Immunosuppressant PG490 (Triptolide) Inhibits T-cell Interleukin-2 Expression at the Level of Purine-box/Nuclear Factor of Activated T-cells and NF-κB Transcriptional Activation
1999; Elsevier BV; Volume: 274; Issue: 19 Linguagem: Inglês
10.1074/jbc.274.19.13443
ISSN1083-351X
AutoresDaoming Qiu, Guohua Zhao, Yosuke Aoki, Lingfang Shi, Anne Uyei, Saman Nazarian, James C.-H. Ng, Peter N. Kao,
Tópico(s)Signaling Pathways in Disease
ResumoPG490 (triptolide) is a diterpene triepoxide with potent immunosuppressive and antiinflammatory properties. PG490 inhibits interleukin(IL)-2 expression by normal human peripheral blood lymphocytes stimulated with phorbol 12-myristate 13-acetate (PMA) and antibody to CD3 (IC50 of 10 ng/ml), and with PMA and ionomycin (Iono, IC50 of 40 ng/ml). In Jurkat T-cells, PG490 inhibits PMA/Iono-stimulated IL-2 transcription. PG490 inhibits the induction of DNA binding activity at the purine-box/antigen receptor response element (ARRE)/nuclear factor of activated T-cells (NF-AT) target sequence but not at the NF-κB site. PG490 can completely inhibit transcriptional activation at the purine-box/ARRE/NF-AT and NF-κB target DNA sequences triggered by all stimuli examined (PMA, PMA/Iono, tumor necrosis factor-α). PG490 also inhibits PMA-stimulated activation of a chimeric transcription factor in which the C-terminal TA1 transactivation domain of NF-κB p65 is fused to the DNA binding domain of GAL4. In 16HBE human bronchial epithelial cells, IL-8 expression is regulated predominantly by NF-κB, and PG490 but not cyclosporin A can completely inhibit expression of IL-8. The mechanism of PG490 inhibition of cytokine gene expression differs from cyclosporin A and involves nuclear inhibition of transcriptional activation of NF-κB and the purine-box regulator operating at the ARRE/NF-AT site at a step after specific DNA binding. PG490 (triptolide) is a diterpene triepoxide with potent immunosuppressive and antiinflammatory properties. PG490 inhibits interleukin(IL)-2 expression by normal human peripheral blood lymphocytes stimulated with phorbol 12-myristate 13-acetate (PMA) and antibody to CD3 (IC50 of 10 ng/ml), and with PMA and ionomycin (Iono, IC50 of 40 ng/ml). In Jurkat T-cells, PG490 inhibits PMA/Iono-stimulated IL-2 transcription. PG490 inhibits the induction of DNA binding activity at the purine-box/antigen receptor response element (ARRE)/nuclear factor of activated T-cells (NF-AT) target sequence but not at the NF-κB site. PG490 can completely inhibit transcriptional activation at the purine-box/ARRE/NF-AT and NF-κB target DNA sequences triggered by all stimuli examined (PMA, PMA/Iono, tumor necrosis factor-α). PG490 also inhibits PMA-stimulated activation of a chimeric transcription factor in which the C-terminal TA1 transactivation domain of NF-κB p65 is fused to the DNA binding domain of GAL4. In 16HBE human bronchial epithelial cells, IL-8 expression is regulated predominantly by NF-κB, and PG490 but not cyclosporin A can completely inhibit expression of IL-8. The mechanism of PG490 inhibition of cytokine gene expression differs from cyclosporin A and involves nuclear inhibition of transcriptional activation of NF-κB and the purine-box regulator operating at the ARRE/NF-AT site at a step after specific DNA binding. Extracts of the Chinese herb Tripterygium Wilfordii hook have potent antiinflammatory and immunosuppressive properties and have been used successfully in traditional Chinese medicine for the treatment of rheumatoid arthritis (1Tao X.L. Sun Y. Dong Y. Xiao Y.L. Hu D.W. Shi Y.P. Zhu Q.L. Dai H. Zhang N.Z. Chin. Med. J. 1989; 102: 327-332PubMed Google Scholar). One active component of Tripterygium extracts is the diterpene triepoxide, triptolide, which possesses antileukemic activities (2Kupchan S.M. Court W.A. Dailey Jr., R. Gilmore C.J. Bryan R.F. J. Am. Chem. Soc. 1972; 94: 7194-7195Crossref PubMed Scopus (451) Google Scholar) and also inhibits proliferation of transformed cell lines (3Wei Y.S. Adachi I. Chung-Kuo Yao Li Hsueh Pao. 1991; 12: 406-410PubMed Google Scholar, 4Shamon L.A. Pezzuto J.M. Graves J.M. Mehta R.R. Wangcharoentrakul S. Sangsuwan R. Chaichana S. Tuchinda P. Cleason P. Reutrakul V. Cancer Lett. 1997; 112: 113-117Crossref PubMed Scopus (172) Google Scholar). Kupchan and Shubert (5Kupchan S.M. Schubert R.M. Science. 1974; 185: 791-793Crossref PubMed Scopus (114) Google Scholar) described that triptolide possesses a 9,11-epoxy-14β-hydroxy system, which is important for biologic activity, and proposed that this system may be involved in selective alkylation by nucleophilic groups such as thiols present in key target enzymes involved in growth regulation. A refined extract of Tripterygium, PG27, which contains PG490 (triptolide) as its active compound, prolongs heart and kidney allograft survival in rat transplantation models and, furthermore, displays synergy with the immunosuppressant cyclosporin A (CsA) 1The abbreviation used is: CsA, cyclosporin A; ARRE, antigen receptor response element; EMSA, electrophoretic mobility shift assay; GAPDH, glyceraldehyde phosphate dehydrogenase; IL, interleukin; Iono, ionomycin; NF-AT, nuclear factor of activated T-cells; TNF, tumor necrosis factor; PG490, Pharmagenesis 490, 97% pure triptolide; PMA, phorbol 12-myristate 13-acetate; PBL, peripheral blood lymphocytes; bp, base pair(s); RLU, relative light units; IFN, interferon; P/I, PMA + ionomycin; ELISA, enzyme-linked immunosorbent assay; NS, nonstimulated.1The abbreviation used is: CsA, cyclosporin A; ARRE, antigen receptor response element; EMSA, electrophoretic mobility shift assay; GAPDH, glyceraldehyde phosphate dehydrogenase; IL, interleukin; Iono, ionomycin; NF-AT, nuclear factor of activated T-cells; TNF, tumor necrosis factor; PG490, Pharmagenesis 490, 97% pure triptolide; PMA, phorbol 12-myristate 13-acetate; PBL, peripheral blood lymphocytes; bp, base pair(s); RLU, relative light units; IFN, interferon; P/I, PMA + ionomycin; ELISA, enzyme-linked immunosorbent assay; NS, nonstimulated. in preventing cardiac and renal allotransplant rejection. 2J. Fidler, Pharmagenesis, Palo Alto, CA, unpublished data.2J. Fidler, Pharmagenesis, Palo Alto, CA, unpublished data. The combination of PG27 with CsA substantially prolongs hamster cardiac xenograft survival in rat recipients and inhibits the production of serum anti-hamster IgM and IgG xenoantibodies where single drug therapies are ineffective.2 In addition, PG27 suppresses the development of graft versus host disease associated with allogeneic bone marrow transplantation. 3N. Chao, Duke University, and J. Fidler, Pharmagenesis, Palo Alto, CA, unpublished data.3N. Chao, Duke University, and J. Fidler, Pharmagenesis, Palo Alto, CA, unpublished data. The chloroform methanol extract of Triptyergium, T2 (6Zheng J.R. Fang J.L. Gu K.X. Xu L.F. Gao J.W. Guo H.Z. Yu Y.H. Sun H.Z. Chung-Kuo I Hsueh Ko Hsueh Yuan Hsueh Pao. 1987; 9: 317-322Google Scholar), has been studied recently (7Tao X. Davis L.S. Lipsky P.E. Arthritis Rheum. 1991; 34: 1274-1281Crossref PubMed Scopus (115) Google Scholar, 8Yang S.X. Xie S.S. Gao H.L. Ma D.L. Long Z.Z. Int. J. Immunopharmacol. 1994; 16: 895-904Crossref PubMed Scopus (32) Google Scholar, 9Tao X. Cai J.J. Lipsky P.E. J. Pharmacol. Exp. Ther. 1995; 272: 1305-1312PubMed Google Scholar) and was shown to block mitogen-induced early cytokine gene transcription in T-cells (10Tao X. Davis L.S. Hashimoto K. Lipsky P.E. J. Pharmacol. Exp. Ther. 1996; 276: 316-325PubMed Google Scholar). An early cytokine transcribed during T-cell activation is IL-2 (reviewed in Ref. 11Serfling E. Avots A. Neumann M. Biochim. Biophys. Acta. 1995; 1263: 181-200Crossref PubMed Scopus (200) Google Scholar). IL-2 transcription involves specific DNA binding and transcriptional activation of a purine-box transcriptional regulator operative at the antigen receptor response element (ARRE)/NF-AT target DNA sequence, and of NF-κB, AP-1, and Oct-1 (reviewed in Ref. 11Serfling E. Avots A. Neumann M. Biochim. Biophys. Acta. 1995; 1263: 181-200Crossref PubMed Scopus (200) Google Scholar). The T-cell immunosuppressants, CsA and FK506, inhibit transcription of the IL-2 gene through mechanisms that may involve the serine/threonine protein phosphatase, calcineurin (reviewed in Ref. 11Serfling E. Avots A. Neumann M. Biochim. Biophys. Acta. 1995; 1263: 181-200Crossref PubMed Scopus (200) Google Scholar). Cyclosporin A and FK506 interfere with the induction of sequence-specific DNA binding activity at the purine-box/ARRE/NF-AT target DNA sequence (reviewed in Ref. 11Serfling E. Avots A. Neumann M. Biochim. Biophys. Acta. 1995; 1263: 181-200Crossref PubMed Scopus (200) Google Scholar). We previously purified to homogeneity a CsA- and FK506-sensitive sequence-specific purine-box DNA binding complex that contains NF45 and NF90 proteins (12Corthesy B. Kao P.N. J. Biol. Chem. 1994; 269: 20682-20690Abstract Full Text PDF PubMed Google Scholar, 13Kao P.N. Chen L. Brock G. Ng J. Kenny J. Smith A.J. Corthesy B. J. Biol. Chem. 1994; 269: 20691-20699Abstract Full Text PDF PubMed Google Scholar). Recently, we showed that NF45 and NF90 associate tightly with the catalytic subunit of DNA-dependent protein kinase and serve to stabilize the association of the catalytic subunit of DNA-dependent protein kinase with DNA-targeting proteins, Ku80 and Ku70 (14Ting N.S.Y. Kao P.N. Chan D.W. Lintott L.G. Lees-Miller S.P. J. Biol. Chem. 1998; 273: 2136-2145Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). The catalytic subunit of DNA-dependent protein kinase and Ku have been shown to bind with sequence specificity to purine-rich target DNA sequences and to mediate sequence-specific transcriptional repression (15Giffin W. Torrance H. Rodda D.J. Prefontaine G.G. Pope L. Hache R.J. Nature. 1996; 380: 265-268Crossref PubMed Scopus (197) Google Scholar). We have recently shown that the specific CsA-sensitive purine-box/ARRE DNA binding complex in human bronchial epithelial cells involves NF45, NF90, Ku80, and Ku70 with no evidence for NF-ATp or NF-ATc proteins (16Aoki Y. Zhao G. Qiu D. Shi L. Kao P.N. Am. J. Physiol. 1998; 275: L1164-L1172PubMed Google Scholar). Activation of IL-2 transcription triggered through costimulatory receptors such as CD28 involves NF-κB (17Los M. Schenk H. Hexel K. Baeuerle P.A. Droge W. Schulze-Osthoff K. EMBO J. 1995; 14: 3731-3740Crossref PubMed Scopus (297) Google Scholar) and is largely resistant to inhibition by CsA (17Los M. Schenk H. Hexel K. Baeuerle P.A. Droge W. Schulze-Osthoff K. EMBO J. 1995; 14: 3731-3740Crossref PubMed Scopus (297) Google Scholar, 18June C.H. Ledbetter J.A. Gillespie M.M. Lindsten T. Thompson C.B. Mol. Cell. Biol. 1987; 7: 4472-4481Crossref PubMed Scopus (475) Google Scholar). The activation of transcription by NF-κB involves stimulation-induced degradation of IκB, which serves to release NF-κB p65 for translocation from the cytoplasm into the nucleus. In the nucleus, p65 binds as a heterodimer to its target DNA sequence and then interacts with transcriptional regulatory components to signal initiation of transcription by RNA polymerase II (reviewed in Ref. 19Baeuerle P.A. Henkel T. Annu. Rev. Immunol. 1994; 12: 141-179Crossref PubMed Scopus (4591) Google Scholar). NF-κB signaling is regulated by phosphorylation in the cytoplasm at the level of IκB in the nucleus at the level of specific DNA binding and also at the level of transcriptional activation (reviewed in Ref. 20Schmitz M.L. Baeuerle P.A. Immunobiology. 1995; 193: 116-127Crossref PubMed Scopus (58) Google Scholar). We investigated the immunosuppressive and antiinflammatory properties of PG490 (pure triptolide) in human peripheral blood lymphocytes, Jurkat T-cells, and human bronchial epithelial cells and show that the mechanism of inhibition by PG490 differs fundamentally from that of CsA and involves inhibition of transcriptional activation of the purine-box regulator of the ARRE/NF-AT site and of NF-κB at a step after specific binding to DNA. PG490 (triptolide, molecular weight 360) was obtained from Pharmagenesis (Palo Alto, CA). The material was composed of white to off-white crystals, had a melting point of 226–240 °C, conformed to standard triptolide preparation by proton nuclear magnetic resonance (2Kupchan S.M. Court W.A. Dailey Jr., R. Gilmore C.J. Bryan R.F. J. Am. Chem. Soc. 1972; 94: 7194-7195Crossref PubMed Scopus (451) Google Scholar), and was 97% pure by reverse phase high pressure liquid chromatography evaluation using acetonitrile:methanol:water (18:9:73). 4J. Fidler and R.-L. Jin, Pharmagenesis, private communication. Human peripheral blood lymphocytes (PBL) were prepared by centrifugation on a gradient of sodium diatrizoate/Ficoll (Sigma) of a buffy coat obtained from the Stanford Hospital blood bank. Monocytes were depleted by adherence to plastic culture dishes for 30 min at room temperature and then the PBLs were stimulated for 12 h at a density of 1 × 107cells/ml in RPMI 1640 supplemented with 10% fetal bovine serum. Jurkat T-cells (clone E6–1) were obtained from American Type Culture Collection (Manassas, VA), and cultured in RPMI 1640 (Mediatech, Herndon, VA) supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 mg/ml streptomycin (BioWhittaker, Walkersville, MD). An SV-40 large T-antigen transformed human bronchial epithelial cell line 16HBE14o- (16HBE), which retains differentiated morphology and function of normal human airway epithelia (21Cozens A.L. Yezzi M.J. Kunzelmann K. Ohrui T. Chin L. Eng K. Finkbeiner W.E. Widdicombe J.H. Gruenert D.C. Am. J. Resp. Crit. Care Med. 1994; 10: 38-47Google Scholar), was cultured in Eagle's minimum essential medium (BioWhittaker) supplemented with 10% heat-inactivated fetal bovine serum, 100 units/ml penicillin, and 100 mg/ml streptomycin (BioWhittaker) as described (22Aoki Y. Qiu D. Uyei A. Kao P.N. Am. J. Physiol. 1997; 272: L272-L284Google Scholar). Human PBLs, Jurkat T-cells, or monolayer 16HBE cells were stimulated for the indicated times in culture media containing 20 ng/ml phorbol 12-myristate 13-acetate (PMA; Calbiochem), PMA + antibody to CD3 (clone HIT3a, Pharmingen, San Diego, CA), PMA + antibody to CD28 (YTH913.12, BIOSOURCE, Camarillo, CA), PMA + 2 μm ionomycin (Iono; Calbiochem), or 20 ng/ml TNF-α (BIOSOURCE) in the presence of PG490, CsA (Sandoz), or FK506 (Fujisawa). Total RNA was isolated and analyzed by Northern hybridization as described (22Aoki Y. Qiu D. Uyei A. Kao P.N. Am. J. Physiol. 1997; 272: L272-L284Google Scholar). Complementary DNA probes for human IL-2 (738 bp). IL-8 (289 bp), IκBα (883 bp), NF90 (2,008 bp), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 0.6-kilobase XbaI-HindIII fragment of cDNA) were labeled with [α-32P]dCTP using a random hexamer labeling kit (Stratagene). Jurkat T-cells were stimulated for 3 h, pelleted, and washed, and cytosolic and nuclear extracts were prepared as described (12Corthesy B. Kao P.N. J. Biol. Chem. 1994; 269: 20682-20690Abstract Full Text PDF PubMed Google Scholar). Briefly, nuclear proteins were extracted from chromatin using 0.3m (NH4)2SO4, and then soluble nuclear proteins were precipitated using 1.5 m(NH4)2SO4, followed by dialysis into DNA binding buffer. Protein concentrations were determined by Bradford assay (Bio-Rad). Transcription factor DNA binding activities in Jurkat T-cell nuclear extracts were assayed using electrophoretic mobility shift assays (EMSA). 10 μg of nuclear proteins were incubated for 30 min at 25 °C in 20 μl of binding buffer (25 mm HEPES, pH 7.6, 0.1 mm EDTA, 10% glycerol, 50 mm KCl, 0.05 mm dithiothreitol) containing 1–2 μg of poly(dI-dC) and 2.5 pg of 32P-labeled oligonucleotide probe (approximately 1 × 105 cpm). The sequences of oligonucleotide probes used were agctAAAGAGGGACTTTCCCTAAA for the immunoglobulin κ light chain NF-κB site (19Baeuerle P.A. Henkel T. Annu. Rev. Immunol. 1994; 12: 141-179Crossref PubMed Scopus (4591) Google Scholar), and aagaAAGGAGGAAAAACTGTTTCATA (−259 to −284 in the human IL-2 enhancer) for the purine-box/NF-AT site (11Serfling E. Avots A. Neumann M. Biochim. Biophys. Acta. 1995; 1263: 181-200Crossref PubMed Scopus (200) Google Scholar). Probes were labeled by filling in overhanging ends (identified by lowercase letters) using Klenow DNA polymerase (New England Biolabs) and [α-32P]dCTP (Amersham Pharmacia Biotech), and nonradioactive dGTP, dATP, and dTTP. Protein-DNA complexes were resolved from free probe using 4% nondenaturing polyacrylamide gels in 0.5× Tris borate EDTA (pH 8.3) and visualized by fluorography. Luciferase reporter gene constructs were under the control of the IL-2 enhancer (nucleotides −326 to +48, pCLN15deltaCX, prepared by D. Durand, Stanford University), or three copies of the purine-box/NF-AT regulatory sequence sequence (−285 to −255 of the human IL-2 enhancer) in the context of the minimal IL-2 promoter, or the immunoglobulin κ light chain NF-κB sequence monomer in the context of the minimal IL-8 promoter (−45 to +40 of the human IL-8 promoter). These plasmids also contain a neomycin resistance gene under control of the constitutively active SV40 promoter, and this allows G418 antibiotic (Life Technologies, Inc.) selection of cell lines that stably express the luciferase reporter constructs. The GAL4-luciferase reporter contains five copies of the GAL4 target DNA sequence upstream of the minimal IL-2 promoter and was prepared by J. Riegel at Stanford University. For normalizing the transient transfection assays, plasmid pEF Renilla luciferase was generated by cloning the elongation factor 1α promoter (23Uetsuki T. Naito A. Nagata S. Kaziro Y. J. Biol. Chem. 1989; 264: 5791-5798Abstract Full Text PDF PubMed Google Scholar) into the pRL null vector (Promega) between theEcoRI and HindIII sites. Expression plasmids pEF CNA and pEF CNB contain the elongation factor 1α promoter (23Uetsuki T. Naito A. Nagata S. Kaziro Y. J. Biol. Chem. 1989; 264: 5791-5798Abstract Full Text PDF PubMed Google Scholar) upstream of rat calcineurin A and calcineurin B cDNAs (24Kincaid R.L. J. Allergy Clin. Immunol. 1995; 96: 1170-1177Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). Expression plasmids GAL4p65TA1 and GAL4p65{TA1+TA2} utilize the Rous sarcoma virus promoter to drive expression of a chimeric protein with the GAL4 DNA binding domain (amino acids 1–147) fused to the p65 transactivating domains and were generated by Schmitz and Baeuerle (25Schmitz M.L. Baeuerle P.A. EMBO J. 1991; 10: 3805-3817Crossref PubMed Scopus (664) Google Scholar) and obtained through A. Baldwin (University of North Carolina, Chapel Hill). Jurkat T-cells were transfected by electroporation with reporter and expression plasmids as described (13Kao P.N. Chen L. Brock G. Ng J. Kenny J. Smith A.J. Corthesy B. J. Biol. Chem. 1994; 269: 20691-20699Abstract Full Text PDF PubMed Google Scholar, 26Aoki Y. Kao P.N. Biochem. Biophys. Res. Commun. 1997; 234: 424-431Crossref PubMed Scopus (22) Google Scholar). T-cells were stimulated for 6–12 h (24 h after electroporation in the case of transient transfections) and then cells were washed in phosphate-buffered saline and pelleted by centrifugation. Cell pellets were resuspended in 50 μl of lysis buffer (1% Triton X-100, 0.1 mm HEPES, pH 7.6, 1 mm dithiothreitol, and 2 mm EDTA, pH 8.0) for 10 min at 4 °C, then the cell lysates were centrifuged at 13,000 rpm for 10 min. The supernatants were collected as whole cell extracts, and the Bradford reagent (Bio-Rad) was used to measure protein concentration. 20 μg of protein was mixed with 200 μl of luciferase reaction mixtures (1 mg/ml bovine serum albumin, 5 mm ATP, pH 7.6, 25 mm glycylglycine, and 15 mm MgSO4) and 100 μl of 1 mm d-luciferin (Analytical Luminescence Laboratory, San Diego, CA). Triplicate determinations of luminescence were each read for 20 s using a MonolightTM 2010 luminometer (Analytical Luminescence Laboratory), and were measured in relative light units (RLU). In transient transfection experiments that incorporated the pEF Renilla luciferase normalizing plasmid, 20-μl aliquots of whole cell extracts were analyzed sequentially for firefly and Renilla luciferase activities using a dual luciferase assay kit (Promega), and the ratio of firefly to Renilla RLU was taken to represent the normalized (firefly) luciferase activity. Cytosolic extracts (10 μg of protein) were fractionated by SDS-polyacrylamide gel electrophoresis (8% separating gel) and transferred to nitrocellulose membranes (Schleicher and Schuell). IκBα was detected using rabbit polyclonal IgG primary antibody (Santa Cruz Biotechnology) at 1:500 dilution for 2 h at 37 °C and horseradish peroxidase-conjugated goat anti-rabbit secondary antibody at 1:3,000 dilution for 1 h at room temperature. Detection was with enhanced chemiluminsescence, according to the manufacturer's directions (Amersham Pharmacia Biotech). Significance of the differences between the experimental conditions were determined by paired two sample Student's t test (Microsoft EXCEL). The data presented are the means ± S.D. PG490 potently inhibits IL-2 expression by human PBLs stimulated by PMA + anti-CD3 (PMA/αCD3, Fig. 1A, rows 5–8 versus row 4), and by PMA + ionomycin (Fig. 1A, rows 15–17 versus row 14). The IC50 for PG490 inhibition of PMA/αCD3-stimulated IL-2 expression is approximately 10 ng/ml (28 nm). PG490 at 200 ng/ml (560 nm) causes more complete inhibition of PMA/αCD3-stimulated IL-2 expression than CsA at 1,000 ng/ml (832 nm, Fig. 1A, row 7 versus row 11). Ionomycin stimulation alone causes minimal induction of IL-2 expression, which is inhibited by the lowest dose of 4 ng/ml PG490 (Fig. 1A, rows 12 and 13). The IC50 for PG490 inhibition of PMA/Iono-stimulated IL-2 expression is approximately 40 ng/ml (112 nm). Similar to the result in human PBLs, we show that PG490 potently inhibits Jurkat T-cell expression of IL-2 (Fig. 1B, panel a). Stimulation with PMA/Iono bypasses membrane signaling events of T-cell activation (27Weiss A. Imboden J.B. Adv. Immunol. 1987; 41: 1-38Crossref PubMed Scopus (225) Google Scholar) and strongly induces IL-2 protein and mRNA expression (Fig. 1B, panels a and b, lane 6). PG490 at 20 ng/ml (56 nm) causes over 80% inhibition of PMA/Iono-stimulated IL-2 protein and mRNA expression (Fig. 1B, panel a and b, lane 7 versus lane 6). PG490 at 200 ng/ml more completely suppresses Jurkat T-cell IL-2 protein and mRNA expression stimulated by PMA and by PMA/Iono than 1,000 ng/ml of CsA (Fig. 1B, panels a and b, lanes 3and 5 versus lane 2, and lanes 8 and 10 versus lane 6). Using Northern hybridization analysis, we show that PG490 has distinct effects on the mRNA expression of cytokine regulators IκBα and NF90 (Fig. 1B, panels c and d). We observe constitutive expression of IκBα mRNA (28Haskill S. Beg A.A. Tompkins S.M. Morris J.S. Yurochko A.D. Sampson-Johannes A. Mondal K. Ralph P. Baldwin Jr., A. Cell. 1991; 65: 1281-1289Abstract Full Text PDF PubMed Scopus (586) Google Scholar) (Fig. 1B, panel c, lane 1), and this expression is further stimulated by PMA and PMA/Iono (Fig. 1B, panel c, lanes 2 and 6). PG490 potently inhibits IκBα mRNA expression (Fig. 1B, panel c, lanes 3,4, and 7-9), whereas 1,000 ng/ml CsA shows only limited inhibition (Fig. 1B, panel c,lanes 5 and 10). In contrast, mRNA expression for NF90, a transcriptional regulator of IL-2 gene expression (13Kao P.N. Chen L. Brock G. Ng J. Kenny J. Smith A.J. Corthesy B. J. Biol. Chem. 1994; 269: 20691-20699Abstract Full Text PDF PubMed Google Scholar), is significantly induced in the presence of 200 and 1,000 ng/ml PG490 (Fig. 1B, panel d, lanes 3 and 4 versus lane 2 and lanes 8and 9 versus lane 6). Finally, the levels of GAPDH mRNA are unaltered in response to stimulation and drug treatment (Fig. 1B, panel e). Our observations that PG490 causes more complete inhibition of IL-2 expression than CsA and that PG490 but not CsA inhibits IκBα mRNA expression and enhances NF90 mRNA expression establish that PG490 modulates gene expression through distinctly different mechanisms than CsA. We next demonstrated that PG490 inhibits activation of an IL-2 luciferase reporter gene transfected transiently and stably into Jurkat T-cells (Fig. 2), and this result implies that PG490 inhibits IL-2 expression at the level of transcriptional activation of the IL-2 gene. We used the IL-2 luciferase assay to test the ability of PG490 to inhibit the CsA-resistant pathway of T-cell activation achieved by stimulation with PMA in combination with anti-CD28 monoclonal antibody (18June C.H. Ledbetter J.A. Gillespie M.M. Lindsten T. Thompson C.B. Mol. Cell. Biol. 1987; 7: 4472-4481Crossref PubMed Scopus (475) Google Scholar). We stimulated Jurkat T-cells that stably express the IL-2 luciferase reporter gene with either PMA + anti-CD28 antibody (PMA/αCD28), or with PMA/Iono, each in the presence of PG490 or CsA (Fig. 2A). For T-cells stimulated with PMA/Iono (Fig. 2A, left panel), the IC50 is approximately 20 ng/ml (56 nm) for PG490 and approximately 3 ng/ml (2.5 nm) for CsA inhibition of IL-2 transcription. In contrast, for T-cells stimulated with PMA/αCD28 (Fig. 2B, right panel), the IC50 is approximately 50 ng/ml for PG490 inhibition of IL-2 transcription, and CsA is completely ineffective in inhibiting this pathway of stimulation. At the two lower doses of PG490 but not of CsA, we observed modest increases in IL-2 luciferase activity before inhibition occurs at the higher doses. These results demonstrate that although CsA is more potent than PG490 in inhibiting PMA/Iono-stimulated IL-2 luciferase activity, PG490 is capable of inhibiting T-cell activation triggered through a costimulatory receptor, a situation in which CsA is ineffective. To explore further the mechanistic differences between PG490 and CsA and FK506 inhibition of IL-2 transcription, we tested the effects of calcineurin overexpression on sensitivity to inhibition by these immunosuppressant drugs (Fig. 2B) Overexpression of calcineurin in Jurkat T-cells confers relative resistance to the inhibitory effects of CsA and FK506 upon transcriptional activation of the IL-2 gene (29O'Keefe S.J. Tamura J. Kincaid R.L. Tocci M.J. O'Neill E.A. Nature. 1992; 357: 692-694Crossref PubMed Scopus (788) Google Scholar, 30Clipstone N.A. Crabtree G.R. Nature. 1992; 357: 695-697Crossref PubMed Scopus (1474) Google Scholar). We transiently cotransfected Jurkat T-cells with the IL-2 luciferase reporter plasmid together with either an empty expression vector or with expression vectors encoding calcineurin A and B subunits (Fig. 2B). The transfected T-cells were stimulated with PMA/Iono in the presence of increasing doses of PG490 (Fig. 2B, left panel), CsA (Fig. 2B,center panel), or FK506 (Fig. 2B, right panel). There is no shift in the dose-inhibition curve of IL-2 transcription by PG490 conferred by overexpression of calcineurin (Fig. 2B, left panel). In contrast, overexpression of calcineurin shifts the IC50 for CsA inhibition of IL-2 transcription from approximately 2 to 10 ng/ml (Fig. 2B,center panel), and shifts the IC50 for FK506 inhibition of IL-2 transcription from approximately 0.2 to 1 ng/ml (Fig. 2B, right panel). These results demonstrate that PG490 inhibits IL-2 transcription through mechanisms distinct from CsA and FK506, and which probably do not involve calcineurin. After demonstrating that PG490 inhibits IL-2 expression at the level of transcription, we next investigated the effects of PG490 on the induction of DNA binding activity and transcriptional activation of specific factors controlling the purine-box/ARRE/NF-AT and NF-κB target sequences in the IL-2 enhancer (reviewed in Ref.11Serfling E. Avots A. Neumann M. Biochim. Biophys. Acta. 1995; 1263: 181-200Crossref PubMed Scopus (200) Google Scholar). We used EMSA to analyze the effects of PG490 on the PMA/Iono-induced Jurkat T-cell purine-box/ARRE/NF-AT DNA binding activity (Fig. 3A). PG490 inhibits the induction of the purine-box/ARRE·EMSA complex with an IC50 slightly below 200 ng/ml (Fig. 3A,lane 4 versus lane 2). At 1,000 ng/ml PG490, the PMA/Iono-induced purine-box/ARRE·EMSA complex is undetectable (Fig. 3A, lane 5) and is therefore weaker than the EMSA complex constitutively present in nonstimulated cells (Fig. 3A, lane 1), and weaker than the complex induced in the presence of 1,000 ng/ml CsA (Fig. 3A,lane 6). PG490 is more potent in inhibiting PMA/Iono-stimulated transcriptional activation of a purine-box/NF-AT luciferase reporter gene with an IC50 of approximately 20 ng/ml (Fig. 3B, lane 3 versus lane 2). Taken together, these results demonstrate that PG490 inhibits both transcriptional activation and also DNA binding of the regulator operating at the purine-box/ARRE/NF-AT target sequence and that the predominant site of signaling inhibition is at the level of transcription after specific binding to DNA. We investigated the effects of PG490 on NF-κB signaling in Jurkat T-cells (Fig. 4). We show constitutive expression of the NF-κB p65 anchoring protein, IκBα, in the cytoplasm of nonstimulated Jurkat T-cells (Fig. 4A,lane 1), and IκBα expression decreases following stimulation with PMA (Fig. 4A, lane 2 versus lane 1). PG490 at 200 and 1,000 ng/ml causes nearly complete inhibition of IκBα protein expression in PMA-stimulated T-cells (Fig. 4A, lanes 4 and 5), and this result correlates with PG490 inhibition of IκBα mRNA expression (Fig. 1B, panel c, lanes 3and 4). At the level of NF-κB DNA binding, stimulation of Jurkat T-cells with PMA induces the appearance of a new band (NF-κB complex, Fig. 4B, lanes 2–6). The presence of the NF-κB p65 subunit within this complex is established by the quantitative supershift of the inducible complex with an antibody to p65 (Fig. 4B, lanes 7–12). PG490 at 20 ng/ml causes no significant effect on the NF-κB·EMSA complex (Fig. 4B,lane 3 versus lane 2), and PG490 at 200 and 1,000 ng/ml causes a significant increase in the strength of the NF-κB complex (Fig. 4B, lanes 4 and 5 versus lanes 2 and 10,lane 11 versus lane 8). We propose that the increase in NF-κB DNA binding activity which we observe at 200 and 1,000 ng/ml PG490 occurs as a consequence of PG490 inhibition of IκBα expression, which likely allows increased nuclear translocation of p65. Despite the development of a strong NF-κB DNA binding complex in the nucleus of T-cells s
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