甲状旁腺素通过磷脂酶C非依赖途径激活蛋白激酶C

Parathyroid hormone activates protein kinase C via phospholipase C-independent signaling pathway

目的利用基于荧光共振能量转移(Fluorescence resonance energy transfer,FRET)技术的检测PKC激活或PKC-delta激活的报告分子来确定PTH是否可以通过PLC非依赖途径激活PKC和PKC-delta。方法将表达PKC激活报告分子(CKAR)的质粒和表达PKC-delta激活报告分子的质粒转染HEK293细胞,培养72h后通过共聚焦显微镜检测FRET的改变,并以此判断佛波酯(TPA)是否激活PKC和PKC-delta。将表达甲状旁腺素1型受体(PTHR1)的质粒与CKAR质粒或PKC-delta质粒共转染HEK293细胞,培养72h后通过共聚焦显微镜检测FRET的改变,并判断PTH(1-34)、G1R19(1-34)和0.1%的三氟乙酸(TFA)对PKC和PKC-delta的作用。结果在转染CKAR质粒或PKC-delta质粒的HEK293细胞,TPA均使青色荧光与黄色荧光的强度之比(C/Y)增加。在共转染PTH1R质粒与CKAR质粒或PKC-delta质粒的HEK293细胞,PTH(1-34)和G1R19(1-34)均增加了C/Y的值,而0.1%TFA未引起C/Y的改变。结论 PTH与PTHR1结合后通过PLC非依赖途径激活PKC/PKC-delta。

Objective To determine whether PTH activates PKC/PKC-delta via PLC-independent pathway using the reporter of PKC or PKC-delta activation based on fluorescence resonance energy transfer（FRET）.Methods HEK293 cells were transfected with C kinase activity reporter（CKAR） or C-delta kinase activity reporter（PKC-delta）.After incubation for 72h,FRET was measured with or without stimulation of phorbol esters（TPA）.The activation of PKC or PKC-delta could be judged by the change in FRET.HEK293 cells were co-transfected with either CKAR or PKC-delta plasmid or PTHR1 plasmid.Stimulation of PKC or PKC-delta by PTH（1-34） or G1R19（1-34） or 0.1% trifluoroacetic acid（TFA） were demonstrated by monitoring the change in FRET after 72 h.Results TPA increased the CFP/YFP emission ratio（C/Y） in HEK293 cells transfected with CKAR or PKC-delta plasmid.PTH（1-34） and G1R19（1-34）,but not 0.1% TFA,increased C/Y in HEK293 cells co-transfected with either CKAR or PKC-delta plasmid or PTHR1 plasmid.Conclusion PTH binding to PTHR1 can activate PKC and PKC-delta via PLC-independent signaling pathway.