The complex of FK506-binding protein 12 and FK506 inhibits calcineurin phosphatase activity and IgE activation-induced cytokine transcripts, but not exocytosis, in mouse mast cells.
FK506 and cyclosporin A (CsA) are immunosuppressive agents that inhibit IL-2 production by activated T cells, but only CsA inhibits IgE activation-induced cytokine transcripts in mouse IL-3-dependent, bone marrow-derived mast cells (BMMC). We previously associated the resistance of BMMC to FK506 with a deficiency in the expression of FK506 binding protein (FKBP) 12, a molecule that forms a complex with FK506 capable of inhibiting calcineurin phosphatase activity in vitro. In this report, we establish that FKBP12 mediates FK506 inhibition of both calcineurin phosphatase activity and IgE activation-induced cytokine transcripts in a Kirsten murine sarcoma virus-immortalized mast cell line that is FKBP12 deficient. Overexpression of FKBP12 by transfection enhanced the ability of FK506 to inhibit calcineurin phosphatase activity (IC50 = 2 nM), compared with cells transfected with the expression vector alone (IC50 > 30 nM). The IC50 value for FK506 inhibition of IgE activation-induced transcripts for TNF-alpha decreased from 40 nM in vector control cells to 10 nM in FKBP12 transfectants. Similarly, the IC50 value for inhibition of IL-6 transcripts decreased from > 1000 nM in vector control cells to 35 nM in FKBP12 transfectants. In contrast, activation-elicited release of the secretory granule mediator beta-hexosaminidase was only partially inhibited by FK506 at 1000 nM, regardless of the levels of FKBP12 expressed by the cells. Thus, FKBP12 is the dominant cytosolic protein that mediates FK506 inhibition of TNF-alpha and IL-6 transcripts.
Fruman
D A
DA
Committee on Immunology, Dana-Farber Cancer Institute, Boston, MA.
Bierer
B E
BE
Benes
J E
JE
Burakoff
S J
SJ
Austen
K F
KF
Katz
H R
HR
D000818:Animals; D019703:Calcineurin; D002148:Calmodulin-Binding Proteins / Q000037:antagonists & inhibitors*; D002352:Carrier Proteins / Q000378:metabolism*; D002461:Cell Line, Transformed; D003594:Cytoplasmic Granules / Q000187:drug effects / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005089:Exocytosis / Q000187:drug effects*; D005786:Gene Expression Regulation / Q000187:drug effects*; D006360:Heat-Shock Proteins / Q000378:metabolism*; D007073:Immunoglobulin E / Q000276:immunology*; D015850:Interleukin-6 / Q000096:biosynthesis* / Q000235:genetics; D008407:Mast Cells / Q000187:drug effects* / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013601:T-Lymphocytes / Q000276:immunology; D016559:Tacrolimus / Q000378:metabolism / Q000494:pharmacology*; D022021:Tacrolimus Binding Proteins; D014162:Transfection; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis* / Q000235:genetics; D001619:beta-N-Acetylhexosaminidases / Q000378:metabolism
D002148:Calmodulin-Binding Proteins; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D006360:Heat-Shock Proteins; D015850:Interleukin-6; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; D007073:Immunoglobulin E; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases; D001619:beta-N-Acetylhexosaminidases; D022021:Tacrolimus Binding Proteins; D016559:Tacrolimus