Ca^(2+)和CaN凋亡信号通路参与皮质酮诱导的Leydig细胞凋亡中FasL的表达

Involvement of Ca^(2+) and CaN signaling pathway in CORT-induced activation of fasl during apoptosis in rat leydig cell

目的观察在皮质酮诱导的大鼠Leydig细胞凋亡中,Ca2+和钙调神经磷酸酶(CaN)依赖的信号通路是否参与FasL表达的调控。方法利用钙定性探针Fluo-3/AM检测皮质酮作用下的Leydig细胞中Ca2+浓度变化。通过酶底物法测定CaN活性。以Westernblot检测FasL表达。用Annexin-Ⅴ-FITC和PI双标评价Leydig细胞凋亡率。结果经超生理剂量皮质酮处理的Leydig细胞中出现Ca2+浓度升高,CaN活性增加及FasL表达增加。环孢菌素A可抑制CaN活性,使FasL表达下调,细胞凋亡率下降。结论Ca2+和CaN依赖的信号通路参与了皮质酮诱导的大鼠Leydig细胞凋亡;CaN介导了由Ca2+引发的FasL表达,Ca2+和CaN在大鼠Leydig细胞凋亡过程中起重要作用。

Objectives To elucidate whether calcium and calcineurin （CAN） regulate FasL expression in response to CORT in Leydig ceUs. Methods Changes in intraceUular calcium were measured by Fluo-3/AM under laser scanning confocal microscopy （LSCM）. Calcineurin phosphatase activity in Leydig ceUs was measurexi using a specific orthophosphate substrate. The activation of FasL in Leydig cell was analyzed by Western blot and Annexin- V staining was used to detect the frequency of apoptotic Leydig cells. Results LCSM analysis of Leydig cells labeling with Fluo-3/AM showed that CORT can result in a sharp increase of Ca^2＋ level in cultured rat Leydig cells. CORT significantly （P〈0.05） increase CaN activity within 12h, but co-incubation with CsA completely blocked enzyme stimulation. After incubation with 100nM CORT, the expression of FasL was enhanced greatly （P〈0.05）, and the frexluency of apoptotic cells increased from （16.8±4.1）% in the medium control to （43.86±10.5）% in CORT-treated Leydig cells. By contrast, after simultaneous treatment with CORT and CsA, the product of FasL was only weakly expressed and the number of Annexin- V -positive cells was reduced significantly. Conclusion These results suggested that the Ca^2＋ and CaN signaling pathway is involved in CORT-induced activation of the FasL during apoptosis in rat Leydig cell.