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

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.