Notch2 inhibition diminishes differentiation-dependent prolactin secretion in HDSC.

<p>Cells were incubated with Notch2 blocking antibodies (A) or Notch2 siRNAs (B) and differentiated for 6 days in the presence of cAMP/E2P4. Supernatants were collected and prolactin (PRL) concentrations were measured by ELISA. Normalization to cellular protein concentrations was performed as mentioned in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112723#s2" target="_blank">materials and methods</a>. Bars represent mean values ± S.D. of each 3 different experiments performed in duplicates. * indicates p≤0.05 compared to non-stimulated control (n.c.); Significant changes (*, p≤0.05) between Notch2 ab or si-Notch2-treated cells (open bars) and untreated (black bars) cells are indicated by brackets. IgG ctrl, IgG control; si-ntc, si-non targeting control.</p

Notch2 Controls Prolactin and Insulin-Like Growth Factor Binding Protein-1 Expression in Decidualizing Human Stromal Cells of Early Pregnancy

<div><p>Decidualization, the transformation of the human uterine mucosa into the endometrium of pregnancy, is critical for successful implantation and embryonic development. However, key regulatory factors controlling differentiation of uterine stromal cells into hormone-secreting decidual cells have not been fully elucidated. Hence, we herein investigated the role of the Notch signaling pathway in human decidual stromal cells (HDSC) isolated from early pregnancy samples. Immunofluorescence of first trimester decidual tissues revealed expression of Notch2 receptor and its putative, membrane-anchored interaction partners Jagged1, Delta-like (DLL) 1 and DLL4 in stromal cells whereas other Notch receptors and ligands were absent from these cells. During in vitro differentiation with estrogen/progesterone (E2P4) and/or cyclic adenosine monophosphate (cAMP) HDSC constitutively expressed Notch2 and weakly downregulated Jagged1 mRNA and protein, measured by quantitative PCR (qPCR) and Western blotting, respectively. However, increased levels of DLL1 and DLL4 were observed in the decidualizing cultures. Transfection of a Notch luciferase reporter and qPCR of the Notch target gene hairy and enhancer of split 1 (HES1) revealed an induction of canonical Notch activity during in vitro differentiation. In contrast, treatment of HDSC with a chemical Notch/γ-secretase inhibitor decreased cAMP/E2P4-stimulated Notch luciferase activity, HES1 transcript levels and mRNA expression of the decidual marker genes prolactin (PRL) and insulin-like growth factor binding protein 1 (IGFBP1). Similarly, siRNA-mediated gene silencing or antibody-mediated blocking of Notch2 diminished HES1, PRL and IGFBP1 mRNA levels as well as secreted PRL protein. In summary, the data suggest that canonical, Notch2-dependent signaling plays a role in human decidualization.</p></div

Differentiation-dependent protein expression of Notch2 receptor and Notch ligands in HDSC.

<p>Cells were stimulated with cAMP and/or E2P4 for 3 and 6 days. Total protein lysates were analyzed by western blotting and quantitated using densitometry as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112723#s2" target="_blank">Materials and methods</a>. A representative example out of 3 different experiments is shown. n.c., non-stimulated controls; (A) Western blot showing specific signals for Notch2, Jagged1, DLL1 and DLL4 (marked by arrows). GAPDH was used as a loading control, * indicates unspecific band. (B) Densitometrical quantification of western blot signals normalized to GAPDH. Bars (n = 3) represent mean values ± S.D. For relative quantification of protein expression, values of n.c. at day 3 were arbitrarily set to 100%. * indicates p≤0.05 compared to n.c. of day 3; n.s., not significant.</p

qPCR measuring mRNA expression of Notch2 receptor and Notch ligands (A) and decidual markers IGFBP1 and PRL (B) in differentiating HDSC.

<p>Cultures were incubated for 3 and 6 days with cAMP, E2P4 or cAMP/E2P4. Cells without stimuli were cultivated in parallel representing non-stimulated controls (n.c.). For relative quantification of mRNA expression, n.c. of day 3 was arbitrarily set to 1. Bars depict mean values ± S.D. of 4 different experiments. PCR reactions were performed in duplicates. * depicts p≤0.05 compared to the n.c. of day 3; n.s., not significant. ** indicates p≤0.05 between cAMP and cAMP/E2P4-treated cultures at day 6.</p

Downregulation of canonical Notch activity and Notch2 protein expression in HDSC.

<p>Cells were cultured in the absence or presence of Notch2 blocking antibodies (Notch2 ab) (A) or si-RNAs targeting Notch2 (si-Notch2) (B). (A) Luciferase activity of the canonical Notch reporter in the presence of Notch2 ab or IgG controls. Reporter expression was normalized to constitutive β-Gal activity. Mean values ± S.D. of 3 experiments performed in duplicates are shown. IgG ctrl, IgG control; * indicates p≤0.05 compared to IgG-treated n.c. Significant changes (*, p≤0.05) between DAPT-treated (open bars) and untreated (black bars) cells are indicated by brackets. (B) Western blot showing Notch2 protein expression in siRNA-treated HDSC. Specific Notch2 signals are marked by an arrow (110 kDa). GAPDH was used as a loading control. Representative examples of 3 independent experiments are depicted. Lower panel shows the densitometrical quantification calculated from 3 independent experiments. si-ntc, si-non targeting control. * indicates p≤0.05 compared to si-ntc of the same day.</p

Inhibition of canonical Notch signalling impairs decidualization of HDSC.

<p>Stimulation with cAMP/E2P4 in the absence or presence of the γ-secretase inhibitor DAPT, transfection of the RBPJκ luciferase-reporter plasmid and quantitative real time PCR were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112723#s2" target="_blank">materials and methods</a>. n.c. non-stimulated controls. (A) Luciferase activity of a canonical Notch reporter plasmid harboring RBPJκ cognate sequences normalized to constitutive β-Gal expression after treatment with decidualizing stimulus and DAPT. Mean values ± S.D. of 3 experiments performed in duplicates are shown. Values of n.c. transfected with RBPJκ plasmids in the absence of DAPT were arbitrarily set at 100%. (B, C) Quantitative real-time PCR analyses evaluating mRNA expression of the Notch target gene HES1 (B) or marker genes of differentiation, IGFBP1 and PRL (C), in decidualizing cultures in the absence or presence of DAPT. For relative quantification of mRNA expression n.c. at day 3 (absence of DAPT) was arbitrarily set to 1. Bars indicate mean values ± S.D. of 3 different experiments. PCR reactions were performed in duplicates. * indicates p≤0.05 compared to n.c. of day 3. Significant changes (*, p≤0.05) between DAPT-treated (open bars) and untreated (black bars) cells are indicated by brackets.</p

Expression pattern of Notch ligands and receptors in first trimester decidual tissue.

<p>Serial sectioning of paraffin-embedded tissues and immunofluorescence were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112723#s2" target="_blank">materials and methods</a>. Representative examples (8^th week of pregnancy) of 5 different deciduae analyzed are depicted. DSC, decidual stromal cells; GEC, glandular epithelial cell; LC, leukocytes; Scale bars represent 100 µm. (A) Localization of Notch family members in human deidual stromal cells. Double staining with antibodies recognizing vimentin (Vim, green), Notch2 (green) and CD45 (red) mark decidual stromal cells and leukocytes, respectively. The respective counterstaining with DAPI is depicted on the right hand side. Double stainings with the appropriate isotype-specific monoclonal (mAb, green, insert picture) and polyclonal (pAb, green, insert picture) controls with vimentin (Vim, red) are shown. (B) Glandular expression of Notch receptors and ligands. Double staining with antibodies recognizing vimentin (Vim, green) or cytokeratin 7 (KRT7, red) was used to depict DSC and GEC, respectively. Co-staining of Notch receptor or ligand (both green) with nuclear staining (DAPI, blue) is shown.</p

Signaling pathways activated by PDGF-AA, PDGF-BB, HB-EGF, TCM, or VECM.

<p>(<b>A</b>) Decidualized St-T1b were starved in Opti-MEM and then treated for 5 or 30 min with PDGF-BB, HB-EGF, TCM or PDGF-AA. Levels of phosphorylated (p-) or total ERK1/2, Akt, and p38 were determined by Western blotting. (<b>B</b>) Decidualized hESC were starved in Opti-MEM and then treated for 5 min with PDGF-BB, HB-EGF, TCM, 5 individual VECM preparations, villous explant control medium (VECM-Co), or PDGF-AA. Western blotting was performed as above.</p

Chemokinetic response of St-T1b cells or primary hESCs to PDGF-BB, HB-EGF, TCM and trophoblast secretory products identified by proteome profiling.

<p>St-T1b cells (<i>upper panel</i>) or hESC (<i>lower panel</i>), non-decidualized (ND) or decidualized (D), were subjected to Oris migration assay to monitor random motility, in the presence of PDGF-BB, HB-EGF, TCM, PDGF-AA, PLGF-1, or VEGF-165 at the indicated concentrations. Numbers of cells that had migrated into the detection zone after 18 h incubation were determined and normalized to unstimulated controls within each ND or D group. Shown are the means±SEM of n = 3 independent experiments. Results were analyzed by ANOVA and Dunnett test within each group. ***, <i>P</i><0.001; **, <i>P</i><0.01 compared to untreated controls (white columns).</p

Effect of pathway inhibitors on chemokinetic motility of St-T1b cells.

<p>(<b>A</b>) Decidualized St-T1b cells were seeded in Oris migration plates and preincubated with inhibitors for 1 h: PD98059 (50 µM), Y27632 (100 µM), NSC23766 (50 µM), SB202190 (10 µM) or Wortmannin (200 nM). Then PDGF-BB was added to the indicated final concentration, or the equivalent volume of control medium, and cells allowed to migrate into the detection zone for 18 h. Numbers of migrated cells were normalized to the control in the absence of stimulus and inhibitor (left white column). Results represent the means±SEM of n = 3 independent experiments. Data were analyzed by ANOVA and Dunnett test within each group (unstimulated or treated with PDGF-BB) and revealed significant differences after inhibitor treatment compared to the respective controls without inhibitor: *, <i>P</i><0.05; **, <i>P</i><0.01; *** <i>P</i><0.001. (<b>B</b>) Representative images of detection zones for the treatments evaluated in panel (<b>A</b>), showing cells that had migrated into the cell-free zone within 18 h. Cells were stained with Diff-Quik. Images of each detection zone were reassembled from 4 microphotographs taken with a 4× objective and graphically overlaid with the black mask.</p