Progesterone-mediated remodeling of the maternal-fetal interface by a PGRMC1-dependent mechanism

Abstract

Implantation and maintenance of pregnancy involve intricate immunological processes that enable the developing fetus to coexist with the maternal immune system. Progesterone, a critical hormone during pregnancy, is known to promote immune tolerance and prevent preterm labor. However, the mechanism by which progesterone mediates these effects remains unclear. In this study, we investigated the role of the non-classical progesterone receptor membrane component 1 (PGRMC1) in progesterone signaling at the maternal-fetal interface. Using JEG3 cells, a trophoblast model cell line, we observed that progesterone stimulation increased the expression of human leukocyte antigen-C (HLA-C) and HLA-G, key molecules involved in immune tolerance. We also found that progesterone upregulated the expression of the transcription factor ELF3, which is known to regulate trophoblast-specific HLA-C expression. Interestingly, JEG3 cells lacked expression of classical progesterone receptors (PRs) but exhibited high expression of PGRMC1, a finding we confirmed in primary trophoblasts by mining sc-RNA seq data from human placenta. To investigate the role of PGRMC1 in progesterone signaling, we used CRISPR/Cas9 technology to knockout PGRMC1 in JEG3 cells. PGRMC1-deficient cells showed a diminished response to progesterone stimulation. Furthermore, we found that the progesterone antagonist RU486 inhibited ELF3 expression in a PGRMC1-dependent manner, suggesting that RU486 acts as a progesterone antagonist by competing for receptor binding. Additionally, we found that RU486 inhibited cell invasion, an important process for successful pregnancy, and this inhibitory effect was dependent on PGRMC1. Our findings highlight the crucial role of PGRMC1 in mediating the immunoregulatory effects of progesterone at the maternal-fetal interface.

Keywords: ELF3; HLA-C; HLA-G; Immune tolerance; Maternal-fetal interface; Mifepristone; PGRMC1; Pregnancy; Progesterone; RU486; Trophoblasts.

Fig. 1.. Expression of HLA-G, HLA-C, and ELF3 in JEG3 after treatment with progesterone.

(A) Progesterone (P4) dose-escalation. P4 is the standard abbreviation for progesterone and is used throughout the Fig. s. JEG3 cells were stimulated for 4 hrs with the indicated concentrations of P4 (10nM-10uM) and target gene expression (HLA-G, HLA-C, and ELF3) was assessed by qRT-PCR performed in triplicates. Data are reported as mean ± SEM. Unpaired t-test. *p < 0.05; ***p < 0.001. n.s.: not significant. B) Western blot analysis. HLA-C and ELF3 protein levels after 4 hours of low (1 μM) or high dose (10 μM) P4 administration. C) Time course. HLA-G, HLA-C, and ELF3 protein levels in JEG3 increase over the course of 12 hrs following 1uM P4 administration; HSP70 was used as loading control.

Fig. 2.. Generation of PGRMC1 KO JEG3 cells.

(A) Expression of non-classical progesterone receptors in JEG3 trophoblast cell line. RNA seq was performed in triplicates and gene expression is given as FPKM. Error bars represent the mean ± SD. (B) PGRMC1 targeting strategy. CRISPR-Cas9 cutting sites are indicated by scissors. The position of the primers used for PCR screening are indicated by red arrows. (C) PCR Screening identified PGRMC1 KO clones. (D) Western blot confirmation of PGRMC1 KO in WT JEG3 (clone 2C9). HSP70 was used as loading control.

Fig. 3.. Absence of progesterone response in PGRMC1 KO JEG3.

(A) Quantification of HLA-G mRNA levels by qRT-PCR in JEG3 cells after 4 hrs of P4 administration. (B) Stimulation of the PGRMC1 KO JEG3 with P4 failed to upregulate HLA-G expression. qRT-PCR was done in triplicates. Data are reported as the mean ± SEM. Unpaired t-test. ***p < 0.001. n.s: not significant.

Fig. 4.. RU486 inhibition decreased ELF3 expression in JEG3.

Effect of RU486 on HLA-C, and ELF3 expression in JEG3 cells. Cells were treated for 48hrs with the indicated RU486 concentrations (0.1–5uM). (A) qRT-PCR was performed in triplicates using the indicated gene-specific primers. Data are reported as mean ± SEM. Unpaired t-test. ***p < 0.01. (n.s: not significant. B, C) Western blot analyses of RU486 dose escalation in JEG3 treated for 48 hrs. HSP70 was used as a loading control.

Fig. 5.. Progesterone can restore ELF3 levels following RU486 treatment.

High dose progesterone can antagonize the effect of RU486 on ELF3 expression. JEG3 cells were pretreated with 1uM RU486 for 48 hrs (A) and subsequently either treated with ETH as a control, or with progesterone at the indicated concentrations (1 or 10uM P4) for 4 hrs (B) or 8 hrs (C). qRT-PCR; ELF3 transcript levels were normalized to GAPDH expression. Data represents n = 3, independent experiments. ***p < 0.01; n.s: not significant.

Fig. 6.. Transwell invasion of JEG3 is inhibited by RU486 in an PGRMC1-dependent manner.

Crystal violet staining depicting JEG3 WT cells (A), or PGRMC1 KO JEG3 (B) that transgressed an artificial membrane in a transwell migration assay in the presence or absence of 5 uM RU486. The solvent DMSO was used as a negative control. Bar graphs represent quantification of transwell invasion of WT JEG3 or the PGRMC1 KO clone 2C9. n = 3, independent experiments. Graphs show mean ± SEM. ***p < 0.001. n.s: not significant.