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. 2012 Oct;97(10):3672-83.
doi: 10.1210/jc.2012-2075. Epub 2012 Aug 6.

Down-regulation of S100A11, a calcium-binding protein, in human endometrium may cause reproductive failure

Xin-Mei Liu  1 , Guo-Lian Ding, Ying Jiang, Hong-Jie Pan, Dan Zhang, Ting-Ting Wang, Run-Ju Zhang, Jing Shu, Jian-Zhong Sheng, He-Feng Huang
Affiliations

Down-regulation of S100A11, a calcium-binding protein, in human endometrium may cause reproductive failure

Xin-Mei Liu et al. J Clin Endocrinol Metab. 2012 Oct.

Abstract

Background: Low expression levels of S100A11 proteins were demonstrated in the placental villous tissue of patients with early pregnancy loss, and S100A11 is a Ca2+-binding protein that interprets the calcium fluctuations and elicits various cellular responses.

Objectives: The objective of the study was to determine S100A11 expression in human endometrium and its roles in endometrial receptivity and embryo implantation.

Methods: S100A11 expression in human endometrium was analyzed using quantitative RT-PCR, Western blot, and immunohistochemical techniques. The effects of S100A11 on embryo implantation were examined using in vivo mouse model, and JAr (a human choriocarcinoma cell line) spheroid attachment assays. The effects of endometrial S100A11 on factors related to endometrial receptivity and immune responses were examined. Using a fluorescence method, we examined the changes in cytosolic Ca2+ and Ca2+ release from intracellular stores in epidermal growth factor (EGF)-treated endometrial cells transfected with or without S100A11 small interfering RNA.

Results: S100A11 was expressed in human endometrium. S100A11 protein levels were significantly lower in endometrium of women with failed pregnancy than that in women with successful pregnancy outcomes. The knockdown of endometrial S100A11 not only reduced embryo implantation rate in mouse but also had adverse effects on the expression of factors related to endometrial receptivity and immune responses in human endometrial cells. Immunofluorescence analysis showed that S100A11 proteins were mainly localized in endoplasmic reticulum. The EGF up-regulated endometrial S100A11 expression and promoted the Ca2+ uptake and release from Ca2+ stores, which was inhibited by the knockdown of S100A11.

Conclusions: Endometrial S100A11 is a crucial intermediator in EGF-stimulated embryo adhesion, endometrium receptivity, and immunotolerance via affecting Ca2+ uptake and release from intracellular Ca2+ stores. Down-regulation of S100A11 may cause reproductive failure.

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Figures

Fig. 1.
Fig. 1.
Association of low expression levels of S100A11 with poor pregnancy outcomes in humans and mice. Immunohistochemical analysis showed expression and localization of S100A11 in the glandular and luminal epithelia of human midsecretory endometrium. Aa, Negative control, ×200; Ab, ×100; Ac, ×200; Ad, isotype control. B and C, Expression of S100A11protein in human endometria from infertile women who were stratified into successful (n = 15) and failed pregnancy (n = 23) subgroups according to IVF outcomes. D and E, S100A11 protein levels in endometria of mice injected with scrambled siRNA (n = 5) and specific siRNA targeting S100A11 (n = 5), respectively. F and G, Embryo implantation rate in mice injected with scrambled siRNA (n = 5) and specific siRNA targeted S100A11 (n = 5), respectively. Data are presented as mean ± se. **, P < 0.01 compared with the corresponding controls.
Fig. 2.
Fig. 2.
Effects of S100A11 knockdown in endometrial cells on JAr spheroid attachment and factors related to endometrial receptivity and immune responses. S100A11 mRNA levels (A) and protein (B and C) levels in Ishikawa cells treated with scrambled siRNA or targeted specific siRNA S100A11. D and E, JAr spheroid attachment rate in Ishikawa cells treated with scrambled siRNA and specific siRNA targeting S100A11. Arrow in D indicates the JAr spheroid attached to human endometrial cells. F, The mRNA levels of factors related to endometrial receptivity in Ishikawa cells treated with scrambled siRNA or specific siRNA targeting S100A11. G, The mRNA levels of factors related to immune responses. H and I, The protein levels of selected factors related to embryo implantation in endometrial cells treated with scrambled siRNA or specific siRNA targeting S100A11. Data are presented as mean ± se. * or **, P < 0.05 or P < 0.01, compared with the corresponding controls. N, Number of repeated experiments.
Fig. 3.
Fig. 3.
Effects of different hormones and EGF on S100A11 mRNA expression levels in human endometrial cells. The expression levels of S100A11 mRNA (A) and protein (B and C) in Ishikawa cells treated with E2 (7.14 nm/liter), P4 (63.5 nm/liter), and E2 plus P4 for 24 h, respectively. S100A11 mRNA (C) and protein (D) levels in human endometrial cells treated with hCG at concentrations of 0.04–40 IU/ml for 24 h. E, S100A11 mRNA levels in human endometrial cells treated with EGF (20 ng/ml) for 3–24 h, respectively. F and G, S100A11 protein levels in human endometrial cells treated with EGF (20 ng/ml) for 12–24 h. H, Effects of EGF on JAr spheroid attachment rate in human endometrial cells treated with or without specific siRNA targeting S100A11. Data are presented as mean ± se. * or **, P < 0.05 or P < 0.01, compared with the corresponding controls. N, Number of repeated experiments.
Fig. 4.
Fig. 4.
Subcellular distribution of S100A11 and effects of S100A11 on cytosolic Ca2+ homeostasis in human endometrial cells. A, Double staining with a rabbit antibody to S100A11 (Ab) and a mouse antibody to PDIA3 (Ac) in human endometrial cells. Aa, Nuclear staining (blue); Ad, merge of images. B, Subcellular localization of S100A11 in human endometrial cells (a and c). Bb and Bd, The region of higher magnification of Ba and Bc, respectively. Arrows indicate S100A11. Scale bars, 0.1 nm at ×65,000. C and D, Cytosolic free Ca2+ measurement in endometrial cells treated with scrambled siRNA alone, EGF plus scrambled siRNA, and specific siRNA targeting S100A11 alone and EGF plus specific siRNA targeting S100A11 for 24 h, respectively. Fluorescence was recorded from single Ishikawa cells loaded with 5 μm fluo-4-AM dye. Exposure of human endometrial cells to 10 μm ATP induced a Ca2+ transient (C). Effects of S100A11 on thapsigargin-induced Ca2+ depletion (E and F) and ryanodine-induced Ca2+ release (G and H) in human endometrial cells treated with scrambled siRNA, EGF plus scrambled siRNA, specific siRNA targeting S100A11 and EGF plus specific siRNA targeting S100A11 for 24 h, respectively, are shown. In F/F0, F is fluorescence intensity at each recording point, and F0 is fluorescence intensity at the beginning point. Data are presented as mean ± se. **, P < 0.01 compared with the corresponding controls. N, Number of repeated experiments.
Fig. 5.
Fig. 5.
The schematic diagram depicting the potential interactions between EGF, S100A11, and Ca2+ signaling pathway in human endometrial cell. EGF, acting through their receptor, up-regulates S100A11, which exerts its roles in calcium uptake and release from calcium store in ER. This cross talk influences some of the calcium-trigged signaling pathways to promote the expression of genes including EGFR, IL-15, and LIF, etc., and further affect immunotolerance, cell proliferation, and endometrial receptivity to implantation, ensuring the successful execution of the implantation process. CaM, Calmodulin; IP3R, inositol trisphosphate receptor; RyR, ryanodine receptor; SERCA, sarco/endoplamic reticulum Ca2+-ATPase.
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