotein abrogated acetylation of both histones and STAT6 and strongly suppressed transcriptional activation of the 15-LOX-1 gene. Moreover, we found that the inhibition by sodium butyrate of histone deacetylases, which apparently suppress 15-LOX-1 gene transcription, synergistically enhanced the IL-4-stimulated 15-LOX-1 expression. These data suggest that both phosphorylation and acetylation of STAT6 as well as acetylation of nuclear histones are involved in transcriptional activation of the 15-LOX-1 gene, although these reactions follow differential kinetics. STAT6 phosphorylation proceeds within the first hour of IL-4 stimulation. In contrast, CBP/p300-mediated acetylation requires 9–11 h, and similar kinetics were observed for the expression of the active enzyme. Thus, our results suggest that in the absence of IL-4, nuclear histones may be bound to regulatory elements of the 15-LOX-1 gene, preventing its transcription. IL-4 stimulation causes rapid phosphorylation of STAT6, but its binding to the promoter appears to be prevented by nonacetylated histones. After 9–11 h, when histones become acetylated, STAT6 binding sites may be demasked so that the phosphorylated and acetylated transcription factor can bind to activate gene transcription. reticulocyte-type 15-lipoxygenase interleukin 4 signal transducer and activator of transcription 6 histone acetyltransferases histone deacetylase cAMP response element-binding protein CREB-binding protein wild-type E1A oncoprotein mutant for CBP binding domain electrophoretic mobility shift assay phosphate-buffered saline polyacrylamide gel electrophoresis polymerase chain reaction reverse transcription Lipoxygenases constitute a family of widely distributed non-heme-containing enzymes, which dioxygenate polyenoic fatty acids to their corresponding hydroperoxide derivatives (1Yamamoto S. Biochim. Biophys. Acta. 1992; 1128: 117-131Crossref PubMed Scopus (565) Google Scholar, 2Lehmann W.D. Free Radic. Biol. Med. 1994; 16: 241-253Crossref PubMed Scopus (44) Google Scholar). Among the members of the lipoxygenase family, the reticulocyte-type 15-lipoxygenase (15-LOX-1)1 is of particular interest because of its ability to oxygenate complex substrates, such as phospholipids (3Murray J.J. Brash A.R. Arch. Biochem. Biophys. 1988; 265: 514-523Crossref PubMed Scopus (83) Google Scholar), biomembranes (4Kühn H. Belkner J. Wiesner R. Brash A.R. J. Biol. Chem. 1990; 265: 18351-18361Abstract Full Text PDF PubMed Google Scholar), and lipoproteins (5Belkner J. Wiesner R. Rathman J. Barnett J. Sigal E. Kühn H. Eur. J. Biochem. 1993; 213: 251-261Crossref PubMed Scopus (136) Google Scholar). The enzyme has been implicated in the programmed breakdown of mitochondria during red blood cell maturation (6Rapoport S.M. Schewe T. Biochim. Biophys. Acta. 1986; 864: 471-495Crossref PubMed Scopus (112) Google Scholar), in the development of fiber cells in the eye lens (7Van Leyen K. Duvoisin R.M. Engelhardt H. Wiedmann M.A. Nature. 1998; 395: 392-395Crossref PubMed Scopus (255) Google Scholar), and recently in actin polymerization during phagocytosis of apoptotic cells (8Miller Y.I. Chang M.K. Funk C.D. Feramisco J.R. Witztum J.L. J. Biol. Chem. 2001; 276: 19431-19439Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). 15-LOX-1 is also expressed in lipid-laden macrophages of atherosclerotic lesions (9Kühn H. Chan L. Curr. Opin. Lipidol. 1997; 8: 111-117Crossref PubMed Scopus (91) Google Scholar) and in human bronchial epithelial cells (10Hill E.M. Eling T. Nettesheim P. Am. J. Respir. Cell Mol. Biol. 1998; 18: 662-669Crossref PubMed Scopus (43) Google Scholar). Expression of the 15-LOX-1 gene is highly regulated. In young rabbit reticulocytes, a regulatory protein, which binds to a repetitive sequence element in the 3′-untranslated region of the 15-LOX mRNA, prevents its translation (11Ostareck-Lederer K. Ostareck D.H. Wilm M. Thiele B.J. Man M. Hentze M.W. Cell. 1997; 89: 597-605Abstract Full Text Full Text PDF PubMed Scopus (430) Google Scholar). In human monocytes (12Conrad D.J. Kuhn H. Mulkins M. Highland E. Sigal E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 217-221Crossref PubMed Scopus (347) Google Scholar), alveolar macrophages (13Levy B.D. Romano M. Chapman H.A. Reilly J.J. Drazen J. Serhan C.N. J. Clin. Invest. 1993; 92: 1572-1579Crossref PubMed Scopus (176) Google Scholar), A549 lung epithelial carcinoma cells (14Brinckmann R. Topp M.S. Zalan I. Heydeck D. Ludwig P. Kuhn H. Berdel W.E. Habenicht J.R. Biochem. J. 1996; 318: 305-312Crossref PubMed Scopus (83) Google Scholar), human tracheo-bronchial epithelial cells (15Jayawickreme S.P. Gray T. Nettesheim P. Eling T. Am. J. Physiol. 1999; 276: L596-L603PubMed Google Scholar), human colorectal carcinoma HTB 38 cells (16Ikawa H. Kamitani H. Calvo B.F. Foley J.F. Eling T.E. Cancer Res. 1999; 59: 360-366PubMed Google Scholar), and Caco-2 cells (17Kamitani H. Kameda H. Kelavkar U.P. Eling T.E. FEBS Lett. 2000; 467: 341-347Crossref PubMed Scopus (32) Google Scholar), it is up-regulated by interleukin-4 (IL-4), IL-13, or both. Recently, it has been demonstrated that IL-4 also induces the expression of peroxisome proliferator-activated receptor-γ and transcription of theCD36 gene (18Huang J.T. Welch J.S. Ricote M. Binder C.J. Willson T.M. Kelly C. Witztum J.L. Funk C.D. Conrad D. Glass C.K. Nature. 1999; 400: 378-382Crossref PubMed Scopus (775) Google Scholar). IL-4 binding to its cell surface receptor leads to tyrosine phosphorylation of the intracellular part of the IL-4 receptor and to an activation of Janus kinases 1 and 3 (19Ihle J.N. Kerr I.M. Trends Genet. 1995; 11: 69-74Abstract Full Text PDF PubMed Scopus (821) Google Scholar). These kinases may be involved in phosphorylation of signal transducer and activator of transcription 6 (STAT6) (19Ihle J.N. Kerr I.M. Trends Genet. 1995; 11: 69-74Abstract Full Text PDF PubMed Scopus (821) Google Scholar). After phosphorylation, STAT6 dimerizes and translocates to the nucleus, where it may bind to a particular sequence elements (20Mikita T. Campbell D. Wu P. Williamson K. Schindler U. Mol. Cell. Biol. 1996; 16: 5811-5820Crossref PubMed Scopus (226) Google Scholar) in the promoter of IL-4-responsible genes. The involvement of STAT6 in IL-4-induced 15-LOX-1 expression was underlined by the fact that no induction of 12/15-LOX-1 activity was observed in macrophages from homozygous STAT6-deficient mice (21Heydeck D. Thomas L. Schnurr K. Trebus F. Thierfelder W.E. Ihle J.N. Kuhn H. Blood. 1998; 92: 2503-2510Crossref PubMed Google Scholar). Unfortunately, the detailed mechanism by which STAT6 acts as a transcriptional activator is far from clear. Mutagenesis studies suggested that the biological activity of the transcription factor requires an intact carboxyl terminus (22Lu B. Reichel M. Fisher D.A. Smith J.F. Rothman P. J. Immunol. 1997; 159: 1255-1264PubMed Google Scholar). Moreover, several STAT proteins are known to recruit coactivators possessing histone acetyltransferase (HAT) activity for the stimulation of gene expression (23Imhof A. Yang X.J. Ogryzko V.V. Nakatani Y. Wolffe A.P. Ge H. Curr. Biol. 1997; 7: 689-692Abstract Full Text Full Text PDF PubMed Scopus (535) Google Scholar, 24Kouzarides T. EMBO J. 2000; 19: 1176-1179Crossref PubMed Scopus (1007) Google Scholar). For instance, STAT6 was shown to interact with the CREB-binding protein (CBP) and an associated protein, named p300, which exhibit HAT activity (25McDonald C. Reich N.C. J. Interferon Cytokine Res. 1999; 19: 711-722Crossref PubMed Scopus (59) Google Scholar). This interaction required an intact carboxyl-terminal region of STAT6 (26Gingras S. Simard J. Groner B. Pfitzner E. Nucleic Acids Res. 1999; 27: 2722-2729Crossref PubMed Scopus (109) Google Scholar). In the present study, we have investigated the mechanism of IL-4-induced expression of the 15-LOX-1 gene in A549 cells and found that acetylation of histone proteins and STAT6 is required for transcriptional activation of this particular gene. From our data, it may be concluded that the acetylation of histones, which block STAT6 binding at the 15-LOX-1 promoter if they are present as nonacetylated proteins, enables promoter binding of phosphorylated and acetylated STAT6, which in turn may lead to transcriptional activation of the 15-LOX gene. A549 cells (Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Recombinant IL-4 (human) was purchased from Biomol Research Laboratory (Hamburg, Germany). Anti-STAT6 and anti-acetyl antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA), and anti-histone H3 and anti-acetylhistone H3 were from Upstate Biotechnology (Lake Placid, NY). Anti-15-LOX-1 antibody was purchased from Cayman Chemicals (Ann Arbor, MI). Genistein, cycloheximide, and sodium butyrate were supplied by Sigma. Immunoprecipitations were performed by incubating the nuclear extracts with 2 μg of the primary antibody for 1 h, and the immune complexes were bound to protein A-agarose. The beads were then washed three times with radioimmunoprecipitation assay buffer (1% Nonidet P-40, 0.5% deoxycholate, and 0.1% SDS dissolved in phosphate-buffered saline (PBS) along with phenylmethylsulfonyl fluoride, leupeptin, and pepstatin as protease inhibitors), and the immune complex was released by SDS sample buffer. After separation of proteins by SDS-polyacrylamide gel electrophoresis (PAGE), they were transferred onto Immobilon nitrocellulose membranes (Millipore) by semi-dry blotting. The membranes were then probed with various antibodies and developed using the ECL detection system (Amersham Pharmacia Biotech). For the detection of acetyl-STAT6 among abundantly present acetylhistone proteins a double immunoprecipitation strategy was used. The first immunoprecipitation was performed with anti-acetylhistone H3 antibody. Subsequently, proteins were extracted with elution buffer (1% SDS and 0.1 m NaHCO3) from the immunoprecipitate and subjected to a second immunoprecipitation in radioimmunoprecipitation assay buffer using anti-STAT6 antibody. The so obtained immunoprecipitate was electrophoresed, blotted on a nylon membrane, and probed with an antibody raised against acetylated proteins (Santa Cruz Biotechnology, Heidelberg, Germany). This anti-acetyl antibody apparently exhibits higher affinity for acetyl-STAT6 than for acetylhistone H3, giving a more intense band of acetyl-STAT6 than that of acetylhistone H3. Formaldehyde was added to the IL-4-treated cells at a final concentration of 1% and incubated for 20 min at room temperature. The reaction was stopped by the addition of glycine to a final concentration of 0.125 m. The cells were washed with cold PBS and harvested. The soluble chromatin was prepared according to the method of Dignam et al. (27Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9160) Google Scholar) and sonicated at maximal power for 30 s twice to shear the genomic DNA. Immunoprecipitations were performed with anti-histone, anti-acetylhistone, and anti-STAT6 antibodies. Cross-linking was reversed in the immunoprecipitate complexes by the addition of NaCl to a final concentration of 200 mm and incubation at 65 °C for 6 h. The DNA was purified by proteinase K treatment (150 μg/ml) for 1 h, followed by phenol-chloroform extraction and precipitation by ethanol. The polymerase chain reaction (PCR) analysis was performed for the presence of the 15-LOX-1 promoter using specific primers. The extract aliquoted before the immunoprecipitations was used to prepare control input genomic DNA, which was also used for PCR analysis. For Western blotting, protein was directly denatured by electrophoresis sample buffer and applied to SDS-PAGE. Double-stranded oligonucleotide containing the STAT6 binding element, present at −963 base pairs counted from the start of transcription of the human 15-LOX-1 gene, was used as probe in the gel shift assays. The assays were performed with nuclear extracts from IL-4-treated cells as described previously (25McDonald C. Reich N.C. J. Interferon Cytokine Res. 1999; 19: 711-722Crossref PubMed Scopus (59) Google Scholar). The reaction mixture was electrophoresed on 6% PAGE and visualized by autoradiography. Transfections were performed using Transfectase reagent (Life Technologies, Inc.). 1.5 μg of each mammalian expression plasmid containing wild-type E1A oncoprotein (wtE1A) and a mutant for the CBP binding domain (E1AmCBP), which were kind gifts from Dr. A. Hecht, Freiburg, Germany, were cotransfected together with 0.1 μg of control plasmid PRSVLACZ to normalize for transfection efficiency. A titration was performed with varying amounts of wtE1A plasmid to determine the quantity of DNA required for maximal transfection efficiency. A 1-kilobase fragment of the 15-LOX-1 promoter proximal to the coding sequence was amplified by PCR. 100 pmol of this DNA were end-labeled with biotin 16-dUTP. The end-labeled DNA was purified and bound to streptavidin-coated magnetic beads (Roche Molecular Biochemicals). The beads were washed with PBS and incubated with the nuclear protein extracts and 10 μg of poly(dI-dC), a nonspecific competitor of DNA, in electrophoretic mobility shift assay (EMSA) buffer for 1 h at 4 °C. The beads were washed three times with PBS, and proteins were eluted with buffer containing 2 m NaCl. The eluted protein was desalted and analyzed by SDS-PAGE. Silver staining was performed to visualize the proteins. Filter binding assays were performed as described (28Schreiber E. Matthias P. Muller M.M. Schaffner W. Nucleic Acids Res. 1989; 17: 6419-6423Crossref PubMed Scopus (3917) Google Scholar) with minor modifications. Core histones were isolated from acid-solubilized nuclear proteins after trichloroacetic acid-acetone precipitation. 3.3 mg/ml histones were acetylated in a reaction buffer containing 50 mm Tris-HCl, pH 8.0, 100 mm NaCl, 10% glycerol, and 1 mm proteinase inhibitor phenylmethylsulfonyl fluoride. [3H]Acetyl-CoA (Amersham Pharmacia Biotech) and 6 μg of protein extract for 30–60 min at 30 °C. The reaction mixture was spotted onto P81 phosphocellulose paper (Upstate Biotechnology) and washed for 30 min with 0.2 m carbonate buffer, pH 9.2. The filter paper was dried and used for liquid scintillation counting. Similar experiments were performed using nonradioactive acetyl-CoA. The reaction mixture was denatured and loaded onto SDS-PAGE. The Western blot was probed with anti-acetylhistone H3 antibodies and developed using the ECL detection system. A549 cells were cocultured in the presence of 670 pm of IL-4 for 24 h. The cells were trypsinized, washed, and resuspended in 500 μl of PBS. After addition of arachidonic acid (100 μm), the reaction was allowed to proceed at 37 °C for 20 min. Reduction of hydroperoxy fatty acids to their corresponding hydroxy derivatives was achieved by the addition of a molar excess of sodium borohydride. The reaction mixture was acidified to pH 3.0, and lipids were extracted with an equal volume of ethyl acetate. A defined amount of 13-hydroxyoctadecaenoic acid, which is absent in cells, was added as an internal standard before extraction. High performance liquid chromatography analysis was performed on a Supelco-SIL column (250 × 4.6 mm, 5 μm) using n-hexane/2-propanol/acetic acid (100:2:0.1 v/v/v) as a mobile phase at a flow rate of 1 ml/min. 15-Hydroxyeicosatetraenoic acid and 13-hydroxyoctadecaenoic acid were detected and quantified at 235 nm. Similar experiments were performed with A549 cells transfected with wtE1A and E1AmCBP oncoproteins. A549 cells were cultured for various periods in the presence of 670 pm IL-4. The cells were harvested, and the lysates were analyzed for the expression of 15-LOX-1 mRNA by reverse transcription (RT)-PCR using 15-LOX-1-specific primers. The highest mRNA concentration was detected after a 12-h incubation period (Fig. 1 A). After longer incubation periods, the mRNA levels dropped perceptibly. Similar kinetics were observed when the expression of the 15-LOX-1 protein was followed by Western blot analysis (data not shown). To find out whether IL-4 has to be present during the entire incubation period or whether a single cytokine stimulus may be sufficient to induce 15-LOX-1 expression, the following experiment was carried out. A549 cells were exposed to IL-4 for various periods, the cytokine was washed away, and incubation was resumed for a total of 24 h. Finally, the expression of 15-LOX-1 mRNA was analyzed by RT-PCR. As shown in Fig. 1 B, 15-LOX-1 expression in A549 cells required a minimum of 11 h of continuous exposure to IL-4. These data indicate that a single IL-4 stimulus is not sufficient to up-regulate expression of the 15-LOX-1 mRNA. Moreover, it was concluded that the IL-4-induced intracellular signal transduction cascade leading to 15-LOX-1 expression is a time-requiring process and may involve yet unidentified regulatory elements (21Heydeck D. Thomas L. Schnurr K. Trebus F. Thierfelder W.E. Ihle J.N. Kuhn H. Blood. 1998; 92: 2503-2510Crossref PubMed Google Scholar, 29Roy B. Cathcart M.K. J. Biol. Chem. 1998; 273: 32023-32029Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). To find out whether IL-4-induced 15-LOX-1 expression involves de novo synthesis of STAT6-dependent regulatory proteins, additional transcription factors, or both, experiments were carried out in the presence of cycloheximide, a protein synthesis inhibitor. As shown in Fig. 1 C, cycloheximide did not affect the activation of 15-LOX-1. Activation of cellular acetyltransferases may constitute an additional regulatory element in the intracellular signal transduction cascade (24Kouzarides T. EMBO J. 2000; 19: 1176-1179Crossref PubMed Scopus (1007) Google Scholar). Acetylation of histones causes conformational changes of nuclear proteins, leading to demasking of potential transcription factor binding sites, so that the transcription factor may bind to the promoter of target genes. Because histone acetylation has recently been implicated in the induction of 15-LOX-1 expression in CaCo-2 cells (31Kamitani H. Taniura S. Ikawa H. Watanabe T. Kelavkar U.P. Eling T.E. Carcinogenesis. 2001; 22: 187-191Crossref PubMed Scopus (46) Google Scholar), we investigated the effect of IL-4 on HAT activity in our cellular model. For this purpose, cells were exposed to IL-4 (670 pm final concentration) for 3 h, and the cell lysates were assayed for HAT activity. We found that IL-4 significantly up-regulated HAT activity even after a relatively short incubation periods (Fig. 2). The HAT activity is the sum of several catalytic processes and involves the activity of various proteins. One of these enzymes is the transactivating protein CBP/p300, which exhibits strong HAT activity. To find out whether CBP/p300 is involved in IL-4-induced up-regulation of acetyltransferase activity in A549 cells, we transfected the cells with the viral oncoprotein wtE1A, which has been identified as an endogenous inhibitor of CBP/p300. After IL-4 treatment, the transfected cells exhibited significantly reduced HAT activity (Fig. 2). When different amounts of cDNA were transfected, the suppression was found to be dose-dependent (Fig. 2 B). However, this suppression of IL-4-induced HAT activity could be reversed by transfecting the cells with E1AmCBP, a mutant of E1A protein incapable of binding to CBP/p300. These data indicate that the augmented acetyltransferase activity is mainly due to activation of CBP/p300. The acetylation degree of nuclear histones depends on cellular HAT activity but also on the activity state of histone deacetylases (HDACs). In resting cells, there appears to be a steady state of acetylating and deacetylating events and inhibitors, or activators of either process may shift the equilibrium in either direction. Recently, it has been reported that activation of HDAC may cause alterations in the chromatin state and may inhibit gene transcription (30Kao H.-Y. Downes M. Ordentlich P. Evans R.M. Genes Dev. 2000; 14: 55-66PubMed Google Scholar). If our working hypothesis (CBP/p300-catalyzed histone acetylation is important for IL-4 induced 15-LOX-1 expression) is true, inhibitors of cellular HDAC are likely to act synergistically to IL-4 or may even be capable of inducing 15-LOX-1 expression in the absence of IL-4. To test this conclusion, A549 cells were incubated with suboptimal doses (335 pm) of IL-4 in the presence of sodium butyrate, and the expression of 15-LOX was assayed by Western blotting. From Fig.3 it can be seen that IL-4 at suboptimal concentrations did not induce 15-LOX-1 expression. However, in the presence of sodium butyrate, a strong LOX signal was observed. Interestingly, sodium butyrate did also induce 15-LOX-1 expression in the absence of IL-4. Similar results have recently been reported for other cellular systems (31Kamitani H. Taniura S. Ikawa H. Watanabe T. Kelavkar U.P. Eling T.E. Carcinogenesis. 2001; 22: 187-191Crossref PubMed Scopus (46) Google Scholar). Because histone acetylation may be important for binding of transcription factors to the promoter of the 15-LOX-1 gene, we carried out transcription factor binding assays in vitro. A549 cells were incubated with IL-4 for different periods; then nuclear extracts were prepared, and binding studies of nuclear proteins to the 15-LOX-1 promoter were performed. In cells that were cultured in the absence of IL-4, we did not detect any promoter-binding proteins (Fig. 4 A, first lane). In contrast, a variety of 15-LOX-1 promoter-binding proteins were present in the nucleus of IL-4-treated cells (Fig.4 A, second and third lanes). As expected, STAT6 was one of the major components, and its identity was confirmed by Western blots using commercially available anti-STAT6 antibodies and by EMSA (data not shown). Interestingly, the pattern of the binding proteins was very similar when cells were treated with IL-4 for 1 or 12 h (Fig. 4 A, second and third lanes). These data indicate that under in vitro conditions, the transcription factors including STAT6 are capable of binding to the immobilized 15-LOX-1 promoter and that 1 h incubation is sufficient for maximal in vitro binding. Combining these data (rapid in vitro binding) with the results shown in Fig.1 (delayed 15-LOX-1 expression), one may conclude that in vivo the binding of phosphorylated STAT6 may be prevented. Alternatively, coactivators exhibiting a prolonged time dependence may be required for transcriptional regulation of the 15-LOX-1 gene. It has been reported for other cell types that tyrosine phosphorylation is involved in IL-4- and IL-13-induced 15-LOX-1 expression (29Roy B. Cathcart M.K. J. Biol. Chem. 1998; 273: 32023-32029Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Thus, we examined the effect of genistein, a potent tyrosine kinase inhibitor, on protein phosphorylation and on the binding activity of nuclear proteins to the 15-LOX-1 promoter. A549 cells were treated with genistein (25 μg/ml) for 30 min. After washing away the inhibitor, 670 pm IL-4 was added, and the cells were cultured for additional 12 h. Subsequently, the nuclear extracts were analyzed for the presence of 15-LOX-1 promoter-binding proteins. From Fig.4 A, fourth lane, it can be seen that genistein completely blocked the binding of proteins to the promoter. Surprisingly, genistein did also abrogate STAT6 acetylation (Fig. 4 B). These data suggests that tyrosine phosphorylation in A549 cells may be a prerequisite for STAT6 acetylation and STAT6 binding to the 15-LOX-1 promoter. It is well known that IL-4-induced intracellular signal transduction cascade bifurcates in various cellular systems and may initiate the Janus kinase/STAT6 pathway, the mitogen-activated protein kinase/protein kinase C route, or both (32Wery S. Letourneur M. Bertoglio J. Pierre J. J. Biol. Chem. 1996; 271: 8529-8532Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 33Chuang L.M. Tai T.Y. Kahn R.C. Wu H.P. Lee S.C. Lin B.J. J. Biochem. 1996; 120: 111-116Crossref PubMed Scopus (16) Google Scholar, 34Hirasawa N. Sato Y. Fujita Y. Ohuchi K. Biochim. Biophys. Acta. 2000; 1456: 45-55Crossref PubMed Scopus (40) Google Scholar). Because we found that the protein kinase C inhibitor bisindolylmaleamide and calphostin C failed to inhibit IL-4-induced 15-LOX-1 expression (data not shown), this pathway may not be relevant for the regulatory mechanism in A549 cells. These data confirm earlier observations in a different cellular model (35Wick K.R. Berton M.T. Mol. Immunol. 2000; 37: 641-652Crossref PubMed Scopus (41) Google Scholar). From Fig. 4 it was concluded that under in vitro conditions, phosphorylated STAT6 is capable of binding to the 15-LOX-1 promoter. The next series of experiments were aimed at addressing the question of whether such a binding may actually occurin vivo. For this purpose, A549 cells were cultured in the presence of IL-4 for various periods, and DNA-binding proteins were cross-linked to the nucleic acid by formaldehyde treatment; then STAT6 was immunoprecipitated with a specific antibody, and the cross-linked DNA was analyzed by PCR using 15-LOX-1 promoter-specific primers. We found that the earliest binding of STAT6 was detected after 11 h of IL-4 exposure (Fig. 5, upper row). These data were somewhat surprising, because both STAT6 phosphorylation and its in vitro binding were rapid processes. Thus, it was concluded that the binding of STAT6 to the 15-LOX-1 promoter in vivo was inhibited during early phases of the incubation period. Similar in vivo binding studies were performed using anti-histone H3 (Fig. 5, second row) and anti-acetylhistone H3 antibodies (Fig. 5, third row). Here we observed that nonacetylated histones are bound at the early phases of the induction process. In contrast, STAT6 and acetylated histones were bound exclusively at later stages. These data indicate an inverse correlation between the binding of nonacetylated histone and the activation of the 15-LOX-1 gene. At early time points when histones are bound to the promoter (Fig. 5), we did not observe any 15-LOX-1 expression (Fig. 1). In contrast, after long-term incubations (≥11 h), when we observed promoter binding of acetylated histones and STAT6, the 15-LOX-1 mRNA was also expressed. Immunoprecipitations were performed to obtain experimental evidence for a physical interaction between STAT6 and histones on IL-4 stimulation. To check for in vivo interaction, the cells were treated with 1% formaldehyde to cross-link the proteins, followed by immunoprecipitation in the protein extract, applying a dual immunoprecipitation strategy. After the first immunoprecipitation with the anti-acetylhistone H3 antibody, the protein" @default.
• W2039883000 created "2016-06-24" @default.
• W2039883000 creator A5029615335 @default.
• W2039883000 creator A5040140560 @default.
• W2039883000 creator A5064301762 @default.
• W2039883000 creator A5088541092 @default.
• W2039883000 date "2001-11-01" @default.
• W2039883000 modified "2023-10-07" @default.
• W2039883000 title "Acetylation by Histone Acetyltransferase CREB-binding Protein/p300 of STAT6 Is Required for Transcriptional Activation of the 15-Lipoxygenase-1 Gene" @default.
• W2039883000 cites W1505047300 @default.
• W2039883000 cites W1511815964 @default.
• W2039883000 cites W1549591697 @default.
• W2039883000 cites W1562310953 @default.
• W2039883000 cites W1818471554 @default.
• W2039883000 cites W1895481850 @default.
• W2039883000 cites W1923015860 @default.
• W2039883000 cites W1965199529 @default.
• W2039883000 cites W1976763304 @default.
• W2039883000 cites W1981661975 @default.
• W2039883000 cites W1993243592 @default.
• W2039883000 cites W1997785224 @default.
• W2039883000 cites W2005505618 @default.
• W2039883000 cites W2006384611 @default.
• W2039883000 cites W2008057379 @default.
• W2039883000 cites W2016730049 @default.
• W2039883000 cites W2019835417 @default.
• W2039883000 cites W2019884726 @default.
• W2039883000 cites W2028913495 @default.
• W2039883000 cites W2030571655 @default.
• W2039883000 cites W2038554263 @default.
• W2039883000 cites W2040256969 @default.
• W2039883000 cites W2050414776 @default.
• W2039883000 cites W2051152566 @default.
• W2039883000 cites W2054798367 @default.
• W2039883000 cites W2071980946 @default.
• W2039883000 cites W2083923544 @default.
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