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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 knockdown decreases ES pluripotency genes and upregulates differentiation genes. (a) Real-time PCR of mRNA levels of pluripotency genes (Oct4, Nanog and Sox2) after treatment of ES cells with control siRNA or NPR-A siRNA. (b) RT-PCR analysis of ES cells treated as described in panel a, showing expression of stem cell marker genes. GAPDH was used as the internal control. (c) Real-time PCR of ES cells treated as described in panel a, showing mRNA levels of the early differentiation genes, GATA-4 (GATA-binding protein 4), GATA-6 (GATA-binding protein 6), AFP (α-fetoprotein), Brachyury, nestin, Cdx2 (caudal-type homeobox 2), Hand1 (heart and neural crest derivatives expressed transcript 1) and Eomes (eomesodermin homolog). (d) RT-PCR analysis of ES cells treated as described in panel a, showing mRNA levels of the early differentiation genes. GAPDH was used as the internal control. Data represent mean±S.D. (n=3); *P<0.05 (two-tailed t-test)
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fig3: NPR-A knockdown decreases ES pluripotency genes and upregulates differentiation genes. (a) Real-time PCR of mRNA levels of pluripotency genes (Oct4, Nanog and Sox2) after treatment of ES cells with control siRNA or NPR-A siRNA. (b) RT-PCR analysis of ES cells treated as described in panel a, showing expression of stem cell marker genes. GAPDH was used as the internal control. (c) Real-time PCR of ES cells treated as described in panel a, showing mRNA levels of the early differentiation genes, GATA-4 (GATA-binding protein 4), GATA-6 (GATA-binding protein 6), AFP (α-fetoprotein), Brachyury, nestin, Cdx2 (caudal-type homeobox 2), Hand1 (heart and neural crest derivatives expressed transcript 1) and Eomes (eomesodermin homolog). (d) RT-PCR analysis of ES cells treated as described in panel a, showing mRNA levels of the early differentiation genes. GAPDH was used as the internal control. Data represent mean±S.D. (n=3); *P<0.05 (two-tailed t-test)

Mentions: To assess the differentiation state of NPR-A knockdown cells, we assayed for changes in the expression of key pluripotency genes (such as Oct4, Nanog and Sox2) that are considered a part of the core set of factors associated with the maintenance of pluripotency and self-renewal in ES cells.4, 5, 6, 7 RT-PCR and real-time PCR analyses of all these genes showed a decreased level of expression in NPR-A-deficient cells when compared with control siRNA-treated cells (Figures 3a and b). These results indicate that NPR-A has a role in regulating ES cell pluripotency genes.


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

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

NPR-A knockdown decreases ES pluripotency genes and upregulates differentiation genes. (a) Real-time PCR of mRNA levels of pluripotency genes (Oct4, Nanog and Sox2) after treatment of ES cells with control siRNA or NPR-A siRNA. (b) RT-PCR analysis of ES cells treated as described in panel a, showing expression of stem cell marker genes. GAPDH was used as the internal control. (c) Real-time PCR of ES cells treated as described in panel a, showing mRNA levels of the early differentiation genes, GATA-4 (GATA-binding protein 4), GATA-6 (GATA-binding protein 6), AFP (α-fetoprotein), Brachyury, nestin, Cdx2 (caudal-type homeobox 2), Hand1 (heart and neural crest derivatives expressed transcript 1) and Eomes (eomesodermin homolog). (d) RT-PCR analysis of ES cells treated as described in panel a, showing mRNA levels of the early differentiation genes. GAPDH was used as the internal control. Data represent mean±S.D. (n=3); *P<0.05 (two-tailed t-test)
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig3: NPR-A knockdown decreases ES pluripotency genes and upregulates differentiation genes. (a) Real-time PCR of mRNA levels of pluripotency genes (Oct4, Nanog and Sox2) after treatment of ES cells with control siRNA or NPR-A siRNA. (b) RT-PCR analysis of ES cells treated as described in panel a, showing expression of stem cell marker genes. GAPDH was used as the internal control. (c) Real-time PCR of ES cells treated as described in panel a, showing mRNA levels of the early differentiation genes, GATA-4 (GATA-binding protein 4), GATA-6 (GATA-binding protein 6), AFP (α-fetoprotein), Brachyury, nestin, Cdx2 (caudal-type homeobox 2), Hand1 (heart and neural crest derivatives expressed transcript 1) and Eomes (eomesodermin homolog). (d) RT-PCR analysis of ES cells treated as described in panel a, showing mRNA levels of the early differentiation genes. GAPDH was used as the internal control. Data represent mean±S.D. (n=3); *P<0.05 (two-tailed t-test)
Mentions: To assess the differentiation state of NPR-A knockdown cells, we assayed for changes in the expression of key pluripotency genes (such as Oct4, Nanog and Sox2) that are considered a part of the core set of factors associated with the maintenance of pluripotency and self-renewal in ES cells.4, 5, 6, 7 RT-PCR and real-time PCR analyses of all these genes showed a decreased level of expression in NPR-A-deficient cells when compared with control siRNA-treated cells (Figures 3a and b). These results indicate that NPR-A has a role in regulating ES cell pluripotency genes.

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
Related in: MedlinePlus