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Notch2 controls prolactin and insulin-like growth factor binding protein-1 expression in decidualizing human stromal cells of early pregnancy.

Otti GR, Saleh L, Velicky P, Fiala C, Pollheimer J, Knöfler M - PLoS ONE (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Fetal-Maternal Medicine, Reproductive Biology Unit, Medical University of Vienna, Vienna, Austria.

ABSTRACT
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.

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Expression pattern of Notch ligands and receptors in first trimester decidual tissue.Serial sectioning of paraffin-embedded tissues and immunofluorescence were performed as described in materials and methods. Representative examples (8th 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.
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pone-0112723-g001: Expression pattern of Notch ligands and receptors in first trimester decidual tissue.Serial sectioning of paraffin-embedded tissues and immunofluorescence were performed as described in materials and methods. Representative examples (8th 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.

Mentions: Immunofluorescence of first trimester decidua revealed expression of Notch2, Jagged1, DLL1 and DLL4 in human decidual stromal cells (Fig. 1A). The latter could easily be distinguished from decidual leukocytes as shown by double-staining with antibodies recognizing vimentin and CD45, respectively. By utilization of a Notch2 antibody recognizing both the membrane bound and translocated NICD, Notch2 was detectable at the cell surface as well as in nuclei of stromal cells, suggesting active, canonical Notch signaling. The Notch ligands Jagged1, DLL1, and DLL4 displayed cytoplasmic staining in stromal cells whereas Jagged1 and DLL4 additionally localized to nuclei. Notch1, 3 and 4 as well as the ligands Jagged2 and DLL3 were absent from these cells, but could be detected in the glandular epithelium expressing all Notch receptors and ligands (Fig. 1B). Compared to stromal cells, glandular epithelial cells showed stronger immunofluorescence signals, particularly for Notch2 and Jagged1. Besides Notch2, Jagged1, DLL1 and DLL4 (Fig. 1A), CD45-positive leukocytes additionally expressed Notch1, 3, and 4 but lacked Jagged2 and DLL3 (Fig. S1).


Notch2 controls prolactin and insulin-like growth factor binding protein-1 expression in decidualizing human stromal cells of early pregnancy.

Otti GR, Saleh L, Velicky P, Fiala C, Pollheimer J, Knöfler M - PLoS ONE (2014)

Expression pattern of Notch ligands and receptors in first trimester decidual tissue.Serial sectioning of paraffin-embedded tissues and immunofluorescence were performed as described in materials and methods. Representative examples (8th 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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4232464&req=5

pone-0112723-g001: Expression pattern of Notch ligands and receptors in first trimester decidual tissue.Serial sectioning of paraffin-embedded tissues and immunofluorescence were performed as described in materials and methods. Representative examples (8th 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.
Mentions: Immunofluorescence of first trimester decidua revealed expression of Notch2, Jagged1, DLL1 and DLL4 in human decidual stromal cells (Fig. 1A). The latter could easily be distinguished from decidual leukocytes as shown by double-staining with antibodies recognizing vimentin and CD45, respectively. By utilization of a Notch2 antibody recognizing both the membrane bound and translocated NICD, Notch2 was detectable at the cell surface as well as in nuclei of stromal cells, suggesting active, canonical Notch signaling. The Notch ligands Jagged1, DLL1, and DLL4 displayed cytoplasmic staining in stromal cells whereas Jagged1 and DLL4 additionally localized to nuclei. Notch1, 3 and 4 as well as the ligands Jagged2 and DLL3 were absent from these cells, but could be detected in the glandular epithelium expressing all Notch receptors and ligands (Fig. 1B). Compared to stromal cells, glandular epithelial cells showed stronger immunofluorescence signals, particularly for Notch2 and Jagged1. Besides Notch2, Jagged1, DLL1 and DLL4 (Fig. 1A), CD45-positive leukocytes additionally expressed Notch1, 3, and 4 but lacked Jagged2 and DLL3 (Fig. S1).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Fetal-Maternal Medicine, Reproductive Biology Unit, Medical University of Vienna, Vienna, Austria.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus