NLRP7 contributes to in vitro decidualization of endometrial stromal cells

Abstract

Background: Nucleotide-binding oligomerization domain (NACHT), leucine rich repeat (LRR) and pyrin domain (PYD) 7 containing protein, NLRP7, is a member of the NLR family which serves as innate immune sensors. Mutations and genetic variants of NLRP7 have been found in women with infertility associated conditions, such as recurrent hydatidiform mole, recurrent miscarriage, and preeclampsia. Decidualization of endometrial stromal cells is a hallmark of tissue remodeling to support embryo implantation and proper placental development. Given defective decidualization has been implicated in miscarriage as well as preeclampsia, we aimed to explore the link between the NLRP7 gene and decidualization.

Methods: Endometrial samples obtained from pregnant women in the first trimester and non-pregnant women were used to study NLRP7 expression pattern. The human telomerase reverse transcriptase (hTERT)-immortalized human endometrial stromal cells (T-HESCs) were used to study the effect of NLRP7 on decidualization. Decidualization of T-HESCs was induced with 1 μM medroxyprogesterone acetate (MPA) and 0.5 mM 8-bromoadenosine 3':5'-cyclic monophosphate (8-Br-cAMP). siRNA was used to knock down NLRP7 while lentiviral vectors were used to overexpress NLRP7 in cells. NLRP7 expression was detected by immunofluorescence, qRT-PCR, and Western blotting. Decidualization markers, Insulin-like growth factor-binding protein 1 (IGFBP-1) and prolactin (PRL), were detected by qRT-PCR and ELISA. Nuclear translocation of NLRP7 was detected by the subcellular fractionation and confocal microscopy. The effect of NLRP7 on progesterone receptor (PR) activity was evaluated by a reporter system.

Results: NLRP7 was up-regulated in the decidual stromal cells of human first-trimester endometrium. After in vitro decidualization, T-HESCs presented with the swollen phenotype and increased expressions of IGFBP-1 and PRL. Knockdown or over-expression of NLRP7 reduced or enhanced the decidualization, respectively, according to the expression level of IGFBP-1. NLRP7 was found to translocate in the nucleus of decidualized T-HESCs and able to promote PR activity.

Conclusions: NLRP7 was upregulated and translocated to the nucleus of the endometrial stromal cells in an in vitro decidualization model. Overexpressed NLRP7 promoted the IGFBP-1 expression and PR reporter activation. IGFBP-1 expression decreased with the knockdown of NLRP7. Therefore, we suggest that NLRP7 contributes to in vitro decidualization of endometrial stromal cells.

Keywords: Decidualization; Endometrial stromal cells; NLRP7; Progesterone receptor.

Fig. 1

NLRP7 expressed in the decidualized stromal cells of the human endometrium during the first trimester. The tissue sections of the non-pregnant endometrium (n = 5) or the first trimester endometrium (n = 5) were deparaffinized, rehydrated and stained with NLRP7 antibody. Representative images of NLRP7 immunohistochemistry in the endometrium are shown. The NLRP7 signal was developed with the anti-rabbit HRP antibody and AEC substrate. Staining with the 2nd antibody only served as the negative control. Arrows point to the endometrial stromal cells. NLRP7 dominantly appeared in the swollen decidualized stromal cells of pregnant endometrium, but not in the stromal cells of non-pregnant endometrium (magnification 200X)

Fig. 2

Increasing NLRP7 expression with in-vitro decidualization of T-HESCs. T-HESCs were treated with 1 μM MPA and 0.5 mM 8-Br-cAMP on day 0 and day 3 to induce decidualization. Untreated cells served as the control. a The cell morphology was evaluated using microscopy. The PRL and IGFBP-1 transcripts were detected by qRT-PCR on day 3 and day 6. The treated cells displayed a swollen phenotype and produced more transcripts of two decidual markers, compared with the untreated cells. b The NLRP7 transcript and protein in the treated or untreated cells was detected by qRT-PCR and Western blotting, respectively, on day 3 and day 6. The NLRP7 transcript amount is shown in fold changes. Both NLRP7 transcript amount and protein level were significantly higher in treated cells than in untreated cells. In the treated cells, the NLRP7 transcript and protein level were also higher on day 6 than on day 3. The expressions of both NLRP7 isoforms were higher in the treated cells. The statistical differences were calculated from three independent experiments. p-values less than 0.05 are marked with “*”

Fig. 3

The expression level of NLRP7 is positively correlated with the decidualization degree of T-HESCs. a T-HESCs transfected with siNLRP7 or negative siCtrl were subjected to in-vitro decidualization. The NLRP7 transcript and protein were analyzed on day 6. The total transcript as well as the short isoform of NLRP7 protein was suppressed by siRNA. Less abundant IGFBP-1 was produced by T-HESCs (siNLRP7), compared to T-HESCs (siCtrl). b T-HESCs infected with LV-NLRP7 or LV-RFP were subjected to in-vitro decidualization. T-HESCs (LV-NLRP7) had a significantly higher expression level of NLRP7 than T-HESCs (LV-RFP), as detected by Western blotting. The IGFBP-1 level was significantly higher in T-HESCs (LV-NLRP7), compared to T-HESCs (LV-RFP). The statistics were calculated from three individual experiments. p-values less than 0.05 are marked with “*”

Fig. 4

The location of NLRP7 protein in the decidualized human endometrial stromal cells. a The proteins of NLRP7, Lamin B, and α-tubulin in different subcellular fractions were analyzed by Western blotting. The short isoform of NLRP7 protein abundantly presents in the nuclear fraction after decidualization is induced. C: cytoplasmic protein; M: membrane protein; N: nuclear soluble protein; CB: chromatin-bound protein; CP: cytoskeletal protein. b The immunofluorescence of NLRP7. The co-localized fluorescent signals of NLRP7 and DAPI are observed under a confocal fluorescent microscope. The cytoplasmic juxtanuclear aggregates of NLRP7 are observed in un-decidualized T-HESCs. Intense NLRP7 signals appear in the nucleus after induced decidualization. DAPI was used as the counter stain

Fig. 5

NLRP7 promoted progesterone receptor activity. T-HESCs were co-transfected with PR reporter and NLRP7-expressed vector or RFP-expressed vector. Cells co-transfected with control reporter and NLRP7-expressed vector or RFP-expressed vector served as the control. The transfected cells were subjected to in-vitro decidualization. After 24 h, the firefly and Renilla luminescence were detected. The results are shown as the relative ratio of PR reporter activity to control reporter activity. NLPR7-expressed PR reporter group has higher progesterone receptor activity than that of RFP-expressed group. The statistics were calculated from three individual experiments. p-values less than 0.05 are marked with “*”