Volume 36, Issue 6, Pages 652-660 (June 2015) NOD1 and NOD2 control the invasiveness of trophoblast cells via the MAPK/p38 signaling pathway in human first-trimester pregnancy  Z. Wang, M. Liu, X. Nie, Y. Zhang, Y. Chen, L. Zhu, X. Chen, L. Chen, H. Chen, J. Zhang  Placenta  Volume 36, Issue 6, Pages 652-660 (June 2015) DOI: 10.1016/j.placenta.2015.03.004 Copyright © 2015 Elsevier Ltd Terms and Conditions

Fig. 1 Expression of NOD1 and NOD2 in human first-trimester villi and decidua. NOD1 and NOD2 expression were confirmed for protein and RNA levels. (A) Immunohistochemical characterization of NOD1 and NOD2 in human first-trimester villi tissues and decidua tissues. Villi tissues were CK7 positive and vimentin negative, but dicidua tissues were CK7 negative and vimentin positive. Specific brown staining for NOD1 and NOD2 was observed in the cytoplasm. (B) NOD1 and NOD2 mRNA expressions in decidua tissues and villi tissues were compared by real-time RT-PCR. (C) Primary trophoblast cells were seeded on the cell culture plates and processing by immunocytochemical staining (CK7, vimentin, HLA-G, or the isotype control antibody). Data represent the mean ± standard error (SE) of 3 independent experiments.*P < 0.05, vs placebo group. **P < 0.01, vs placebo group. Placenta 2015 36, 652-660DOI: (10.1016/j.placenta.2015.03.004) Copyright © 2015 Elsevier Ltd Terms and Conditions

Fig. 2 The specific and nonspecific ligands of NOD1 and NOD2 upregulated the expression of their receptors in trophoblast cells. A and B: The mRNA (A) and protein (B) expression of NOD1 can be upregulated by the specific ligand, iE-DAP. C and D: LPS also upregulated the expression of NOD1 on mRNA levels (C) and protein levels (D). E and F: The specific ligand of NOD2, MDP, also enhanced the expression of the receptor on the transcriptional level (E) and the protein level (F), but this regulation was remarkable only when the concentration of MDP was 10 μg/mL. (G) and (H): LPS expanded the expression of NOD2. Data are the mean ± SE of triplicate values. *P < 0.05. **P < 0.01, compared to the control. Placenta 2015 36, 652-660DOI: (10.1016/j.placenta.2015.03.004) Copyright © 2015 Elsevier Ltd Terms and Conditions

Fig. 3 The effects of ligands of NOD1 and NOD2 on the invasion of primary human trophoblasts. A Matrigel-based transwell assay was carried out to test the effects of ligands on human trophoblast invasion. After different concentrations of iE-DAP (A), MDP (B), and LPS (C) treatment for 48 h, microscopic morphologies of the trophoblasts after invasion through the Matrigel-coated membranes were taken at 200× magnifications. The invasive index of each group was calculated. Data represent the mean ± standard error (SE) of 3 independent experiments. *P < 0.05. **P < 0.01 compared to the untreated control group. Placenta 2015 36, 652-660DOI: (10.1016/j.placenta.2015.03.004) Copyright © 2015 Elsevier Ltd Terms and Conditions

Fig. 4 The specific ligands of NOD1 and NOD2 promoted MMP9 secretion and transcription but not MMP2, but their nonspecific ligands upregulated both MMP2 and MMP9 secretion and mRNA levels. Trophoblast cells were seeded at 2 × 105 cells/well in 24-well tissue culture plates and were stimulated with different concentrations of iE-DAP (A1 and B1), MDP (A2 and B2), or LPS (A3 and B3) for 48 h. After that, the culture supernatants were collected. The concentrations of MMP9 (A1, A2, and A3) and MMP2 (B1, B2, and B3) in these supernatants were detected by ELISA. Trophoblast cells were seeded at 5 × 105 cells/well in cell culture plates and were stimulated with different concentrations of: iE-DAP (C1 and D1), MDP and (C2 and D2), LPS (C3 and D3) for 24 h. The cells were harvested and subjected to RNA extraction and real-time PCR for detection of mRNA levels of MMP9 (C1, C2 & C3) and MMP2 (D1, D2 & 3). Data are the mean ± SE of triplicate values. *P < 0.05. **P < 0.01 compared to the control; NS, no significance compared to controls. Placenta 2015 36, 652-660DOI: (10.1016/j.placenta.2015.03.004) Copyright © 2015 Elsevier Ltd Terms and Conditions

Fig. 5 IE-DAP and MDP-activated MAPK/p38 signaling pathways in trophoblasts and they inhibited the invasion of trophoblasts via the MAPK/p38 signal pathway. The primary-cultured trophoblasts were treated with 10 μg/mL iE-DAP (A) or MDP (B) for different times. The levels of phosphor- and total-p38 were determined by in-cell Western analysis (see text for details). The pictures were typical from 3 individual experiments. Red represents phospho-p38, and green represents total p38. The MAPK/p38 inhibitor SB203580 (10 μM) was added 1 h before the stimulation with 10 μg/mL iE-DAP or MDP. (C): SB203580 downregulated the basal invasion of trophoblast cells. When co-stimulated with ligands, SB203580 partially reversed the downregulation of iE-DAP or MDP on trohoplast invasion. After we collected the culture supernatant, MMP9 (D) and MMP2 (E), we measured secretion levels by ELISA. Data represent the mean ± SEM of triplicate wells. *P < 0.05. **P < 0.01. ***P < 0.001 compared to the untreated group. ##P < 0.01. ###P < 0.001 compared to the group treated with iE-DAP. ΔΔP < 0.01. ΔΔΔP < 0.001 compared to the group treated with MDP. NS, no significance. Placenta 2015 36, 652-660DOI: (10.1016/j.placenta.2015.03.004) Copyright © 2015 Elsevier Ltd Terms and Conditions

Fig. 6 Differential NOD1 and NOD2 expression level in villi tissues among patients with normal pregnancy and those with RSA. The NOD1 and NOD2 expression in villi from the normal pregnancy or RSA were evaluated by real-time RT-PCR, immunohistochemistry, and Western blot. (A): NOD1 and NOD2 mRNA expressions in villous tissues from normal pregnancy and RSA were compared by real-time PCR. Both NOD1 and NOD2 mRNA levels in villi from RSA were higher than those from normal pregnancy. Data are from 6 repeated experiments, N is normal pregnancy, and R is RSA. (B): Sections of villous tissues from normal early pregnancy and RSA were stained for NOD1 or NOD2. The pictures are representatives from 4 repeated experiments, respectively. Original magnification in the representative results is 200-fold. (C): Villi tissues from normal pregnancies and RSA were subjected to Western blot analysis to determine the protein levels of NOD1 and NOD2, semiquantified by densitometry and normalized by tubulin. *P < 0.05. **P < 0.05. ***P < 0.001vs the normal pregnancy group. Placenta 2015 36, 652-660DOI: (10.1016/j.placenta.2015.03.004) Copyright © 2015 Elsevier Ltd Terms and Conditions