title: Calcineurin phosphatase activity: activation by glucocorticoids and role of intracellular calcium.
abstract: BACKGROUND
  Glucocorticoids stimulate release of intracellular calcium in peripheral lymphocytes, but their effects on calcineurin phosphatase activity are unknown.
  
  
  METHODS
  Calcineurin phosphatase activity was measured in permeabilized Jurkat T cells using a specific orthophosphate substrate. Changes in intracellular calcium were measured by FURA-2 fluorescence. Inositol triphosphate levels were measured by radioimmunoassay. Transfection with luciferase reporter plasmids linked to glucocorticoid response elements were used to evaluate glucocorticoid receptor function in Jurkat T cells.
  
  
  RESULTS
  Dexamethasone significantly (P<0.004) increased calcineurin activity within 15 sec, peaking at 10 min (P<0.001) and returning to basal levels by 180 min. Inhibition of DNA transcription with actinomycin D failed to block calcineurin activation, but co-incubation with RU-486 completely blocked enzyme stimulation. To determine whether Jurkat T cells express active glucocorticoid receptors, cells were transfected with a luciferase reporter plasmid linked to a glucocorticoid response element (GRE). Jurkat T cells incubated with dexamethasone (10 microM) for 24 hr failed to stimulate luciferase activity, whereas cells co-transfected with a transcriptionally active glucocorticoid receptor resulted in a doubling of luciferase activity. Dexamethasone rapidly increases intracellular inositol triphosphate (IP3) and intracellular calcium within 15 sec. Cells incubated with U-73122 (a nonspecific phospholipase C [PLC] antagonist) completely blocked dexamethasone-induced activation of calcineurin, whereas U-73343 failed to block enzyme activation. Dexamethasone-induced activation of calcineurin activity stimulates dephosphorylation of the proapoptotic protein BAD and augments apoptosis through a calcineurin-dependent pathway.
  
  
  CONCLUSIONS
  Dexamethasone rapidly increases calcineurin activity through a transcription-independent mechanism involving activation of phospholipase C and the release of IP3-dependent calcium stores.
authors.affiliation: Renal Division, Emory University School of Medicine, 1364 Clifton Road, N.E., Atlanta, GA 30322, USA.
authors.forename: David C
authors.initials: DC
authors.lastname: Tong
authors.forename: Sydney M
authors.initials: SM
authors.lastname: Buck
authors.forename: Brian R
authors.initials: BR
authors.lastname: Roberts
authors.forename: Janet D
authors.initials: JD
authors.lastname: Klein
authors.forename: James A
authors.initials: JA
authors.lastname: Tumlin
mesh_terms: D017304:Annexin A5 / Q000378:metabolism; D002118:Calcium / Q000502:physiology*; D003609:Dactinomycin / Q000494:pharmacology; D003907:Dexamethasone / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004963:Estrenes / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D005938:Glucocorticoids / Q000494:pharmacology*; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D019169:Jurkat Cells; D007700:Kinetics; D015735:Mifepristone / Q000494:pharmacology; D010744:Phosphoric Monoester Hydrolases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D011760:Pyrrolidinones / Q000494:pharmacology; D013601:T-Lymphocytes / Q000201:enzymology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D017304:Annexin A5; D004791:Enzyme Inhibitors; D004963:Estrenes; D005938:Glucocorticoids; D011760:Pyrrolidinones; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; D003609:Dactinomycin; D015735:Mifepristone; D003907:Dexamethasone; D015544:Inositol 1,4,5-Trisphosphate; C120817:calcineurin phosphatase; D010744:Phosphoric Monoester Hydrolases; D010738:Type C Phospholipases; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium