Initially, we examined the expression of BNP and its receptor, NPR-A, in murine ES cells grown under self-renewal and differentiation conditions (Fig. 1). Polymerase chain reaction with reverse transcription (RT-PCR) (Fig. 1A), Western blotting (Fig. 1B), double-immunofluorescence (Fig. 1C, D), and flow cytometry (Fig. 1E, F) analyses showed that BNP and NPR-A were highly expressed in pluripotent ES (Oct-4-positive) cells that were cultured in the presence of LIF, and that expression was down-regulated upon differentiation induced by culturing ES cells without LIF for 5 days (Fig. 1). The differentiated cells, which were negative for oct-4 expression, were also negative for BNP and NPR-A expressions (Fig. 1C, D). These results indicate that BNP and NPR-A are specifically expressed in self-renewing ES cells.

To determine whether BNP, NPR-A and NPR-B are expressed in pre-implantation embryos, 3.5-day-old murine blastocysts were subjected to double-immunofluorescence analysis using anti-BNP, anti-NPR-A, or anti-NPR-B antibodies, as well as an antibody against Oct-4 (self-renewal marker). Consistent with their presence in the undifferentiated ES cells, BNP, NPR-A and NPR-B were co-expressed with the self-renewal regulator Oct-4 in the blastocyst (Fig. 2A–C). These findings suggest that BNP exerts regulatory functions prior to embryo implantation and in undifferentiated ES cells.

BNP expression in undifferentiated ES cells and repression upon differentiation suggested that it may play a role in maintaining pluripotency. We investigated this possibility by examining the effect of BNP knockdown in ES cells. A small interfering RNA (siRNA)-based technique was employed to specifically knockdown the BNP gene in undifferentiated ES cells that were maintained in a feeder-free culture. Before siRNA transfection, the undifferentiated status of the murine ES cells used for transfection was confirmed by flow cytometry and immunofluorescence (for Oct-4-positive cells). RT-PCR revealed a marked reduction in the level of BNP mRNA at 48 h post-transfection in the ES cells that were transfected with BNP-targeting siRNA (BNP siRNA), as compared with the ES cells that were transfected with a non-targeting siRNA (control siRNA) (Fig. 3A). This finding was confirmed by the reductions in protein levels observed in Western blots (Fig. 3B, C). Knockdown of BNP significantly reduced the number and colony size of the ES cells, as compared with the ES cells that were treated with the control siRNA (Fig. 3D, E).

To validate further the observed decrease in ES cell proliferation after BNP knockdown, siRNA-transfected cells were exposed to bromo-deoxyuridine (BrdU), and its incorporation into ES cells was quantified by immunofluorescence and flow cytometry. ES cells were examined 48 h post-transfection and following pulsed incorporation of BrdU (45 min). Flow cytometric analysis of BrdU, which was performed concurrent with the analysis of the cellular DNA content, showed a marked reduction in BrdU incorporation in ES cells treated with BNP siRNA (Fig. 3F). Likewise, we observed a significant reduction in the number of BrdU-positive cells in the BNP siRNA-treated cells, as compared to the control siRNA-treated cells, as shown by immunofluorescence (Fig. 3G, H). These results suggest that knockdown of BNP signaling has a significant effect in decreasing the rate of DNA synthesis.

To assess directly the effect of BNP knockdown on the cell cycle profile, we analyzed the phases of the cell cycle 48 h after siRNA transfection. There was a significant reduction in the proportion of cells in S phase, and an increase in the proportion of cells in G1 and G2/M phases in ES cells treated with BNP siRNA (Fig. 4A, B). In contrast, there were no significant changes in the cell cycle profile of differentiated ES cells after BNP knockdown (Fig. 4C). These results suggest that inhibition of endogenous BNP suppresses DNA synthesis as a result of the reduction of the cells in the S phase in undifferentiated ES cells.

Furthermore, we examined the effect of exogenous BNP on ES cell proliferation. The presence of NPR-A in ES cells (Fig. 1A, D), suggests that BNP signaling can be transduced in ES cells via NPR-A. BNP addition to ES cells in low-density ES cell cultures, as previously reported [15], resulted in increased ES cell proliferation, as evidenced by the ES cell morphology (Supplemental Fig. 1A). However, the proliferative potency of exogenous BNP appeared to be lower than that of endogenously produced BNP, suggesting that the BNP/NPR-A signaling produced by ES cells masks the effect of exogenous BNP on ES cell proliferation.

The reduction in ES cell proliferation caused by abrogation of BNP signaling had no effect on the undifferentiated status of the ES cells, as determined by morphologic examination (Fig. 3E) and confirmed by measurements of alkaline phosphatase activity, which revealed that the levels of self-renewal in the control siRNA- and BNP siRNA–treated cells were identical (Fig. 5A). In addition, examination of the pluripotent ES cell markers Oct-4 and nanog 48 h post-transfection confirmed these results (Fig. 3A, 5B, C). Thus, ES cells treated with BNP siRNA expressed nanog mRNA (Fig. 3A, 5B) and Oct-4 mRNA and protein (Fig. 3A, 5C) at levels equivalent to those in ES cells treated with the control siRNA. Also, addition of BNP to ES cell cultures promotes ES cell propagation (Fig. S1A) without loss of pluripotent markers, as assessed by alkaline phosphatase activity 4 days after BNP addition (Fig. S1B). These findings indicate that reducing BNP does not affect ES cell pluripotent markers, and underline the role of BNP in undifferentiated murine ES cells rather than in spontaneously differentiated progeny.

In addition, we measured the intracellular levels of cGMP, to determine whether the cGMP pathway is involved in the effects of BNP knockdown on ES cells. The levels of cGMP were reduced significantly in ES cells treated with BNP siRNA, as compared to the levels in ES cells treated with the control siRNA (Fig. 6A). It is known that the natriuretic peptides -induced cGMP production is mainly due to the activation of NPR-A and/or NPR-B. Examination of NPR-A and NPR-B genes showed that NPR-B mRNA was markedly reduced in ES cells treated with BNP siRNA, as compared to the ES cells treated with the control siRNA, and no change was observed in the levels of NPR-A mRNA (Fig. 6B). These results suggest that the cGMP reduction was mainly due to the suppression of NPR-B gene.

To elucidate the mechanisms underlying the effects of BNP signaling on ES cell proliferation, we used Real-Time PCR to analyze the expression level of GABA_AR 48 h after siRNA transfection. We found that the expression levels of the GABA_AR α1 and β3 subunits, which are the major units of GABA_AR in ES cells, were significantly up-regulated (Fig. 7A) in response to the suppression of BNP in ES cells.

To determine whether the elevated levels of the GABA_AR subunits in BNP-knockdown ES cells are associated with the activation of γ-H2AX, as previously described [16], we examined the level of γ-H2AX by Western blotting. As expected for the presence of GABA_AR up-regulation, γ-H2AX was expressed more abundantly in ES cells treated with BNP siRNA than in those treated with control siRNA (Fig. 7B). Furthermore, immunofluorescence analysis revealed the accumulation of γ-H2AX nuclear foci in response to BNP knockdown (Fig. 7C).

Furthermore, activation of endogenous GABA_AR with muscimol (a GABA_AR agonist) in low-density cultures, significantly reduced the BNP mRNA (Fig. 7D) and protein (Fig. 7E) levels, which are associated with reductions in ES cell proliferation, suggesting a regulatory mechanism between endogenous BNP and GABA_AR signaling for the control of ES cell proliferation.

The reduced cell numbers and colony size that we observed in ES cells after BNP knockdown could be caused by increased cell death and/or decreased cell proliferation. Therefore, the apoptosis assay was performed using Annexin V as a marker for apoptotic cells. Flow cytometric analysis showed that the percentage of cells undergoing apoptosis significantly increased in the BNP siRNA-treated cells, as compared to the control siRNA-treated cells (Fig. 7F). These data indicate that BNP plays an important role in maintaining ES cell survival. However, it is known that GABA_AR activation does not cause apoptosis or DNA damage in ES cells. Therefore, we examined the phosphoinositide 3-kinase (PI3K) pathway, which is known to be involved in proliferation and growth of ES cells by stimulating the G1-S phase transition, and its inhibition induces apoptosis in ES cells [17] . Western blot (Fig. S2A–B) and flow cytometry (Fig. S2C) analyses revealed no change in the level of phospho-Akt (ser 473), a marker for PI3K, suggesting that BNP signaling does not interfere with PI3K pathway. Furthermore, we examined the mRNA levels of the transcription factor Ets-1, which is required for normal survival of cells. Using RT-PCR, we found that the expression levels of the Ets-1 significantly down-regulated (Fig. 7G) at 48 h and 72 h from transfection in ES cells treated with BNP siRNA, indicating that BNP is required for the activation of Ets-1 in ES cells.

Murine ES cells (E14TG2a) (CRL-1821; American Type Culture Collection, Manassas, VA, USA) were maintained in DMEM/F-12 medium (Sigma) that was supplemented with 1000 U/ml LIF (Chemicon), 11% FBS, 2 mM glutamine (Nacalai Tesque, Japan), 1 mM sodium pyruvate (Sigma), 1% MEM nonessential amino acids (GIBCO), 0.1 mM 2-mercaptoethanol (Sigma) and 1% penicillin-streptomycin. For siRNA transfection, ES cells were cultured in the same medium, except that FBS was replaced with 15% Knockout Serum Replacement (KSR; GIBCO). Murine ES cells were cultured under feeder-free conditions in the presence of LIF. For differentiation, embryoid bodies were grown in bacteriologic dishes without LIF and β-mercaptoethanol for 5 days, as described previously [33]. Murine BNP (American Peptide) and muscimol (Tocris) [16] were added at 1 µM and 100 µM, respectively, to the cultured ES cells, while the control cells received vehicle alone.

ES cells grown on glass coverslips were rinsed briefly with PBS and fixed for 20 min in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Blastocysts were collected from ICR mice at 3.5 dpc and fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). All experimental procedures and protocols were reviewed and approved by the Institutional Animal Care and Use Committee of the Shiga University of Medical Science, Otsu, Japan and conformed to the NIH Guide for the Care and Use of Laboratory Animals.

The ES cells and blastocysts were permeabilized for 10 min with 0.1% Triton X-100 in PBS, and blocked for 40 min with 4% BSA in PBS at room temperature (RT). They were then incubated at 4°C overnight with the following antibodies; anti-BNP (1∶1000; Chemicon), anti-NPR-A (1∶300; Abcam), anti-NPR-B (1∶200, Abcam), anti-Oct-4 (1∶200, sc-5279; Santa Cruz Biotechnology), and anti-γ-H2AX IgG (1: 500; Upstate). This was followed by incubation with the following secondary antibodies; Chromeo 546-labelled anti-mouse IgG (1∶1000; Active Motif Chromeon GmbH, Tegernheim, Germany), Alexa Fluor 488-labeled anti-mouse IgG, and Alexa Fluor 488-labeled anti-rabbit IgG (1∶500; Molecular Probes). Nuclei were counterstained with Hoechst 33342 (1 µg/ml) (Invitrogen). The slides were examined by confocal laser microscopy (C1si; Nikon, Japan) and the images were processed using the Nikon EZ-C1 viewer software.

Immunofluorescence analysis of BrdU incorporation was performed as described previously [34]. BrdU (1∶100; Invitrogen) was added to the culture medium 48 h after transfection, and the incubation was continued for an additional 45 min. Cells were incubated with Alexa Fluor 488-conjugated mouse anti-BrdU antibody (1∶200; Molecular Probes) in 2% BSA-PBS overnight at 4°C. The percentage of BrdU-positive nuclei relative to the total number of nuclei per field of vision was determined. A minimum of five fields of vision per coverslip was counted.

Total RNA was extracted using the RNeasy Mini kit (Qiagen) according to the manufacturer's instructions. cDNA synthesis was performed with 1 µg of total RNA using the Superscript III first-stand cDNA synthesis kit (Invitrogen). The cDNA (0.5 µg) was used as a template in a mixture that contained Ampli Taq Gold polymerase (Applied Biosystems). RT-PCR was performed with an annealing temperature of 58°C.

The cDNA samples were analyzed by Real-Time PCR in a LightCycler Real-Time PCR system (Roche Diagnostics) using the SYBR Premix Ex Taq II (Takara, Japan). Standard curves were generated for each Real-Time PCR run using serial 5-fold dilutions of samples that contained the gene of interest. Following PCR, the specificities and identities of the RT-PCR products were verified using melting curve analysis; this analysis distinguished specific PCR products from non-specific PCR products that arose from primer-dimer formation. The reactions were carried out in triplicates. The relative mRNA levels were determined from the appropriate standard curves and corrected for the β-actin mRNA levels.

The primer sequences (forward and reverse) were as follows: BNP, 5′-CTGAAGGTGCTGTCCCAGATG-3′ and 5′- GACGGATCCGATCCGGTC-3′; NPR-A, 5′-AGTACGCCAACAACCTGGAG-3′ , and AAGAGCTGTAAAGCCCACGA-3′; Oct-4, 5′-GGATGCTGTGAGCCAAGG-3′, and 5′-GAACAAAATGATGAGTGACAGACAG-3′ ; Nanog, 5′-CACCCACCCATGCTAGTCTT-3′ and 5′-ACCCTCAAACTCCTGGTCCT-3′ ; Ets-1, 5′-CATATCAGGTTAATGGAGCC-3′ and 5′-GTAGTCGAAGCTGTCATAGG-3′ ; GABA_AR α1, 5′-TTTGGGAGAGCGTGTAACTGAA-3′ and 5′-ACTCCATATCGTGGTCTGAAACTG-3′; GABA_AR β3, 5′-AATCAAAATCCCTGATCTAACCGA-3′ and 5′-AAGAGAGAAAAGGTGAATGGAAACA-3′; NPR-B, 5′-TCATGACAGCCCATGGGAAA-3′ and 5′-GGTGACAATGCAGATGTTGG-3′; β-actin, 5′-TGGTGGGCATGGGTCAGAAGGATTC-3′ and 5′-CATGGCTGGGGTGTTGAAGGTCTCA-3′.

RNA interference in murine ES cells was carried out according to the manufacturer's protocol using Lipofectamine 2000 (Invitrogen) in 6-well plates. Two pairs of siRNAs (Invitrogen) were designed for BNP (NM_008726) using the BLOCK-iT RNAi Designer software. BNP siRNA1 gave higher level of BNP knockdown and therefore used for all subsequent experiments. The appropriate siRNA negative control Duplex (Cat. No. 12935-300; Invitrogen) was selected based on the percentage G/C. The BNP siRNA and control siRNA were transfected at a final concentration of 40 µM for 24 h in triplicate for each treatment. At 48 h post-transfection, BNP knockdown was confirmed by RT-PCR and Western blotting. The sequences of BNP siRNAs were as follows:

BNP siRNA1: sense, 5′-CCCAGAGACAGCUCUUGAATT-3′; antisense, 5′- UUCAAGAGCUGUCUCUGGGTT-3′.

BNP siRNA2: sense, 5′-GGCACAAGAUAGACCGGAUTT-3′; antisense, 5′- AUCCGGUCUAUCUUGUGCCTT-3′.

Total protein extracts were prepared from ES cells, dissolved in SDS-PAGE buffer, and transferred to nitrocellulose membranes (Amersham Biosciences, Germany). Proteins were detected using antibodies against BNP (1∶2000; Chemicon), γ-H2AX (1: 1000; Upstate), phospho-Akt (Ser 473) (1: 1000, Cell Signaling), and β-actin (1∶6000, sc-47778; Santa Cruz Biotechnology). The secondary antibodies were: Histofine anti-rabbit IgG complex or Histofine anti-mouse IgG complex (1∶50; Nichirei, Japan). The blots were developed using the SuperSignal West Pico Chemiluminescent substrate (Pierce), and visualized using a LAS-3000 FujiFilm Lumino-Image Analyzer (FujiFilm, Tokyo, Japan).

Cells were fixed in 4% paraformaldehyde for 15 min at RT, and washed with PBS that contained 2% FBS. Cells were incubated for 3 min in 100% ethanol, and blocked in 2% BSA for 20 min at RT. For staining for Oct-4 or double-labeling for BNP and Oct-4 or NPR-A and Oct-4, the cells were incubated for 1 h on ice with the following primary antibodies: anti-BNP (1∶1000; Chemicon); anti-NPR-A (1∶200; Abcam); and anti-Oct-4 (1∶200; Santa Cruz Biotechnology). This was followed by incubation for 1 h at RT with Alexa Fluor 488-conjugated anti-rabbit antibody and/or Alexa Fluor 647-conjugated anti-mouse antibody (1∶200; Molecular Probes). Data acquisition was performed using a FACSCalibur (BD Biosciences) and the data were analyzed using the CellQuest program. At least 10,000 events were collected for each sample.

Staining for alkaline phosphatase (ALP) was performed at room temperature using an alkaline phosphatase kit (Chemicon), according to the manufacturer's instructions. During reaction, culture dishes were protected from drying and direct light. Images were observed under an inverted microscope (Nikon, Japan).

In order to determine the number of cells, the cells were washed with PBS and trypsinized from the culture dishes. The cell suspension was mixed with trypan blue solution, and the number of live cells was determined using a homocytometer. Cells failing to exclude the dye were considered nonviable.

Annexin V/propidium iodide (PI) staining was performed using flow cyometry according to the manufacturer's guidelines (MBL, USA). Briefly, ES cells were washed in ice-cold PBS. The cells were then resuspended in 500 µl of binding buffer and incubated with 5 µl of Annexin V-EGFP and 5 µl of PI for 10 min in the dark at room temperature. Flow cytometric analysis was immediately performed using a FACSCalibur (BD Biosciences).

Cells were lyzed with 0.1 M HCl containing 0.1% Triton X-100 at RT for 20 min. The lysates were centrifuged, and the levels of cGMP in the supernatants were measured with a cGMP enzyme immunoassay kit (Assay Designs).

The results are expressed as mean s.d., as indicated in the figure legends. Statistical significance was determined using the paired Student's t-test.