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• W2022374442 abstract "Tumor necrosis factor a (TNF-α) is a potent proinflammatory cytokine and plays a crucial role in early events of inflammation. TNF-α is primarily produced by monocytes and T lymphocytes. In particular, T-cell-derived TNF-α plays a critical role in autoimmune inflammation and superantigen-induced septic shock. However, little is known about the intracellular signaling pathways that regulate TNF expression in T cells. Here we show that extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38-mitogen-activated protein kinase (MAPK) pathways control the transcription and synthesis of TNF-α in A3.01 T cells that produce the cytokine upon T cell activation by costimulation with 12-O-tetradecanoylphorbol-13-acetate (TPA) and ionomycin. Selective activation of each of the distinct MAPK pathways by expression of constitutively active kinases is sufficient for TNF-α promoter induction. Furthermore, blockage of all three pathways almost abolishes TPA/ionomycin-induced transcriptional activation of the TNF-α promoter. Selective inhibition of one or more MAPK pathways impairs TNF-α induction by TPA/ionomycin, indicating a cooperation between these signal transduction pathways. Our approach revealed that the MAPK kinase 6 (MKK6)/p38 pathway is involved in both transcriptional and posttranscriptional regulation of TNF expression. Moreover, analysis of the progressive 5′ deletion mutants of the TNF-α promoter indicates that distinct promoter regions are targeted by either ERK-, JNK-, or p38-activating pathways. Thus, unlike what has been reported for other TNF-α-producing cells, all three MAPK pathways are critical and cooperate to regulate transcription of theTNF-α gene in T lymphocytes, suggesting a T-cell-specific regulation of the cytokine. Tumor necrosis factor a (TNF-α) is a potent proinflammatory cytokine and plays a crucial role in early events of inflammation. TNF-α is primarily produced by monocytes and T lymphocytes. In particular, T-cell-derived TNF-α plays a critical role in autoimmune inflammation and superantigen-induced septic shock. However, little is known about the intracellular signaling pathways that regulate TNF expression in T cells. Here we show that extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38-mitogen-activated protein kinase (MAPK) pathways control the transcription and synthesis of TNF-α in A3.01 T cells that produce the cytokine upon T cell activation by costimulation with 12-O-tetradecanoylphorbol-13-acetate (TPA) and ionomycin. Selective activation of each of the distinct MAPK pathways by expression of constitutively active kinases is sufficient for TNF-α promoter induction. Furthermore, blockage of all three pathways almost abolishes TPA/ionomycin-induced transcriptional activation of the TNF-α promoter. Selective inhibition of one or more MAPK pathways impairs TNF-α induction by TPA/ionomycin, indicating a cooperation between these signal transduction pathways. Our approach revealed that the MAPK kinase 6 (MKK6)/p38 pathway is involved in both transcriptional and posttranscriptional regulation of TNF expression. Moreover, analysis of the progressive 5′ deletion mutants of the TNF-α promoter indicates that distinct promoter regions are targeted by either ERK-, JNK-, or p38-activating pathways. Thus, unlike what has been reported for other TNF-α-producing cells, all three MAPK pathways are critical and cooperate to regulate transcription of theTNF-α gene in T lymphocytes, suggesting a T-cell-specific regulation of the cytokine. Tumor necrosis factor α (TNF-α) 1The abbreviations used are: TNF, tumor necrosis factor; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; SAPK, stress-activated protein kinase; JNK, c-Jun N-terminal kinase; MEK, MAPK/ERK kinase; MKK6, MAPK kinase 6; MLK3, mixed-lineage kinase 3; TPA, 12-O-tetradecanoylphorbol-13-acetate; CsA, cyclosporin A; JIP-1, JNK-interacting protein 1; CRE, cAMP responsive element; LPS, lipopolysaccharide; HA, hemagglutinin; GST, glutathioneS-transferase; MBP, myelin basic protein. is primarily produced by cells of hematopoietic origin, such as lymphocytes, monocytes, and mast cells. T lymphocytes produce the cytokine when they are activated via their antigen receptor, and cells of the monocyte/macrophage lineage generate it upon lipopolysaccharide (LPS) stimulation (1Pauli U. Crit. Rev. Eukaryotic Gene Expression. 1994; 4: 323-344Crossref PubMed Scopus (38) Google Scholar). Mast cells also secrete TNF-α after high-affinity IgE receptor aggregation (2Zhang C. Baumgartner R.A. Yamada K. Beaven M.A. J. Biol. Chem. 1997; 272: 13397-13402Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). TNF-α is among the earliest activated cytokines in inflammation, and its production is crucial for the development of an early defense against many pathogens (reviewed in Ref. 1Pauli U. Crit. Rev. Eukaryotic Gene Expression. 1994; 4: 323-344Crossref PubMed Scopus (38) Google Scholar). However, these beneficial effects of TNF-α are dependent on the strength and duration of its expression. High systemic levels of TNF-α induced by stimulation of T cells with bacterial superantigen (3Miethke T. Wahl C. Heeg K. Echtenacher B. Krammer P.H. Wagner H. J. Exp. Med. 1992; 175: 91-98Crossref PubMed Scopus (515) Google Scholar) or by LPS stimulation of macrophages (1Pauli U. Crit. Rev. Eukaryotic Gene Expression. 1994; 4: 323-344Crossref PubMed Scopus (38) Google Scholar, 4Jongeneel C.V. Prog. Clin. Biol. Res. 1994; 388: 367-381PubMed Google Scholar) cause subsequent septic shock. Furthermore, the critical role of TNF-α in the generation of autoimmune inflammation has been defined by the targeted disruption of the TNF gene (5Körner H. Riminton D.S. Strickland D.H. Lemckert T.A. Pollard J.D. Sedgwick J.D. J. Exp. Med. 1997; 186: 1585-1590Crossref PubMed Scopus (203) Google Scholar). In addition to autoimmune diseases (6Taupin V. Renno T. Bourbonniere L. Peterson A.C. Rodriguez M. Owens T. Eur. J. Immunol. 1997; 27: 905-913Crossref PubMed Scopus (130) Google Scholar) and superantigen-induced septic shock (7Kramer B. Machleidt T. Wiegmann K. Kronke M. J. Inflamm. 1995; 45: 183-192PubMed Google Scholar), the pivotal role of T-cell-produced TNF-α in the modulation of inflammatory responses is further reflected in the observation that T-cell membrane-bound TNF-α is of particular importance for the regulation of monocytic interleukin 10 and TNF-α production (8Parry S.L. Sebbag M. Feldmann M. Brennan F.M. J. Immunol. 1997; 158: 3673-3681PubMed Google Scholar). The diverse stimuli that up-regulate TNF-α expression in different cells are known to be activators of MAPK-activating signaling pathways. Indeed, in monocytes, macrophages, and mast cells, it was shown that MAPK activation plays a central role in the induced TNF-α expression (2Zhang C. Baumgartner R.A. Yamada K. Beaven M.A. J. Biol. Chem. 1997; 272: 13397-13402Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 9Ishizuka T. Terada N. Gerwins P. Hamelmann E. Oshiba A. Fanger G.R. Johnson G.L. Gelfand E.W. Proc. Natl. Acad. Sci. U. S. A. 1997; 10: 6358-6363Crossref Scopus (131) Google Scholar, 10Lee J.C. Laydon J.T. McDonnell P.C. Gallagher T.F. Kumar S. Green D. McNulty D. Blumenthal M.J. Heys J.R. Landvatter S.W. Strickler J.E. McLaughlin M.M. Siemens I.R. Fisher S.M. Livi G.P. White J.R. Adams J.L. Young P.R. Nature. 1994; 372: 739-746Crossref PubMed Scopus (3147) Google Scholar, 11Geppert T.D. Whitehurst C.E. Thompson P. Beutler B. Mol. Med. 1994; 1: 93-103Crossref PubMed Google Scholar, 12Swantek J.L. Cobb M.H. Geppert T.D. Mol. Cell. Biol. 1997; 17: 6274-6282Crossref PubMed Google Scholar), whereas little is known about the regulation of this cytokine in T lymphocytes. The family of MAPKs consists of at least three subgroups: (a) the extracellular signal-regulated kinase (ERK), (b) the Jun N-terminal kinase, which is also known as stress-activated protein kinase (JNK/SAPK), and (c) the p38 subgroup of MAPKs (for a review, see Ref. 13Robinson M.J. Cobb M.H. Curr. Opin. Cell Biol. 1997; 9: 180-186Crossref PubMed Scopus (2286) Google Scholar). The human homolog of p38 designated CSBP has been identified as the binding protein of the pyridinyl-imidazole compound SB203580 that was shown to have an inhibitory effect on LPS-stimulated TNF-α production by human monocytes (10Lee J.C. Laydon J.T. McDonnell P.C. Gallagher T.F. Kumar S. Green D. McNulty D. Blumenthal M.J. Heys J.R. Landvatter S.W. Strickler J.E. McLaughlin M.M. Siemens I.R. Fisher S.M. Livi G.P. White J.R. Adams J.L. Young P.R. Nature. 1994; 372: 739-746Crossref PubMed Scopus (3147) Google Scholar). JNK and p38 differ from ERK in that they are predominantly regulated by cellular stress inducers and proinflammatory cytokines (14Kyriakis J.M. Banerjee P. Nikolakaki E. Dai T. Rubie E.A. Ahmad M.F. Avruch J. Woodgett J.R. Nature. 1994; 369: 156-160Crossref PubMed Scopus (2415) Google Scholar, 15Raingeaud J. Gupta S. Rogers J.S. Dickens M. Han J. Ulevitch R.J. Davis R.J. J. Biol. Chem. 1995; 270: 7420-7426Abstract Full Text Full Text PDF PubMed Scopus (2046) Google Scholar). Whereas in T cells, T cell receptor (TCR) ligation or TPA treatment is sufficient to maximally induce ERK activity, JNK and p38 activation requires a costimulatory signal such as CD28 ligand binding or ionomycin cotreatment, respectively (16Dickens M. Rogers J.S. Cavanagh J. Raintano A. Xia Z. Halpern J.R. Greenberg M.E. Sawyers C.L. Davis R.J. Science. 1997; 277: 693-696Crossref PubMed Scopus (629) Google Scholar, 17Su B. Jacinto E. Hibi M. Kallunki T. Karin M. Ben-Neriah Y. Cell. 1994; 77: 727-736Abstract Full Text PDF PubMed Scopus (849) Google Scholar, 18Hoffmeyer A. Avots A. Flory E. Weber C.K. Serfling E. Rapp U.R. J. Biol. Chem. 1998; 273: 10112-10119Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). MAPKs are activated by other kinases functioning in a kinase cascade. The direct upstream kinase of ERK is MEK, which is regulated via phosphorylation by Raf (19Daum G. Eisenmann-Tappe I. Fries H.-W. Troppmair J. Rapp U.R. Trends Biochem. Sci. 1994; 19: 474-480Abstract Full Text PDF PubMed Scopus (488) Google Scholar). JNK is activated by SAPK/ERK kinase (SEK, also known as MKK4) as well as by the recently identified kinase MKK7 (20Fanger G.R. Gerwins P. Widmann C. Jarpe M.B. Johnson G.L. Curr. Biol. 1997; 7: 67-74Abstract Full Text Full Text PDF Google Scholar, 21Holland P.M. Suzanne M. Campbell J.S. Noselli S. Cooper J.A. J. Biol. Chem. 1997; 272: 24994-24998Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 22Tournier C. Whitmarsh A.J. Cavanagh J. Barrett T. Davis R.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7337-7342Crossref PubMed Scopus (342) Google Scholar). The activation of JNK is further controlled by a putative scaffold protein, JNK-interacting protein 1 (JIP-1), which binds to JNK and several other components of the JNK pathway (23Whitmarsh A.J. Cavanagh J. Tournier C. Yasuda J. Davis R.J. Science. 1998; 281: 1671-1674Crossref PubMed Scopus (589) Google Scholar). Overexpression of JIP-1 or the JNK-binding domain of JIP-1 leads to the cytoplasmic retention of JNK and the inhibition of JNK-dependent gene expression (16Dickens M. Rogers J.S. Cavanagh J. Raintano A. Xia Z. Halpern J.R. Greenberg M.E. Sawyers C.L. Davis R.J. Science. 1997; 277: 693-696Crossref PubMed Scopus (629) Google Scholar). One of the SAPK/ERK kinase activators (reviewed by Fanger et al., Ref. 20Fanger G.R. Gerwins P. Widmann C. Jarpe M.B. Johnson G.L. Curr. Biol. 1997; 7: 67-74Abstract Full Text Full Text PDF Google Scholar) is the mixed lineage kinase 3 (MLK3) also known as the SH3 domain-containing proline-rich kinase (SPRK) (24Rana A. Gallo K. Godowski P. Hirai S. Ohno S. Zon L. Kyriakis J.M. Avruch J. J. Biol. Chem. 1996; 271: 19025-19028Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). MAPK kinase 6 (MKK6) functions as an activating kinase for all known p38 isoforms (25Han J. Lee J.D. Jiang Y. Li Z. Feng L. Ulevitch R.J. J. Biol. Chem. 1996; 271: 2886-2891Crossref PubMed Scopus (482) Google Scholar, 26Raingeaud J. Whitmarsh A.J. Barrett T. Derijard B. Davis R.J. Mol. Cell. Biol. 1996; 16: 1247-1255Crossref PubMed Scopus (1150) Google Scholar, 27Enslen H. Raingeaud J. Davis R.J. J. Biol. Chem. 1998; 273: 1741-1748Abstract Full Text Full Text PDF PubMed Scopus (476) Google Scholar), whereas MKK3 predominantly activates the isoform p38δ (28Wang X.S. Diener K. Manthey C.L. Wang S. Rosenzweig B. Bray J. Delaney J. Cole C.N. Chan-Hui P.Y. Mantlo N. Lichenstein H.S. Zukowski M. Yao Z. J. Biol. Chem. 1997; 272: 23668-23674Crossref PubMed Scopus (295) Google Scholar). Until this time, a specific physiological activator of MKK6 has not been identified. Whereas MAPK-activating pathways have been implicated in LPS-induced TNF-α expression by monocytes and macrophages at diverse control levels (10Lee J.C. Laydon J.T. McDonnell P.C. Gallagher T.F. Kumar S. Green D. McNulty D. Blumenthal M.J. Heys J.R. Landvatter S.W. Strickler J.E. McLaughlin M.M. Siemens I.R. Fisher S.M. Livi G.P. White J.R. Adams J.L. Young P.R. Nature. 1994; 372: 739-746Crossref PubMed Scopus (3147) Google Scholar, 11Geppert T.D. Whitehurst C.E. Thompson P. Beutler B. Mol. Med. 1994; 1: 93-103Crossref PubMed Google Scholar, 12Swantek J.L. Cobb M.H. Geppert T.D. Mol. Cell. Biol. 1997; 17: 6274-6282Crossref PubMed Google Scholar), and two reports show that the JNK and ERK pathways play a role in TNF-α expression by mast cells (2Zhang C. Baumgartner R.A. Yamada K. Beaven M.A. J. Biol. Chem. 1997; 272: 13397-13402Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 9Ishizuka T. Terada N. Gerwins P. Hamelmann E. Oshiba A. Fanger G.R. Johnson G.L. Gelfand E.W. Proc. Natl. Acad. Sci. U. S. A. 1997; 10: 6358-6363Crossref Scopus (131) Google Scholar), the contribution of MAPKs to the regulation of TNF-α expression in T lymphocytes is still unclear. Two findings suggest a cell type-specific involvement of intracellular signaling pathways inducing TNF-α expression: (a) in lymphocytes versus monocytes, different sets of transcription factors are recruited to the promoter of the TNF-α gene (29Zagariya A. Mungre S. Lovis R. Birrer M. Ness S. Thimmapaya B. Pope R. Mol. Cell. Biol. 1998; 18: 2815-2824Crossref PubMed Google Scholar, 30Tsai E.Y. Yie J. Thanos D. Goldfeld A.E. Mol. Cell. Biol. 1996; 16: 5232-5244Crossref PubMed Scopus (178) Google Scholar), and (b) extracellular stimuli with a cell type-specific function trigger TNF-α expression in different cells, such as LPS in monocytes, FcεRI receptor aggregation in mast cells, or activation of the antigenic receptor in T lymphocytes. Antigenic activation of T lymphocytes, which can be mimicked by costimulation with a phorbol ester such as TPA and a calcium ionophore such as ionomycin (for a review, see Ref. 31Rao A. Crit. Rev. Immunol. 1991; 10: 495-519PubMed Google Scholar), leads to a rapid induction of TNF-α transcription that does not require new protein biosynthesis (32Goldfeld A.E. McCaffrey P.G. Strominger J.L. Rao A. J. Exp. Med. 1993; 178: 1365-1379Crossref PubMed Scopus (191) Google Scholar). To investigate T-cell-specific regulation of the TNF-α gene, we analyzed the involvement of distinct MAPK pathways in TNF-α transcription and biosynthesis upon activation of the human T-cell line A3.01. We demonstrate that ERK, JNK, and p38 pathways that are activated upon stimulation with TPA and ionomycin (TPA/ionomycin) are critical for and cooperatively contribute to the induction of TNF-α expression in these T cells. A3.01 human T lymphoma cells were grown in RPMI 1640 medium supplemented with 10% fetal calf serum to a density of 8 × 105 cells/ml. Cells were incubated at 37 °C in humidified air with 7% CO2. Antibodies raised against ERK2 (sc-154), JNK1 (sc-474), and p38 (sc-535) were purchased from Santa Cruz Biotechnology, Inc. The monoclonal antibodies against the HA tag (12CA5) were produced and purified according to a standard protocol. The TNF-α monoclonal antibody was purchased from PharMingen, Inc. The human TNF-α promoter (−1057/+131; a generous gift of Dr. S. A. Nedospasov, Laboratory of Molecular Immunoregulation, PRI/DynCorp; National Cancer Institute, Frederick Cancer Research and Development Center, MD) was cloned into the HindIII site of pGL3 basic luciferase expression vector (Promega). The diverse 5′ deletion mutants of the TNF-α promoter were produced by restriction digestion or PCR amplification of regions of the human TNF-α promoter from nucleotide +131 relative to the transcriptional start site to various deletion end points described in Fig. 6. The promoter fragments were cloned into the pGL3 basic luciferase expression vector, and successful cloning was confirmed by sequencing. The eukaryotic expression vector for HA-SAPKβ and the prokaryotic expression vector pGEX-KG-c-Jun(1–135) were gifts from J. Kyriakis and L. Zon. The pRSPA vector system was used for the expression of all cDNAs in eukaryotic cells. pRSPA is an expression vector with the Rous sarcoma virus promoter and the simian virus 40 polyadenylation signal region in a pBluescript backbone (33Dorn P.L. DaSilva L. Matarano L. Derse D. J. Virol. 1990; 64: 1616-1624Crossref PubMed Google Scholar). The cDNA of MLK3 and the corresponding kinase inactive mutant were kindly provided by K. Gallo and P. Godowski (34Gallo K.A. Mark M.R. Scadden D.T. Wang Z. Gu Q. Godowski P.J. J. Biol. Chem. 1994; 269: 15092-15100Abstract Full Text PDF PubMed Google Scholar). Raf-BXB-CX (constitutively active Raf) lacks the N-terminal negative regulatory domain and contains the C-terminal membrane targeting 17 amino acids of Ki-Ras fused to the kinase domain of c-Raf l (18Hoffmeyer A. Avots A. Flory E. Weber C.K. Serfling E. Rapp U.R. J. Biol. Chem. 1998; 273: 10112-10119Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 35Flory E. Weber C.K. Chen P. Hoffmeyer A. Jassoy C. Rapp U.R. J. Virol. 1998; 72: 2788-2794Crossref PubMed Google Scholar). MKK6(EE) is a constitutively active mutant of MKK6 with two serines involved in the activation of the kinase replaced by glutamic acid (26Raingeaud J. Whitmarsh A.J. Barrett T. Derijard B. Davis R.J. Mol. Cell. Biol. 1996; 16: 1247-1255Crossref PubMed Scopus (1150) Google Scholar). The interfering mutants of ERK2, SAPKβ, MKK6, Raf-BXB-CX, and MLK3 are ATP-binding site mutants generated by the replacement of lysine with arginine (ERK2(B3), SAPKβ(K-R), and Raf-BXB-CX375) or alanine (MKK6(A) and MLK3 K144A) (26Raingeaud J. Whitmarsh A.J. Barrett T. Derijard B. Davis R.J. Mol. Cell. Biol. 1996; 16: 1247-1255Crossref PubMed Scopus (1150) Google Scholar, 34Gallo K.A. Mark M.R. Scadden D.T. Wang Z. Gu Q. Godowski P.J. J. Biol. Chem. 1994; 269: 15092-15100Abstract Full Text PDF PubMed Google Scholar, 36Ludwig S. Engel K. Hoffmeyer A. Sithanandam G. Neufeld B. Palm D. Gaestel M. Rapp U.R. Mol. Cell. Biol. 1996; 16: 6687-6697Crossref PubMed Scopus (154) Google Scholar, 37Robbins D.J. Zhen E. Owaki H. Vanderbilt C.A. Ebert D. Geppert T.D. Cobb M.H. J. Biol. Chem. 1993; 268: 5097-5106Abstract Full Text PDF PubMed Google Scholar). JIP-1 is a cytoplasmic protein that was identified as a putative scaffold protein that binds to several components of the JNK pathway and regulates JNK activity (23Whitmarsh A.J. Cavanagh J. Tournier C. Yasuda J. Davis R.J. Science. 1998; 281: 1671-1674Crossref PubMed Scopus (589) Google Scholar). Overexpression of JIP-1 or the JNK-binding domain of JIP-1 inhibits JNK activity by causing cytoplasmic retention of JNK that leads to the subsequent inhibition of JNK-regulated gene expression (16Dickens M. Rogers J.S. Cavanagh J. Raintano A. Xia Z. Halpern J.R. Greenberg M.E. Sawyers C.L. Davis R.J. Science. 1997; 277: 693-696Crossref PubMed Scopus (629) Google Scholar). The JIP-1 cDNA used in this study consists of the JNK-binding domain fused to a Flag-Tag and was kindly provided by R. Davies. All cDNAs were subcloned in the pRSPA vector. Cells were split to a density of 4 × 105 cells/ml 1 day before transfection. A DMRIETM-C based transfection protocol was used according to the manufacturer's instructions (Life Technologies). Cells were seeded in 6-well plates (7 × 105cells/well) in 1.5 ml of Opti-MEM (Life Technologies) containing 3 μl of DMRIETM and up to 3 μg of vector DNA. Transfections for luciferase assays were performed with 0.5 μg of reporter construct plus 2 μg of pRSPA containing diverse cDNAs. Unless otherwise indicated, cells in each well were harvested in 100 μl of lysis buffer (50 mmNa-2-(N-morpholino)ethanesulfonic acid, pH 7.8, 50 mm Tris-HCl, pH 7.8, 10 mm dithiothreitol, and 2% Triton X-100) 24 h after transfection. The crude cell lysates were cleared by centrifugation, and 50 μl of precleared cell extracts were added to 50 μl of luciferase assay buffer (125 mmNa-2-(N-morpholino)ethanesulfonic acid, pH 7.8, 125 mm Tris-HCl, pH 7.8, 25 mm magnesium acetate, and 2 mg/ml ATP). Immediately after the injection of 50 μl of 1 mmd-luciferin (AppliChem) into each sample, the luminescence was measured for 5 s in a luminometer (Berthold). The luciferase activities were normalized on the basis of protein content as well as on the β-galactosidase activity of cotransfected Rous sarcoma virus LTR β-gal vector. The β-galactosidase assay was performed with 20 μl of precleared cell lysate according to a standard protocol (38Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, New York1989Google Scholar). Mean and standard deviations of at least three independent experiments are shown in the figures. A3.01 T cells were stimulated with 10 ng/ml TPA (Sigma) or 0.5 μm ionomycin (Sigma) for up to 24 h. The MEK-specific inhibitor PD098059 (Calbiochem) was used in a 20 μm concentration of a 20 mm stock solution in DMSO. The p38-specific inhibitor SB203580 (Calbiochem) was used at a concentration of 2–20 μm of a 20 mm stock solution in DMSO. Actinomycin D (Sigma) was used at a concentration of 2 μg/ml of a 0.4 mg/ml stock solution in 10% ethanol, and cyclosporin A (CsA) (Sigma) was used at a concentration of 200 ng/ml of a 10 mg/ml stock solution in DMSO. Cells were preincubated with these inhibitors 30 min before stimulation. To determine the expression of TNF-α, an intracellular immunostaining procedure and subsequent flow cytometry analysis were applied. A3.01 T cells were split 24 h before stimulation. The stimulation was carried out in the presence of 2 mm monensin (Sigma), which prohibits the secretion of proteins, thereby leading to intracellular retention of the produced protein. Treatment of monensin did not affect the basal or induced MAPK activity (data not shown). After a stimulation time of 2 or 10 h, cells were harvested, washed once in phosphate-buffered saline, fixed with 4% (w/v) paraformaldehyde in phosphate-buffered saline at 4 °C for 20 min, and subject to the incubation and washing steps described below in permeabilization buffer containing 1% fetal calf serum and 0.1% (w/v) saponin in phosphate-buffered saline. According to the manufacturer's instructions (PharMingen), cells were then incubated with the primary antibody in permeabilization buffer supplemented with 2% goat serum. A mouse IgG1 antiserum (Dako) was used as an isotype-specific control for the monoclonal mouse anti-human TNF-α antibody of isotype IgG1 (PharMingen). After two washing steps, cells were exposed to biotin-SP-conjugated goat anti-mouse IgG F(ab′)2 (Dianova), washed again, and stained with streptavidin-Cy-chrome (PharMingen). Fluorescence was measured on 10,000 cells/sample using a FACScan (Beckton Dickinson). Cells were lysed in radioimmunoprecipitation buffer (25 mmTris-HCl, pH 8, 137 mm NaCl, 10% glycerol, 0.1% SDS, 0.5% deoxycholate, 1% Nonidet P-40, 2 mm EDTA, 1 mm Pefabloc, 1 mm sodium orthovanadate, 5 mm benzamidine, 5 μg/ml aprotinin, and 5 μg/ml leupeptin), and cell debris was removed by centrifugation. Supernatants were incubated with different antisera for 2 h at 4 °C. The immunocomplexes were precipitated with protein A-agarose (Boehringer) and washed twice with high-salt radioimmunoprecipitation buffer containing 500 mm NaCl. Immunocomplexes were used forin vitro kinase assays as described previously (36Ludwig S. Engel K. Hoffmeyer A. Sithanandam G. Neufeld B. Palm D. Gaestel M. Rapp U.R. Mol. Cell. Biol. 1996; 16: 6687-6697Crossref PubMed Scopus (154) Google Scholar) with myelin basic protein (MBP), 3pK(K-M), and glutathioneS-transferase (GST)-c-Jun(1–135) as substrates for ERK, p38, and JNK, respectively. Proteins were separated by SDS-polyacrylamide gel electrophoresis, blotted onto polyvinylidene difluoride membranes, and detected with a BAS 2000 Bio Imaging Analyzer (Fuji) and by autoradiography. The appropriate primary antibodies and peroxidase-coupled protein A were used for detection of the immunoprecipitated proteins in immunoblots, followed by a standard enhanced chemiluminescence reaction (Amersham). To investigate the role of the ERK, JNK, or p38 signaling pathways as mediators of induced TNF-α transcription, we performed transient cotransfection experiments and measured the human TNF-α promoter activity as promoter-dependent luciferase expression in the human T-cell line A3.01. Previously, we have established an approach to selectively activate ERK, JNK, or p38 in A3.01 T cells by expressing constitutively active versions of corresponding upstream kinases (18Hoffmeyer A. Avots A. Flory E. Weber C.K. Serfling E. Rapp U.R. J. Biol. Chem. 1998; 273: 10112-10119Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Briefly, a constitutively active kinase mutant of Raf (Raf-BXB-CX) serves as a specific ERK activator. Overexpression of MLK3 results in a strong activation of JNK without affecting ERK and p38 activities. Finally, an active mutant of MKK6 (MKK6(EE)) is a specific activator of p38 (18Hoffmeyer A. Avots A. Flory E. Weber C.K. Serfling E. Rapp U.R. J. Biol. Chem. 1998; 273: 10112-10119Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Expression of each of these kinases in A3.01 cells is sufficient to induce strong TNF-α promoter activity in a concentration-dependent manner (Fig.1 A–C, see also Fig. 6), although they exert no effect on a nonspecific thymidine kinase minimal promoter (data not shown). The corresponding catalytically inactive kinase versions showed no significant effect on the TNF-α promoter, even at the highest input (Fig. 1, A–C). Moreover, combining MKK6(EE) with either Raf-BXB-CX or MLK3 synergistically enhanced the promoter activity (Fig. 1 D), suggesting cooperation between these signaling pathways in the regulation of TNF-α-specific transcription. We next investigated the role of MAPK pathways in TNF-α gene expression of T cells activated by TPA/ionomycin in more detail. To characterize the regulation of TNF-α expression, we stimulated A3.01 T cells with TPA, ionomycin, or a combination of both. The inducibility of TNF-α expression was determined at both the translational and transcriptional levels by TNF-α-specific flow cytometry analysis and a TNF-α promoter reporter gene assay, respectively. Reporter gene analysis allows for the assessment of TNF-α promoter activity independent of TNF-α mRNA stability, another control level of expression. Unstimulated A3.01 cells do not produce any detectable amount of TNF-α (Fig. 5 A). Stimulation of these cells with TPA results in a weak induction of TNF-α transcription (Fig.2 B) and synthesis (Fig.2 A). A high induction of both transcription and protein synthesis was observed by cotreatment with TPA and ionomycin (Fig. 2). TPA/ionomycin-induced TNF-α production is sensitive to cyclosporin A, whereas TPA-induced protein synthesis is unaffected (Fig. 2 A). TNF-α protein synthesis was detectable as early as 2 h after TPA/ionomycin stimulation only in the absence of the transcriptional inhibitor actinomycin D (Fig. 2 A), indicating that TPA/ionomycin-induced TNF-α synthesis requiresde novo transcription of the TNF-α gene.Figure 2Expression of TNF-α by A3.01 T cells. A, to measure TNF-α production upon stimulation, A3.01 T cells were stained intracellularly with an anti-TNF-α monoclonal antibody and analyzed by fluorescence-activated cell-sorting analysis (see “Experimental Procedures”). The cells were stimulated with TPA (T) or TPA/ionomycin (T+I) or left untreated (w/o) for the times indicated. Some of the cells were pretreated with CsA or the transcriptional inhibitor actinomycin D. The mean of the TNF-α specific fluorescence intensity of stimulated cells is based on that of unstimulated cells and is given in fold stimulation. The experiment was repeated three times. B, to analyze the induction of TNF-α promoter-dependent transcription, the promoter construct TP(−1057) was cotransfected with a Rous sarcoma virus LTR-driven β-galactosidase expression vector. Cells were stimulated with TPA (T), ionomycin (I), or both (T+I) for 16 h or left untreated (w/o). The relative luciferase activity equalized on β-galactosidase activity is given in fold stimulation based on untreated cells (w/o). The figure shows a mean of three independent transfections and is representative of four independent experiments performed in triplicates.View Large Image Figure ViewerDownload (PPT) Therefore, we analyzed the time dependence of TNF-α transcription compared with TNF-α production. TNF-α promoter activity as well as protein synthesis is induced after 2 h of TPA/ionomycin stimulation and reaches maximal induction levels after 6 and 8 h, respectively (Fig. 3, A andB). These results indicate that the regulation of TNF-α expression in A3.01 cells is similar to that" @default.
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• W2022374442 title "Different Mitogen-activated Protein Kinase Signaling Pathways Cooperate to Regulate Tumor Necrosis Factor α Gene Expression in T Lymphocytes" @default.
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