上海交通大学学报（医学版）

上海交通大学学报（医学版）

• 论著（基础研究） • 上一篇    下一篇

蛋白激酶A部分通过环磷腺苷效应元件结合蛋白信号防止足细胞凋亡

向芃，谢可炜，姜金星，倪兆慧，顾乐怡   

  1. 上海交通大学 医学院附属仁济医院肾脏科 分子细胞肾病实验室, 上海 200127
  • 出版日期:2016-01-28 发布日期:2016-02-26
  • 通讯作者: 顾乐怡, 电子信箱: guleyi@aliyun.com。
  • 作者简介:向芃（1990—）， 女， 硕士生; 电子信箱: 744040378@qq.com。
  • 基金资助:

    国家自然科学基金（30971363， 81270781）

Prevention of apoptosis of podocytes by protein kinase A via cAMP response element binding protein signaling pathway

XIANG Peng, XIE Ke-wei, JIANG Jin-xing, NI Zhao-hui, GU Le-yi   

  1. Department of Nephrology, Molecular Cell Laboratory for Kidney Disease, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200127, China
  • Online:2016-01-28 Published:2016-02-26
  • Supported by:

    National Natural Science Foundation of China, 30971363， 81270781。

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摘要：

目的 探讨转录因子环磷腺苷效应元件结合蛋白（CREB）在蛋白激酶A（PKA）信号防止足细胞损伤中的作用。方法 使用条件永恒的小鼠足细胞株（5P12）14~25代分化的足细胞进行研究，细胞分为对照组、阿霉素（ADR）处理组（ADR组）和PKA特异性激动剂(pCPT-cAMP)预孵育+ADR刺激组（pCPT-cAMP+ADR组）。CCK-8法检测细胞毒性； Western blotting检测足细胞内被切割的半胱氨酸蛋白酶-3（cleaved caspase-3）表达；siRNA抑制细胞内CREB表达，免疫荧光共聚焦显微镜和Western blotting检测磷酸化CREB（p-CREB）的定位和表达。结果 ADR诱导足细胞内cleaved caspase-3表达升高，pCPT-cAMP预孵育可显著降低ADR诱导的cleaved caspase-3表达上升。ADR使足细胞减少为原来的（40.45±6.78）%，pCPT-cAMP预孵育使细胞数量恢复为原来的（69.8±5.48）%。Western blotting检测结果显示：pCPT-cAMP分别预孵育5、15 min可使足细胞总蛋白中p-CREB表达分别上升（105.64±38.21）%和(98.61±41.03)%，细胞核内p-CREB的表达上升（114.52±11.28）%和（118.84±14.44）%。免疫荧光共聚焦显微镜检测显示pCPT-cAMP主要引起细胞核内p-CREB表达上升。siRNA可使足细胞CREB蛋白表达下降为原来的（25.1±2.44）%，且pCPT-cAMP预孵育不能防止ADR诱导的cleaved caspase-3表达升高。结论 转录因子CREB介导了PKA信号对足细胞的保护作用。

关键词: 足细胞, 环磷酸腺苷, 蛋白激酶 A, 环磷腺苷效应元件结合蛋白, 凋亡

Abstract:

Objective To explore the effects of cAMP response element binding protein (CREB) signaling pathway on the protection of podocytes by the protein kinase A (PKA). Methods The 14-25 generations of mouse podocytes 5P12 differentiated under constant condition were selected and divided into the control group, adriamycin (ADR) treatment group (ADR group), and PKA specific agonist (pCPT-cAMP) pre-culture+ADR stimulation group (pCPT-cAMP+ADR group). Cell toxicity was detected by CCK-8 method. The expression of cleaved caspase-3 in podocytes was detected by Western blotting. The expression of CREB in podocytes was inhibited by siRNA and the localization and expression of phosphorylated CREB (p-CREB) were detected by immunofluorescence confocal microscopy and Western blotting. Results ADR induced up-regulation of the expression of cleaved caspase-3 in podocytes and pre-culture by pCPT-cAMP could significantly decrease the elevated expression of cleaved caspase-3 induced by ADR. ADR decreased the number of podocytes to (40.45±6.78)% of its original number and pre-culture by pCPT-cAMP increased the number of podocytes to (69.8±5.48)% of its original number. Results of Western blotting showed that pre-culture by pCPT-cAMP for 5 and 15 min could increase the expressions of p-CREB in total protein of podocytes by (105.64±38.21)% and (98.61±41.03)% and those in nuclei by (114.52±11.28)% and (118.84±14.44)%. Immunofluorescence confocal microscopy showed that pCPT-cAMP mainly increased the expression of p-CREB in nuclei. siRNA could decreased the protein expression of CREB in podocytes to (25.1±2.44)% of its original expression and pre-culture by pCPT-cAMP could not prevent the ADR-induced up-regulation of the expression of cleaved caspase-3. Conclusion Transcription factor CREB mediates the protective effect of PKA signaling pathway on podocytes.

Key words: podocyte, cyclic adenosine monophosphate, protein kinase A, cAMP response element binding protein, apoptosis