上海交通大学学报(医学版)

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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。

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