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NPR-A regulates self-renewal and pluripotency of embryonic stem cells.

Abdelalim EM, Tooyama I - Cell Death Dis (2011)

Bottom Line: NPR-A knockdown also resulted in a marked downregulation of phosphorylated Akt.Interestingly, we found that ANP was expressed in self-renewing ES cells, whereas its level was reduced after ES cell differentiation.These findings provide a novel role for NPR-A in the maintenance of self-renewal and pluripotency of ES cells.

View Article: PubMed Central - PubMed

Affiliation: Molecular Neuroscience Research Center, Shiga University of Medical Science, Setatsukinowa-cho, Otsu, Shiga 520-2192, Japan. essam_abdelalim@yahoo.com

ABSTRACT
Self-renewal and pluripotency of embryonic stem (ES) cells are maintained by several signaling cascades and by expression of intrinsic factors, such as Oct4, Nanog and Sox2. The mechanism regulating these signaling cascades in ES cells is of great interest. Recently, we have demonstrated that natriuretic peptide receptor A (NPR-A), a specific receptor for atrial and brain natriuretic peptides (ANP and BNP, respectively), is expressed in pre-implantation embryos and in ES cells. Here, we examined whether NPR-A is involved in the maintenance of ES cell pluripotency. RNA interference-mediated knockdown of NPR-A resulted in phenotypic changes, indicative of differentiation, downregulation of pluripotency factors (such as Oct4, Nanog and Sox2) and upregulation of differentiation genes. NPR-A knockdown also resulted in a marked downregulation of phosphorylated Akt. Furthermore, NPR-A knockdown induced accumulation of ES cells in the G1 phase of the cell cycle. Interestingly, we found that ANP was expressed in self-renewing ES cells, whereas its level was reduced after ES cell differentiation. Treatment of ES cells with ANP upregulated the expression of Oct4, Nanog and phosphorylated Akt, and this upregulation depended on NPR-A signaling, because it was completely reversed by pretreatment with either an NPR-A antagonist or a cGMP-dependent protein kinase inhibitor. These findings provide a novel role for NPR-A in the maintenance of self-renewal and pluripotency of ES cells.

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ANP upregulates Oct4 and Nanog through the NPR-A/PKG-dependent pathway. (a) Representative protein blot of Oct4 and Nanog proteins in ES cells treated with vehicle, ANP alone or ANP and anantin. (b) Quantitative analysis of the western blots as shown in panel a. (c) Representative protein blot of Oct4 and Nanog proteins in ES cells treated with vehicle, ANP alone or ANP and a PKG inhibitor (KT5823). (d) Quantitative analysis of the western blots as shown in panel c. (e) Western blot analysis of Oct4 and Nanog in ES cells treated with anantin for 3 days. (f) Western blot analysis of Oct4 and Nanog in ES cells treated with KT5823 for 3 days. Data represent mean±S.D. (n=3). *P<0.05 or **P<0.01 (two-tailed t-test)
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fig6: ANP upregulates Oct4 and Nanog through the NPR-A/PKG-dependent pathway. (a) Representative protein blot of Oct4 and Nanog proteins in ES cells treated with vehicle, ANP alone or ANP and anantin. (b) Quantitative analysis of the western blots as shown in panel a. (c) Representative protein blot of Oct4 and Nanog proteins in ES cells treated with vehicle, ANP alone or ANP and a PKG inhibitor (KT5823). (d) Quantitative analysis of the western blots as shown in panel c. (e) Western blot analysis of Oct4 and Nanog in ES cells treated with anantin for 3 days. (f) Western blot analysis of Oct4 and Nanog in ES cells treated with KT5823 for 3 days. Data represent mean±S.D. (n=3). *P<0.05 or **P<0.01 (two-tailed t-test)

Mentions: Involvement of ANP receptor-mediated signaling in the upregulation of Oct4 and Nanog was confirmed using anantin (a specific NPR-A antagonist), which blocked the effect of ANP on the expression of Oct4 and Nanog when ES cells were treated with 300 nM anantin before ANP exposure (Figures 6a and b). Furthermore, to test the possibility of the involvement of PKG signaling in mediating ANP effect on the expression of Oct4 and Nanog, we used KT5823 (a specific inhibitor of PKG). Pretreatment with 2 μM of the PKG inhibitor 30 min before ANP treatment, blocked the upregulation of Oct4 and Nanog (Figures 6c and d), suggesting the involvement of the NPR-A/PKG pathway in this process.


NPR-A regulates self-renewal and pluripotency of embryonic stem cells.

Abdelalim EM, Tooyama I - Cell Death Dis (2011)

ANP upregulates Oct4 and Nanog through the NPR-A/PKG-dependent pathway. (a) Representative protein blot of Oct4 and Nanog proteins in ES cells treated with vehicle, ANP alone or ANP and anantin. (b) Quantitative analysis of the western blots as shown in panel a. (c) Representative protein blot of Oct4 and Nanog proteins in ES cells treated with vehicle, ANP alone or ANP and a PKG inhibitor (KT5823). (d) Quantitative analysis of the western blots as shown in panel c. (e) Western blot analysis of Oct4 and Nanog in ES cells treated with anantin for 3 days. (f) Western blot analysis of Oct4 and Nanog in ES cells treated with KT5823 for 3 days. Data represent mean±S.D. (n=3). *P<0.05 or **P<0.01 (two-tailed t-test)
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3101807&req=5

fig6: ANP upregulates Oct4 and Nanog through the NPR-A/PKG-dependent pathway. (a) Representative protein blot of Oct4 and Nanog proteins in ES cells treated with vehicle, ANP alone or ANP and anantin. (b) Quantitative analysis of the western blots as shown in panel a. (c) Representative protein blot of Oct4 and Nanog proteins in ES cells treated with vehicle, ANP alone or ANP and a PKG inhibitor (KT5823). (d) Quantitative analysis of the western blots as shown in panel c. (e) Western blot analysis of Oct4 and Nanog in ES cells treated with anantin for 3 days. (f) Western blot analysis of Oct4 and Nanog in ES cells treated with KT5823 for 3 days. Data represent mean±S.D. (n=3). *P<0.05 or **P<0.01 (two-tailed t-test)
Mentions: Involvement of ANP receptor-mediated signaling in the upregulation of Oct4 and Nanog was confirmed using anantin (a specific NPR-A antagonist), which blocked the effect of ANP on the expression of Oct4 and Nanog when ES cells were treated with 300 nM anantin before ANP exposure (Figures 6a and b). Furthermore, to test the possibility of the involvement of PKG signaling in mediating ANP effect on the expression of Oct4 and Nanog, we used KT5823 (a specific inhibitor of PKG). Pretreatment with 2 μM of the PKG inhibitor 30 min before ANP treatment, blocked the upregulation of Oct4 and Nanog (Figures 6c and d), suggesting the involvement of the NPR-A/PKG pathway in this process.

Bottom Line: NPR-A knockdown also resulted in a marked downregulation of phosphorylated Akt.Interestingly, we found that ANP was expressed in self-renewing ES cells, whereas its level was reduced after ES cell differentiation.These findings provide a novel role for NPR-A in the maintenance of self-renewal and pluripotency of ES cells.

View Article: PubMed Central - PubMed

Affiliation: Molecular Neuroscience Research Center, Shiga University of Medical Science, Setatsukinowa-cho, Otsu, Shiga 520-2192, Japan. essam_abdelalim@yahoo.com

ABSTRACT
Self-renewal and pluripotency of embryonic stem (ES) cells are maintained by several signaling cascades and by expression of intrinsic factors, such as Oct4, Nanog and Sox2. The mechanism regulating these signaling cascades in ES cells is of great interest. Recently, we have demonstrated that natriuretic peptide receptor A (NPR-A), a specific receptor for atrial and brain natriuretic peptides (ANP and BNP, respectively), is expressed in pre-implantation embryos and in ES cells. Here, we examined whether NPR-A is involved in the maintenance of ES cell pluripotency. RNA interference-mediated knockdown of NPR-A resulted in phenotypic changes, indicative of differentiation, downregulation of pluripotency factors (such as Oct4, Nanog and Sox2) and upregulation of differentiation genes. NPR-A knockdown also resulted in a marked downregulation of phosphorylated Akt. Furthermore, NPR-A knockdown induced accumulation of ES cells in the G1 phase of the cell cycle. Interestingly, we found that ANP was expressed in self-renewing ES cells, whereas its level was reduced after ES cell differentiation. Treatment of ES cells with ANP upregulated the expression of Oct4, Nanog and phosphorylated Akt, and this upregulation depended on NPR-A signaling, because it was completely reversed by pretreatment with either an NPR-A antagonist or a cGMP-dependent protein kinase inhibitor. These findings provide a novel role for NPR-A in the maintenance of self-renewal and pluripotency of ES cells.

Show MeSH