癌基因p21^(ras)、raf和细胞外信号调节蛋白激酶参与刺激Jurkat细胞产生TNF-β和IL-2

Stimulation of TNF-β and IL-2 production by PMA/PHA requires p21^(ras), raf and ERK

目的 明确p2 1ras 细胞外信号调节蛋白激酶 (theextracellularsignal regulatedproteinki nase,ERK)系列是否参与调节Jurkat细胞产生TNF β和IL 2。方法 用负向突变基因p2 1ras(ras17N或ras15A) ,raf 1(raf1～ 130 )及ERK1(erk1K71R)或活性结构p2 1ras(H ras6 1L)和raf(raf2 2W)转染细胞 ,以PMA PHA刺激细胞 ,ELISA法检测细胞因子。结果 PMA PHA刺激细胞产生较正常对照细胞约 2倍的TNF β和IL 2 (P <0 .0 0 1)。负向突变基因去除了PMA PHA增加细胞因子分泌的作用。活性突变基因本身不影响TNF β和IL 2产生 ,但明显增强细胞对PMA PHA的应答 (P <0 .0 5 )。结论 p2 1ras ERK系列在调节Jurkat细胞在PMA PHA刺激下产生TNF β和IL 2中起重要的作用。

Objective To investigate whether the p21 ras /ERK (MAPK) cascade is involved in regulating the production of TNF β and IL 2 in Jurkat cells. Methods Cells were transiently transfected with a dominant negation construct of p21 ras (ras15A or ras17N), raf (raf 1 130) or ERK1 (erk1K71R), or transforming p21 ras (H ras61L) or constitutively active raf (raf22W) using lipofectamine. Control cells received a plasmid carrying β galactosidease. Transfectant cells were stimulated with PMA/PHA and released TNF β and IL 2 quantitated by ELISA. Results In control cells, the production of TNF β and IL 2 was approx. 2 fold by PMA/PHA stimulation ( P <0.001), while transfection with a dominant negative mutant construct abrogated the effects of PMA/PHA. Transfection with a constitutively active mutant had no effect on basal production of TNF β or IL 2, but all of the constitutively active mutants significantly enhanced the response of the cells to PMA/PHA ( P <0.05). MEK1 inhibitor PD98059 inhibited the effects of PMA/PHA and active construct genes on ERK activities and cytokine production. Conclusion These data demonstrate that the p21 ras ERK cascade plays an important role in regulating the production of both TNF β and IL 2 in Jurkat cells stimulated with PMA/PHA.