BNP Signaling Is Crucial For Embryonic Stem Cell Proliferation

Abstract

BackgroundEmbryonic stem (ES) cells have unlimited proliferation potential, and can differentiate into several cell types, which represent ideal sources for cell-based therapy. This high-level proliferative ability is attributed to an unusual type of cell cycle. The Signaling pathways that regulate the proliferation of ES cells are of great interest.Methodology/Principal FindingsIn this study, we show that murine ES cells specifically express brain natriuretic peptide (BNP), and its signaling is essential for ES cell proliferation. We found that BNP and its receptor (NPR-A, natriuretic peptide receptor-A) were highly expressed in self-renewing murine ES cells, whereas the levels were markedly reduced after ES cell differentiation by the withdrawal of LIF. Targeting of BNP with short interfering RNA (siRNA) resulted in the inhibition of ES cell proliferation, as indicated by a marked reduction in the cell number and colony size, a significant reduction in DNA synthesis, and decreased numbers of cells in S phase. BNP knockdown in ES cells led to the up-regulation of gamma-aminobutyric acid receptor A (GABAAR) genes, and activation of phosphorylated histone (γ-H2AX), which negatively affects ES cell proliferation. In addition, knockdown of BNP increased the rate of apoptosis and reduced the expression of the transcription factor Ets-1.Conclusions/SignificanceAppropriate BNP expression is essential for the maintenance of ES cell propagation. These findings establish BNP as a novel endogenous regulator of ES cell proliferation.

BNP is a Novel Regulator of Embryonic Stem Cell Proliferation

“…Immunofluorescence and flow-cytometry analyses showed a significant reduction in the number of BrdU-positive cells in the BNP siRNA-treated cells compared with control siRNA-treated cells. These findings suggest that knockdown of BNP signaling has a significant effect in decreasing the rate of DNA synthesis (Abdelalim and Tooyama, 2009). The role of BNP in proliferation has been observed in other studies Ogawa et al, 2004).…”

“…We found that NPR-A and NPR-B are expressed in undifferentiated ES cells and preimplantation embryos (Abdelalim and Tooyama, 2009), suggesting the involvement of particulate GCs in the signaling pathway of BNP in murine ES cells. The intracellular levels of cGMP were measured to determine whether the cGMP pathway is involved in the effects of BNP on ES cells.…”

“…5), which revealed that levels of self-renewal in the control siRNA-and BNP siRNA-treated cells were identical. Also, the expression levels of Oct4 mRNA and Nanog mRNA did not differ between control siRNA-treated cells and BNP siRNA-treated cells, suggesting that BNP signaling is not involved in the pluripotency of ES cells (Abdelalim and Tooyama, 2009). …”

“…2). The expression of BNP and NPR-A were co-localized to those of Oct4 (Abdelalim and Tooyama, 2009). Oct4 expression is required to maintain the pluripotent-cell population of the ICM and epiblast (Nichols et al, 1998).…”

“…Since ES cells are derived from the ICM of pre-implantation embryos, the expression of BNP and its receptor were examined in pre-implantation embryos (Abdelalim and Tooyama, 2009). The blastocyst stage of murine pre-implantation development occurs approximately 3.5 days post-fertilization.…”