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

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

3\u27-Phosphoadenosine 5\u27-Phosphosulfate Transporter (PAPST) Enhanced Sulfation of Keratan Sulfate Proteoglycan to Reduce Radiation-Induced Apoptosis in a Human Burkitt Lymphoma Cell Line

[Objectives] Sulfation of various molecules plays an essential role in biological and pathological processes.Two members of 3\u27-Phosphoadenosine 5\u27-phosphosulfate transporters (PAPSTs), PAPST1 and PAPAT2, arecurrently identified in humans. However, the influence of sulfate transporter on radiosensitvity of malignanttumors remains unknown. This study aimed at clarifying the role of sulfate transporter in radiation-inducedapoptosis of lymphoma cells.[Methods] A human Burkitt\u27 s lymphoma cell line, Namalwa, was transfected with an expression vector ofhuman PAPST1 or PAPST2, or siRNA targeted against each PAPST. Apoptosis was evaluated by Hoechst 33258staining 24 h after irradiation with X-rays or C-ion.[Results] The level of PAPST1 transcripts was approximately 5-fold higher than that of PAPST2 in Namalwacells. Overexpression of each PAPST reduced radiation-induced apoptosis, whereas the repression of PAPSTexpression enhanced apoptosis. In contrast, keratan sulafate proteoglycan (KSPG) was expressed on the cellsurface of Namalwa cells, and depletion of KS with keratanase significantly increased radiation-inducedapoptosis. Three (CHST2, 6, and 7) of five sulfotransferases involved in KS synthesis were expressed inNamalwa cells and the sulfation catalyzed by all three sulfotransferases promoted anti-apoptotic effects ofKSPG. PAPST1 enhanced phosphorylation of p38 MAPK and Akt in Namalwa cells, whereas inhibition of p38MAPK or PI-3K increased radiation-induced apoptosis.[Conclussions] PAPST inhibited radiation-induced apoptosis through sulfation of KSPG. These findingssuggest that KSPG plays an important role in anti-apoptotic signaling in human Burkitt\u27 s lymphoma, and PAPSTis useful to increase the efficacy of radiotherapy and decrease side effects through the regulation of KSPG.The 2nd Japan-China Symposium on Cancer Researc

Sulfation of keratan sulfate proteoglycan reduces radiation-induced apoptosis in human Burkitt\u27s lymphoma cell lines

This study focuses on clarifying the contribution of sulfation to radiation-induced apoptosis in human Burkitt\u27s lymphoma cell lines, using 3\u27-phosphoadenosine 5\u27-phosphosulfate transporters (PAPSTs). Overexpression of PAPST1 or PAPST2 reduced radiation-induced apoptosis in Namalwa cells, whereas the repression of PAPST1 expression enhanced apoptosis. Inhibition of PAPST slightly decreased keratan sulfate (KS) expression, so that depletion of KS significantly increased radiation-induced apoptosis. In addition, the repression of all three N-acetylglucosamine-6-O-sulfotransferases (CHST2, CHST6, and CHST7) increased apoptosis. In contrast, PAPST1 expression promoted the phosphorylation of p38 MAPK and Akt in irradiated Namalwa cells. These findings suggest that 6-O-sulfation of GlcNAc residues in KS reduces radiation-induced apoptosis of human Burkitt\u27s lymphoma cells

3-<em>O</em>-Sulfated Heparan Sulfate Recognized by the Antibody HS4C3 Contribute to the Differentiation of Mouse Embryonic Stem Cells via Fas Signaling

<div><p>Maintenance of self-renewal and pluripotency in mouse embryonic stem cells (mESCs) is regulated by the balance between several extrinsic signaling pathways. Recently, we demonstrated that heparan sulfate (HS) chains play important roles in the maintenance and differentiation of mESCs by regulating extrinsic signaling. Sulfated HS structures are modified by various sulfotransferases during development. However, the significance of specific HS structures during development remains unclear. Here, we show that 3-<em>O</em>-sulfated HS structures synthesized by HS 3-<em>O</em>-sulfotransferases (3OSTs) and recognized by the antibody HS4C3 increase during differentiation of mESCs. Furthermore, expression of Fas on the cell surface of the differentiated cells also increased. Overexpression of the HS4C3-binding epitope in mESCs induced apoptosis and spontaneous differentiation even in the presence of LIF and serum. These data showed that the HS4C3-binding epitope was required for differentiation of mESCs. Up-regulation of the HS4C3-binding epitope resulted in the recruitment of Fas from the cytoplasm to lipid rafts on the cell surface followed by activation of Fas signaling. Indeed, the HS4C3-binding epitope interacted with a region that included the heparin-binding domain (KLRRRVH) of Fas. Reduced self-renewal capability in cells overexpressing <em>3OST</em> resulted from the degradation of Nanog by activated caspase-3, which is downstream of Fas signaling, and was rescued by the inhibition of Fas signaling. We also found that knockdown of <em>3OST</em> and inhibition of Fas signaling reduced the potential for differentiation into the three germ layers during embryoid body formation. This is the first demonstration that activation of Fas signaling is mediated by an increase in the HS4C3-binding epitope and indicates a novel signaling pathway for differentiation in mESCs.</p> </div