期刊
TRANSPLANTATION
卷 77, 期 2, 页码 259-267出版社
LIPPINCOTT WILLIAMS & WILKINS
DOI: 10.1097/01.TP.0000099267.05131.11
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资金
- NHLBI NIH HHS [1 R01 HL59978-01-A1] Funding Source: Medline
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 muM) 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. Conclusion. Dexamethasone rapidly increases calcineurin activity through a transcription-independent mechanism involving activation of phospholipase C and the release of IP3-dependent calcium stores.
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