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. 2020 May 15;26(5):327-339.
doi: 10.1093/molehr/gaaa021.

Abnormal Cullin1 neddylation-mediated p21 accumulation participates in the pathogenesis of recurrent spontaneous abortion by regulating trophoblast cell proliferation and differentiation

Xiaohe Sun  1 , Xiaomei Tong  1 , Yanqing Hao  1 , Chao Li  1 , Yinli Zhang  1 , Yibin Pan  1 , Yongdong Dai  1 , Liu Liu  1 , Tai Zhang  1 , Songying Zhang  1
Affiliations

Abnormal Cullin1 neddylation-mediated p21 accumulation participates in the pathogenesis of recurrent spontaneous abortion by regulating trophoblast cell proliferation and differentiation

Xiaohe Sun et al. Mol Hum Reprod. .

Abstract

The study explores the role of neddylation in early trophoblast development and its alteration during the pathogenesis of recurrent spontaneous abortion (RSA). Immunofluorescence and western blot were conducted to evaluate the expression pattern of NEDD8 protein in the first-trimester placentas of healthy control and RSA patients. Neddylated-cullins, especially neddylated-cullin1, were downregulated and their substrate, p21, was accumulated in RSA samples. NEDD8 cytoplasmic recruitment was observed in extravillous trophoblast (EVT) progenitors of RSA placentas. Consistent with the results of clinical samples, neddylation inhibition using MLN4924 in trophoblast cell lines caused obvious p21 accumulation and free NEDD8 cytoplasmic recruitment VSports手机版. Further in vitro study demonstrated neddylation inhibition attenuated proliferation of Jeg-3 cells via p21 accumulation. Moreover, when trophoblast stem (TS) cells derived from first-trimester placentas were cultured for differentiation analyses. MLN4924 impaired the differentiation of TS cells towards EVTs by downregulating HLA-G and GATA3. p21 knockdown could partly rescue MLN4924-suppressed HLA-G and GATA3 expression. In conclusion, cullin1 neddylation-mediated p21 degradation is required for trophoblast proliferation and can affect trophoblast plasticity by affecting HLA-G and GATA3 expression. The results provide insights into the pathological mechanism of RSA and the biological regulation of trophoblast development. .

Keywords: NEDD8; neddylation; p21; recurrent spontaneous abortion; trophoblast stem cell V体育安卓版. .

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Figure 1
Figure 1. Expression pattern of NEDD8 in first trimester placentas and change in subcellular localization of NEDD8 protein in recurrent spontaneous abortion (RSA) placentas. (A–B) Nedd8 immunofluorescence (IF) in first trimester placentas: sections of healthy control (HC) (n = 4) and RSA (n = 4) early pregnancy placentas from 6–8-week pregnancies were stained with antibodies against NEDD8 (red), HGCβ (green) or HLA-G (green). Nuclei were stained with DAPI. For negative controls, primary antibodies were replaced with rabbit monoclonal isotype IgG (mAb IgG). VS, villus stroma; CTB, cytotrophoblast; STB, syncytiotrophoblast; pCCT: proximal cell column trophoblast; dCCT: distal cell column trophoblast. Scale bars: 50 μm. (A) Top: representative images of NEDD8 staining in the first-trimester floating villus. HCGβ (green) was costained to indicate STB. Bottom: representative images of NEDD8 staining in the first trimester anchoring villus. HLA-G (green) was costained to indicate dCCTs. (B) Representative images of NEDD8 subcellular localization of the anchoring villus of HC and RSA first trimester placentas. (C) Scatter plot of nuclear/cytoplasmic ratio of NEDD8 measured by ImageJ. Sixty single nuclear HLA-G (+) cells from four first trimester villus were analyzed for each group. Means ± interquartile range (IQR) are shown. ****P < 0.0001.
Figure 2
Figure 2. Neddylation inhibition causes cytoplasmic recruitment of NEDD8 in trophoblasts. (A) Representative western blotting (WB) of neddylated cullins (NEDD8-Cullins), Cullin1 (CUL1) and p21 in the recurrent spontaneous abortion (RSA) early pregnancy villus (n = 6) compared with healthy controls (HCs) (n = 5). Bar graph on the left is quantification of Nedd8 and p21. Means ± SD normalized to β-actin are shown. **P = 0.0013; *P = 0.0225. (B) Representative WB of Jeg-3 cell line treated with MLN4924 at increasing concentrations. (C–F) Jeg-3 cells were treated with 1 μM MLN4924 for 48 h. (C) Representative WB of NEDD8-Cullins (90 kD) and free NEDD8 (12 kD). WCL: whole cell lysis. (D) RT-PCR analysis of NEDD8 mRNA expression in Jeg-3 cells after MLN4924 treatment. Means ± SD normalized to 18 s rRNA are shown. (E) Representative WB of Jeg-3 cells separated into cytoplasmic and nuclear fractions. (F) Representative immunofluorescence (IF) images and nuclear/cytoplasmic ratio analysis showing the subcellular localization change of NEDD8 after MLN4924 treatment. Scale bars: 50 μm. Nuclear/cytoplasm ratio was calculated from 30 single cells for each group of three independent assays. Means ± IQR are shown. ****P < 0.0001.
Figure 3
Figure 3. Proliferation restriction in recurrent spontaneous abortion (RSA) placentas is related to p21 accumulation caused by neddylation inhibition. (A) Representative immunohistochemical staining of Ki67 in healthy control (HC) and RSA floating and anchoring villus. n = 4 for each group. Scale bars: 50 μm. Bar graph shows the means ± SD of the HSCORE. ****P < 0.0001. (B) Representative images of HC villus explants culture (4 days) after MLN4924 (1 μM) or DMSO treatment for 48 h. Scale bars: 500 μm. Bar graph on the left is quantification of the villus outgrowth distance. Fifteen explants for each condition derived from four different HC placentas were measured. Means ± SD are shown. ***P = 0.006. (C–F) Jeg-3 cells were treated with 1 μM MLN4924 or DMSO for 48 h following p21 silencing: (C) western blot (WB) and (D) RT-PCR results showing p21 knockdown efficiency after siRNA transfection. Bar graph of the mRNA level shows means ± SD normalized to 18 s rRNA. *P = 0.0266; ***P = 0.001. (E) CCK8 assay measuring the proliferation rate of Jeg-3 cells. (F) IF staining of Ki67 (red) and p-H3 (red). Nuclei were stained with DAPI. Scale bars: 50 μm. (G) Jeg-3 cells were synchronized overnight before treated with 1 μM MLN4924 or DMSO for 48 h following p21 silencing. The cell cycle distributions were analyzed by flow cytometry. Bar graph on the left indicates the percentage of cells in G1, S or G2/M phase. Means ± SD are shown, t test was conducted to compare the G2 phase ratio between each group. **P = 0.0023; *P = 0.00187.
Figure 4
Figure 4. Neddylation inhibition hinders trophoblast stem cell differentiation toward extravillous trophoblasts (EVTs). (A) Phase-contrast image of trophoblast stem (TS) cells. Scale bar: 100 μm. (B) Representative immunofluorescence (IF) images showing CK7 (trophoblast marker), CDH1 (CTB marker) and VIM (vimentin, a stromal marker) in trophoblast stem cells (TS). Scale bars: 50 μm. (C) Flow cytometry histogram of CK7 and IGTA6 expression in TS cells. (D–F) TS cells, TS cells treated with MLN4924, differentiating EVT-Day 5 (EVT-D5) and EVT-D5 treated with MLN4924 (EVT-D5 + MLN4924) were processed for further analysis: (D) RT-PCR analysis of HLA-G (***P = 0.001; ****P < 0.0001), MMP2 (***P = 0.001; ****P < 0.0001), ITGA1 (****P < 0.0001; *P = 0.0419), GATA3 (****P < 0.0001; ***P = 0.0003; *P = 0.0194) and CDH1 (****P < 0.0001; ***P = 0.0006) mRNA expression. Means ± SD normalized to 18 s rRNA are shown. (E) Representative western blot (WB) results and quantification (n = 3) showing the protein expression levels of HLA-G (***P = 0.0002; *P = 0.0211), CHD1 (***P = 0.0008; *P = 0.0102; **P = 0.0049) and GATA3 (****P < 0.0001; *P = 0.0211). NEDD8-Cullins were regarded as an indicator of Neddylation inhibition. Mean values ± SD normalized to β-actin are shown. (F) Representative IF images showing expression of HLA-G, GATA3 and CDH1. Scale bars: 20 μm.
Figure 5
Figure 5. HLA-G and GATA3 downregulation caused by MLN4924 is related to p21 accumulation. (A) Representative western blot (WB) results of HLA-G and GATA3 proteins in Jeg-3 cells after treatment with various concentration of MLN4924 for 48 h. (B) Representative WB results of HLA-G, GATA3 and p21 expression in Jeg-3 cells after MLN4924 treatment for indicated hours. (C) Representative WB results of Jeg-3 cells after CUL1 silencing. (D) Representative phase contrast image of SA-β-GAL staining of senescent Jeg-3 cells. Jeg-3 cells were treated with 1 μM MLN4924 or DMSO following p21 silencing. Scale bars: 50 μm. (E) Representative WB of Jeg-3 cells separated into cytoplasmic and nuclear fractions after MLN4924 treatment for 48 h. (F) Representative WB results of Jeg-3 cells after p21 silencing followed by MLN4924 treatment for 24 h.
Figure 6
Figure 6. Schematic of the present study. Neddylation inhibition in recurrent spontaneous abortion (RSA) trophoblasts causes cytoplasmic retention of free NEDD8 and p21 accumulation, thereby causing proliferation restriction and plasticity impairment.
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References

    1. Baek KH, Lee EJ, Kim YS. Recurrent pregnancy loss: the key potential mechanisms. Trends Mol Med 2007;13:310–317. - PubMed
    1. Bischof P, Irminger-Finger I. The human cytotrophoblastic cell, a mononuclear chameleon. Int J Biochem Cell Biol 2005;37:1–16. - PubMed (VSports app下载)
    1. Brownell JE, Sintchak MD, Gavin JM, Liao H, Bruzzese FJ, Bump NJ, Soucy TA, Milhollen MA, Yang X, Burkhardt AL et al. . Substrate-assisted inhibition of ubiquitin-like protein-activating enzymes: the NEDD8 E1 inhibitor MLN4924 forms a NEDD8-AMP mimetic in situ. Mol Cell 2010;37:102–111. - PubMed
    1. Carrington B, Sacks G, Regan L. Recurrent miscarriage: pathophysiology and outcome. Curr Opin Obstet Gynecol 2005;17:591–597. - PubMed
    1. Chen P, Hu T, Liang Y, Li P, Chen X, Zhang J, Ma Y, Hao Q, Wang J, Zhang P et al. . Neddylation inhibition activates the extrinsic apoptosis pathway through ATF4-CHOP-DR5 Axis in human esophageal cancer cells. Clin Cancer Res 2016;22:4145–4157. - VSports手机版 - PubMed

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