小脑颗粒神经元撤钾凋亡中JNK/c-Jun通路对促凋亡基因Hrk的转录调控

Regulation of Proapoptotic Gene Hrk Transcription by JNK/c-Jun Pathway in Potassium Deprivation-induced Apoptosis in Cerebellar Granule Neurons

目的研究小脑颗粒神经元(CGNs)撤钾凋亡模型中,BH3-only促凋亡基因Hrk的转录,及JNK/c-Jun通路对Hrk基因转录的调控。方法新生7 d Sprague-Dawley大鼠CGNs在含10%胎牛血清和25 mmol/L KCl培养液中培养。培养第7天,分别用不含胎牛血清的25或5 mmol/L KCl培养液处理2、4、8 h,或5 mmol/L KCl+JNK通路抑制剂处理细胞4 h;或在培养第5天用表达负显性c-Jun突变体的腺病毒载体(Ad-FLAGΔ169)感染CGNs 36 h后,用5mmol/L KCl处理细胞4 h。用RT-PCR方法检测各组CGNs促凋亡基因Hrk mRNA的表达。结果在CGNs撤钾凋亡模型中促凋亡基因Hrk mRNA转录上调;JNK通路抑制剂CEP-11004或SP600125均部分地下调了Hrk的转录;负显性c-Jun突变体抑制了Hrk mRNA的表达。结论在CGNs撤钾凋亡模型中促凋亡基因Hrk转录上调;JNK/c-Jun信号通路介导了Hrk的转录。

Objective To investigate the transcriptional expression of proapoptotic gene Hrk in potassium deprivation-induced apoptosis in cerebellar granule neurons（CGNs）,and to explore the role of JNK/c-Jun pathway in the regulation of Hrk transcription.Methods The CGNs dissociated from the 7-day-old Sprague-Dawley rat pups were cultured in basal medium Eagle（BME）containing 10% fetal bovine serum and 25 mmol/L KCl in vitro.CGNs were switched into serum-free medium containing 25 or 5 mmol/L KCl for 2,4 or 8 h,or treated with 5 mmol/L KCl in the presence of JNK pathway inhibitors for 4 h at the 7th day,or transfected with an adenovirus vector expressing Dominant-negative c-Jun（Ad-FLAGΔ169）for 36 h at the 5th day and followed by 5 mmol/L KCl treatment for 4 h.Reverse transcriptase-PCR was applied to detect the expression of Hrk mRNA of each group.Results The level of proapoptotic gene Hrk transcription was up-regulated significantly in potassium deprivation-induced apoptosis in CGNs.The expression of Hrk was incompletely blocked by the inhibitors of JNK signaling pathway,and obviously inhibited by Ad-FLAGΔ169.Conclusion The transcription of Hrk is elevated in CGNs undergoing potassium deprivation-induced apoptosis and mediated by JNK/c-Jun pathway.