Osteoprotegerin interacts with syndecan-1 to promote human endometrial stromal decidualization by decreasing Akt phosphorylation
Authors: Jiang Yufei , Li Jianing , Li Gaizhen , Liu Songting , Lin Xinjie , He Yan , Lu Jinhua , Zhang Ying , Wu Jinxiang , Yang Zhiping , Jiang Yaling , Wang Haibin , Kong Shuangbo , Shi Guixiu 
Published: August 2020

Abstract

STUDY QUESTION
Does osteoprotegerin (OPG) promote human endometrial stromal decidualization?

SUMMARY ANSWER
OPG is essential for human endometrial stromal decidualization through its interaction with syndecan-1 to decrease Akt phosphorylation.

WHAT IS KNOWN ALREADY
OPG (a cytokine receptor) levels are significantly increased in the circulation of pregnant women. However, the role and mechanism of OPG in human endometrial stromal cell (ESC) decidualization remain elusive.

STUDY DESIGN, SIZE, DURATION
We analyzed the endometrial expression of OPG in endometrial tissue samples collected from women with regular menstrual cycles (ranging from 25 to 35 days), and decidual tissue samples collected from woman with normal early pregnancy or recurrent pregnancy loss (RPL) who visited the Department of Gynecology and Obstetrics at a tertiary care center from January to October 2018. None of the subjects had hormonal treatment for at least 3 months prior to the procedure. In total, 16 women with normal early pregnancy and 15 with RPL were selected as subjects for this study. The function of OPG in decidualization was explored in a human endometrial stromal cell (HESC) line and primary cultures of HESCs.

PARTICIPANTS/MATERIALS, SETTING, METHODS
We collected endometrial tissues (by biopsy) from the subjects during their menstrual cycle and decidual tissues from subjects with a normal early pregnancy and those with RPL at the time of dilation and curettage. The control group comprised randomly selected women who underwent termination of an apparently normal early pregnancy. The endometrial OPG expression was analyzed using immunohistochemical staining and quantitative RT–PCR (qRT–PCR). Immunofluorescence staining and western blot, and qRT–PCR were used to explore the mRNA and protein expression, respectively, of OPG in an immortalized HESC line and in primary cultures of HESC during proliferation and decidualization. siRNA-mediated knockdown experiments were performed to examine the function of OPG in HESC proliferation and decidualization. Flow cytometry and the cell proliferation MTS assay were performed to further examine the role of OPG in HESC proliferation. We also analyzed decidual marker gene expression by qRT–PCR to assess the consequences of OPG loss for HESC decidualization. A co-immunoprecipitation (IP) assay was used to determine the potential interaction between the OPG and Syndecan-1. Western blot analysis of the rescue experiments performed using the phosphatidylinositol 3-kinase (PI3K) signaling-specific inhibitor LY294002 was used to investigate the downstream signaling pathways through which OPG could mediate HESC decidualization.

MAIN RESULTS AND THE ROLE OF CHANCE
OPG was expressed in both the human endometrium and in vitro decidualized ESCs. Knockdown experiments revealed that OPG loss impaired the expression of IGF-binding protein-1 (IGFBP-1) (P &lt; 0.05) and prolactin (PRL) (P &lt; 0.05), two specific markers of decidualization, in HESC undergoing decidualization. We also uncovered that OPG knockdown induced the aberrant activation of Akt (protein kinase B) during HESC decidualization (P &lt; 0.05). The inhibition of Akt activation could rescue the impaired expression of the decidual markers PRL (P &lt; 0.05) and IGFBP-1 (P &lt; 0.05) in response to OPG knockdown. Syndecan-1 was considered a potential receptor candidate, as it was expressed in both the endometrium and in vitro cultured stromal cells. Subsequent co-IP experiments demonstrated the interaction between OPG and Syndecan-1 during decidualization. In addition, Syndecan-1 knockdown not only clearly attenuated the decidualization markers PRL (P &lt; 0.05) and IGFBP-1 (P &lt; 0.05) but also induced the aberrant enhancement of Akt phosphorylation in decidualized cells, consistent with the phenotype of OPG knockdown cells. Finally, we revealed that the transcript and protein expression of both OPG and Syndecan-1 was significantly lower in the decidual samples of women with RPL than in those of women with normal pregnancy (P &lt; 0.05).

LARGE SCALE DATA
N/A

LIMITATIONS, REASONS FOR CAUTION
In this study, based on a number of approaches, it was demonstrated that OPG mediated the repression of Akt that occurs during human stromal cell decidualization, however, the molecular link between OPG and Akt signaling was not determined, and still requires further exploration.

WIDER IMPLICATIONS OF THE FINDINGS
OPG is required for decidualization, and a decrease in OPG levels is associated with RPL. These findings provide a new candidate molecule for the diagnosis and potential treatment of RPL.

STUDY FUNDING/COMPETING INTEREST(S)
This work was supported in part by the National Natural Science Foundation of China U1605223 (to G.S.), 81701457 (to Y.J.) and 81601349 (to Y.J.). The authors have no conflicts of interest to disclose.