Tong David C, Buck Sydney M, Roberts Brian R, Klein Janet D, Tumlin James A
Renal Division, Emory University School of Medicine, 1364 Clifton Road, N.E., Atlanta, GA 30322, USA.
Transplantation. 2004 Jan 27;77(2):259-67. doi: 10.1097/01.TP.0000099267.05131.11.
Glucocorticoids stimulate release of intracellular calcium in peripheral lymphocytes, but their effects on calcineurin phosphatase activity are unknown.
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.
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.
Dexamethasone rapidly increases calcineurin activity through a transcription-independent mechanism involving activation of phospholipase C and the release of IP3-dependent calcium stores.
糖皮质激素可刺激外周淋巴细胞释放细胞内钙，但它们对钙调神经磷酸酶活性的影响尚不清楚。
使用特定的正磷酸盐底物在通透的Jurkat T细胞中测量钙调神经磷酸酶活性。通过FURA-2荧光测量细胞内钙的变化。通过放射免疫测定法测量肌醇三磷酸水平。用与糖皮质激素反应元件相连的荧光素酶报告质粒转染来评估Jurkat T细胞中的糖皮质激素受体功能。
地塞米松在15秒内显著（P<0.004）增加钙调神经磷酸酶活性，在10分钟时达到峰值（P<0.001），并在180分钟时恢复到基础水平。用放线菌素D抑制DNA转录未能阻断钙调神经磷酸酶的激活，但与RU-486共同孵育可完全阻断酶的刺激。为了确定Jurkat T细胞是否表达活性糖皮质激素受体，用与糖皮质激素反应元件（GRE）相连的荧光素酶报告质粒转染细胞。用10μM地塞米松孵育24小时的Jurkat T细胞未能刺激荧光素酶活性，而与转录活性糖皮质激素受体共转染的细胞导致荧光素酶活性加倍。地塞米松在15秒内迅速增加细胞内肌醇三磷酸（IP3）和细胞内钙。用U-73122（一种非特异性磷脂酶C [PLC]拮抗剂）孵育的细胞完全阻断了地塞米松诱导的钙调神经磷酸酶激活，而U-73343未能阻断酶的激活。地塞米松诱导的钙调神经磷酸酶活性激活刺激促凋亡蛋白BAD的去磷酸化，并通过钙调神经磷酸酶依赖性途径增强细胞凋亡。
地塞米松通过涉及磷脂酶C激活和IP3依赖性钙储存释放的转录非依赖性机制迅速增加钙调神经磷酸酶活性。
