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 interleukin-2 (IL-2) are produced by stimulation with phorbol-12-myristate acetate (PMA) and calcium ionophore (A23187) in human T cell leukemia Jurkat cells. The expression of GM-CSF and IL-2 is inhibited by immunosuppressive 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 transcription from the IL-2 promoter through the NF-AT-binding site. In this study, we obtained evidence that transfection of the cDNAs for CN A (catalytic) and CN B (regulatory) subunits also augments transcription from the GM-CSF promoter and recovers the transcription inhibited by CsA. The constitutively active type of the CN A subunit, which lacks 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 synergy with PMA to induce expression of endogenous GM-CSF and IL-2. By multimerizing the regulatory elements of the GM-CSF promoter, we found that one of the target sites for the CN action is the conserved lymphokine element 0 (CLE0), located at positions between -54 and -40. Mobility shift assays showed that the CLE0 sequence has an AP1-binding site and is associated with an NF-AT-like factor, termed NF-CLE0 gamma. NF-CLE0 gamma binding is induced by PMA/A23187 and is inhibited by treatment with CsA. These results suggest that CN is involved in the coordinated 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 activation.
Tsuboi
A
A
Department of Molecular Biology, DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, California 94304-1104.
Masuda
E S
ES
Naito
Y
Y
Tokumitsu
H
H
Arai
K
K
Arai
N
N
D000818:Animals; D001483:Base Sequence; D000001:Calcimycin / Q000494:pharmacology; D019703:Calcineurin; D002148:Calmodulin-Binding Proteins / Q000494:pharmacology*; D016572:Cyclosporine / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects*; D005796:Genes; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000096:biosynthesis / Q000235:genetics*; D006801:Humans; D007376:Interleukin-2 / Q000096:biosynthesis / Q000235:genetics; D015458:Leukemia, T-Cell / Q000473:pathology; D008213:Lymphocyte Activation; D051379:Mice; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000494:pharmacology*; D011401:Promoter Regions, Genetic; D012045:Regulatory Sequences, Nucleic Acid; D012689:Sequence Homology, Nucleic Acid; D013601:T-Lymphocytes / Q000187:drug effects* / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects; D014407:Tumor Cells, Cultured / Q000187:drug effects
D002148:Calmodulin-Binding Proteins; D007376:Interleukin-2; D009363:Neoplasm Proteins; D014157:Transcription Factors; D000001:Calcimycin; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D016572:Cyclosporine; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases