NLRP7 contributes to in vitro decidualization of endometrial stromal cells

Abstract

BackgroundNucleotide-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.MethodsEndometrial 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.ResultsNLRP7 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.ConclusionsNLRP7 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.

A Gelatin Hydrogel to Study Endometrial Angiogenesis and Trophoblast Invasion

“…HESCs maintained good viability over the course of the study, as indicated by live/dead staining, and no obvious differences were observed in viability for HESCs cultured in HESC growth medium or EGM (Supplemental Figure 1). Taken together, these results demonstrate that decidualization of endometrial stromal cells can be induced via hormonal stimulation in 3D cultures in a manner consistent with the established 2D decidualization assays [30][31][32][33][34].…”

“…We demonstrated that the GelMA hydrogel is capable of supporting endometrial stromal cell differentiation for at least 6 days in response to hormonal cues of 1 μM MPA and 0.5 mM 8-Br-cAMP. This decidualization protocol has been used extensively in literature primarily for 2D cultures [30][31][32]35], though some success has been reported inducing stromal differentiation with hormonal cues in 3D environments formed from thermally gelled collagen or crosslinked polyethylene glycol macromers [17,30,35,55,56]. In our study, stromal cells encapsulated in GelMA hydrogels maintained a decidual phenotype for 6 days in culture, as indicated by their epithelial-like morphology as well as increased secretion of characteristic decidual markers prolactin and IGFBP-1.…”

“…Decidualization was induced by culturing HESCs in the presence of 1 μM medroxyprogesterone acetate (MPA; Sigma-Aldrich M1629) and 0.5 mM 8-Bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP; Sigma-Aldrich B5386) in EGM for 6 days [30][31][32][33][34]. For assays of decidualization in conventional 2D culture, HESCs were seeded in 6 well tissue culture plates at an initial seeding density of 36,000 cells/well.…”

ALK3-SMAD1/5 Signaling Mediates the BMP2-Induced Decrease in PGE2 Production in Human Endometrial Stromal Cells and Decidual Stromal Cells

“…A telomerase-immortalized HESC line was purchased from the American Type Culture Collection (ATCCR CRL-4003TM) and cultured according to the manufacturer’s instructions ( Krikun et al, 2004 ; Huang et al, 2017 ). Briefly, I-HESCs were cultured in phenol red-free Dulbecco’s modified Eagle’s medium/Ham’s F-12 (DMEM/F-12; Sigma-Aldrich Corp, United States) supplemented with 1.5 g/L sodium bicarbonate, 1% ITS + Premix (CORNING, NY, United States), 5 μg/μL puromycin (Invitrogen, Life Technologies, NY) and 10% charcoal-stripped FBS (HyClone, Logan, UT, United States).…”

Human Amnion Epithelial Cells (AECs) Respond to the FSL-1 Lipopeptide by Engaging the NLRP7 Inflammasome

“…NLRP7 is a member of NLRP subfamily and is especially known for the involvement of its mutated forms in recurrent hydatidiform moles ( 52 – 54 ). Several studies have also demonstrated a role for NLRP7 in different mechanisms related to pregnancy, including the in vitro decidualization of endometrial stromal cells ( 55 ), trophoblast lineage differentiation ( 56 ), and trophoblast proliferation, migration, and invasion ( 57 ). Khare et al ( 58 ) who demonstrated an ASC-dependent caspase-1 activation by NLRP7 in response to microbial acetylated lipopeptides, have first characterized the NLRP7 inflammasome.…”