The Regulation of Trophoblastic p53 Homeostasis by the p38-Wip1 Feedback Loop is Disturbed in Placentas from Pregnancies Complicated by Preeclampsia.

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. METHODS: 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. RESULTS: 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-p53Ser15 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. CONCLUSION: 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.