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

E M Abdelalim,I Tooyama

doi:10.1038/cddis.2011.10

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.

Highlights

The pluripotent identity of Embryonic stem (ES) cells is controlled by a group of transcription factors.[4,5,6,7,8] Within this group, the transcription factors Oct[4], Nanog and Sox[2] contribute to the hallmark characteristics of ES cells by activation of target genes that encode pluripotency and self-renewal mechanisms, as well as by repression of signaling pathways that promote differentiation.[8]
Double-immunofluorescence analysis showed that Natriuretic peptide receptor A (NPR-A) was highly expressed in undifferentiated ES (Oct4-positive) cells, which were cultured in the presence of leukemia-inhibitory factor (LIF), and that expression was downregulated upon differentiation induced by culturing ES cells without LIF for 5 days
We investigated the effect of NPR-A knockdown on the phosphoinositide 3-kinase (PI3K) signaling pathway, which is critical for maintenance of murine ES cell pluripotency and selfrenewal,[25,26] by analyzing phosphorylation levels of Akt

Summary

Introduction

The pluripotent identity of ES cells is controlled by a group of transcription factors.[4,5,6,7,8] Within this group, the transcription factors Oct[4], Nanog and Sox[2] contribute to the hallmark characteristics of ES cells by activation of target genes that encode pluripotency and self-renewal mechanisms, as well as by repression of signaling pathways that promote differentiation.[8] In addition, ES cell pluripotency requires inputs from extrinsic factors and their downstream effectors.[9] the molecular mechanisms that control pluripotency and differentiation of ES cells are largely unknown. We showed that NPR-A is expressed at high levels in pre-implantation embryos and in undifferentiated murine ES cells, which is rapidly downregulated after their differentiation,[24] suggesting involvement of NPR-A in regulating early development. As no role has been established for NPR-A in ES cells, we aimed to selectively knock down NPR-A expression in murine

Objectives

Methods

Results

Conclusion