The 3′-Phosphoadenosine 5′-Phosphosulfate Transporters, PAPST1 and 2, Contribute to the Maintenance and Differentiation of Mouse Embryonic Stem Cells

Norihiko Sasaki,Hidenao Toyoda,Kazumi Hirano,Shoko Nishihara,Yasuo Suda,Tomomi Ichimiya,Akiko Kinoshita-Toyoda,Masahiro Wakao,Takuya Hirano,Jose Alberola-Ila

doi:10.1371/journal.pone.0008262

Abstract

Recently, we have identified two 3′-phosphoadenosine 5′-phosphosulfate (PAPS) transporters (PAPST1 and PAPST2), which contribute to PAPS transport into the Golgi, in both human and Drosophila. Mutation and RNA interference (RNAi) of the Drosophila PAPST have shown the importance of PAPST-dependent sulfation of carbohydrates and proteins during development. However, the functional roles of PAPST in mammals are largely unknown. Here, we investigated whether PAPST-dependent sulfation is involved in regulating signaling pathways required for the maintenance of mouse embryonic stem cells (mESCs), differentiation into the three germ layers, and neurogenesis. By using a yeast expression system, mouse PAPST1 and PAPST2 proteins were shown to have PAPS transport activity with an apparent Km value of 1.54 µM or 1.49 µM, respectively. RNAi-mediated knockdown of each PAPST induced the reduction of chondroitin sulfate (CS) chain sulfation as well as heparan sulfate (HS) chain sulfation, and inhibited mESC self-renewal due to defects in several signaling pathways. However, we suggest that these effects were due to reduced HS, not CS, chain sulfation, because knockdown of mouse N-deacetylase/N-sulfotransferase, which catalyzes the first step of HS sulfation, in mESCs gave similar results to those observed in PAPST-knockdown mESCs, but depletion of CS chains did not. On the other hand, during embryoid body formation, PAPST-knockdown mESCs exhibited abnormal differentiation, in particular neurogenesis was promoted, presumably due to the observed defects in BMP, FGF and Wnt signaling. The latter were reduced as a result of the reduction in both HS and CS chain sulfation. We propose that PAPST-dependent sulfation of HS or CS chains, which is regulated developmentally, regulates the extrinsic signaling required for the maintenance and normal differentiation of mESCs.

Highlights

Embryonic stem cells (ESCs) [1,2] are promising tools for biotechnology and possess key features that should allow their exploitation in the development of cell replacement therapies [3]
The P100 membrane fractions prepared from yeast cells that expressed PAPST1 or PAPST2 showed phosphoadenosine 59-phosphosulfate (PAPS) transport activity that was significantly higher than that observed in the control cells (Figure 1B)
We conclude that the defects observed in PAPS transporter (PAPST)-KD cells are due to a reduction in heparan sulfate (HS) chain sulfation. The knockdown of both PAPST1 and PAPST2 together had an additive effect on self-renewal, and it is likely that the further reduction of HS sulfation observed in the PAPST1+2-KD cells, as shown in Figure 2B, was responsible for this additive effect. These results demonstrate that the reduction of PAPST1- and PAPST2-dependent sulfation inhibit both selfrenewal and proliferation of mouse ESCs (mESCs), and this is presumably due to reduced levels of HS chain-dependent signaling

Summary

Introduction

Embryonic stem cells (ESCs) [1,2] are promising tools for biotechnology and possess key features that should allow their exploitation in the development of cell replacement therapies [3]. Leukemia inhibitory factor (LIF) [7,8], which is one of the known extrinsic factors, plays an important role in maintaining the self-renewal of mESCs via the activation of STAT3 [9,10,11,12] and induction of c-Myc [13]. Wnt/b-catenin signaling plays a role in the regulation of self-renewal of both mESCs and hESCs and this signaling is independent of LIF/STAT3 signaling [16,17,18,19]. The activation of Nanog by Wnt/b-catenin signaling can sustain ESC self-renewal without the use of feeder cells or treatment with LIF [16,17]

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Results

Conclusion