Tan Bin, Tong Chao, Yuan Yu, Xu Ping, Wen Li, Zhang Chen, Zheng Yangxi, Lin Li, Zhu Fangyu, Gui Shunping, Wang Lianlian, Gao Rufei, Li Jie, Qi Hongbo, Baker Philip N
Department of Obstetrics, The First Affiliated Hospital of Chongqing Medical University, Chongqing, China.
International Collaborative Laboratory of Reproduction and Development, Ministry of Education, Chongqing, China.
Cell Physiol Biochem. 2019;52(2):315-335. doi: 10.33594/000000023. Epub 2019 Feb 28.
BACKGROUND/AIMS: Excessive apoptosis of trophoblasts, induced by sustained hypoxia, leads to abnormal placentation and is strongly linked to pregnancy complications such as preeclampsia (PE). Wild-type p53-induced phosphatase (Wip1) positively regulates cellular survival in tumor cells through the p38 and p53 pathways, but its expression pattern and effects in trophoblasts have yet to be reported. This study clarified the effect of Wip1 on the regulatory mechanism of p53-dependent apoptosis in trophoblasts, and thus increases understanding of the etiology of PE.
In normal and PE placentas, Wip1 mRNA and protein levels were determined by RT-qPCR and Western blotting respectively, while localization of Wip1 in placental tissues and in HTR8/SVneo cells was determined by immunohistochemistry and immunofluorescence. Two in vitro trophoblastic PE models were established by subjecting HTR8/SVneo cells to either hypoxia intervention in incubator (HII) or simulated ischemic buffer (SIB). Wip1 was suppressed in the aforementioned PE models by specific inhibitor or shRNA, and apoptosis was then assessed by flow cytometry, while further validation was done by measurement of cleaved-caspase 9 expression by Western blotting. The p38 inhibitor SB202190, Mdm2 inhibitor NVP-CGM097, and proteasome inhibitor MG-132 were administered in PE models, either in combination or alone, to determine the regulatory order of the component signal molecules of the feedback loop. The impact of Wip1 on p53-Mdm2 interaction was examined by coimmunoprecipitation. Lastly, the upregulation of the p38-Wip1 loop was confirmed in human placentas from pregnancies complicated by PE, using Western blotting.
Wip1 expression was significantly elevated in human PE placentas and in vitro trophoblastic PE models; this is opposite to the pattern observed in tumor cells. Inhibition of Wip1 rescued hypoxia-induced p38 activation, cleavage of caspase 9 and apoptosis but significantly compromised p53-Mdm2 binding, while p-p53 was increased. Inhibition of Mdm2 degradation resulted in p53 destabilization and p38-Wip1 loop down-regulation, while degradation of the p53-Mdm2 complex resulted in p53 accumulation and p38-Wip1 loop hyperactivation. However, the p53-Mdm2 interaction was found to be more important in the regulation of the p38-Wip1 loop than Mdm2 stability.
Trophoblastic p53 homeostasis is maintained by the p38-Wip1 feedback regulatory loop in response to hypoxic stress, which is dysregulated in the placentas of pregnancies complicated by PE, and thereby leads to excessive apoptosis.
背景/目的：持续缺氧诱导的滋养层细胞过度凋亡会导致胎盘形成异常，并与子痫前期（PE）等妊娠并发症密切相关。野生型p53诱导磷酸酶（Wip1）通过p38和p53途径正向调节肿瘤细胞的存活，但它在滋养层细胞中的表达模式和作用尚未见报道。本研究阐明了Wip1对滋养层细胞中p53依赖性凋亡调节机制的影响，从而加深了对PE病因的理解。
分别通过RT-qPCR和蛋白质免疫印迹法检测正常胎盘和PE胎盘中Wip1 mRNA和蛋白质水平，同时通过免疫组织化学和免疫荧光法确定Wip1在胎盘组织和HTR8/SVneo细胞中的定位。通过在培养箱中对HTR8/SVneo细胞进行缺氧干预（HII）或模拟缺血缓冲液（SIB）建立两种体外滋养层细胞PE模型。在上述PE模型中通过特异性抑制剂或短发夹RNA（shRNA）抑制Wip1，然后通过流式细胞术评估细胞凋亡，同时通过蛋白质免疫印迹法检测裂解的半胱天冬酶9表达进行进一步验证。在PE模型中联合或单独给予p38抑制剂SB202190、Mdm2抑制剂NVP-CGM097和蛋白酶体抑制剂MG-132，以确定反馈环中信号分子组分的调节顺序。通过免疫共沉淀检测Wip1对p53-Mdm2相互作用的影响。最后，通过蛋白质免疫印迹法在患有PE的妊娠妇女的人胎盘中证实p38-Wip1环的上调。
Wip1在人PE胎盘和体外滋养层细胞PE模型中的表达显著升高；这与在肿瘤细胞中观察到的模式相反。抑制Wip1可挽救缺氧诱导的p38激活、半胱天冬酶9的裂解和细胞凋亡，但显著损害p53-Mdm2结合，而磷酸化p53增加。抑制Mdm2降解导致p53不稳定和p38-Wip1环下调，而p53-Mdm2复合物的降解导致p53积累和p38-Wip1环过度激活。然而，发现p53-Mdm2相互作用在p^38-Wip1环的调节中比Mdm2稳定性更重要。
滋养层细胞中的p53稳态通过p38-Wip1反馈调节环来维持，以应对缺氧应激，而在患有PE妊娠的胎盘中该调节环失调，从而导致过度凋亡。
