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

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.

Figure 1

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)

Figure 2

Knockdown of NPR-A reduces the potential for ES cell self-renewal. (a) Morphology of ES cells treated with control siRNA or NPR-A siRNAs at 2 days after transfection in the presence of LIF. (b) Self-renewal assay, AP-staining of ES cells treated as described in panel a, 4 days after transfection. (c) Immunofluorescence images of ES cells treated as in panel a for 72 h, stained with antibodies against Oct4 and counterstained with Hoechst reagent. (d) Western blot analysis of ES cells treated as in panel a, showing reduced protein levels of Oct4 and Nanog 72 h after siRNA transfection. β-Actin was used as a loading control. (e) Left panels, photographs of the formed colonies after 6 days from culturing at low density in the presence of LIF, stained with AP. AP staining of cells treated as in panel a. It must be noted that the staining intensity of NPR-A siRNA-treated cells is decreased. Right panels, high-magnification images of AP staining. (f) Graph showing quantification of the percentages of AP-positive colonies treated as described in panel e. Data represent mean±S.D. (n=2) and significant values are indicated; ^**P<0.01, compared with control (two-tailed t-test). Scale bar=50 μm

Figure 3

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)

Figure 4

NPR-A knockdown reduced ES cell propagation, prolonged the G1 phase and upregulated phosphorylated Akt in murine ES cells. (a) Quantification of ES cells 48 h after transfection with control siRNA or NPR-A siRNA (n=4). (b) Effect of NPR-A knockdown on cell-cycle progression, showing the percentages of cells in the G1, S and G2/M phases of the cell cycle. (c) Quantification of cell-cycle phases (n=3). (d) RT-PCR analysis of p21 and cyclin D1 mRNA 48 h after ES cell transfection with control siRNA or NPR-A siRNA. (e) Real-time PCR analysis of ES cells treated as described in panel d. (f) Western blot analysis for p21 48 h after siRNA transfection. (g) Western blot analysis for phosphorylated Akt (Ser473; p-Akt) and total Akt 48 h after siRNA transfection. β-Actin was used as a loading control. (h) Relative levels of the p-Akt protein were quantified after normalization to total Akt. Data represent mean±S.D. (n=3); ^*P<0.05 or ^**P<0.01 (two-tailed t-test)

Figure 5

ANP is expressed in murine ES cells, and exogenous ANP stimulates ES cell pluripotency factors. (a) Upper panels, double-immunofluorescence images of ES cells cultured in the presence of LIF, stained with antibodies against the ES cell markers, Oct4 and ANP. Lower panels, double-immunofluorescence images of ES cells cultured in the absence of LIF for 5 days, stained with antibodies against Nanog and ANP. It must be noted that ANP signals are downregulated upon differentiation. (b) Western blot analysis for ANP of ES cells treated as described in panels a and b. (c) Real-time PCR analysis of mRNA levels for pluripotency genes 12 h after treatment of ES cells with 2.5 μ ANP. (d) Western blot analysis of Oct4 and Nanog in ES cells treated with ANP for 12 or 24 h. β-Actin is shown as a control for loading. (e) Western blot analysis for phosphorylated Akt (Ser473; p-Akt) and total Akt 12 h after treatment of ES cells with ANP. (f) Relative levels of the p-Akt protein were quantified after normalized to total Akt. (g) ES cells were treated with LY294002, ANP or ANP+LY294002 for 3 days, followed by staining for alkaline phosphatase (AP). (h) Western blot analysis of Nanog in ES cells treated with LY294002 or ANP+LY294002 for 18 h. (i) Quantitative analysis of the western blots as shown in panel h. Data represent mean±S.D. (n=3). ^*P<0.05 or ^**P<0.01 (two-tailed t-test)

Figure 6

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)

Figure 7

Proposed model of NPR-A signaling-mediated maintenance of ES cell self-renewal and pluripotency. Activation of NPR-A by ANP stimulates the cGMP/PKG signaling pathway, leading to increased expressions of Oct4 and Nanog, and reduced expression of p21, which in turn suppresses ES cell differentiation and maintains ES cell pluripotency. In another pathway, binding of ANP to NPR-A activates the phosphoinositide 3-kinase (PI3K) signaling pathway, which enhances G1-to-S progression through induction of cyclin D1, and maintains pluripotency by activation of Nanog expression