Calcineurin phosphatase activity: activation by glucocorticoids and role of intracellular calcium.
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.
Tong
David C
DC
Renal Division, Emory University School of Medicine, 1364 Clifton Road, N.E., Atlanta, GA 30322, USA.
Buck
Sydney M
SM
Roberts
Brian R
BR
Klein
Janet D
JD
Tumlin
James A
JA
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
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