nm23 regulates decidualization through the PI3K-Akt-mTOR signaling pathways in mice and humans

Abstract

Study question: Does nm23 have functional significance in decidualization in mice and humans?

Summary answer: nm23 affects decidualization via the phosphoinositide 3 kinase/mammalian target of rapamycin (PI3K-Akt-mTOR) signaling pathways in mouse endometrial stromal cells (ESCs; mESCs) and human ESCs.

What is known already: The function of nm23 in suppressing metastasis has been demonstrated in a variety of cancer types. nm23 also participates in the control of DNA replication and cell proliferation and differentiation.

Study design, size and duration: We first analyzed the expression profile of nm23 in mice during early pregnancy (n = 6/group), pseudopregnancy (n = 6/group) and artificial decidualization (n = 6/group) and in humans during the menstrual cycle phases and the first trimester. We then used primary cultured mESCs and a human ESC line, T-HESC, to explore the hormonal regulation of nm23 and the roles of nm23 in in vitro decidualization, and as a possible mediator of downstream PI3K-Akt-mTOR signaling pathways.

Participants/materials, settings and methods: We evaluated the dynamic expression of nm23 in mice and humans using immunohistochemistry, western blot and real-time quantitative RT-PCR (RT-qPCR). Regulation of nm23 by steroid hormones was investigated in isolated primary mESCs and T-HESCs by western blot. The effect of nm23 knockdown (using siRNA) on ESC proliferation was analyzed by 5-ethynyl-2'-deoxyuridine staining (EdU) and proliferating cell nuclear antigen protein (PCNA) expression. The influence of nm23 expression on the differentiation of ESCs was determined by RT-qPCR using the mouse differentiation markers decidual/trophoblast PRL-related protein (dtprp, also named prl8a2) and prolactin family 3 subfamily c member 1 (prl3c1) and the human differentiation markers insulin-like growth factor binding protein 1 (IGFBP1) and prolactin (PRL). The effects of nm23 siRNA (si-nm23) and the PI3K inhibitor LY294002 on the downstream effects of nm23 on the PI3K-Akt-mTOR signaling pathway were estimated by western blot.

Main results and the role of chance: NM23-M1 was specifically expressed in the decidual zone during early pregnancy and in artificially induced deciduoma, and NM23-H1 was strongly expressed in human first trimester decidua. The expression of nm23 was upregulated by oestradiol and progesterone (P < 0.05 versus control) in vitro in mESCs and T-HESC, and this was inhibited by their respective receptor antagonists, ICI 182,780 and RU486. Mouse and human nm23 knockdown decreased ESC proliferation and differentiation (P < 0.05 versus control). The PI3K-Akt-mTOR signaling pathways were downstream mediators of nm23 in mESCs and T-HESCs decidualization.

Limitations and reasons for caution: Whether the nm23 regulates decidualization via the activation of AMPK, RAS, PKA, STAT3 or other signaling molecules remains to be determined. The role of nm23 in decidualization was tested in vitro only.

Wider implications of the findings: Results demonstrate that nm23 plays a vital role in decidualization in mice and humans and that nm23 gene expression is hormonally regulated. The downregulation of nm23 in decidua during the first trimester may be associated with infertility in women.

Study funding/competing interests: This study was supported by the National Natural Science Foundation of China (grant nos. 81370731, 31571551 and 31571190), the Science and Technology Project of Chongqing Education Committee (KJ130309), open funding by the Chongqing Institute for Family Planning (1201) and the Excellent Young Scholars of Chongqing Medical University (CQYQ201302). The authors have no conflicts of interest to declare.

Keywords: decidualization; differentiation; mammalian target of rapamycin pathway; nm23; phosphoinositide 3 kinase; proliferation.