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A Novel ets-2-Related Nuclear Factor Is Involved in Transcriptional Activation of the Human Interleukin-12 p40 Gene Promoter in Response to Interferon- ? and LPS Stimulation of Monocytic Cells

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A Novel ets-2-Related Nuclear Factor Is Involved in Transcriptional Activation of the Human Interleukin-12 p40 Gene Promoter in Response to Interferon- ? and LPS Stimulation of Monocytic Cells
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  PDFlib PLOP: PDF Linearization, Optimization, ProtectionPage inserted by evaluation versionwww.pdflib.com – sales@pdflib.com  A Novel ets-2-Related Nuclear Factor Is Involved in Transcriptional Activation of the Human Interleukin-12 p40 Gene Promoter in Response to Interferon- and LPS Stimulation of Monocytic Cells XIAOJING MA, GIORGIA GRI, AND GIORGIO TRINCHIERI Wistar Institute 3601 Spruce Street Philadelphia Pennsylvania 19104 Interleukin- 12 (IL-12) is a proinflammatory cytokine produced by phagocytic cells, B cells, and other antigen-presenting cells (APC) that modulate adaptive immune responses by favoring the generation of T helper type 1 (Thl) cells.’ IL-12 mediates some of its physiological activities by acting as a potent inducer of interferon-y (IFN-y) production by T and NK cells. The IFN-y induced by IL-12 acts not only as a stimulator of macrophages in the inflamed tissue, increasing their phagocytic and bacteriocidal activity, but also enhances the ability of macrophages to produce IL-12 in a powerful positive feedback loop.2 We have previously shown that the IL-12 p40 and p35 genes are transcription- ally and differentially regulated by IFN-y and LPS.3 To further elucidate the molecular mechanisms by which the IL-12 genes are modulated during immune responses, we cloned the IL-12 p40 gene promoter as a 3.3-kb genomic fragment, determined the transcription initiation site by primer extension, and fully se- quenced it. There is a classical TATA box located 27 bp upstream of the cap site. A number of other putative cis elements were also identified. They include an NFKB “half site” located between -116 and -106 (TGAAATTCCCC or GGGGAATTTCA for the complement), which has been suggested by Murphy t ~1 ~ s a critical element that interacts with members of the NFKB family and is responsible principally for the induction of the murine IL-12 p40 gene promoter by LPS and IFN-y. Five base pairs upstream of the NFKB half site is a PU. 1 site (AAGGAA) whose functional importance has not been clearly demonstrated. Using transient transfection in a number of cell lines that either express or do not express IL-12 coupled with mutagenesis studies, we have characterized the p40 promoter and established the following: The promoter as a 3.3-kb genomic fragment linked to a luciferase reporter gene is cell type specific in that it is inducible in monocytic (THP-1 of human srcin and RAW of murine srcin) and constitutively active in EBV-transformed B cells (RPMI-8866 and CESS) but inactive in T-cell lines (Jurkat and Molt-13), suggesting that this promoter contains sufficient sequence information to confer cell type-specific expression in analogy to its endogenous counterpart. The promoter is responsive to LPS stimulation fairly modestly in RAW cells, but the response is greatly augmented by priming RAW cells with IFN-y for 8 hours before LPS stimulation, similar to previous observations in human PBMC 357  358 ANNALS NEW YORK ACADEMY OF SCIENCES 25 -265 edium EZ23 IFNy LPS EiEZZJ IFNy LPS ~AAAAGTCA~TTCCTJCTT -222 ets -2d4 FIGURE 1. The -222 and -204 region of the human IL-12 p40 promoter is critical for the induction by IFN-yand LPS. The human IL-12 p40 promoter was sequentially deleted from the 5’ end, starting with the 3.3-kb promoter linked at the 3‘ end with the luciferase gene as a reporter. The numbers on the X axis refer to the coordinates of the promoter deletions. “TATA” indicates that the promoter only contains the TATA box. All constructs were then transiently transfected into RAW cells by electroporation. One day after transfection, cells were treated with 1.2 DMSO for 24 hr, IFN-y 1000 phi for 8 hr before stimulation with LPS 1 pg/ml). Cell lysates were prepared and assayed for luciferase activity which was then corrected for transfection efficiency with an internal control (p-galactosidase). The results shown are the means of at least three independent experiments with standard devi- ation. or pure monocyte~.~,~ horbol diesters such as TPA have no effect on the promoter in RPMI-8866 or CESS cells. This observation apparently differs from our data on the measurement of transcription rate of the endogenous IL-12 p40 gene, which showed that p40 transcription is induced approximately twofold upon TPA stimulation. A possible explanation for this discrepancy is that the p40 promoter construct used in transfection studies does not contain any intron sequence where we have observed potential “transcriptional pausing” in the endogenous gene. By excluding the downstream sequences from the promoter construct, the transgene may have been exempted from transcriptional arrest, therefore becoming more constitutively active (X. Ma and G. Trinchieri, unpublished results). ’ Promoter truncation studies indicated that there are two main regions respon- sive to LPS and IFN y induction. One is between 2 and 3.3 kb upstream, since removal of this region resulted in the loss of 40 to 50 of the overall inducibility of the promoter. A second region appears to lie between -222 and -204 FIG. . In this region, there is an ets-like motif TTTCCT (AGGAAA for the complement) located between -212 and -207 from the transcription start site. This element,  MA el af : HUMAN IL 12 p40 GENE PROMOTER 359 Reporter Sequence when deleted from the 3.3-kb promoter, resulted in dramatic loss of promoter activity induced by IFN-y and LPS and also in the inability of the promoter to be activated by a co-expressed recombinant ets-2 FIG. ). The preeminence of this element holds true for both monocytic and EBV-B cells. Furthermore, the -222 p40 promoter construct still retains cell type specificity in that it is, like the 3.3-kb promoter, inducible in RAW cells, constitutively active in RPMI-8866 cells, and inactive in Jurkat and CESS cells (G. Gri, X. Ma, and G. Trinchieri, unpub- lished results). Our preliminary data indicate that there is a nuclear complex that interacts with the ets-like element in a sequence-specific and spatial manner. This nuclear complex is inducible by either IFN-y or LPS, and there is no synergistic effect when both inducers are used together. Its interaction with the promoter at the ets-2 site is rather complex in that it requires a core motif and quite extensive but nonspecific flanking sequences, in order to stabilize the complex. Alteration at the ets-2 site or shortening of the fragment from either the 5 or the 3 end by a small number of nucleotides adversely affect this complex formation, but would result in some other complexes that show patterns of differential induction by IFN-y and LPS. Using a combination of DNA affinity purification, Western blot, 3300 3300 ets-2d) AGTCATTTCCTCTT AGTCA- TCTT I Ic 60 50 40 3 20 10 0 FIGURE 2. Role of ets 2 in the activation of the human IL 12 40 promoter in RAW cells. RAW cell transfection was performed exactly as described in FIGUR 1, with the 3.3-kb p40 promoter-luciferase construct (-3300), or with an identical construct except for the 5-bp deletion in the ets-2 site located between -212 and -207 [-3300(ets-2d)]. Cotransfection was performed with a murine-ets-2 expression vector under CMV (effector).  360 ANNALS NEW YORK ACADEMY OF SCIENCES metabolic labeling, electrophoretic gel mobility shift assay (EMSA), supershift assay, DNAseI footprint, and methylation interference assay, this nuclear factor was shown to consist of several components including ets-2 and a related novel protein of about 100 kDa as well as a yet-to-be-characterized form of NFKB p50. The novel protein was identified by virtue of its cross reactivity with an anti- mouse ets-2 polyclonal IgG (affinity purified). This protein is considered novel because of the fact that the epitope to which the antibody was directed shares no homology with any other known realistic proteins or hypothetical proteins avail- able in the current databases and that the size of this reduced protein is much larger than any known member of the ets family of transcription factor^.^. A second element that might also play a role in p40 regulation is the NFKB half-site recently reported by Murphy t ai.4 in the mouse IL-12 p40 promoter. This site alone, as shown by the above workers by transient transfection in 5774 cells a murine macrophage-like cell line), appears able to activate the promoter in response to IFN-y and LPS stimulation. This site in the human promoter does interact with NFKB p50 homodimer primarily, but is not directly responsible for the induction of the promoter, although its presence is required for the activation. To summarize, the responsiveness of the p40 promoter to IFN-y and LPS seems difficult to separate, although in both the human and the mouse systems, IFN-y-inducible factors that interact with the p40 promoter have been identified (X. Ma and G. Trinchieri, unpublished ob~ervation).~ here is a reasonable amount of evidence to suggest that both IFN-y and LPS induce distinct alterations in the repertoire of nuclear factors, which by themselves alone are not sufficient to activate the p40 promoter in any significant way but which cooperate to produce a synergistic induction of the p40 promoter. REFERENCES 1. TRINCHIERI, . 1995. Interleukin- 12: A proinflammatory cytokine with immunoregula- tory functions that bridge innate resistance and antigen-specific adaptive immunity. Annu. Rev. Immunol. 13: 251-276. KUBIN, M., J. M. CHOW G. TRINCHIERI. 994. Differential regulation of interleukin- 12 (IL-12), tumor necrosis factor-a, and IL-Ip production in human myeloid leukemia cell lines and peripheral blood mononuclear cells. Blood 83: 1847-1855. 3. MA, X., J. M. CHOW, G. GRI, G. CARRA, F. GEROSA, S. F. WOLF, R. DZIALO G. TRINCHIERI. 996. The interleukin-12 p40 gene promoter is primed by interferon- in monocytic cells. J. Exp. Med. 183: 147-157. 4. MURPHY, . L., M. G. CLEVELAND, . KULESZA, . MAGRAM K. M. MURPHY. 995. Regulation of interleukin 12 p40 expression through an NK-kB half-site. Mol. Cell. Biol. 15: 52.58-5267. HAYES, M. P., J. WANG M. A. NORCROSS. 995. Regulation of interleukin-I2 expres- sion in human monocytes: Selective priming by IFN-y of LPS-inducible p35 and p40 genes. Blood 86: 646-650. MACLEOD, K., D. LEPRINCE D. STEHELIN. 992. The ets gene family. Trends Bio- chem. Sci. 17: 251-256. JANKNECHT, . A. NORDHEIM. 993. Gene regulation by Ets proteins. Biochim. Biophys. Acta 1155: 346-356. 2. 5 6. 7.
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