CALCINEURIN MEDIATES HUMAN TUMOR-NECROSIS-FACTOR-ALPHA GENE INDUCTIONIN STIMULATED T-CELLS AND B-CELLS

The tumor necrosis factor alpha (TNF-alpha) gene is rapidly transcribe d in activated T cells via a calcium-dependent pathway that does not r equire de novo protein synthesis, but is completely blocked by the imm unosuppressive drugs cyclosporin A (CsA) and FK506. Here we show that calcineurin phosphatase activity is both necessary and sufficient for TNF-alpha gene transcription in T cells, and identify the factor that binds to the kappa 3 element of the the TNF-alpha gene promoter as the target for calcineurin action. The ability of analogues of CsA and FK 506 to block calcineurin phosphatase activity correlates completely wi th their ability to inhibit induction of TNF-alpha mRNA, induction of a TNF-alpha promoter reporter plasmid in transiently transfected T cel ls, and induction of the kappa 3 binding factor in an electrophoretic mobility shift assay. Moreover, a cDNA encoding the constitutively act ive form of calcineurin is sufficient to activate the TNF-alpha promot er and the kappa 3 element. TNF-alpha gene transcription is also highl y inducible, CsA-sensitive, and protein synthesis-independent in B cel ls stimulated through their surface immunoglobulin receptors. Using th e panel of CsA and FK506 analogues, we show that calcineurin participa tes in the induction of TNF-alpha transcription in activated B cells. These results extend our previous demonstration that the kappa 3 bindi ng factor is related to NFATp, the preexisting subunit of nuclear fact or of activated T cells, and suggest that calcineurin-mediated modific ation of the kappa 3 binding factor in T cells is of key importance in the induction of TNF-alpha transcription.