Limits...
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.

Show MeSH

Related in: MedlinePlus

Differentiation-dependent protein expression of Notch2 receptor and Notch ligands in HDSC.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 Materials and methods. 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.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4232464&req=5

pone-0112723-g003: Differentiation-dependent protein expression of Notch2 receptor and Notch ligands in HDSC.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 Materials and methods. 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.

Mentions: To verify the decidual stromal cell-specific expression observed in tissue sections, Notch receptors and ligands were studied in isolated, primary HDSC which, similar to endometrial stromal cells, differentiate in vitro in the presence of estrogen/progesterone (E2P4) and/or cAMP [21]. To monitor the dynamic expression of Notch receptors and ligands, transcript and protein levels of Notch2, Jagged1, DLL1 and DLL4 were analyzed at day 3 and 6 of decidualization using qPCR (Fig. 2A) and Western blotting (Fig. 3A), respectively. Additionally, mRNA expression of the decidual markers IGFBP1 and PRL was assessed to ensure proper in vitro decidualization. As expected, IGFBP1 and PRL transcript levels were strongly induced upon treatment with cAMP, however treatment of cAMP in combination with E2P4 proved to be the most effective stimulus for in vitro decidualization (Fig. 2B). Quantitative real time analyses showed constitutively expressed Notch2 mRNA levels after 3 and 6 days of treatment with E2P4 and/or cAMP (Fig. 2A). Compared to untreated cultures, Jagged1 was weakly downregulated in the presence of cAMP alone or in combination with E2P4, but not upon sole hormonal treatment. In contrast, addition of cAMP and the combined treatment increased DLL1 mRNA levels 5.1 and 5.3 fold at day 3 of cultivation, whereas DLL4 was elevated 94 and 95 fold, respectively. The presence of E2P4 alone did not affect mRNA expression of these ligands. Protein levels of Notch family members mirrored mRNA expression patterns in differentiating HDSC (Fig. 3). While Notch2 did not show stimuli- or time-dependent regulation, Jagged1 was faintly diminished upon incubation with cAMP or cAMP/E2P4 (Fig. 3B). In contrast, cAMP or the combined treatment increased protein expression of DLL1 and DLL4 in HDSC. Moreover, cAMP- or cAMP/E2P4-induced DLL1 protein levels differed significantly between 3 and 6 days of cultivation, respectively. However, Notch1, 3 and 4 as well as Jagged2 and DLL3 mRNA and protein were undetectable in HDSC using real-time PCR and Western blotting (unpublished observation), confirming results obtained in the immunofluorescence analyses (Fig. 1B and Fig. S2).


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)

Differentiation-dependent protein expression of Notch2 receptor and Notch ligands in HDSC.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 Materials and methods. 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.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0112723-g003: Differentiation-dependent protein expression of Notch2 receptor and Notch ligands in HDSC.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 Materials and methods. 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.
Mentions: To verify the decidual stromal cell-specific expression observed in tissue sections, Notch receptors and ligands were studied in isolated, primary HDSC which, similar to endometrial stromal cells, differentiate in vitro in the presence of estrogen/progesterone (E2P4) and/or cAMP [21]. To monitor the dynamic expression of Notch receptors and ligands, transcript and protein levels of Notch2, Jagged1, DLL1 and DLL4 were analyzed at day 3 and 6 of decidualization using qPCR (Fig. 2A) and Western blotting (Fig. 3A), respectively. Additionally, mRNA expression of the decidual markers IGFBP1 and PRL was assessed to ensure proper in vitro decidualization. As expected, IGFBP1 and PRL transcript levels were strongly induced upon treatment with cAMP, however treatment of cAMP in combination with E2P4 proved to be the most effective stimulus for in vitro decidualization (Fig. 2B). Quantitative real time analyses showed constitutively expressed Notch2 mRNA levels after 3 and 6 days of treatment with E2P4 and/or cAMP (Fig. 2A). Compared to untreated cultures, Jagged1 was weakly downregulated in the presence of cAMP alone or in combination with E2P4, but not upon sole hormonal treatment. In contrast, addition of cAMP and the combined treatment increased DLL1 mRNA levels 5.1 and 5.3 fold at day 3 of cultivation, whereas DLL4 was elevated 94 and 95 fold, respectively. The presence of E2P4 alone did not affect mRNA expression of these ligands. Protein levels of Notch family members mirrored mRNA expression patterns in differentiating HDSC (Fig. 3). While Notch2 did not show stimuli- or time-dependent regulation, Jagged1 was faintly diminished upon incubation with cAMP or cAMP/E2P4 (Fig. 3B). In contrast, cAMP or the combined treatment increased protein expression of DLL1 and DLL4 in HDSC. Moreover, cAMP- or cAMP/E2P4-induced DLL1 protein levels differed significantly between 3 and 6 days of cultivation, respectively. However, Notch1, 3 and 4 as well as Jagged2 and DLL3 mRNA and protein were undetectable in HDSC using real-time PCR and Western blotting (unpublished observation), confirming results obtained in the immunofluorescence analyses (Fig. 1B and Fig. S2).

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