Decidualization and syndecan-1 knock down sensitize endometrial stromal cells to apoptosis induced by embryonic stimuli.

Human embryo invasion and implantation into the inner wall of the maternal uterus, the endometrium, is the pivotal process for a successful pregnancy. Whereas disruption of the endometrial epithelial layer was already correlated with the programmed cell death, the role of apoptosis of the subjacent endometrial stromal cells during implantation is indistinct. The aim was to clarify whether apoptosis plays a role in the stromal invasion and to characterize if the apoptotic susceptibility of endometrial stromal cells to embryonic stimuli is influenced by decidualization and Syndecan-1. Therefore, the immortalized human endometrial stromal cell line St-T1 was used to first generate a new cell line with a stable Syndecan-1 knock down (KdS1), and second to further decidualize the cells with progesterone. As a replacement for the ethically inapplicable embryo all cells were treated with the embryonic factors and secretion products interleukin-1β, interferon-γ, tumor necrosis factor-α, transforming growth factor-β1 and anti-Fas antibody to mimic the embryo contact. Detection of apoptosis was verified via Caspase ELISAs, PARP cleavage and Annexin V staining. Apoptosis-related proteins were investigated via antibody arrays and underlying signaling pathways were analyzed by Western blot. Non-decidualized endometrial stromal cells showed a resistance towards apoptosis which was rescinded by decidualization and Syndecan-1 knock down independent of decidualization. This was correlated with an altered expression of several pro- and anti-apoptotic proteins and connected to a higher activation of pro-survival Akt in non-differentiated St-T1 as an upstream mediator of apoptotis-related proteins. This study provides insight into the largely elusive process of implantation, proposing an important role for stromal cell apoptosis to successfully establish a pregnancy. The impact of Syndecan-1 in attenuating the apoptotic signal is particularly interesting in the light of an already described influence on pregnancy disorders and therefore might provide a useful clinical tool in the future to prevent pregnancy complications provoked by inadequate implantation

Expression of apoptosis-related proteins before and after IITT+F treatment.

<p>Antibody array analysis of apoptosis-related proteins in ESCs of (A) untreated, non-differentiated (St-T1, red bar; KdS1, blue bar) and decidualized (dSt-T1, bright red bar; dKdS1, bright blue bar) ESCs, n = 4 ± SEM, *p<0.05 Sdc-1 wildtype vs. Sdc-1 kd cells, #p<0.05 non-differentiated vs. decidualized cells. (B) Antibody Array with protein from IITT+F treated, non-differentiated (St-T1, red bar; KdS1, blue bar) and decidualized (dSt-T1, bright red bar; dKdS1, bright blue bar) ESCs. Pixel density is given as mean±SEM of n = 4 independent experiments, *p<0.05 Sdc-1 wildtype vs. Sdc-1 kd cells, #p<0.05 non-differentiated vs. decidualized cells, ✝p<0.05 untreated vs. IITT+F treated.</p

Activation of the pro-apoptotic JNK pathway after IITT and F treatment.

<p>Western blot analysis of pJNK and JNK in non-differentiated (St-T1, first line; KdS1, second line) and decidualized (dSt-T1, third line; dKdS1, fourth line) ESCs after treatments with F 15min, IITT 15min and IITT 24h + F 15min vs. untreated controls. (A) Representative blot of pJNK (46/54kDa), JNK (46/54kDa) and β-Actin (42kDa) as loading control. (b) Pixel densitiy evaluation of pJNK normalized to JNK is given as mean±SEM of n = 6 independent experiments, *p<0.05 wildtype vs. Sdc-1 kd cells, ✝p<0.05 untreated control vs. treated.</p

Activation of the pro-survival protein Akt after IITT and F treatment.

<p>Western blot analysis of pAkt and Akt in non-differentiated (St-T1, first line; KdS1, second line) and decidualized (dSt-T1, third line; dKdS1, fourth line) ESCs after treatments with F 15min, IITT 15min and IITT 24h + F 15min vs. untreated controls. (A) Representative blot of pAkt (60kDa), Akt (60kDa) and β-Actin (42kDa) as loading control. (B) Pixel densitiy evaluation of pAkt normalized to Akt is given as mean±SEM of n = 6 independent experiments, *p<0.05 wildtype vs. Sdc-1 kd cells, #p<0.05 undifferentiated vs. decidualized cells, ✝p<0.05 untreated control vs. treated.</p

Investigation of the extrinsic and intrinsic apoptosis pathway after IITT+F treatment.

<p>Analysis of Caspase-8 and -9 activation of treated cells vs. untreated controls in non-differentiated (St-T1, red bar; KdS1, blue bar) and decidualized dSt-T1, bright red bar; dKdS1, bright blue bar) ESCs after treatment with IITT+F. Untreated controls were assigned being 1 and enzymatic activity of caspases after treatment was determined as fold induction vs. controls and given as mean±SEM of n = 3 independent experiments, *p<0.05 Sdc-1 wildtype vs. Sdc-1 kd cells, ✝p<0.05 untreated controls vs. treated.</p

Induction of FasR expression after IITT treatment.

<p>Fold change of FasR mRNA in non-differentiated (St-T1, red bar; KdS1, blue bar) and decidualized (dSt-T1, bright red bar; dKdS1, bright blue bar) ESCs after treatment with IITT 24h. 2^-ΔΔCt is are given as mean±SEM of n = 5 independent experiments, ✝p<0.05 untreated vs. IITT treated.</p

Loss of membrane asymmetry after IITT+F treatment.

<p>Non-differentiated (St-T1, KdS1) and decidualized (dSt-T1, dKdS1) ESCs were treatmed with IITT+F and loss of membrane asymmetry was visualized with Annexin V FITC staining (green), n = 3; (A)–(D) untreated controls: (A) St-T1, (B) KdS1, (C) dSt-T1, (D) dKdS1. (E)-(H) IITT+F treated: (E) St-T1, (F) KdS1, (G) dSt-T1, (H) dKdS1; blue nuclei are stained with Hoechst 33342. Scale bars indicate 100μm.</p

Activation of the pro-survival NFκB pathway after IITT and F treatment.

<p>Western blot analysis of NFκB members p65 and IκBα in non-differentiated (St-T1, first line; KdS1, second line) and decidualized (dSt-T1, third line; dKdS1, fourth line) ESCs after treatments with F 15min, IITT 15min and IITT 24h + F 15min vs. untreated controls. (A) Representative blot of pp65 (65kDa), p65 (65kDa) and β-Actin (42kDa) as loading control. (B) Pixel densitiy evaluation of pp65 normalized to p65 is given as mean±SEM of n = 6 independent experiments, *p<0.05 wildtype vs. Sdc-1 kd cells, #p<0.05 undifferentiated vs. decidualized cells, ✝p<0.05 untreated control vs. treated. (C) Representative blot of IκBα (39kDa) and β-Actin (42kDa) as loading control. (D) Pixel densitiy evaluation of IκBα normalized to β-Actin is given as mean±SEM of n = 6 independent experiments, *p<0.05 wildtype vs. Sdc-1 kd cells, #p<0.05 undifferentiated vs. decidualized cells, ✝p<0.05 untreated control vs. treated.</p

Quantification of active Caspase-3 in ESCs treated with embryonic stimuli.

<p>Non-differentiated (St-T1, red bar; KdS1, blue bar) and decidualized (dSt-T1, bright red bar; dKdS1, bright blue bar) ESCs were treated with IITT and F for 24h individually or in combination as indicated and the amount of active Caspase-3 was analyzed in ng/ml and displayed as mean±SEM of n = 3 independent experiments; *p<0.05 Sdc-1 wildtype vs. Sdc-1 kd cells, #p<0.05 non-differentiated vs. decidualized cells, ✝p<0.05 untreated controls vs. treated cells.</p