CALCINEURIN POTENTIATES ACTIVATION OF THE GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR GENE IN T-CELLS - INVOLVEMENT OF THE CONSERVED LYMPHOKINE ELEMENT-0

Granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleu kin-2 (IL-2) are produced by stimulation with phorbol-12-myristate ace tate (PMA) and calcium ionophore (A23187) in human T cell leukemia Jur kat cells. The expression of GM-CSF and IL-2 is inhibited by immunosup pressive drugs such as cyclosporin A (CsA) and FK506. Earlier studies on the IL-2 gene expression showed that overexpression of calcineurin (CN), a Ca2+/calmodulin-dependent protein phosphatase, can stimulate t ranscription from the IL-2 promoter through the NF-AT-binding site. In this study, we obtained evidence that transfection of the cDNAs for C N A (catalytic) and CN B (regulatory) subunits also augments transcrip tion from the GM-CSF promoter and recovers the transcription inhibited by CsA. The constitutively active type of the CN A subunit, which lac ks the auto-inhibitory and calmodulin-binding domains, acts in synergy with PMA to activate transcription from the GM-CSF promoter. We also found that the active CN partially replaces calcium ionophore in syner gy with PMA to induce expression of endogenous GM-CSF and IL-2. By mul timerizing the regulatory elements of the GM-CSF promoter, we found th at one of the target sites for the CN action is the conserved lymphoki ne element 0 (CLE0), located at positions between -54 and -40. Mobilit y shift assays showed that the CLE0 sequence has an AP1-binding site a nd is associated with an NF-AT-like factor, termed NF-CLE0 gamma. NF-C LE0 gamma binding is induced by PMA/A23187 and is inhibited by treatme nt with CsA. These results suggest that CN is involved in the coordina ted induction of the GM-CSF and IL-2 genes and that the CLE0 sequence of the GM-CSF gene is a functional analogue of the NF-AT-binding site in the IL-2 promoter, which mediates signals downstream of T cell acti vation.