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• W2032695847 abstract "Interleukin-5 (IL-5), expressed primarily by type-2 T helper (Th2) cells, plays an important role in the development of allergic diseases, such as allergic asthma. Studying the regulation of IL-5 gene expression by Ets transcription factors, we found that Ets1 and Ets2, but not Elf-1, were able to activate the human IL-5 promoter in Jurkat T-cells. This required the presence of either phorbol 12-myristate acetate (PMA) plus ionomycin or PMA plus the viral protein HTLV-I Tax1. By mutation studies, it could be shown that Ets1 and Ets2 exerted their effects on the IL-5 promoter through a GGAA motif within the Cle0 element. In myeloid Kasumi cells, Ets1 and Ets2 failed to stimulate IL-5 promoter activity, unless the T-cell specific transcription factor GATA3 was added. These results show, for the first time, that Ets1 and Ets2 are able to cooperate with GATA3. Both ionomycin and Tax1 increased the combined effect of GATA3 with Ets1 and Ets2 in the presence of PMA. The data further demonstrate that, in addition to Ets1, Ets2 is also able to functionally cooperate with Tax1. The synergism of GATA3 with either Ets1 or Ets2 may play an important role in calcium- or Tax1-dependent regulation of IL-5 expression in Th2 cells or in HTLV-I transformed adult T-cell leukemia cells, respectively. Interleukin-5 (IL-5), expressed primarily by type-2 T helper (Th2) cells, plays an important role in the development of allergic diseases, such as allergic asthma. Studying the regulation of IL-5 gene expression by Ets transcription factors, we found that Ets1 and Ets2, but not Elf-1, were able to activate the human IL-5 promoter in Jurkat T-cells. This required the presence of either phorbol 12-myristate acetate (PMA) plus ionomycin or PMA plus the viral protein HTLV-I Tax1. By mutation studies, it could be shown that Ets1 and Ets2 exerted their effects on the IL-5 promoter through a GGAA motif within the Cle0 element. In myeloid Kasumi cells, Ets1 and Ets2 failed to stimulate IL-5 promoter activity, unless the T-cell specific transcription factor GATA3 was added. These results show, for the first time, that Ets1 and Ets2 are able to cooperate with GATA3. Both ionomycin and Tax1 increased the combined effect of GATA3 with Ets1 and Ets2 in the presence of PMA. The data further demonstrate that, in addition to Ets1, Ets2 is also able to functionally cooperate with Tax1. The synergism of GATA3 with either Ets1 or Ets2 may play an important role in calcium- or Tax1-dependent regulation of IL-5 expression in Th2 cells or in HTLV-I transformed adult T-cell leukemia cells, respectively. The cytokine interleukin-5 (IL-5) 1The abbreviations used are: IL-5, interleukin-5; Th2 cells, type-2 T-helper cells; Cle0, conserved lymphokine element 0; PMA, phorbol 12-myristate 13-acetate; EMSA, electromobility shift assay; HTLV-I, human lymphotropic leukemia virus I; CHAPS, 3-[(3-cholamidopropyl) dimethyl ammonio]-propanesulfonate1The abbreviations used are: IL-5, interleukin-5; Th2 cells, type-2 T-helper cells; Cle0, conserved lymphokine element 0; PMA, phorbol 12-myristate 13-acetate; EMSA, electromobility shift assay; HTLV-I, human lymphotropic leukemia virus I; CHAPS, 3-[(3-cholamidopropyl) dimethyl ammonio]-propanesulfonateactivates eosinophiles (1Lopez A.F. Sanderson C.J. Gamble J.R. Campbell H.D. Young I.G. Vadas M.A. J. Exp. Med. 1988; 167: 219-224Crossref PubMed Scopus (808) Google Scholar, 2Yamaguchi Y. Hayashi Y. Sugama Y. Miura Y. Kasahara T. Kitamura S. Torisu M. Mita S. Tominaga A. Takatsu K. J. Exp. Med. 1988; 167: 1737-1742Crossref PubMed Scopus (564) Google Scholar) and basophiles (3Bischoff S.C. Brunner T. De Weck A.L. Dahinden C.A. J. Exp. Med. 1990; 172: 1577-1582Crossref PubMed Scopus (187) Google Scholar, 4Hirai K. Yamaguchi M. Misaki Y. Takaishi T. Ohta K. Morita Y. Ito K. Miyamoto T. J. Exp. Med. 1990; 172: 1525-1528Crossref PubMed Scopus (81) Google Scholar) and seems to be responsible for the development of eosinophilia associated with a number of diseases, including asthma and HTLV-I transformed adult T-cell leukemia (5Murata K. Yamada Y. Kamihira S. Atogami S. Tsukasaki K. Momita S. Amagasaki T. Sadamori N. Tomonaga M. Kinoshita K. Ichimaru M. Cancer. 1992; 69: 966-971Crossref PubMed Scopus (55) Google Scholar, 6Noma T. Nakakubo H. Sugita M. Kumagai S. Maeda M. Shimizu A. Honjo T. J. Exp. Med. 1989; 169: 1853-1858Crossref PubMed Scopus (64) Google Scholar, 7Sanderson C.J. Blood. 1992; 79: 3101-3109Crossref PubMed Google Scholar, 8Hogan S.P. Koskinen A. Matthaei K.I. Young I.G. Foster P.S. Am. J. Respir. Crit. Care Med. 1998; 157: 210-218Crossref PubMed Scopus (175) Google Scholar, 9Foster P.S. Hogan S.P. Matthaei K.I. Young I.G. Mem. Inst. Oswaldo Cruz. 1997; 92: 55-61Crossref PubMed Scopus (11) Google Scholar). Primarily, IL-5 is synthesized by Th2 cells, a subset of T helper cells, following stimulation with antigens or mitogens (10Warren D.J. Sanderson C.J. Immunology. 1985; 54: 615-623PubMed Google Scholar, 11Bohjanen P.R. Okajima M. Hodes R.J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5283-5287Crossref PubMed Scopus (80) Google Scholar). Regulation of IL-5 expression is likely to involve calcium and protein kinase C-dependent signaling pathways, as a calcium ionophore and phorbol ester synergistically increase IL-5 expression and IL-5 promoter activity (12Tominaga A. Matsumoto M. Harada N. Takahashi T. Kikuchi Y. Takatsu K. J. Immunol. 1988; 140: 1175-1181PubMed Google Scholar, 13Gruart-Gouilleux V. Engels P. Sullivan M. Eur. J. Immunol. 1995; 25: 1431-1435Crossref PubMed Scopus (45) Google Scholar). Activation of Th2 cells is accompanied by increased activities of phorbol ester responsive AP1 and calcium responsive NFAT as well as of GATA3 (14Rincon M. Flavell R.A. Curr. Biol. 1997; 7: R729-R732Abstract Full Text Full Text PDF PubMed Google Scholar). All three transcription factors have been found to be able to activate the IL-5 promoter (15Zhang M. Maass N. Magit D. Sager R. Cell Growth Differ. 1997; 8: 179-186PubMed Google Scholar, 16Yamagata T. Nishida J. Sakai R. Tanaka T. Honda H. Hirano N. Mano H. Yazaki Y. Hirai H. Mol. Cell. Biol. 1995; 15: 3830-3839Crossref PubMed Scopus (80) Google Scholar, 17Yamagata T. Mitani K. Ueno H. Kanda Y. Yazaki Y. Hirai H. Mol. Cell. Biol. 1997; 17: 4272-4281Crossref PubMed Scopus (46) Google Scholar, 18Stranick K.S. Zambas D.N. Uss A.S. Egan R.W. Billah M.M. Umland S.P. J. Biol. Chem. 1997; 272: 16453-16465Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), suggesting that they may play an important role in controlling IL-5 expression. In retrovirally transformed adult T-cell leukemia cells, the viral protein Tax1 may contribute to the production of IL-5 (17Yamagata T. Mitani K. Ueno H. Kanda Y. Yazaki Y. Hirai H. Mol. Cell. Biol. 1997; 17: 4272-4281Crossref PubMed Scopus (46) Google Scholar). Tax1 has been shown to deregulate a variety of cellular promoters (for a review, see Ref. 19Gitlin S.D. Dittmer J. Reid R.L. Brady J.N. Cullen B.R. Human Retroviruses. Oxford University Press, New York1993: 159-192Google Scholar) by interacting with various transcription factors, such as CREB, NF-κB, SRF, NF-Y, and Ets1 (20Fujii M. Tsuchiya H. Chuhjo T. Akizawa T. Seiki M. Genes Dev. 1992; 6: 2066-2076Crossref PubMed Scopus (217) Google Scholar, 21Rousset R. Desbois C. Bantignies F. Jalinot P. Nature. 1996; 381: 328-331Crossref PubMed Scopus (126) Google Scholar, 22Sun S.C. Elwood J. Beraud C. Greene W.C. Mol. Cell. Biol. 1994; 14: 7377-7384Crossref PubMed Google Scholar, 23Hirai H. Suzuki T. Fujisawa J. Inoue J. Yoshida M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3584-3588Crossref PubMed Scopus (150) Google Scholar, 24Kanno T. Brown K. Franzoso G. Siebenlist U. Mol. Cell. Biol. 1994; 14: 6443-6451Crossref PubMed Google Scholar, 25Kwok R.P. Laurance M.E. Lundblad J.R. Goldman P.S. Shih H. Connor L.M. Marriott S.J. Goodman R.H. Nature. 1996; 380: 642-646Crossref PubMed Scopus (308) Google Scholar, 26Ruben S.M. Perkins A. Rosen C.A. New Biol. 1989; 1: 275-284PubMed Google Scholar, 27Yin M.J. Gaynor R.B. Mol. Cell. Biol. 1996; 16: 3156-3168Crossref PubMed Scopus (83) Google Scholar, 28Pise-Masison C.A. Dittmer J. Clemens K.E. Brady J.N. Mol. Cell. Biol. 1997; 17: 1236-1243Crossref PubMed Scopus (32) Google Scholar, 29Dittmer J. Pise-Masison C.A. Clemens K.E. Choi K.S. Brady J.N. J. Biol. Chem. 1997; 272: 4953-4958Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), and with basal factors, such as TBP, TFIIA, and TAFII 28 (30Caron C. Rousset R. Beraud C. Moncollin V. Egly J.M. Jalinot P. EMBO J. 1993; 12: 4269-4278Crossref PubMed Scopus (101) Google Scholar, 31Caron C. Mengus G. Dubrowskaya V. Roisin A. Davidson I. Jalinot P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3662-3667Crossref PubMed Scopus (34) Google Scholar, 32Clemens K.E. Piras G. Radonovich M.F. Choi K.S. Duvall J.F. DeJong J. Roeder R. Brady J.N. Mol. Cell. Biol. 1996; 16: 4656-4664Crossref PubMed Scopus (47) Google Scholar). Ets proteins are transcription factors that share a unique DNA-binding domain, the Ets domain, allowing these proteins to interact specifically with GGA(A/T)-based recognition sites (for a review, see Ref. 33Ghysdael J. Boureux A. Yaniv M. Ghysdael J. Oncogenes as Transcriptional Regulators. 1. Birkhaeuser Verlag, Basel1997: 29-88Google Scholar). Ets transcription factors have been found to play a crucial role in controlling transcription of a variety of genes involved in important cellular processes, such as proliferation or differentiation. They have also been shown to contribute to the development of certain human diseases (for review, see Ref. 34Dittmer J. Nordheim A. Biochim. Biophys. Acta. 1998; 1377: F1-F11PubMed Google Scholar). Some Ets proteins, such as Ets1, Elf-1, and Fli-1, are primarily expressed in T-cells where they fulfill important functions, e.g. Ets1 has been reported to play a crucial role in T-cell survival (35Bories J.C. Willerford D.M. Grevin D. Davidson L. Camus A. Martin P. Stehelin D. Alt F.W. Nature. 1995; 377: 635-638Crossref PubMed Scopus (289) Google Scholar, 36Muthusamy N. Barton K. Leiden J.M. Nature. 1995; 377: 639-642Crossref PubMed Scopus (286) Google Scholar). The IL-5 promoter shares with other cytokine promoters, such as the granulocyte macrophage-colony stimulating factor promoter, the so-called conserved lymphokine element (Cle0) (37Wang C.Y. Bassuk A.G. Boise L.H. Thompson C.B. Bravo R. Leiden J.M. Mol. Cell. Biol. 1994; 14: 1153-1159Crossref PubMed Scopus (132) Google Scholar). It is a composite AP1/Ets element, which supports activation of the granulocyte macrophage-colony stimulating factor promoter by Ets1 in an AP1/NF-κB dependent manner (37Wang C.Y. Bassuk A.G. Boise L.H. Thompson C.B. Bravo R. Leiden J.M. Mol. Cell. Biol. 1994; 14: 1153-1159Crossref PubMed Scopus (132) Google Scholar, 38Thomas R.S. Tymms M.J. Seth A. Shannon M.F. Kola I. Oncogene. 1995; 11: 2135-2143PubMed Google Scholar, 39Thomas R.S. Tymms M.J. McKinlay L.H. Shannon M.F. Seth A. Kola I. Oncogene. 1997; 14: 2845-2855Crossref PubMed Scopus (136) Google Scholar). This raises the question of whether Ets proteins may also be able to regulate the IL-5 promoter. In order to address this possibility, we performed transient transfection studies with Jurkat T-cells and the myeloid Kasumi cell line. We found that Ets1 and Ets2, but not Elf-1, were able to activate the IL-5 promoter in Jurkat cells in the presence of either PMA plus ionomycin or PMA plus Tax1. Using Kasumi cells, we could show that Ets1 and Ets2 needed to cooperate with GATA3 for their stimulatory activities on the IL-5 promoter. These synergisms were enhanced in the presence of ionomycin or HTLV-I Tax1. The data suggest an important role for Ets proteins in the regulation of the IL-5 expression in Th2 lymphocytes and HTLV-I transformed leukemic cells. Jurkat T-cells and myeloid Kasumi cells were maintained in RPMI medium supplemented with 10% fetal calf serum in the absence of any antibiotic. Plasmids pKCR3-c-ets1, ΔEBets1 Δ243–330, pCTax, pCM5-Tax, and pCM7-Tax for expression of p54ets1, p46ets1, WT-Tax1, M5-Tax1, or M7-Tax1, respectively, are described elsewhere (40Pognonec P. Boulukos K.E. Ghysdael J. Oncogene. 1989; 4: 691-697PubMed Google Scholar, 41Gegonne A. Punyammalee B. Rabault B. Bosselut R. Seneca S. Crabeel M. Ghysdael J. New Biol. 1992; 4: 512-519PubMed Google Scholar, 42Rimsky L. Hauber J. Dukovich M. Malim M.H. Langlois A. Cullen B.R. Greene W.C. Nature. 1988; 335: 738-740Crossref PubMed Scopus (132) Google Scholar, 43Smith M.R. Greene W.C. Genes Dev. 1990; 4: 1875-1885Crossref PubMed Scopus (347) Google Scholar). The plasmids ΔEBets2 and ΔEBelf-1 for expression of Ets2 or Elf-1, respectively, were generous gifts from Jacques Ghysdael. The plasmid RSV/hG3 for expression of human GATA3 was kindly provided by James D. Engel. For construction of the −494/+44 IL-5 promoter/luciferase plasmid (pIL5P.luc), the IL-5 promoter fragment was first amplified by polymerase chain reaction using the primers as described previously (13Gruart-Gouilleux V. Engels P. Sullivan M. Eur. J. Immunol. 1995; 25: 1431-1435Crossref PubMed Scopus (45) Google Scholar), followed by cloning this fragment into a luciferase-containing vector (44Annweiler A. Muller-Immergluck M. Wirth T. Mol. Cell. Biol. 1992; 12: 3107-3116Crossref PubMed Scopus (59) Google Scholar). For generation of the deletion mutant −106/+44 IL-5 promoter, the internal HindIII/BsmI fragment, containing the IL-5 promoter sequence from −494 to −82, was replaced by an oligonucleotide bearing the IL-5 specific sequence from −106 to −82. For creating the G to A mutation at position −44 of the antisense strand, we polymerase chain reaction amplified an approximately 500-base pair long fragment containing the IL-5 promoter fragment from −81 to +44 and part of the luciferase gene by using the following primers: 5′-GGCATTCTCTATCTGATTGTTAGAAATTATTCATTTCTTCAAAGACAG-3′ (the mutated nucleotide is underlined) and 5′-AATTGAAGAGAGTTTTCACTGCATACGACGATTCTGTGATTTGTATTC-3′. The fragment was cloned into the polymerase chain reaction 2.1 vector (Invitrogen) and, after cutting with BsmI andXbaI, inserted into the −106/+44 IL-5 luciferase construct creating the −106/+44 EM IL-5 promoter luciferase plasmid. Jurkat and Kasumi cells were transfected with 5 μg of an IL-5 luciferase construct by electroporation using a Bio-Rad gene pulser under conditions, as described previously (45Dittmer J. Gitlin S.D. Reid R.L. Brady J.N. J. Virol. 1993; 67: 6087-6095Crossref PubMed Google Scholar). For expression of Ets1, Ets2, or Elf-1, 6 μg of the corresponding expression plasmid was added to the transfection mixture, while for expression of Tax1 or GATA3, 4 μg of pCTax or 2 μg of RSV/hG3, respectively, was used. One hour after transfection either PMA (final concentration: 10 ng/ml) and/or ionomycin (final concentration: 2 μm), both dissolved in dimethyl sulfoxide, or dimethyl sulfoxide (control) was added to the cells. After another 6 h cells were harvested. Cell lysates were assayed for luciferase activity according to Ref. 46Janknecht R. Ernst W.H. Houthaeve T. Nordheim A. Eur. J. Biochem. 1993; 216: 469-475Crossref PubMed Scopus (17) Google Scholar. Nuclear extracts from Jurkat cells were prepared essentially as described (28Pise-Masison C.A. Dittmer J. Clemens K.E. Brady J.N. Mol. Cell. Biol. 1997; 17: 1236-1243Crossref PubMed Scopus (32) Google Scholar). Briefly, cells were harvested and washed in phosphate-buffered saline. After resuspension in 1 ml of buffer A (10 mm Hepes, pH 7.9, 10 mm KCl, 0.1 mm EDTA, 0.1 mm EGTA, 1 mm dithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride) and incubation on ice for 15 min, cells were lysed by addition of 120 μl of Nonidet P-40 followed by vortexing for 10 s. The nuclei were pelleted by centrifugation at 13,000 rpm for 30 s at room temperature and extracted by addition of buffer C (20 mm Hepes, pH 7.9, 400 mm NaCl, 1 mmEDTA, 1 mm EGTA, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride). The nuclear lysate was cleared by centrifugation at 13,000 rpm for 5 min at 4 °C and stored at −80 °C. Western blot analysis of Jurkat cell lysates was carried out as described previously (29Dittmer J. Pise-Masison C.A. Clemens K.E. Choi K.S. Brady J.N. J. Biol. Chem. 1997; 272: 4953-4958Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Rabbit anti-Ets1 (C-20), anti-Ets1/2 (C-275), or anti-Elf-1 (C-20) serum, all provided by Santa Cruz Biotechnology, or mouse anti-Tax1 was diluted to 1:7500, 1:1000, 1:4000, or 1:300, respectively, prior to use. Anti-IgG horseradish peroxidase and ECL reagents were obtained from Amersham Corp. Ets1 baculovirus extract (47Bosselut R. Duvall J.F. Gegonne A. Bailly M. Hemar A. Brady J. Ghysdael J. EMBO J. 1990; 9: 3137-3144Crossref PubMed Scopus (141) Google Scholar) was mixed with 400 pg of Klenow α-32P-labeled IL-5 WT (sense strand: 5′-GTTAGAAATTATTCATTTCCTCAAAGA-3′), corresponding to the IL-5 promoter sequence from nucleotides at position −63 to −37, in the presence of 2.5% CHAPS (48Hassanain H.H. Dai W. Gupta S.L. Anal. Biochem. 1993; 213: 162-167Crossref PubMed Scopus (42) Google Scholar), 1 mm Tris, pH 7.5, 3 mmHepes, pH 7.9, 70 mm NaCl, 0.25 mm EDTA, 0.15 mm EGTA, and 0.01% bovine serum albumin, and incubated for 5 min at room temperature. For competition experiments, an IL-5 specific oligonucleotide carrying a C to A mutation at −44 (IL-5 EM) or unlabeled IL-5 WT was used in a 100-fold molar excess over the probe. Electrophoresis was carried out as described (49Dittmer J. Gegonne A. Gitlin S.D. Ghysdael J. Brady J.N. J. Biol. Chem. 1994; 269: 21428-21434Abstract Full Text PDF PubMed Google Scholar). We first studied the ability of Ets1 to regulate the activity of the human IL-5 promoter. Jurkat T-cells were transiently transfected with the luciferase reporter plasmid pIL5P.luc containing a −494/+44 fragment of the IL-5 promoter either in the presence or absence of an expression plasmid coding for p54ets1(pKCR3-c-ets1). As shown in Fig.1 A, Ets1 was able to increase IL-5 promoter activity approximately 3-fold, when cells were simultaneously treated with PMA and ionomycin. Withdrawal of either one or both of these agents prevented activation of the IL-5 promoter by Ets1. In addition to ionomycin, HTLV-I Tax1 was able to act in concert with PMA to stimulate Ets1 activity (Fig. 1 B). For this stimulatory effect on Ets1, Tax1 had to be active, as two inactive Tax1 mutants, M5 and M7 Tax1(43Smith M.R. Greene W.C. Genes Dev. 1990; 4: 1875-1885Crossref PubMed Scopus (347) Google Scholar), failed to support trans-activation of the IL-5 promoter by Ets1 (Fig. 1 C). At the same time, wild-type and mutant Tax1 were expressed at the same level (Fig.1 F). Importantly, p54ets1 expression was not altered by any of these treatments (Fig. 1, D and E). Note, however, that ionomycin affected the production of three smaller proteins (I-III) that could be recognized by the Ets1 antibody (Fig. 1 E). These proteins probably resulted from an exon VII domain-dependent/calcium-triggered degradation of p54ets1 (50Rabault B. Ghysdael J. J. Biol. Chem. 1994; 269: 28143-28151Abstract Full Text PDF PubMed Google Scholar, 51Pognonec P. Boulukos K.E. Gesquiere J.C. Stehelin D. Ghysdael J. EMBO J. 1988; 7: 977-983Crossref PubMed Scopus (72) Google Scholar, 52Fleischman L.F. Pilaro A.M. Murakami K. Kondoh A. Fisher R.J. Papas T.S. Oncogene. 1993; 8: 771-780PubMed Google Scholar, 53Bhat N.K. Thompson C.B. Lindsten T. June C.H. Fujiwara S. Koizumi S. Fisher R.J. Papas T.S. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3723-3727Crossref PubMed Google Scholar), a process that Tax1 apparently could partially prevent (Fig. 1 E, compare lanes 2and 4 with 3 and 5). When we repeated the experiments with the natural ΔVII Ets1 mutant protein p46ets1, we found that exon VII indeed was required for the production of proteins I-III, but was dispensable for Ets1 activation of the IL-5 promoter (data not shown). This demonstrates that proteins I-III were not important for the Ets1 effect on the IL-5 promoter. Collectively, these data show that certain conditions were required to allow Ets1 activation of the IL-5 promoter in Jurkat cells. Tax1 was able to increase IL-5 promoter activity 20-fold, when cells were treated with either PMA alone or PMA plus ionomycin (Fig. 1 B). In the presence of PMA alone, a cooperative effect of Ets1 with Tax1 was observed. Western blot analysis showed that PMA had a strong up-regulating effect on cytomegalovirus-promoter driven Tax1 expression (Fig.1 G) suggesting that PMA was mainly required to obtain Tax1 protein levels sufficient for trans-activation. The −494/+44 IL-5 promoter fragment contains eight GGA(A/T) core motifs (E), all of which could potentially interact with Ets1 (Fig.2 A). To analyze the importance of the Cle0 GGAA motif for Ets1-mediated activation, we first removed the sequence between −494 and −106 (Fig. 2 A). This deletion altered neither Ets1- nor Tax1-dependent activation (Fig. 2 C). This suggests that the seven GGA(A/T) motifs upstream of nucleotide of position −106 were dispensable for stimulation of promoter activity by these proteins. However, compared with the −494/+44 IL-5 promoter, the −106/+44 promoter fragment had a reduced ability to respond to PMA plus ionomycin. This is likely due to the fact that the ionomycin-responsive NFAT site at −116 is missing in the smaller promoter construct (Fig. 2 A). On the other hand, removal of the NFAT site did not impair the ability of the IL-5 promoter to respond to Ets1 in a PMA/ionomycin-dependent manner. This suggests that ionomycin had two different effects on the IL-5 promoter. One was required for activation of the promoter by Ets1, while the other was likely to be mediated by activation of NFAT. We next mutated the GGAA motif of the Cle0 element to AGAA (Fig.2 B), thereby, creating the Ets-binding mutant −106/+44 EM IL-5 promoter (Fig. 2 A). As shown in Fig. 2 D, this mutation completely abrogated the ability of Ets1 to trans-activate the IL-5 promoter. To demonstrate that Ets1 can specifically interact with the GGAA motif of the IL-5 Cle0 element, EMSAs were performed using a32P-labeled oligonucleotide that corresponded to the IL-5 promoter sequence between −63 and −37. By using a p54ets1-enriched baculovirus extract, we could show that Ets1 was able to bind to this probe (Fig. 2 E, lane 2). The formation of the Ets1 complex could be prevented by the addition of unlabeled wild type −63/−37 oligonucleotide (IL-5 WT) (lane 3), but not by adding an Ets mutant version of this oligonucleotide (IL-5 EM) that contained a mutation at the first G of the GGAA motif (lane 4). These results show that Ets1 interacts with the IL-5 Cle0 element by contacting its GGAA motif. These data suggest that responsiveness of the IL-5 promoter to Ets1 relied on the Ets-binding site within the Cle0 element. The G to A mutation of the Cle0 Ets-binding site not only inhibited the activation of the IL-5 promoter by Ets1, but also interfered with the ability of Tax1 to stimulate IL-5 promoter activity (Fig. 2 D). This suggests that the stimulatory effect of Tax1 on the IL-5 promoter required an Ets protein already present in Jurkat T-cells. Full-length Ets1 and ΔVII Ets1 are endogenously expressed in Jurkat cells (Fig.3 A). Treatment with PMA or PMA plus ionomycin, which allowed Tax1 to trans-activate the IL-5 promoter, did not significantly change the expression levels of these Ets1 proteins relative to control conditions (Fig.3 A). Therefore, it is possible that Tax1cooperated with Jurkat cell-derived Ets1, when no Ets1 had been added exogenously. Alternatively, a different Ets transcription factor, such as Ets2 or Elf-1, which are also expressed by Jurkat cells (Fig.3 B), could instead have mediated the effect of Tax1 on the IL-5 promoter. To study this possibility we tested the effects of ectopically expressed Ets2 and Elf-1 on the IL-5 promoter. We found that Ets2 was twice as potent as Ets1 in its ability to activate the IL-5 promoter in Jurkat cells, causing a 6–10-fold increase in promoter activity in the presence of PMA plus ionomycin (Fig. 4 A) or PMA plus Tax1 (Fig. 4 B), respectively. Interestingly, the cooperative effect of Ets2 with Tax1 was stronger than that of Ets1 with Tax1 (Fig. 4 B). Ets2 also had a significant stimulatory effect on the promoter in the presence of PMA alone (Fig. 4 A).Figure 4In addition to Ets1, Ets2, but not Elf-1, is able to activate the IL-5 promoter. Transient transfection experiments were performed with Jurkat cells as described under “Materials and Methods.” Each bar represents the average value of two to three independent experiments.View Large Image Figure ViewerDownload (PPT) Elf-1, although able to activate the IL-5 promoter in cells treated with PMA, failed to stimulate IL-5 promoter activity in the presence of PMA plus ionomycin or PMA plus Tax1 (Fig. 4 A). Interestingly, the PMA/Tax1-mediated activation was even inhibited by Elf-1 (Fig. 4 B). These data show that, in addition to Ets1, Ets2 is able to activate the IL-5 promoter, while Elf-1 lacks this ability and rather seems to have the potential to repress Tax1-induced IL-5 promoter activity. In the myeloid Kasumi cell line, Ets1 and Tax1 were found to be unable to activate the IL-5 promoter under the conditions that allowed these proteins to stimulate promoter activity in Jurkat cells (Fig. 5). This may suggest that a T-cell specific factor may have played a role in Tax1- and Ets1-mediated activation of the IL-5 promoter in Jurkat cells. A potential candidate is the T-cell specific factor GATA3, which is highly expressed in Jurkat T-cells (17Yamagata T. Mitani K. Ueno H. Kanda Y. Yazaki Y. Hirai H. Mol. Cell. Biol. 1997; 17: 4272-4281Crossref PubMed Scopus (46) Google Scholar, 54Ho I-C. Vorhess P. Marin N. Karpinsky-Oakley B. Tsai S-F. Orkin S.H. Leiden J.M. EMBO J. 1991; 10: 1187-1192Crossref PubMed Scopus (260) Google Scholar) and is able to activate the IL-5 promoter through a proximal GATA site (Fig.2 A), shown to be critical for IL-5 promoter activity (15Zhang M. Maass N. Magit D. Sager R. Cell Growth Differ. 1997; 8: 179-186PubMed Google Scholar). By Western blot analyses of unfractionated and fast protein liquid chromatography fractionated nuclear extracts, we confirmed that GATA3 is endogenously expressed in Jurkat cells (data not shown). To test the ability of GATA3 to rescue Ets1/2 and Tax1 stimulatory activity we analyzed the effects of Ets1/2, Elf-1, and Tax1on the −494/+44 IL-5 promoter in myeloid Kasumi cells, when GATA3 was overexpressed. As shown in Fig. 5, overexpression of GATA3 had no effect on the IL-5 promoter (Fig. 5). However, when combined with Ets1 in the presence of PMA a 3-fold increase in promoter activity was observed. This suggests that Ets1 was able to cooperate with GATA3 to trans-activate the IL-5 promoter. Further addition of Tax1or ionomycin strongly increased this effect resulting in a 14- or 11-fold induction of promoter activity, respectively. Neither Ets1 nor GATA3 alone was able to efficiently activate the IL-5 promoter under these conditions. This shows that Tax1 and ionomycin could only exert their effects on Ets1 and GATA3, when both of these transcription factors were present. It suggests that Tax1and ionomycin strengthened the functional interaction between Ets1 and GATA3. Similar to Ets1, Ets2 was unable to activate the IL-5 promoter in Kasumi cells, unless GATA3 was added (Fig. 5). However, the cooperative effect of Ets2 with GATA3 was stronger relative to that of Ets1 leading to an 11-fold induction of IL-5 promoter activity in the presence of PMA, as compared with a 3-fold stimulation by Ets1 under the same conditions. Addition of Tax1 or ionomycin further increased promoter activity 4- or 2-fold, respectively. As found with Jurkat cells, under optimum conditions, Ets2 was twice as effective as Ets1 in activating the IL-5 promoter. Interestingly, compared with Ets1, Ets2 also seemed to be the better partner for Tax1, as Ets2 could synergize with Tax1 even in the absence of GATA3. These data suggest that the interactions of Ets2 with GATA3 and Tax1 may be stronger than those of Ets1 with these proteins. In the presence of PMA alone or PMA plus ionomycin, Elf-1 induced IL-5 promoter activity by approximately 2-fold, while it activated the promoter 3-fold in Kasumi cells treated with PMA plus Tax1. However, under any of these conditions, co-expression with GATA3 did not further increase promoter activity, suggesting that Elf-1 is unable to act in synergy with GATA3. Similar data were obtained, when the experiments were repeated with the −106/+44 IL-5 promoter fragment (data not shown). However, when the Ets mutant 106/+44 EM promoter fragment was used instead, Ets1 and Ets2 failed to synergize with GATA3 and/or Tax1 (data not shown). These results show that the Cle0 Ets-binding site is essential for cooperative effects of Ets1 or Ets2 with GATA3 and/or Tax1. We show here for the first time that in the presence of PMA, Ets1 and Ets2, but not Elf-1, are able to cooperate with GATA3 to synergistically activate the IL-5 promoter. Our results further suggest that Tax1 or ionomycin can enhance this Ets/GATA3 synergy. Based on these data, we propose that IL-5 expression is regulated by the concerted action of at least three transcription factors, Ets1/2, AP1, and GATA3, whose combined activities can be modulated by Tax1 or by a putative ionomycin-regulated cellular factor (Fig. 6). In this model, we suggest PMA to be needed for recruitment of AP1 to the Cle0 element. Both Ets1 and Ets2 are able to cooperate with AP1 to activate a variety of promoters and to mediate PMA-dependent activation (15Zhang M. Maass N. Magit D. Sager R. Cell Growth Differ." @default.
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• W2032695847 title "Regulation of the Human Interleukin-5 Promoter by Ets Transcription Factors" @default.
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