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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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NPR-A small-interfering RNA (siRNA) induces efficient knockdown of NPR-A in murine ES cells. (a) RT-PCR analysis of ES cells transfected with control siRNA or NPR-A siRNAs, showing knockdown of the NPR-A gene 48 h after siRNA transfection. GAPDH was used as the internal control. (b) Real-time PCR analysis of ES cells treated with control siRNA or NPR-A siRNA2. (c) Western blot analysis of ES cells treated as described in panel a, showing a reduced level of the NPR-A protein 48 h after siRNA transfection. β-Actin was used as a loading control. Data represent mean±S.D. (n=3); *P<0.05 (two-tailed t-test)
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fig1: NPR-A small-interfering RNA (siRNA) induces efficient knockdown of NPR-A in murine ES cells. (a) RT-PCR analysis of ES cells transfected with control siRNA or NPR-A siRNAs, showing knockdown of the NPR-A gene 48 h after siRNA transfection. GAPDH was used as the internal control. (b) Real-time PCR analysis of ES cells treated with control siRNA or NPR-A siRNA2. (c) Western blot analysis of ES cells treated as described in panel a, showing a reduced level of the NPR-A protein 48 h after siRNA transfection. β-Actin was used as a loading control. Data represent mean±S.D. (n=3); *P<0.05 (two-tailed t-test)

Mentions: Both reverse transcription-PCR (RT-PCR) and real-time PCR revealed a marked reduction in the level of NPR-A mRNA at 48 h after transfection in ES cells, which were transfected with NPR-A-targeting siRNA (NPR-A siRNA), compared with ES cells that were transfected with a nontargeting siRNA (control siRNA) (Figures 1a and b). This finding was confirmed by a reduction in protein levels observed in western blots (Figure 1c).


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

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

NPR-A small-interfering RNA (siRNA) induces efficient knockdown of NPR-A in murine ES cells. (a) RT-PCR analysis of ES cells transfected with control siRNA or NPR-A siRNAs, showing knockdown of the NPR-A gene 48 h after siRNA transfection. GAPDH was used as the internal control. (b) Real-time PCR analysis of ES cells treated with control siRNA or NPR-A siRNA2. (c) Western blot analysis of ES cells treated as described in panel a, showing a reduced level of the NPR-A protein 48 h after siRNA transfection. β-Actin was used as a loading control. Data represent mean±S.D. (n=3); *P<0.05 (two-tailed t-test)
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: NPR-A small-interfering RNA (siRNA) induces efficient knockdown of NPR-A in murine ES cells. (a) RT-PCR analysis of ES cells transfected with control siRNA or NPR-A siRNAs, showing knockdown of the NPR-A gene 48 h after siRNA transfection. GAPDH was used as the internal control. (b) Real-time PCR analysis of ES cells treated with control siRNA or NPR-A siRNA2. (c) Western blot analysis of ES cells treated as described in panel a, showing a reduced level of the NPR-A protein 48 h after siRNA transfection. β-Actin was used as a loading control. Data represent mean±S.D. (n=3); *P<0.05 (two-tailed t-test)
Mentions: Both reverse transcription-PCR (RT-PCR) and real-time PCR revealed a marked reduction in the level of NPR-A mRNA at 48 h after transfection in ES cells, which were transfected with NPR-A-targeting siRNA (NPR-A siRNA), compared with ES cells that were transfected with a nontargeting siRNA (control siRNA) (Figures 1a and b). This finding was confirmed by a reduction in protein levels observed in western blots (Figure 1c).

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