ETS1 transactivates the human GM-CSF promoter in Jurkat T cells stimulated with PMA and ionomycin.

Activation of T helper cells results in coordinate expression of a number of cytokines involved in differentiation, proliferation and activation of the haematopoietic system. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is one such cytokine whose increased expression results partly from increases in transcription. Cis-acting elements with NF kappa B, AP-1 and ETS-like motifs have been identified in the promoter region of the GM-CSF gene, which are important for transcriptional activity following PMA and ionomycin stimulation. A number of the ETS family of transcription factors are expressed in T cells, including ETS1 and ELF1. Here we describe the ability of these factors to interact with a site (GM5), located within the CLE0 element, -47 to -40 upstream of the GM-CSF transcription initiation site. Exogenous ETS1, but not ELF1, can transactivate GM-CSF, through the GM5 site, in a PMA/ionomycin dependent manner. Other unidentified ETS-like factors present in Jurkat cells are also capable of binding GM5. Mutation of the core ETS binding site from -GGAA- to -GGAT- prevents the binding of ETS-like factors with the exception of ETS1. The GM-CSF promoter, modified in this way to be ETS1 specific, is fully responsive to PMA/ionomycin induction, in addition to ETS1 transactivation in the presence of PMA and ionomycin. Together these data suggest that ETS1 may be involved in mediating the increased GM-CSF production associated with T cell activation.

Thomas RS ，Tymms MJ ，Seth A ，Shannon MF ，Kola I ... -  《ONCOGENE》

ETS1, NFkappaB and AP1 synergistically transactivate the human GM-CSF promoter.

Activation of helper T cells results in coordinate expression of a number of cytokines involved in differentiation, proliferation and activation of the haematopoietic system. Granulocyte-macrophage colony stimulating factor (GM-CSF) is one such cytokine, whose increased expression results mostly from increases in transcription. Cis-acting elements with NFkappaB, AP1 and ETS-like binding motifs have been identified in the promoter region of the GM-CSF gene, and are important or essential for transcriptional activity following T cell activation. ETS1 is a transcription factor of the ETS family that is expressed in T cells. We have previously shown that ETS1 can transactivate GM-CSF in Jurkat T cells, but only after the cells have been stimulated by treatment with PMA and ionomycin, agents that mimic T cell activation. Thus we proposed that ETS1, which is expressed constitutively in Jurkat cells, may act in concert with PMA/ionomycin inducible factors. Here we show that ETS1 can transactivate a GM-CSF reporter construct in unstimulated Jurkat cells, providing that either NFkappaB or AP1 transcription factors are supplied by co-transfection. We confirm that binding of endogenous NFkappaB and AP1 is induced following PMA/ionomycin treatment of T cells. Transactivation by ETS1, NFkappaB and AP1 is synergistic, and mutation of the individual binding sites reveals that the transcriptional activities of these factors are interdependent. Our results suggest that constitutive ETS1, and inducible NFkappaB and AP1, cooperate as part of a higher order transcriptional complex in activated T cells.

Thomas RS ，Tymms MJ ，McKinlay LH ，Shannon MF ，Seth A ，Kola I ... -  《ONCOGENE》

Multiple signals are required for function of the human granulocyte-macrophage colony-stimulating factor gene promoter in T cells.

The human granulocyte-macrophage CSF (GM-CSF) gene is expressed in T cells in response to TCR activation that can be mimicked by treatment of the cells with PMA and Ca2+ ionophore. The gene contains a proximal functional promoter region (-620 to +34), as well as a powerful enhancer located 3 kb upstream, both of which are involved in the response of the gene to TCR activation. The proximal promoter contains a region termed CLEO (-54 to -31) that consists of a purine-rich element abutting an activator protein-1 (AP-1)-like site, as well as an upstream nuclear factor-kappa B (NF-kappa B) site (-85 to -76) and a CK-1 element (-101 to -92). We show in this work that mutations in either the purine-rich region of the CLEO element or the NF-kappa B site result in reduced PMA/Ca2+ activation of a 620-bp human GM-CSF promoter-luciferase reporter construct in Jurkat T cells by 65% and 50%, respectively. The major inducible protein complex that binds to the human CLEO (hCLEO) element is an AP-1-like complex that is inducible by PMA alone, but shows increased binding in response to PMA together with Ca2+ ionophore. Although the binding of this complex is not cyclosporin-sensitive, promoter induction is inhibited by cyclosporin treatment. A second weak inducible complex resembling nuclear factor of activated T cells (NF-AT) was also observed binding to the hCLEO region. By using recombinant proteins, we confirmed that AP-1, NF-ATp, and a higher order NF-ATp/AP-1 complex could all form with the hCLEO element, and we have also defined the sequence requirements for binding of each of these complexes. We found that expression of a constitutively active form of calcineurin could substitute for Ca2+ ionophore and synergize with PMA to activate the GM-CSF promoter, and conversely that mutant-activated Ras could substitute for PMA and cooperate with Ca2+ ionophore. Co-expression of Ras and calcineurin, however, did not activate the GM-CSF promoter, but required the additional expression of NF-kappa B p65. These results imply that at least three signals are required to activate the GM-CSF proximal promoter, and that the signals impinge on distinct transcription factors that bind to the hCLEO and NF-kappa B regions of the promoter.

Jenkins F ，Cockerill PN ，Bohmann D ，Shannon MF ... -  《JOURNAL OF IMMUNOLOGY》

Calcineurin activates transcription from the GM-CSF promoter in synergy with either protein kinase C or NF-kappa B/AP-1 in T cells.

Two cis-acting elements GM-kappa B/GC-box and CLE0, of the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene are required for maximal induction in Jurkat T cells by costimulation with phorbol-12-myristate acetate (PMA) and Ca2+ ionophore (A23187). The GM-kappa B sequence is recognized by NF-kappa B, which is mainly induced by PMA. The CLE0 sequence interacts with factors, related to a PMA-induced AP-1 and a PMA/A23187-induced NF-AT. We examined whether signal transducing components in T cells can activate transcription of the GM-CSF gene. Cotransfection of NF-kappa B (p50/p65)- or AP-1 (c-Jun/c-Fos)-expression vectors into Jurkat cells with a luciferase reporter containing the GM-CSF promoter did not stimulate transcription from the GM-CSF promoter. In contrast, cotransfection with a combination of NF-kappa B and AP-1 significantly augmented transcription from the GM-CSF promoter containing the GM-kappa B/GC-box and the CLE0 (AP-1/NF-AT). Expression of a constitutively active calcineurin (CN), a Ca2+/calmodulin-dependent protein phosphatase, potentiated by two fold the transcriptional activation by NF-kappa B/AP-1. Both constitutively active forms of CN and protein kinase C (PKC) synergistically activated transcription from the GM-CSF promoter. These results suggest that cooperation among NF-kappa B-, AP-1- and NF-AT-binding sequences is required for induction of the GM-CSF gene through PKC- and Ca2+-signaling pathways downstream of T cell activation.

Tsuboi A ，Muramatsu M ，Tsutsumi A ，Arai K ，Arai N ... -  《BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS》

Purification of the 120 kDa component of the human nuclear factor of activated T cells (NF-AT): reconstitution of binding activity to the cis-acting element of the GM-CSF and IL-2 promoter with AP-1.

The cis-acting element of the granulocyte-macrophage colony-stimulating factor (GM-CSF) promoter, CLE0, is required for stimulation dependent expression of the GM-CSF gene by phorbol ester (PMA) and calcium ionophore (A23187) in T cells. We recently obtained evidence that NF-CLE0 gamma, one of the CLE0-binding factors, is similar to the nuclear factor of activated T cells, NF-AT. In the present study, we show that the affinity-purified NF-AT from nuclear extracts of human Jurkat T cells stimulated with both PMA and A23187 bound strongly to the CLE0 element and formed a NF-CLE0 gamma complex. This DNA-protein complex was competitively inhibited by oligonucleotides containing NF-AT and AP-1 binding sites, suggesting that the CLE0 gamma complex is identical to NF-AT and contains AP-1 proteins. Here, one component of NF-AT with an apparent molecular mass of 120 kDa on SDS-polyacrylamide gel electrophoresis was purified to near homogeneity by Mono Q chromatography. The purified 120 kDa protein reconstitutes NF-AT binding in combination with recombinant cJun/cFos heterodimer. Furthermore, we demonstrate that binding of this 120 kDa protein to both the NF-AT and the CLE0 sequences can be reconstituted with the addition of affinity-purified Jurkat AP-1 proteins. These results indicate that NF-AT (NF-CLE0 gamma), which is composed of the 120 kDa nuclear protein and AP-1 proteins, regulates the stimulation-dependent expression of the GM-CSF gene as it does the IL-2 gene.

Tokumitsu H ，Masuda ES ，Tsuboi A ，Arai K ，Arai N ... -  《BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS》